David W Galbraith

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Books

• Galbraith, D. W., Bohnert, H. J., & Bourque, D. P. (1995). Methods in plant cell biology. Academic Press.
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  Subcellular Fractionation and Analysis of Function: D.W. Galbraith, J. Sheen, R.J. Grebenok, G.M. Lambert, and J. Sheen, Flow Cytometric Analysis of Transgene Expression in Higher Plants: Green Fluorescent Protein. W.F.J. Vermaas, Functional Effects of Structural Changes in Photosystem II as Measured by Chlorophyll Fluorescence Kinetics. J.-K. Zhu, R.A. Bressan, and P.M. Hasegawa, Determination of Protein Isoprenylation in Vitro and in Vivo. W.H. Outlaw, Jr., Extraction and Assay of Protein from Single Plant Cells. J. Feldwich, A. Vente, N. Campos, R. Zettl, and K. Palme, Photoaffinity Labeling and Strategies for Plasma Membrane Protein Purification. S. Lucretti and J. Dolezel, Cell Cycle Synchronization, Chromosome Isolation, and Flow-Sorting in Plants. W.L. Crosby and P. Schorr, Principles and Applications of Recombinant Antibody Phage Display Technology to Plant Biology. T.J. Guilfoyle, Isolation and Characterization of Plant Nuclei. J.C. Cushman, Isolation of Nuclei Suitable for in Vitro Transcriptional Studies. G.E. Schaller and N.D. DeWitt, Analysis of H+-ATPase and Other Proteins of the Arabidopsis Plasma Membrane. J.M. Ward and H. Sze, Isolation and Reconstitution of the Vacuolar H+-ATPase. J.M. Gualberto, H. Handa, and J.M. Grienenberger, Isolation and Fractionation of Plant Mitochondria and Chloroplasts: Specific Examples. H. Wein, B. Brady, and W.Z. Cande, Isolating the Plant Mitotic Appratus: A Procedure for Isolating Spindles from the Diatom Cylindrotheca fusiformis. C.A. Price, N. Hadjeb, L.A. Newman, L.A. Newman, and E.M. Reardon, Chromoplasts. E. Davies and S. Abe, Methods for Isolation and Analysis of Polyribosomes. S. Abe and E. Davies, Methods for Isolation and Analysis of the Cytoskeleton. B.L. Epel, B. Kuchuck, G. Kotlizky, S. Shurtz, M. Erlanger, and A. Yahalom, Isolation and Characterization of Plasmodesmata. K. Waegemannand J. Soll, Characterization and Isolation of the Chloroplast Protein Import Machinery. E. Glaser, C. Knorpp, M. Hugosson, and E. Stedingk, Macromolecular Movement into Mitochondria. J.C. Carrington, Targeting of Proteins to the Nucleus. A. Ceriotti, E. Pedrazzini, M. Silvestris, and A. Vitale, Import into the Endoplasmic Reticulum. J.W. Gillikin, E.P.B. Fontes, and R.S. Boston, Protein-Protein Interactions in the Endoplasmic Reticulum. G.J. Lee, Assaying Proteins for Molecular Chaperone Activity. J. Denecke and A. Vitale, The Use of Protoplasts to Study Protein Synthesis and Transport by the Plant Endomembrane System. M.E. Vayda, Assessment of Translational Regulation by Run-Off Translation of Polysomesin Vitro. Molecular Methods for Analysis of Cell Function in Vivo: G.W. Bates, Electroporation of Plant Protoplasts and Tissues. P. Christou, Particle Bombardment. C. Maas, C. Reichel, J. Schell, and H.-H. Steinbisse, Preparation and Transformation of Monocot Protoplasts. R. Fischer and R. Hain, Tobacco Protoplast Transformation and Use for Functional Analysis of Newly Isolated Genes and Gene Constructs. C. Gatz, Novel Inducible/Repressible Gene Systems. C. Suter-Crazzolara, M. Klemm, and B. Reiss, Reporter Genes. M.K. Thorsness and J.B. Nasrallah, Cell-Specific Ablation in Plants. P. Steinecke and P.H. Schreier, Ribozymes. R. Kunze, H. Fusswinkel, and S. Feldmar, Expression of Plant Proteins in Baculoviral and Bacterial Systems. R. Serrano and J.-M. Villalba, Expression and Localization of Plant Membrane Proteins in Saccharomyces. G. Galili, Y. Altschuler, and A. Ceriotti, Synthesis of Plant Proteins in Heterologous Systems: Xenopus laevis Oocytes. J.I. Schroeder, Heterologous Expression of Higher Plant Transport Proteins and Repression of Endogenous Ion Currents in Xenopus Oocytes. Chapter References. Subject Index.

Chapters

• Lambert, G. M., & Galbraith, D. W. (2020). Advances in the Flow Cytometric Characterization of Plant Cells and Tissues. In Flow Cytometry Applications in Cell Culture. CRC Press. doi:10.1201/9781003067467-18
• Galbraith, D. W. (2009). The Wonderland of Global Expression Profiling. In Molecular Genetic Approaches to Maize Improvement. Springer, Berlin, Heidelberg. doi:10.1007/978-3-540-68922-5_18
• Galbraith, D. W., & Bartos, J. (2005). Flow Cytometry and Cell Sorting in Plant Biotechnology. In Flow Cytometry for Biotechnology. Oxford University Press. doi:10.1093/OSO/9780195183146.003.0021
• Galbraith, D. W., & Lucretti, S. (2000). Large Particle Sorting. In Flow Cytometry and Cell Sorting. Springer, Berlin, Heidelberg. doi:10.1007/978-3-662-04129-1_25
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  The design of jet-in-air flow sortes has centered around the optimal analysis of mammalian cells, particularly those of the lymphoid system, for sensible reasons of commerce. Lymphoid cells have small diameters (ca. 10 μm [2, 3]) and are relatively robust. As a general rule, design parameters that are optimal for lymphoid cells are not suited for the analysis and sorting of larger cells (we operationally define large particles as cells or other objects that are larger, often much larger, than 10 μm). Large cells are found in individuals representing all kingdoms of living organisms, including mammalian species. Table 1 lists some examples restricted to the plant and animal kingdoms, although there are many other examples of large cells in the fungi, algae, and protozoa. Since flow cytometry and sorting provide unique ways of examining and quantitatively analyzing cells, it seems inevitable that interest will increase in the application of these techniques to large particles, including transgenic cells (see, for example, [6, 12]).
• Galbraith, D. W. (1991). Fluorescence-activated analysis and sorting of protoplasts and somatic hybrids. In Plant Tissue Culture Manual.. Springer, Dordrecht. doi:10.1007/978-94-009-0103-2_38
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  The first accounts of the application of flow cytometry and cell sorting to higher plant systems were published in 1982 (for reviews, see [3–5]). Since that time, a large variety of further reports have appeared covering such diverse subject areas as somatic hybridization, characterization of cell type, analysis of gene expression, cell cycle measurements, chromosome sorting, and secondary product accumulation. This diversity derives from a recognition of the unique analytical attributes of flow cytometry and cell sorting, which enable this technology to provide insights otherwise impossible using other techniques.
• Galbraith, D. W. (1984). CHAPTER 50 – Selection of Somatic Hybrid Cells by Fluorescence-Activated Cell Sorting. In Cell Culture and Somatic Cell Genetics of Plants. doi:10.1016/B978-0-12-715001-7.50057-3
• Galbraith, D. W. (1984). CHAPTER 83 – Flow Cytometric Analysis of the Cell Cycle. In Cell Culture and Somatic Cell Genetics of Plants. doi:10.1016/B978-0-12-715001-7.50090-1
• Galbraith, D. W. (1983). RAPID FLOW CYTOMETRIC ANALYSIS OF NUCLEAR DNA LEVELS IN HIGHER PLANTS. In Advances in Gene Technology: Molecular Genetics of Plants and Animals. doi:10.1016/B978-0-12-221480-6.50046-3

Journals/Publications

• Antoniadi, I., Skalický, V., Sun, G., Ma, W., Galbraith, D. W., Novák, O., & Ljung, K. (2021). Fluorescence activated cell sorting-A selective tool for plant cell isolation and analysis. Cytometry. Part A : the journal of the International Society for Analytical Cytology.
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  Instrumentation for flow cytometry and sorting is designed around the assumption that samples are single-cell suspensions. However, with few exceptions, higher plants comprise complex multicellular tissues and organs, in which the individual cells are held together by shared cell walls. Single-cell suspensions can be obtained through digestion of the cells walls and release of the so-called protoplasts (plants without their cell wall). Here we describe best practices for protoplast preparation, and for analysis through flow cytometry and cell sorting. Finally, the numerous downstream applications involving sorted protoplasts are discussed.
• Galbraith, D. W. (2021). From mouse to mouse-ear cress: nanomaterials as delivery carriers in plant biotechnology.. Exploration, 1(1), 9-20. doi:https://doi.org/10.1002/EXP.20210002
• Galbraith, D. W. (2021). Shapiro's Laws Revisited: Conventional and Unconventional Cytometry at CYTO2020.. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 99(2), 129-132. doi:10.1002/cyto.a.24228
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  Extracting relevant information from a very large excess of irrelevant debris. Judicious gating of PI-stained Arabidopsis leaf homogenates defines the position of a very minor proportion of nuclei within a two-dimensional frequency distribution, whose properties can then be finely dissected. From: Galbraith (2009). Cytometry Part A. © 2020 International Society for Advancement of Cytometry.
• Galbraith, D. W. (2021). Shapiro's Laws Revisited: Conventional and Unconventional Cytometry at CYTO2020. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 99(2), 129-132.
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  Extracting relevant information from a very large excess of irrelevant debris. Judicious gating of PI-stained Arabidopsis leaf homogenates defines the position of a very minor proportion of nuclei within a two-dimensional frequency distribution, whose properties can then be finely dissected. From: Galbraith (2009). Cytometry Part A. © 2020 International Society for Advancement of Cytometry.
• Galbraith, D. W. (2021). Validation of crowd-sourced plant genome size measurements. Cytometry. Part A : the journal of the International Society for Analytical Cytology.
• Galbraith, D. W., & Sun, G. (2021). Flow Cytometry and Sorting in Arabidopsis. Methods in molecular biology (Clifton, N.J.), 2200, 255-294.
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  Flow cytometry and sorting represents a valuable and mature experimental platform for the analysis of cellular populations. Applications involving higher plants started to emerge around 40 years ago and are now widely employed both to provide unique information regarding basic and applied questions in the biosciences and to advance agricultural productivity in practical ways. Further development of this platform is being actively pursued, and this promises additional progress in our understanding of the interactions of cells within complex tissues and organs. Higher plants offer unique challenges in terms of flow cytometric analysis, first since their organs and tissues are, almost without exception, three-dimensional assemblies of different cell types held together by tough cell walls, and, second, because individual plant cells are generally larger than those of mammals.This chapter, which updates work last reviewed in 2014 [Galbraith DW (2014) Flow cytometry and sorting in Arabidopsis. In: Sanchez Serrano JJ, Salinas J (eds) Arabidopsis Protocols, 3rd ed. Methods in molecular biology, vol 1062. Humana Press, Totowa, pp 509-537], describes the application of techniques of flow cytometry and sorting to the model plant species Arabidopsis thaliana, in particular emphasizing (a) fluorescence labeling in vivo of specific cell types and of subcellular components, (b) analysis using both conventional cytometers and spectral analyzers, (c) fluorescence-activated sorting of protoplasts and nuclei, and (d) transcriptome analyses using sorted protoplasts and nuclei, focusing on population analyses at the level of single protoplasts and nuclei. Since this is an update, details of new experimental methods are emphasized.
• Sliwinska, E., Loureiro, J., Leitch, I. J., Šmarda, P., Bainard, J., Bureš, P., Chumová, Z., Horová, L., Koutecký, P., Lučanová, M., Trávníček, P., & Galbraith, D. W. (2021). Application-based guidelines for best practices in plant flow cytometry. Cytometry. Part A : the journal of the International Society for Analytical Cytology.
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  Flow cytometry (FCM) is currently the most widely-used method to establish nuclear DNA content in plants. Since simple, 1-3-parameter, flow cytometers, which are sufficient for most plant applications, are commercially available at a reasonable price, the number of laboratories equipped with these instruments, and consequently new FCM users, has greatly increased over the last decade. This paper meets an urgent need for comprehensive recommendations for best practices in FCM for different plant science applications. We discuss advantages and limitations of establishing plant ploidy, genome size, DNA base composition, cell cycle activity, and level of endoreduplication. Applications of such measurements in plant systematics, ecology, molecular biology research, reproduction biology, tissue cultures, plant breeding, and seed sciences are described. Advice is included on how to obtain accurate and reliable results, as well as how to manage troubleshooting that may occur during sample preparation, cytometric measurements, and data handling. Each section is followed by best practice recommendations; tips as to what specific information should be provided in FCM papers are also provided.
• Sun, G., & Galbraith, D. W. (2021). Flow Cytometry and Sorting in Arabidopsis.. Methods in molecular biology (Clifton, N.J.), 2200, 255-294. doi:10.1007/978-1-0716-0880-7_12
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  Flow cytometry and sorting represents a valuable and mature experimental platform for the analysis of cellular populations. Applications involving higher plants started to emerge around 40 years ago and are now widely employed both to provide unique information regarding basic and applied questions in the biosciences and to advance agricultural productivity in practical ways. Further development of this platform is being actively pursued, and this promises additional progress in our understanding of the interactions of cells within complex tissues and organs. Higher plants offer unique challenges in terms of flow cytometric analysis, first since their organs and tissues are, almost without exception, three-dimensional assemblies of different cell types held together by tough cell walls, and, second, because individual plant cells are generally larger than those of mammals.This chapter, which updates work last reviewed in 2014 [Galbraith DW (2014) Flow cytometry and sorting in Arabidopsis. In: Sanchez Serrano JJ, Salinas J (eds) Arabidopsis Protocols, 3rd ed. Methods in molecular biology, vol 1062. Humana Press, Totowa, pp 509-537], describes the application of techniques of flow cytometry and sorting to the model plant species Arabidopsis thaliana, in particular emphasizing (a) fluorescence labeling in vivo of specific cell types and of subcellular components, (b) analysis using both conventional cytometers and spectral analyzers, (c) fluorescence-activated sorting of protoplasts and nuclei, and (d) transcriptome analyses using sorted protoplasts and nuclei, focusing on population analyses at the level of single protoplasts and nuclei. Since this is an update, details of new experimental methods are emphasized.
• Čertnerová, D., & Galbraith, D. W. (2021). Best practices in the flow cytometry of microalgae. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 99(4), 359-364.
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  Microalgae are photosynthetic microorganisms with a major influence on global ecosystems. Further, owing to the production of various secondary metabolites, microalgae are also intensively studied for their enormous potential in biotechnology and its applications. While flow cytometry (FCM) is a fast and reliable method particularly suitable for genome size estimation in plant and animal studies, its application to microalgae often comes with many methodological challenges due to specific issues (e.g., cell wall composition, and presence of various secondary metabolites). Sample preparation requires considerable amounts of biomass, chemical fixation, and/or extraction of cellular components. In genome size estimation, appropriate methods for isolation of intact nuclei (using lysis buffers, razor-blade chopping, various enzymes, or bead-beating of cells) are essential for successful and high-quality analyses. Nuclear DNA amounts of microalgae diverge greatly, varying by almost 30,000-fold (0.01 to 286 pg). Even though new algal reference standards for genome size are now being introduced, animal red blood cells and nuclei from plant tissues are still predominantly used. Due to our limited knowledge of microalgal life cycles, particular caution should be taken during 1C/2C-value (or ploidy level) assignments.
• Galbraith, D. W., Shi, Z., Halaly-Basha, T., Zheng, C., Wang, C., Ophir, R., Pang, X., & Or, E. (2020). Identification of post-ethylene events in the signaling cascade induced by dormancy release stimuli.. Plant Journal, 104(5), 1251-1268. doi:DOI:10.1111/tpj.14997.
• Galbraith, D. W., Yan, Q., Bai, S., Li, W., Zhou, Y., Yang, Z., Sun, G., & Wang, B. (2020). Transcriptome analysis reveals key genes involved in the regulation of nicotine biosynthesis at early time points after topping in tobacco (Nicotiana tabacum L.).. B.M.C. Plant Biology, 20, Art. No. 30. doi:https://doi.org/10.1186/s12870-020-2241-9
• Munawar, N., Mahmood, T., & Galbraith, D. W. (2020). Chemical control of rodents and its impact on rodent infestations during subsequent cropping season. International Journal of Pest Management, 1-10. doi:10.1080/09670874.2020.1861362
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  The control of rodent pests with novel rodenticides is an important strategy subsequently they are cost-effective in reduction of agriculture damage in the urban environment. The current field stud...
• Qin, Y., Bai, S., Li, W., Sun, T., Galbraith, D. W., Yang, Z., Zhou, Y., Sun, G., & Wang, B. (2020). Transcriptome analysis reveals key genes involved in the regulation of nicotine biosynthesis at early time points after topping in tobacco (Nicotiana tabacum L.). BMC plant biology, 20(1), 30.
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  Nicotiana tabacum is an important economic crop. Topping, a common agricultural practice employed with flue-cured tobacco, is designed to increase leaf nicotine contents by increasing nicotine biosynthesis in roots. Many genes are found to be differentially expressed in response to topping, particularly genes involved in nicotine biosynthesis, but comprehensive analyses of early transcriptional responses induced by topping are not yet available. To develop a detailed understanding of the mechanisms regulating nicotine biosynthesis after topping, we have sequenced the transcriptomes of Nicotiana tabacum roots at seven time points following topping.
• Shahid, A. A., Rao, A. Q., Latif, A., Jabbar, B., Iqbal, A., Husnain, T., Gul, A., Galbraith, D. W., Ali, M. A., & Ahmed, M. (2020). Structure-based prediction of protein–protein interactions between GhWlim5 Domain1 and GhACTIN-1 proteins: a practical evidence with improved fibre strength. Journal of Plant Biochemistry and Biotechnology, 1-14. doi:10.1007/s13562-020-00603-7
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  Cotton fibre quality is a multigenic trait. Genetic modification of different genes to achieve high quality fibre is difficult without knowing the mechanism lying behind genes interaction. Based on background knowledge an attempt to explore the potential structural interactions between Gossypium hirsutum Wlim5 domain1 and Gossypium hirsutum ACTIN-1 proteins was done in current study. Sequence features of the LIM domain1 of GhWlim5 protein were identified through multiple sequence alignment analysis, and a phylogenetic tree was built to identify evolutionary relationships between sequences. Conservation indicated the evolutionary importance of side chain residues and the presence of several aliphatic and/or bulky residues, which stabilize the protein core and facilitate packing of zinc fingers. The structures of GhWlim5 domain1 and GhACTIN-1 proteins were modelled and validated through computational methods. Validation of GhACTIN-1 and GhWlim5 domain1 structures indicated good structural quality with 99.7% and 100% of the favoured number of residues in allowed regions and Z-score, within the ranges of − 9.87 and − 4.17, respectively. Docking analysis indicated various possible modes of interaction between these two proteins with favourable binding affinities. Based on our strong binding interaction results between GhWlim5 domain1 and GhACTIN-1 proteins, we further investigated the role of over-expression of GhWlim5 by transformation in cotton plants under fibre specific promoter and transgenic plants displayed significant increases in fibre strength.
• Shi, Z., Halaly-Basha, T., Zheng, C., Sharabi-Schwager, M., Wang, C., Galbraith, D. W., Ophir, R., Pang, X., & Or, E. (2020). Identification of potential post-ethylene events in the signaling cascade induced by stimuli of bud dormancy release in grapevine. The Plant journal : for cell and molecular biology, 104(5), 1251-1268.
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  Ethylene signaling appears critical for grape bud dormancy release. We therefore focused on identification and characterization of potential downstream targets and events, assuming that they participate in the regulation of dormancy release. Because ethylene responding factors (ERF) are natural candidates for targets of ethylene signaling, we initially characterized the behavior of two VvERF-VIIs, which we identified within a gene set induced by dormancy release stimuli. As expected, these VvERF-VIIs are localized within the nucleus, and are stabilized upon decreases in oxygen availability within the dormant buds. Less expected, the proteins are also stabilized upon hydrogen cyanamide (HC) application under normoxic conditions, and their levels peak at deepest dormancy under vineyard conditions. We proceeded to catalog the response of all bud-expressed ERFs, and identified additional ERFs that respond similarly to ethylene, HC, azide and hypoxia. We also identified a core set of genes that are similarly affected by treatment with ethylene and with various dormancy release stimuli. Interestingly, the functional annotations of this core set center around response to energy crisis and renewal of energy resources via autophagy-mediated catabolism. Because ERF-VIIs are stabilized under energy shortage and reshape cell metabolism to allow energy regeneration, we propose that: (i) the availability of VvERF-VIIs is a consequence of an energy crisis within the bud; (ii) VvERF-VIIs function as part of an energy-regenerating mechanism, which activates anaerobic metabolism and autophagy-mediated macromolecule catabolism; and (iii) activation of catabolism serves as the mandatory switch and the driving force for activation of the growth-inhibited meristem during bud-break.
• Zheng, C., Wang, C., Shi, Z., Sharabi-schwager, M., Pang, X., Or, E., Ophir, R., Halaly-basha, T., & Galbraith, D. W. (2020). Identification of potential post-ethylene events in the signaling cascade induced by stimuli of bud dormancy release in grapevine.. The Plant journal : for cell and molecular biology, 104(5), 1251-1268. doi:10.1111/tpj.14997
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  Ethylene signaling appears critical for grape bud dormancy release. We therefore focused on identification and characterization of potential downstream targets and events, assuming that they participate in the regulation of dormancy release. Because ethylene responding factors (ERF) are natural candidates for targets of ethylene signaling, we initially characterized the behavior of two VvERF-VIIs, which we identified within a gene set induced by dormancy release stimuli. As expected, these VvERF-VIIs are localized within the nucleus, and are stabilized upon decreases in oxygen availability within the dormant buds. Less expected, the proteins are also stabilized upon hydrogen cyanamide (HC) application under normoxic conditions, and their levels peak at deepest dormancy under vineyard conditions. We proceeded to catalog the response of all bud-expressed ERFs, and identified additional ERFs that respond similarly to ethylene, HC, azide and hypoxia. We also identified a core set of genes that are similarly affected by treatment with ethylene and with various dormancy release stimuli. Interestingly, the functional annotations of this core set center around response to energy crisis and renewal of energy resources via autophagy-mediated catabolism. Because ERF-VIIs are stabilized under energy shortage and reshape cell metabolism to allow energy regeneration, we propose that: (i) the availability of VvERF-VIIs is a consequence of an energy crisis within the bud; (ii) VvERF-VIIs function as part of an energy-regenerating mechanism, which activates anaerobic metabolism and autophagy-mediated macromolecule catabolism; and (iii) activation of catabolism serves as the mandatory switch and the driving force for activation of the growth-inhibited meristem during bud-break.
• Zhou, Y., Yang, Z., Wang, B., Sun, T., Sun, G., Qin, Y., Li, W., Galbraith, D. W., & Bai, S. (2020). Transcriptome analysis reveals key genes involved in the regulation of nicotine biosynthesis at early time points after topping in tobacco (Nicotiana tabacum L.).. BMC plant biology, 20(1), 30. doi:10.1186/s12870-020-2241-9
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  Nicotiana tabacum is an important economic crop. Topping, a common agricultural practice employed with flue-cured tobacco, is designed to increase leaf nicotine contents by increasing nicotine biosynthesis in roots. Many genes are found to be differentially expressed in response to topping, particularly genes involved in nicotine biosynthesis, but comprehensive analyses of early transcriptional responses induced by topping are not yet available. To develop a detailed understanding of the mechanisms regulating nicotine biosynthesis after topping, we have sequenced the transcriptomes of Nicotiana tabacum roots at seven time points following topping..Differential expression analysis revealed that 4830 genes responded to topping across all time points. Amongst these, nine gene families involved in nicotine biosynthesis and two gene families involved in nicotine transport showed significant changes during the immediate 24 h period following topping. No obvious preference to the parental species was detected in the differentially expressed genes (DEGs). Significant changes in transcript levels of nine genes involved in nicotine biosynthesis and phytohormone signal transduction were validated by qRT-PCR assays. 549 genes encoding transcription factors (TFs), found to exhibit significant changes in gene expression after topping, formed 15 clusters based on similarities of their transcript level time-course profiles. 336 DEGs involved in phytohormone signal transduction, including genes functionally related to the phytohormones jasmonic acid, abscisic acid, auxin, ethylene, and gibberellin, were identified at the earliest time point after topping..Our research provides the first detailed analysis of the early transcriptional responses to topping in N. tabacum, and identifies excellent candidates for further detailed studies concerning the regulation of nicotine biosynthesis in tobacco roots.
• Cossarizza, A., Chang, H., Radbruch, A., Acs, A., Adam, D., Adam-Klages, S., Agace, W. W., Aghaeepour, N., Akdis, M., Allez, M., Almeida, L. N., Alvisi, G., Anderson, G., Andrae, I., Annunziato, F., Anselmo, A., Bacher, P., Baldari, C. T., Bari, S., , Barnaba, V., et al. (2019). Guidelines for the use of flow cytometry and cell sorting in immunological studies (second edition). EUROPEAN JOURNAL OF IMMUNOLOGY, 49(10), 1457-1973.
• Santos, B. D., Morones-Ramirez, J. R., Balderas-Renteria, I., Casillas-Vega, N. G., Galbraith, D. W., & Zarate, X. (2019). Optimizing Periplasmic Expression in Escherichia coli for the Production of Recombinant Proteins Tagged with the Small Metal-Binding Protein SmbP. Molecular biotechnology, 61(6), 451-460.
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  We have previously shown that the small metal-binding protein (SmbP) extracted from the gram-negative bacterium Nitrosomonas europaea can be employed as a fusion protein for the expression and purification of recombinant proteins in Escherichia coli. With the goal of increasing the amounts of SmbP-tagged proteins produced in the E. coli periplasm, we replaced the native SmbP signal peptide with three different signal sequences: two were from the proteins CusF and PelB, for transport via the Sec pathway, and one was the signal peptide from TorA, for transport via the Tat pathway. Expression of SmbP-tagged Red Fluorescent Protein (RFP) using these three alternative signal peptides individually showed a considerable increase in protein levels in the periplasm of E. coli as compared to its level using the SmbP signal sequence. Therefore, for routine periplasmic expression and purification of recombinant proteins in E. coli, we highly recommend the use of the fusion proteins PelB-SmbP or CusF-SmbP, since these signal sequences increase periplasmic production considerably as compared to the wild-type. Our work, finally, demonstrates that periplasmic expression for SmbP-tagged proteins is not limited to the Sec pathway, in that the TorA-SmbP construct can export reasonable quantities of folded proteins to the periplasm. Although the Sec route has been the most widely used, sometimes, depending on the nature of the protein of interest, for example, if it contains cofactors, it is more appropriate to consider using the Tat route over the Sec. SmbP therefore can be recommended in terms of its particular versatility when combined with signal peptides for the two different routes.
• Wang, H., Guo, S., Qiao, X., Guo, J., Li, Z., Zhou, Y., Bai, S., Gao, Z., Wang, D., Wang, P., Galbraith, D. W., & Song, C. (2019). BZU2/ZmMUTE controls symmetrical division of guard mother cell and specifies neighbor cell fate in maize. PLOS GENETICS, 15(8).
• Wang, H., Guo, S., Qiao, X., Guo, J., Li, Z., Zhou, Y., Bai, S., Gao, Z., Wang, D., Wang, P., Galbraith, D. W., & Song, C. P. (2019). BZU2/ZmMUTE controls symmetrical division of guard mother cell and specifies neighbor cell fate in maize. PLoS genetics, 15(8), e1008377.
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  Intercellular communication in adjacent cell layers determines cell fate and polarity, thus orchestrating tissue specification and differentiation. Here we use the maize stomatal apparatus as a model to investigate cell fate determination. Mutations in ZmBZU2 (bizui2, bzu2) confer a complete absence of subsidiary cells (SCs) and normal guard cells (GCs), leading to failure of formation of mature stomatal complexes. Nuclear polarization and actin accumulation at the interface between subsidiary mother cells (SMCs) and guard mother cells (GMCs), an essential pre-requisite for asymmetric cell division, did not occur in Zmbzu2 mutants. ZmBZU2 encodes a basic helix-loop-helix (bHLH) transcription factor, which is an ortholog of AtMUTE in Arabidopsis (BZU2/ZmMUTE). We found that a number of genes implicated in stomatal development are transcriptionally regulated by BZU2/ZmMUTE. In particular, BZU2/ZmMUTE directly binds to the promoters of PAN1 and PAN2, two early regulators of protodermal cell fate and SMC polarization, consistent with the low levels of transcription of these genes observed in bzu2-1 mutants. BZU2/ZmMUTE has the cell-to-cell mobility characteristic similar to that of BdMUTE in Brachypodium distachyon. Unexpectedly, BZU2/ZmMUTE is expressed in GMC from the asymmetric division stage to the GMC division stage, and especially in the SMC establishment stage. Taken together, these data imply that BZU2/ZmMUTE is required for early events in SMC polarization and differentiation as well as for the last symmetrical division of GMCs to produce the two GCs, and is a master determinant of the cell fate of its neighbors through cell-to-cell communication.
• Bhosale, R., Boudolf, V., Cuevas, F., Lu, R., Eekhout, T., Hu, Z., Van Isterdael, G., Lambert, G. M., Xu, F., Nowack, M. K., Smith, R. S., Vercauteren, I., De Rycke, R., Storme, V., Beeckman, T., Larkin, J. C., Kremer, A., Höfte, H., Galbraith, D. W., , Kumpf, R. P., et al. (2018). A Spatiotemporal DNA Endoploidy Map of the Arabidopsis Root Reveals Roles for the Endocycle in Root Development and Stress Adaptation. The Plant cell, 30(10), 2330-2351.
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  Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments.
• Galbraith, D. W., Bhosale, R., Boudolf, V., Cuevasa, F., Kumpf, R., Hu, Z., Van Isterdael, G., Lambert, G., Nowack, M., Smith, R., Vercauteren, I., De Rycke, R., Storme, V., Beeckman, T., Larkin, J., Kremer, A., Höfte, H., Kumpf, R., Maere, S., & De Veylder, L. (2018). A spatiotemporal DNA endoploidy map of the Arabidopsis root reveals roles for the endocycle in root development and stress adaptation.. Plant Cell.
• Galbraith, D. W., Shi, Z., Halaly, T., Zheng, C., Weissberg, M., Ophir, R., Pang, X., & Or, E. (2018). Transient induction of a subset of ethylene biosynthesis genes is potentially involved in regulation of grapevine bud dormancy release.. Plant Molecular Biology, 98(Issue 6), 507-523. doi:DOI:10.1007/s11103-018-0793-y (Epub 2018 Nov 03).
• Galbraith, D. W., Sliwinska, E., & Samadder, P. (2018). Nuclear Cytometry: Analysis of the Patterns of DNA Synthesis and Transcription Using Flow Cytometry, Confocal Microscopy, and RNA Sequencing. Methods in molecular biology (Clifton, N.J.), 1678, 371-392.
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  Eukaryotes are defined by cells that contain a nucleus and other membrane-bound organelles. Cytometric analysis in situ, utilizing imaging, provides a useful understanding of the structure and function of the various subcellular components, particularly when combined with methods that preserve the living state. In terms of information provided by the observation of eukaryotic nuclei, imaging has provided a wealth of information about cellular multiplication. When organisms are present in multicellular form (tissues and organs), this property does not generally confound imaging cytometry. Multicellular eukaryotic species present immediate problems when being considered for analysis using flow cytometry which requires suspensions of single particles. Although some eukaryotic cell types exist as natural single cell suspensions (cf. the erythropoietic system), for other tissues and organs, strategies are required to produce single particle suspensions. This chapter illustrates the application of flow cytometry combined with confocal microscopy to analyze complex organs, focusing on properties of the plant nucleus, and then goes on to describe how suspensions of nuclei can be prepared from tissues and organs, and used for flow cytometric analysis of cellular and transcriptional states. The application of these techniques to animal species is also discussed with the implication that this strategy is universally applicable for the characterization of nuclei within tissues that cannot readily be converted into suspensions of cells.
• Shi, Z., Halaly-Basha, T., Zheng, C., Weissberg, M., Ophir, R., Galbraith, D. W., Pang, X., & Or, E. (2018). Transient induction of a subset of ethylene biosynthesis genes is potentially involved in regulation of grapevine bud dormancy release. Plant molecular biology, 98(6), 507-523.
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  Transient increases in ethylene biosynthesis, achieved by tight regulation of transcription of specific ACC oxidase and ACC synthase genes, play a role in activation of grapevine bud dormancy release. The molecular mechanisms regulating dormancy release in grapevine buds are as yet unclear. It has been hypothesized that its core involves perturbation of respiration which induces an interplay between ethylene and ABA metabolism that removes repression and allows regrowth. Roles for hypoxia and ABA metabolism in this process have been previously supported. The potential involvement of ethylene biosynthesis in regulation of dormancy release, which has received little attention so far, is now explored. Our results indicate that (1) ethylene biosynthesis is induced by hydrogen cyanamide (HC) and azide (AZ), known artificial stimuli of dormancy release, (2) inhibitors of ethylene biosynthesis and signalling antagonize dormancy release by HC/AZ treatments, (3) ethylene application induces dormancy release, (4) there are two sets of bud-expressed ethylene biosynthesis genes which are differentially regulated, (5) only one set is transiently upregulated by HC/AZ and during the natural dormancy cycle, concomitant with changes in ethylene levels, and (6) levels of ACC oxidase transcripts and ethylene sharply decrease during natural dormancy release, whereas ACC accumulates. Given these results, we propose that transient increases in ethylene biosynthesis prior to dormancy release, achieved primarily by regulation of transcription of specific ACC oxidase genes, play a role in activation of dormancy release.
• Zheng, C., Acheampong, A. K., Shi, Z., Mugzech, A., Halaly-Basha, T., Shaya, F., Sun, Y., Colova, V., Mosquna, A., Ophir, R., Galbraith, D. W., & Or, E. (2018). Abscisic acid catabolism enhances dormancy release of grapevine buds. Plant, cell & environment, 41(10), 2490-2503.
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  The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It was formerly proposed that dormancy is maintained by abscisic acid (ABA)-mediated repression of bud-meristem activity and that removal of this repression triggers dormancy release. It was also proposed that such removal of repression may be achieved via natural or artificial up-regulation of VvA8H-CYP707A4, which encodes ABA 8'-hydroxylase, and is the most highly expressed paralog in grapevine buds. The current study further examines these assumptions, and its experiments reveal that (a) hypoxia and ethylene, stimuli of bud dormancy release, enhance expression of VvA8H-CYP707A4 within grape buds, (b) the VvA8H-CYP707A4 protein accumulates during the natural transition to the dormancy release stage, and (c) transgenic vines overexpressing VvA8H-CYP707A4 exhibit increased ABA catabolism and significant enhancement of bud break in controlled and natural environments and longer basal summer laterals. The results suggest that VvA8H-CYP707A4 functions as an ABA degrading enzyme, and are consistent with a model in which the VvA8H-CYP707A4 level in the bud is up-regulated by natural and artificial bud break stimuli, which leads to increased ABA degradation capacity, removal of endogenous ABA-mediated repression, and enhanced regrowth. Interestingly, it also hints at sharing of regulatory steps between latent and lateral bud outgrowth.
• Zheng, C., Acheampong, A., Shi, Z., Halaly, T., Kamiya, Y., Ophir, R., Galbraith, D. W., & Or, E. (2018). Distinct functions for gibberellin during and after grape bud dormancy release. Journal of Experimental Botany, 69, 1635-1648.
• Zheng, C., Acheampong, A., Shi, Z., Mugzech, A., Halaly, T., Shaya, F., Colova, V., Ophir, R., Galbraith, D. W., & Or, E. (2018). ABA catabolism enhances dormancy release of grapevine buds.. Plant Cell & Environment, 41, 2490-2503.
• Zheng, C., Kwame Acheampong, A., Shi, Z., Halaly, T., Kamiya, Y., Ophir, R., Galbraith, D. W., & Or, E. (2018). Distinct gibberellin functions during and after grapevine bud dormancy release. Journal of experimental botany, 69(7), 1635-1648.
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  The molecular mechanism regulating dormancy release in grapevine buds is as yet unclear. It has been hypothesized that (i) abscisic acid (ABA) represses bud-meristem activity; (ii) perturbation of respiration induces an interplay between ethylene and ABA metabolism, which leads to removal of repression; and (iii) gibberellin (GA)-mediated growth is resumed. The first two hypothesis have been formally supported. The current study examines the third hypothesis regarding the potential involvement of GA in dormancy release. We found that during natural dormancy induction, levels of VvGA3ox, VvGA20ox, and VvGASA2 transcripts and of GA1 were decreased. However, during dormancy release, expression of these genes was enhanced, accompanied by decreased expression of the bud-expressed GA-deactivating VvGA2ox. Despite indications for its positive role during natural dormancy release, GA application had inhibitory effects on bud break. Hydrogen cyanamide up-regulated VvGA2ox and down-regulated VvGA3ox and VvGA20ox expression, reduced GA1 levels, and partially rescued the negative effect of GA. GA had an inhibitory effect only when applied simultaneously with bud-forcing initiation. Given these results, we hypothesize that during initial activation of the dormant bud meristem, the level of GA must be restricted, but after meristem activation an increase in its level serves to enhance primordia regrowth.
• Galbraith, D. W., & Cossarizza, A. (2017). Guidelines for the use of flow cytometry and cell sorting in immunological studies. European Journal of Immunology, 47, 1584-1797. doi:doi: 10.1002/eji.20164663
• Nicolini, A. M., Toth, T. D., Kim, S. Y., Mandel, M. A., Galbraith, D. W., & Yoon, J. (2017). Mie scatter and interfacial tension based real-time quantification of colloidal emulsion nucleic acid amplification. Advanced Biosystems, 1(10), 1700098. doi:10.1002/adbi.201700098
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  This work demonstrates for the first time rapid, real-time Mie scatter sensing of colloidal emulsion nucleic acid amplification directly from emulsion droplets. Loop-mediated isothermal amplification is used in this study, and, to our knowledge, has not previously been used in a colloidal emulsion platform. Interfacial tension values (γ) associated with bulk protein adsorption and denaturation at the oil–water interface exhibit characteristic changes in the absence or presence of amplification. In the presence of target and amplicon, emulsions maintain a constant 300–400 nm diameter, whereas emulsions formed with no target control show a rapid decrease in droplet diameter to
• Blaszczak, A. G., Galbraith, D. W., Janda, J., Vanier, C., Smith, R., Gutierrez, A., & Wozniak, E. (2016). Molecular mechanism of action for the novel biostimulant CYT31 in plants exposed to drought stress.. Acta Horticulturae, 1148, 85-92. doi:doi: 10.17660/ActaHortic.2016.1148.10
• Cantu-Bustos, J. E., Vargas-Cortez, T., Morones-Ramirez, J. R., Balderas-Renteria, I., Galbraith, D. W., McEvoy, M. M., & Zarate, X. (2016). Expression and purification of recombinant proteins in Escherichia coli tagged with the metal-binding protein CusF. Protein expression and purification, 121, 61-5.
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  Production of recombinant proteins in Escherichia coli has been improved considerably through the use of fusion proteins, because they increase protein solubility and facilitate purification via affinity chromatography. In this article, we propose the use of CusF as a new fusion partner for expression and purification of recombinant proteins in E. coli. Using a cell-free protein expression system, based on the E. coli S30 extract, Green Fluorescent Protein (GFP) was expressed with a series of different N-terminal tags, immobilized on self-assembled protein microarrays, and its fluorescence quantified. GFP tagged with CusF showed the highest fluorescence intensity, and this was greater than the intensities from corresponding GFP constructs that contained MBP or GST tags. Analysis of protein production in vivo showed that CusF produces large amounts of soluble protein with low levels of inclusion bodies. Furthermore, fusion proteins can be exported to the cellular periplasm, if CusF contains the signal sequence. Taking advantage of its ability to bind copper ions, recombinant proteins can be purified with readily available IMAC resins charged with this metal ion, producing pure proteins after purification and tag removal. We therefore recommend the use of CusF as a viable alternative to MBP or GST as a fusion protein/affinity tag for the production of soluble recombinant proteins in E. coli.
• Samadder, P., Weng, N., Doetschman, T., Heimark, R. L., & Galbraith, D. W. (2016). Flow cytometry and single nucleus sorting for Cre-based analysis of changes in transcriptional states. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 89(5), 430-42.
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  The organs of eukaryotic organisms comprise complex interspersions of cell types, whose different molecular activities, and corresponding cellular states, cooperate during development to produce the final, functional organ. Dysfunction of organs in disease, particularly oncogenesis, initiates with changes of state of a minor subset of cells. It therefore is hard to detect early molecular indicators of disease within an overwhelming background of normal cells. Flow cytometry and sorting provides a convenient way to purify minority subpopulations, if a specific fluorophore can be unambiguously and exclusively associated with this subpopulation. We have generated a number of transgenic mouse lines expressing a nuclear-localized version of the Green Fluorescent Protein (GFP), within which the production of a chimeric histone 2B-GFP protein occurs under the control of a constitutively-active, actin-derived promoter, separated by a Floxed-STOP sequence. In the presence of Cre recombinase, within F1 progeny of these mouse lines, excision of the STOP sequence activates transcription which results in the emergence of cells containing green fluorescent nuclei. We describe the characterization of these lines using a combination of microscopic imaging, flow cytometry and sorting, and Reverse-Transcription polymerase chain reaction of transcripts within single sorted nuclei isolated from tissue homogenates. These lines should be particularly useful for analysis of transcriptional changes in oncogenesis. © 2016 International Society for Advancement of Cytometry.
• Galbraith, D. W. (2015). Redrawing the frontiers in the age of post-publication review. Frontiers in genetics, 6, 198.
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  Publication forms the core structure supporting the development and transmission of scientific knowledge. For this reason, it is essential that the highest standards of quality control be maintained, in particular to ensure that the information being transmitted allows reproducible replication of the described experiments, and that the interpretation of the results is sound. Quality control has traditionally involved editorial decisions based on anonymous pre-publication peer review. Post-publication review of individual articles took the lesser role since it did not feed directly back to the original literature. Rapid advances in computer and communications technologies over the last thirty years have revolutionized scientific publication, and the role and scope of post-publication review has greatly expanded. This perspective examines the ways in which pre- and post-publication peer review influence the scientific literature, and in particular how they might best be redrawn to deal with the twin problems of scientific non-reproducibility and fraud increasingly encountered at the frontiers of science.
• Naivar, M. A., & Galbraith, D. W. (2015). Digital data acquisition and processing. Current protocols in cytometry, 71, 10.19.1-10.19.13.
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  A flow cytometer is made up of many different subsystems that work together to measure the optical properties of individual cells within a sample. The data acquisition system (also called the data system) is one of these subsystems, and it is responsible for converting the electrical signals from the optical detectors into list-mode data. This unit describes the inner workings of the data system, and provides insight into how the instrument functions as a whole. Some of the information provided in this unit is applicable to everyday use of these instruments, and, at minimum, should make it easier for the reader to assemble a specific data system. With the considerable advancement of electronics technology, it becomes possible to build an entirely functional data system using inexpensive hobbyist-level electronics. This unit covers both analog and digital data systems, but the primary focus is on the more prevalent digital data systems of modern flow cytometric instrumentation.
• Naivar, M. A., & Galbraith, D. W. (2015). Digital data acquisition and processing.. Current protocols in cytometry, 71(1), 10.19.1-10.19.13. doi:10.1002/0471142956.cy1019s71
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  A flow cytometer is made up of many different subsystems that work together to measure the optical properties of individual cells within a sample. The data acquisition system (also called the data system) is one of these subsystems, and it is responsible for converting the electrical signals from the optical detectors into list-mode data. This unit describes the inner workings of the data system, and provides insight into how the instrument functions as a whole. Some of the information provided in this unit is applicable to everyday use of these instruments, and, at minimum, should make it easier for the reader to assemble a specific data system. With the considerable advancement of electronics technology, it becomes possible to build an entirely functional data system using inexpensive hobbyist-level electronics. This unit covers both analog and digital data systems, but the primary focus is on the more prevalent digital data systems of modern flow cytometric instrumentation.
• Chen, S. C., Cannon, C. H., Kua, C. S., Liu, J. J., & Galbraith, D. W. (2014). Genome size in the Fagaceae and its general implications for trees.. Tree Genetics and Genomics, 10(4), 977-988.
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  AbstractPolyploidization is a major source of diversification among plants, particularly during cladogenesis, but most evidence involves herbaceous temperate species. The prevalence of polyploidy among woody taxa is largely unknown, especially among tropical groups. In this study, we examined genome size variation globally and at several taxonomic levels within the Fagaceae. This family has diversified in the northern temperate zone (Quercus) and at least twice in the Asian tropics (Lithocarpus and Castanopsis), allowing us to examine genomic size evolution across a broad latitudinal range. We compared nuclear DNA contents from 78 species in six genera, including new measurements for 171 individuals from 47 Chinese species using standard flow cytometry methods. No evidence suggests that polyploidization or whole genome duplication has occurred in the family. Genome size varied among genera, but limited variation was present in each genus and species. In general, tropical species had larger genomes than temperate species, but the ancestral state cannot be determined given current evidence. Partial duplication does seem to occur among species as within genus variation was larger than within species variation. A review of the literature suggests that genome size and even chromosome structure is highly conserved among woody plants and trees. We propose that ploidy level and genome size are conserved among trees because they participate in diverse syngameons. This behavior would provide similar benefits to polyploidization but avoid exclusion from the syngameon. This conservatism in genome size and structure should enhance ongoing whole genome studies.
• Galbraith, D. W. (2014). Challenges and Solutions in Cytometric Measurements of Non-Mammalian Species. Cytometry, 85A(10), 831-832.
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  Cytometric instruments and the associated measurement methodologies were generally developed to enable examination of the properties of mammalian cells, tissues, and organs, with a major concern being that of human health. This general statement is particularly applicable to flow cytometric measurements, which were originally used for the study of blood cells, and analysis of these cells and their subsets remains a major fraction of the activities in flow cytometry and cell sorting. Although this statement is less applicable to image cytometry, including microscopy in its various forms, both flow and image cytometry encounter technical difficulties when they are applied outside the field of mammalian biology. Part of this relates to structural issues: cells of non-mammalian origin are often of sizes very different to those of mammals, and may come in the form of tissues and organs that are difficult to convert into single cell suspensions. Cell suspensions obtained from natural non-mammalian ecosystems may also comprise mixtures of cells of very different sizes. Part of this also relates to the functional activities of non-mammalian cells in producing secondary products, or other polymeric components that can interfere with cytometric measurements. This special section describes four recent articles that demonstrate ways in which cytometric methodologies can be improved to ameliorate systematic errors in measurement that are a consequence of the unconventional properties of the cells being studied.
• Galbraith, D. W. (2014). Challenges and solutions in cytometric measurements of non-mammalian species. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 85(10), 831-2.
• Galbraith, D. W. (2014). Endoreduplicative standards for calibration of flow cytometric C-Value measurements. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 85(4), 368-74.
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  It has been estimated that there are, globally, as many as 400,000 species of the angiosperms (the flowering plants). Of these, a minimal proportion has been characterized at the cytological level. Urgency is required in initiating a systematic and comprehensive census, due to species extinction as a consequence of anthropogenic activities. Fundamental to eukaryotes is the 2C-value, the amount of DNA contained within the nucleus of the unreduced gametes. Flow cytometry provides an ideal method for determining C-values, but the values archived in the Kew Plant C-value Database represent
• Galbraith, D. W. (2014). Endoreduplicative standards for calibration of flow cytometric C-value measurements.. Cytometry, 85, 368-374.
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  AbstractIt has been estimated that there are, globally, as many as 400,000 species of the angiosperms (the flowering plants). Of these, a minimal proportion has been characterized at the cytological level. Urgency is required in initiating a systematic and comprehensive census, due to species extinction as a consequence of anthropogenic activities. Fundamental to eukaryotes is the 2C-value, the amount of DNA contained within the nucleus of the unreduced gametes. Flow cytometry provides an ideal method for determining C-values, but the values archived in the Kew Plant C-value Database represent
• Galbraith, D. W. (2014). Flow cytometry and sorting in Arabidopsis. Methods in Molecular Biology, 1062, 509-537.
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  Flow cytometry, and the accompanying technology of cell sorting, represents an established and valuable experimental platform for the analysis of cellular populations. Applications involving higher plants, which started to emerge around 30 years ago, are now widely employed both to provide unique information regarding fundamental questions in basic and applied bioscience and to advance agricultural productivity in practical ways. Further developments of this platform are being actively pursued, promising additional advances in our understanding of the interactions of cells within the complex tissues and organs. Higher plants offer unique challenges in terms of flow cytometric analysis, first since their organs and tissues are, almost without exception, three-dimensional assemblies of different cell types and second that their individual cells are generally larger than those of mammals.This chapter focuses on the use of flow cytometry and cell sorting with the model species Arabidopsis thaliana, in particular addressing (1) fluorescence in vivo labeling of specific cell types, (2) fluorescence-activated sorting of protoplasts and nuclei, and (3) transcriptome analyses using sorted protoplasts and nuclei.
• Mittal, A., Balasubramanian, R., Cao, J., Singh, P., Subramanian, S., Hicks, G., Nothnagel, E. A., Abidi, N., Janda, J., Galbraith, D. W., & Rock, C. D. (2014). TOPOISOMERASE 6B is involved in chromatin remodelling associated with control of carbon partitioning into secondary metabolites and cell walls, and epidermal morphogenesis in Arabidopsis. Journal of Experimental Botany, 65(15), 4217-4239.
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  Plant growth is continuous and modular, a combination that allows morphogenesis by cell division and elongation and serves to facilitate adaptation to changing environments. The pleiotropic phenotypes of the harlequin (hlq) mutant, isolated on the basis of ectopic expression of the abscisic acid (ABA)- and auxin-inducible proDc3:GUS reporter gene, were previously characterized. Mutants are skotomorphogenic, have deformed and collapsed epidermal cells which accumulate callose and starch, cell walls abundant in pectins and cell wall proteins, and abnormal and reduced root hairs and leaf trichomes. hlq and two additional alleles that vary in their phenotypic severity of starch accumulation in the light and dark have been isolated, and it is shown that they are alleles of bin3/hyp6/rhl3/Topoisomerase6B. Mutants and inhibitors affecting the cell wall phenocopy several of the traits displayed in hlq. A microarray analysis was performed, and coordinated expression of physically adjacent pairs/sets of genes was observed in hlq, suggesting a direct effect on chromatin. Histones, WRKY and IAA/AUX transcription factors, aquaporins, and components of ubiquitin-E3-ligase-mediated proteolysis, and ABA or biotic stress response markers as well as proteins involved in cellular processes affecting carbon partitioning into secondary metabolites were also identified. A comparative analysis was performed of the hlq transcriptome with other previously published TopoVI mutant transcriptomes, namely bin3, bin5, and caa39 mutants, and limited concordance between data sets was found, suggesting indirect or genotype-specific effects. The results shed light on the molecular mechanisms underlying the det/cop/fus-like pleiotropic phenotypes of hlq and support a broader role for TopoVI regulation of chromatin remodelling to mediate development in response to environmental and hormonal signals.
• Galbraith, D. W., Grindberg, R. V., Yee-Greenbaum, J. L., McConnell, M. J., Novotny, M., O’ Shaughnessy, A. L., Lambert, G. M., Araúzo-Bravo, M. J., Lee, J., Fishman, M., Lin, X., Robbins, G., Lin, X., Vennepally, P., Badger, J. H., Gage, F. H., & Lasken, R. S. (2013). RNA-Seq from single nuclei. Proceedings of the National Academy of Sciences U.S.A., 110(NA), 19802-19807.
• Galbraith, D. W., Qin, J., Scheuring, C., Wei, G., Zhi, H., Zhang, M., Huang, J., Zhou, X., & Zhang, H. (2013). Identification and characterization of a repertoire of genes differentially expressed in developing top ear shoots between a superior hybrid and its parental inbreds in Zea mays L.. Molecular Genetics and Genomics, 288(NA), 691-705.
• Grindberg, R. V., Yee-Greenbaum, J. L., McConnell, M. J., Novotny, M., O'Shaughnessy, A. L., Lambert, G. M., Araúzo-Bravo, M. J., Lee, J., Fishman, M., Robbins, G. E., Lin, X., Venepally, P., Badger, J. H., Galbraith, D. W., Gage, F. H., & Lasken, R. S. (2013). RNA-sequencing from single nuclei. Proceedings of the National Academy of Sciences of the United States of America, 110(49), 19802-19807.
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  PMID: 24248345;Abstract: It has recently been established that synthesis of double-stranded cDNA can be done from a single cell for use in DNA sequencing. Global gene expression can be quantified from the number of reads mapping to each gene, and mutations and mRNA splicing variants determined from the sequence reads. Here we demonstrate that this method of transcriptomic analysis can be done using the extremely low levels of mRNA in a single nucleus, isolated from a mouse neural progenitor cell line and from dissected hippocampal tissue. This method is characterized by excellent coverage and technical reproducibility.On average,more than 16,000 of the 24,057 mouse protein-coding genes were detected from single nuclei, and the amount of gene-expression variation was similar when measured between single nuclei and single cells. Several major advantages of the method exist: first, nuclei, compared with whole cells, have the advantage of being easily isolated from complex tissues and organs, such as those in the CNS. Second, the method can be widely applied to eukaryotic species, including those of different kingdoms. The method also provides insight into regulatory mechanisms specific to the nucleus. Finally, the method enables dissection of regulatory events at the single-cell level; pooling of 10 nuclei or 10 cells obscures some of the variability measured in transcript levels, implying that single nuclei and cells will be extremely useful in revealing the physiological state and interconnectedness of gene regulation in a manner that avoids the masking inherent to conventional transcriptomics using bulk cells or tissues.
• Qin, J., Scheuring, C. F., Wei, G., Zhi, H., Zhang, M., Huang, J. J., Zhou, X., Galbraith, D. W., & Zhang, H. (2013). Identification and characterization of a repertoire of genes differentially expressed in developing top ear shoots between a superior hybrid and its parental inbreds in Zea mays L.. Molecular Genetics and Genomics, 288(12), 691-705.
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  PMID: 24146224;Abstract: Heterosis has been widely used in crop breeding and production; however, little is known about the genes controlling trait heterosis. The shortage of genes known to function in heterosis significantly limits our understanding of the molecular basis underlying heterosis. Here, we report 748 genes differentially expressed (DG) in the developing top ear shoots between a maize heterotic F1 hybrid (Mo17 × B73) and its parental inbreds identified using maize microarrays containing 28,608 unigene features. Of the 748 DG, over 600 were new for the inbred and hybrid combination. The DG were enriched for 35 of the total 213 maize gene ontology (GO) terms, including those describing photosynthesis, respiration, DNA replication, metabolism, and hormone biosynthesis. From the DG, we identified six genes involved in glycolysis, three genes in the citrate cycle, and four genes in the C4-dicarboxylic acid cycle. We mapped 533 of the 748 DG to the maize B73 genome, 298 (55.9 %) of which mapped to the QTL intervals of 11 maize ear traits. Moreover, we compared the repertoire of the DG with that of 14-day seedlings of the same inbred and hybrid combination. Only approximately 5 % of the DG was shared between the two organs and developmental stages. Furthermore, we mapped 417 (55.7 %) of the 748 maize DG to the QTL intervals of 26 rice yield-related traits. Therefore, this study provides a repertoire of genes useful for identification of genes involved in maize ear trait heterosis and information for a better understanding of the molecular basis underlying heterosis in maize. © 2013 Springer-Verlag Berlin Heidelberg.
• Baisakh, N., Ramana, R. M., Rajasekaran, K., Subudhi, P., Janda, J., Galbraith, D., Vanier, C., & Pereira, A. (2012). Enhanced salt stress tolerance of rice plants expressing a vacuolar H+-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora. Plant Biotechnology Journal, 10, 453-464.
• Baisakh, N., Ramanarao, M. V., Rajasekaran, K., Subudhi, P., Janda, J., Galbraith, D., Vanier, C., & Pereira, A. (2012). Enhanced salt stress tolerance of rice plants expressing a vacuolar H ⁺-ATPase subunit c1 (SaVHAc1) gene from the halophyte grass Spartina alterniflora Löisel. Plant Biotechnology Journal, 10(4), 453-464.
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  PMID: 22284568;Abstract: The physiological role of a vacuolar ATPase subunit c1 (SaVHAc1) from a halophyte grass Spartina alterniflora was studied through its expression in rice. The SaVHAc1-expressing plants showed enhanced tolerance to salt stress than the wild-type plants, mainly through adjustments in early stage and preparatory physiological responses. In addition to the increased accumulation of its own transcript, SaVHAc1 expression led to increased accumulation of messages of other native genes in rice, especially those involved in cation transport and ABA signalling. The SaVHAc1-expressing plants maintained higher relative water content under salt stress through early stage closure of the leaf stoma and reduced stomata density. The increased K +/Na + ratio and other cations established an ion homoeostasis in SaVHAc1-expressing plants to protect the cytosol from toxic Na + and thereby maintained higher chlorophyll retention than the WT plants under salt stress. Besides, the role of SaVHAc1 in cell wall expansion and maintenance of net photosynthesis was implicated by comparatively higher root and leaf growth and yield of rice expressing SaVHAc1 over WT under salt stress. The study indicated that the genes contributing toward natural variation in grass halophytes could be effectively manipulated for improving salt tolerance of field crops within related taxa. © 2012 Louisiana State University Agricultural Center. Plant Biotechnology Journal © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.
• Galbraith, D. (2012). Flow Cytometry and Cell Sorting: the Next Generation. Methods.
• Galbraith, D. (2012). Flow Cytometry and Cell Sorting: the Next Generation: Introduction. Methods, 57, 249-250.
• Galbraith, D. (2012). Flow cytometry and cell sorting: The next generation. Methods, 57(3), 249-250.
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  PMID: 22939984;
• Galbraith, D. W. (2012). Flow cytometry and fluorescence-activated cell sorting in plants: the past, present, and future. Biomedica, 30(0), 65-70. doi:10.7705/biomedica.v30i0.824
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  Introduction: Flow cytometry and cell sorting are powerful technologies for examining the molecular, genetic, and physiological properties of individual cells.Objective: The objective of this article is to provide a historical survey of the development of flow cytometry and cell sorting for use with higher plants, a summary of the state of art at the present day, and a prediction of where the field might progress over the coming years.Methods: Adapting flow cytometry and sorting for use with higher plants requires the production of single cell suspensions, or suspensions of subcellular organelles. It also requires identification of methods for fluorescence labeling of the cells or organelles of interest, such that they can be usefully analyzed and sorting. These methods are identified and outlined.Results and conclusions: Recent advances in molecular and biotechnological methods, platforms, and instrumentation, combined with flow cytometry and sorting, provide increasingly powerful analytical tools for exploring the components and structure of regulatory networks governing plant growth and development, and the interactions of plants with their environments. They also will be invaluable in cataloguing the individual species that comprise the biological diversity of flowering plants.
• Galbraith, D. W., & Lambert, G. M. (2012). High-throughput monitoring of plant nuclear DNA contents via flow cytometry. Methods in Molecular Biology, 918, 311-325.
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  PMID: 22893296;Abstract: Interest in measuring the nuclear holoploid genome sizes of higher plants reflects not just the status of the nucleus as a defining characteristic of eukaryotic organisms. Higher plants also attract interest in that they display an unusually large range of genome sizes, current measurements indicating an almost 2,500-fold difference between the smallest and the largest. Scientists would like to learn more about the significance of nuclear genome sizes, in terms of molecular and cytological mechanisms regulating the interaction of the nucleus with the cytoplasm and regulating the observed increases and decreases of genome sizes observed within and across families, genera, and species. We would like to understand their adaptive significance through charting their distribution within populations and ecosystems. Further, since genome size values are only available for a small minority of the ∼650,000 species of angiosperms (known and yet undiscovered), we would like to systematically survey plant genome sizes globally before their extinction as a consequence of anthropogenic change. Flow cytometry is accepted as the method of choice for genome size measurements, these measurements being based on fluorescent staining of the nuclear DNA. Flow cytometry offers exceptional ease of use, accompanied by high accuracy and reproducibility and low cost. This chapter provides a general discussion of flow cytometric methods for measuring plant genome sizes, and detailed methods for carrying out these analyses. © 2012 Springer Science+Business Media, LLC.
• Galbraith, D., & Lambert, G. (2012). High-throughput monitoring of plant nuclear DNA contents via flow cytometry. Methods in Molecular Biology, 918, 311-325.
• Galbraith, D., Zhang, C., & Galbraith, D. W. (2012). RNA interference-mediated gene knockdown within specific cell types. Plant molecular biology, 80(2).
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  In plants, RNA interference (RNAi)-induced gene silencing can spread from the initiation site to nearby cells. The silencing signal moves from cell-to-cell through plasmodesmata and, over long distances, through the phloem. In this study, we employed a nuclear-localized GFP fusion protein to visualize the pattern of gene silencing induced by three different transgenes expressing double-stranded RNA (dsRNA) in Arabidopsis root tips. In all cases, we found that dsRNA-induced silencing did not spread from the silencing initiation site to adjacent cells. In the first set of experiments, in a transgenic background expressing nuclear-localized GFP within a contiguous cell layer that included endodermis, cortex/endodermis (joint) initial (CEI) cells and the quiescent center (QC) cells, expression of the marker gene was silenced specifically in the QC cells without affecting gene expression in the adjacent CEI and endodermal cells. The next two sets of experiments examined the knockdown of two endogenous genes. We observed that silencing was completely restricted to the QC and endodermal cells within which the dsRNA transgenes were expressed. Overall, these results accentuate one important aspect of RNAi-induced gene silencing, that it can be cell autonomous, and demonstrated the feasibility of selective gene knockdown within specific cell types.
• Wang, Z., Hobson, N., Galindo, L., McDill, J., Hawkins, S., Neutelings, G., Datla, R., Lambert, G., Galbraith, D., Cullis, C., Wong, G., Wang, J., & Deyholos, M. (2012). The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads. Plant Journa, 14, 461-473.
• Wang, Z., Hobson, N., Galindo, L., Zhu, S., Shi, D., McDill, J., Yang, L., Hawkins, S., Neutelings, G., Datla, R., Lambert, G., Galbraith, D. W., Grassa, C. J., Geraldes, A., Cronk, Q. C., Cullis, C., Dash, P. K., Kumar, P. A., Cloutier, S., , Sharpe, A. G., et al. (2012). The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads. PLANT JOURNAL, 72(3), 461-473.
• Wang, Z., Hobson, N., Galindo, L., Zhu, S., Shi, D., McDill, J., Yang, L., Hawkins, S., Neutelings, G., Datla, R., Lambert, G., Galbraith, D. W., Grassa, C. J., Geraldes, A., Cronk, Q. C., Cullis, C., Dash, P. K., Kumar, P. A., Cloutier, S., , Sharpe, A. G., et al. (2012). The genome of flax (Linum usitatissimum) assembled de novo from short shotgun sequence reads. Plant Journal, 72(3), 461-473.
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  PMID: 22757964;Abstract: Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N50 = 694 kb, including contigs with N50 = 20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43 384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (Ks) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species. © 2012 Blackwell Publishing Ltd.
• Zhang, C., & Galbraith, D. (2012). RNA interference-mediated gene knockdown within specific cell types. Plant Molecular Biology, 80, 169-176.
• Zhang, C., & Galbraith, D. W. (2012). RNA interference-mediated gene knockdown within specific cell types. Plant Molecular Biology, 80(2), 169-176.
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  PMID: 22740284;Abstract: In plants, RNA interference (RNAi)-induced gene silencing can spread from the initiation site to nearby cells. The silencing signal moves from cell-to-cell through plasmodesmata and, over long distances, through the phloem. In this study, we employed a nuclear-localized GFP fusion protein to visualize the pattern of gene silencing induced by three different transgenes expressing double-stranded RNA (dsRNA) in Arabidopsis root tips. In all cases, we found that dsRNA-induced silencing did not spread from the silencing initiation site to adjacent cells. In the first set of experiments, in a transgenic background expressing nuclear-localized GFP within a contiguous cell layer that included endodermis, cortex/endodermis (joint) initial (CEI) cells and the quiescent center (QC) cells, expression of the marker gene was silenced specifically in the QC cells without affecting gene expression in the adjacent CEI and endodermal cells. The next two sets of experiments examined the knockdown of two endogenous genes. We observed that silencing was completely restricted to the QC and endodermal cells within which the dsRNA transgenes were expressed. Overall, these results accentuate one important aspect of RNAi-induced gene silencing, that it can be cell autonomous, and demonstrated the feasibility of selective gene knockdown within specific cell types. © 2012 Springer Science+Business Media B.V.
• Bourzac, K. M., Rounseville, M. P., Zarate, X., Maddula, V. S., Henderson, D. C., Luckey, J. A., Seligmann, B., & Galbraith, D. W. (2011). A high-density quantitative nuclease protection microarray platform for high throughput analysis of gene expression. Journal of Biotechnology, 154(1), 68-75.
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  PMID: 21504771;Abstract: The quantitative nuclease protection assay (qNPA) is a very simple and highly sensitive method for measuring mRNA transcripts, can be used on a variety of sample types, and is amenable to high-throughput sample processing. We have combined the power of the qNPA assay with the density of a DNA microarray to create a qNPA Microarray platform. This platform is compatible with common laboratory equipment: it uses fluorescence-based detection, can be analyzed with common microarray scanners, and is in an SBS footprint with 96-well layout for high-throughput applications. Here, we demonstrate the characteristics of a qNPA Microarray slide that contains up to 1700 gene elements per well. We show that the new platform can reliably detect transcripts at levels as low as 10 fM with median CVs below 12%. On a standardized set of samples, the qNPA Microarray detected the same trends in gene expression as the original qNPA technology, real time qPCR, and Affymetrix GeneChip DNA Microarrays. Given its ease of use, compatibility with multiple sample types, high-throughput capabilities, and its integration with standard laboratory equipment, the qNPA Microarray is a powerful new platform for gene expression research. © 2011 Elsevier B.V.
• Bourzac, K., Rounseville, M., Zarate, X., Maddula, V., Henderson, D., Luckey, J., Seligmann, B., & Galbraith, D. (2011). A high-density quantitative nuclease protection microarray platform for high throughput analysis of gene expression. Journal of Biotechnology, 154, 68-75.
• Galbraith, D. (2011). The grand challenges in enabling data-intensive biological research. Frontiers in Genetics, 2(26).
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  doi:10.3389fgene.2011. 00026
• Galbraith, D. W. (2011). Frontiers in genomic assay technologies: The grand challenges in enabling data-intensive biological research. Frontiers in Genetics, 2(JUNE).
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  PMID: 22303322;PMCID: PMC3268581;
• Galbraith, D. W., Janda, J., & Lambert, G. M. (2011). Multiparametric analysis, sorting, and transcriptional profiling of plant protoplasts and nuclei according to cell type.. Methods in molecular biology (Clifton, N.J.), 699, 407-429.
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  PMID: 21116995;Abstract: Flow cytometry has been employed for the analysis of higher plants for approximately the last 30 years. For the angiosperms, ∼500,000 species, itself a daunting number, parametric measurements enabled through the use of flow cytometers started with basic descriptors of the individual cells and their contents, and have both inspired the development of novel cytometric methods that subsequently have been applied to organisms within other kingdoms of life, and adopted cytometric methods devised for other species, particularly mammals. Higher plants offer unique challenges in terms of flow cytometric analysis, notably the facts that their organs and tissues are complex three-dimensional assemblies of different cell types, and that their individual cells are, in general, larger than those of mammals.This chapter provides an overview of the general types of parametric measurement that have been applied to plants, and provides detailed methods for selected examples based on the plant model Arabidopsis thaliana. These illustrate the use of flow cytometry for the analysis of protoplasts and nuclear DNA contents (genome size and the cell cycle). These are further integrated with measurements focusing on specific cell types, based on transgenic expression of Fluorescent Proteins (FPs), and on analysis of the spectrum of transcripts found within protoplasts and nuclei. These measurements were chosen in particular to illustrate, respectively, the issues encountered in the flow analysis and sorting of large biological cells, typified by protoplasts; how to handle flow analyses under conditions that require processing of large numbers of samples in which the individual samples contain only a very small minority of objects of interest; and how to deal with exceptionally small amounts of RNA within the sorted samples.
• Galbraith, D., Bennetzen, J., Kellogg, E., Pires, J., & Soltis, P. (2011). The genomes of all angiosperms: a call for a coordinated global census. Journal of Botany.
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  Article 646198 doi:10.1155/2011/646198
• Galbraith, D., Bourzac, K. M., Rounseville, M. P., Zarate, X., Maddula, V. S., Henderson, D. C., Luckey, J. A., Seligmann, B., & Galbraith, D. W. (2011). A high-density quantitative nuclease protection microarray platform for high throughput analysis of gene expression. Journal of biotechnology, 154(1).
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  The quantitative nuclease protection assay (qNPA) is a very simple and highly sensitive method for measuring mRNA transcripts, can be used on a variety of sample types, and is amenable to high-throughput sample processing. We have combined the power of the qNPA assay with the density of a DNA microarray to create a qNPA Microarray platform. This platform is compatible with common laboratory equipment: it uses fluorescence-based detection, can be analyzed with common microarray scanners, and is in an SBS footprint with 96-well layout for high-throughput applications. Here, we demonstrate the characteristics of a qNPA Microarray slide that contains up to 1700 gene elements per well. We show that the new platform can reliably detect transcripts at levels as low as 10fM with median CVs below 12%. On a standardized set of samples, the qNPA Microarray detected the same trends in gene expression as the original qNPA technology, real time qPCR, and Affymetrix GeneChip DNA Microarrays. Given its ease of use, compatibility with multiple sample types, high-throughput capabilities, and its integration with standard laboratory equipment, the qNPA Microarray is a powerful new platform for gene expression research.
• Galbraith, D., Janda, J., & Lambert, G. (2011). Multiparametric analysis, sorting, and transcriptional profiling of plant protoplasts and nuclei according to cell type. Methods in Molecular Biology, 699, 407-429.
• Kimzey, M. J., Zarate, X., Galbraith, D. W., & Lau, S. S. (2011). Optimizing microarray-based in situ transcription-translation of proteins for matrix-assisted laser desorption ionization mass spectrometry. Analytical Biochemistry, 414(2), 282-286.
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  PMID: 21477576;Abstract: The analysis of self-assembled protein microarrays, using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, combines two high-throughput platforms for investigation of the proteome. In this article, we describe the fabrication in situ of protein arrays optimized for MALDI characterization. Using the green fluorescent protein (GFP) both as an epitope for immobilization and as a gauge for relative protein expression, we were able to generate amounts of protein on the array slides sufficient for MALDI identification. In addition, expression of N-terminal protein constructs fused to GFP demonstrated mass shifts consistent with that of the full-length protein. We envision this technology to be important for the functional screening of protein interactions. © 2011 Elsevier Inc. All rights reserved.
• Kimzey, M., Zarate, X., Galbraith, D., & Lau, S. (2011). Optimizing microarray-based in situ transcription-translation of proteins for MALDI mass spectrometry. Analytical Biochemistr, 414, 282-286.
• Krishnan, A., Sweeney, M., Vasic, J., Galbraith, D., & Vasic, B. (2011). Barcodes for DNA sequencing with guaranteed error correction capability. Electronics Letters, 47, 236-237.
• Liu, Y., Zhang, G., Bernardo, M., Leung, H., Zhu, X. Y., Zhang, S., Liu, B., Edwards, J., Galbraith, D. W., & Leach, J. (2011). Dissecting quantitative resistance against blast disease using heterogeneous inbred family lines in rice. Theoretical and Applied Genetics, 122(2), 341-353.
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  PMID: 20872132;Abstract: SHZ-2 is an indica rice cultivar that exhibits broad-spectrum resistance to rice blast; it is widely used as a resistance donor in breeding programs. To dissect the QTL responsible for broad-spectrum blast resistance, we crossed SHZ-2 to TXZ-13, a blast susceptible indica variety, to produce 244 BC4F3 lines. These lines were evaluated for blast resistance in greenhouse and Weld conditions. Chromosomal introgressions from SHZ-2 into the TXZ-13 genome were identiWed using a single feature polymorphism microarray, SSR markers and gene-speciWc primers. Segregation analysis of the BC4F3 population indicated that three regions on chromosomes 2, 6, and 9, designated as qBR2.1, qBR6.1, and qBR9.1, respectively, was associated with blast resistance and contributed 16.2, 14.9, and 22.3%, respectively, to the phenotypic variance of diseased leaf area (DLA). We further narrowed the three QTL regions using pairs of sister lines extracted from heterogeneous inbred families (HIF). Pairwise comparison of these lines enabled the determination of the relative contributions of individual QTL. The qBR9.1 conferred strong resistance, whereas qBR2.1 or qBR6.1 individually did not reduce disease under Weld conditions. However, when qBR2.1 and qBR6.1 were combined, they reduced disease by 19.5%, suggesting that small eVect QTLs contribute to reduction of epidemics. The qBR6.1 and qBR9.1 regions contain nucleotide-binding sites and leucine rich repeats (NBS-LRR) sequences, whereas the qBR2.1 did not. In the qBR6.1 region, the patterns of expression of adjacent NBS-LRR genes were consistent in backcross generations and correlated with blast resistance, supporting the hypothesis that multiple resistance genes within a QTL region can contribute to non-race-speciWc quantitative resistance. © Springer-Verlag 2010.
• Liu, Y., Zhu, X., Zhang, S., Bernardo, M., Edwards, J., Galbraith, D., Leach, J., Zhang, G., Liu, B., & Leung, H. (2011). Dissecting quantitative resistance against blast using heterogeneous inbred families in rice. Theoretical and Applied Genetics, 122, 341-353.
• Vasic, B., Ravanmehr, V., & Galbraith, D. (2011). Analysis and synthesis of boolean cell-cycle gene regulatory networks. Proc. 2011 Information Theory and Applications Workshop.
• Barthelson, R. A., Qaisar, U., & Galbraith, D. W. (2010). Functional analysis of the gossypium arboreum genome. Plant Molecular Biology Reporter, 28(2), 334-343.
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  Abstract: Gossypium arboreum is an Old World relative of the more commonly cultivated commercial species Gossypium hirsutum, a newer genetic line formed in the New World. G. arboreum has the important property that it can be cultivated in severely hot, dry climates. The genome of G. arboreum has not been completely sequenced, and annotation for the genome is not extensive. We studied the genome of G. arboreum by using cross-species hybridization studies with genomic microarrays for the more annotated species, Arabidopsis thaliana and Oryza sativa. Approximately 30% of the probes on the A. thaliana and O. sativa microarrays were hybridized effectively by target samples prepared from G. arboreum genomic DNA. Many of genes tentatively identified by hybridization function in various levels of the stress response. Cross-species hybridization can provide effective clues as to potentially valuable genes that may be present in a less well-studied species such as G. arboreum. The stress response genes tentatively identified in these studies should provide useful clues for further studies toward the development of hardier strains of cotton. © 2009 Springer-Verlag.
• Galbraith, D. W., & Edwards, J. (2010). Applications of microarrays for crop improvement: Here, there, and everywhere. BioScience, 60(5), 337-348.
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  Abstract: Recent advances in technology and in the ability to acquire, store, and analyze complex biological data sets have provided an unprecedented understanding of the mechanisms regulating living organisms' development and responses to the environment. We are gaining the insights required for rational modification of these organisms toward specific purposes. Microarrays provide an early example of the productive integration of high-throughput technologies with biological inquiry. In this article we discuss the development of this popular platform and its application in crop science and agriculture. © 2010 by American Institute of Biological Sciences. All rights reserved.
• Galbraith, D., Zárate, X., Henderson, D. C., Phillips, K. C., Lake, A. D., & Galbraith, D. W. (2010). Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts. Proteome science, 8.
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  Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The Escherichia coli S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to E. coli, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize in vitro translation for protein microarray assembly.
• Oh, D., Dassanayake, M., Haas, J. S., Kropornika, A., Wright, C., d'Urzo, M. P., Hong, H., Ali, S., Hernandez, A., Lambert, G. M., Inan, G., Galbraith, D. W., Bressan, R. A., Yun, D., Zhu, J., Cheeseman, J. M., & Bohnert, H. J. (2010). Genome structures and halophyte-specific gene expression of the extremophile thellungiella parvula in comparison with Thellungiella salsuginea (Thellungiella halophila) and arabidopsis. Plant Physiology, 154(3), 1040-1052.
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  PMID: 20833729;PMCID: PMC2971586;Abstract: The genome of Thellungiella parvula, a halophytic relative of Arabidopsis (Arabidopsis thaliana), is being assembled using Roche-454 sequencing. Analyses of a 10-Mb scaffold revealed synteny with Arabidopsis, with recombination and inversion and an uneven distribution of repeat sequences. T. parvula genome structure and DNA sequences were compared with orthologous regions from Arabidopsis and publicly available bacterial artificial chromosome sequences from Thellungiella salsuginea (previously Thellungiella halophila). The three-way comparison of sequences, from one abiotic stress-sensitive species and two tolerant species, revealed extensive sequence conservation and microcolinearity, but grouping Thellungiella species separately from Arabidopsis. However, the T. parvula segments are distinguished from their T. salsuginea counterparts by a pronounced paucity of repeat sequences, resulting in a 30% shorter DNA segment with essentially the same gene content in T. parvula. Among the genes is SALT OVERLY SENSITIVE1 (SOS1), a sodium/proton antiporter, which represents an essential component of plant salinity stress tolerance. Although the SOS1 coding region is highly conserved among all three species, the promoter regions show conservation only between the two Thellungiella species. Comparative transcript analyses revealed higher levels of basal as well as salt-induced SOS1 expression in both Thellungiella species as compared with Arabidopsis. The Thellungiella species and other halophytes share conserved pyrimidine-rich 5' untranslated region proximal regions of SOS1 that are missing in Arabidopsis. Completion of the genome structure of T. parvula is expected to highlight distinctive genetic elements underlying the extremophile lifestyle of this species. © American Society of Plant Biologists.
• Zárate, X., Henderson, D. C., Phillips, K. C., Lake, A. D., & Galbraith, D. W. (2010). Development of high-yield autofluorescent protein microarrays using hybrid cell-free expression with combined Escherichia coli S30 and wheat germ extracts. Proteome Science, 8.
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  PMID: 20546627;PMCID: PMC2906421;Abstract: Background: Protein-based microarray platforms offer considerable promise as high-throughput technologies in proteomics. Particular advantages are provided by self-assembling protein microarrays and much interest centers around analysis of eukaryotic proteins and their molecular interactions. Efficient cell-free protein synthesis is paramount for the production of self-assembling protein microarrays, requiring optimal transcription, translation, and protein folding. The Escherichia coli S30 extract demonstrates high translation rates but lacks the protein-folding efficiency of its eukaryotic counterparts derived from rabbit reticulocyte and wheat germ extract. In comparison to E. coli, eukaryotic extracts, on the other hand, exhibit slower translation rates and poor overall protein yields. A cell-free expression system that synthesizes folded eukaryotic proteins in considerable yields would optimize in vitro translation for protein microarray assembly.Results: Self-assembling autofluorescent protein microarrays were produced by in situ transcription and translation of chimeric proteins containing a C-terminal Green Fluorescent Protein tag. Proteins were immobilized as array elements using an anti-GFP monoclonal antibody. The amounts of correctly-folded chimeric proteins were quantified by measuring the fluorescence intensity from each array element. During cell-free expression, very little or no fluorescence was observed from GFP-tagged multidomain eukaryotic plant proteins when in vitro translation was performed with E. coli S30 extract. Improvement was seen using wheat germ extract, but fluorescence intensities were still low because of poor protein yields. A hybrid in vitro translation system, combining S30 and wheat germ extracts, produced high levels of correctly-folded proteins for most of the constructs that were tested.Conclusion: The results are consistent with the hypothesis that the wheat germ extract enhances the protein folding capabilities of the in vitro system by providing eukaryotic ribosomes and chaperones and, at the same time, the E. coli S30 extract, which includes an ATP regeneration system, translates the polypeptides at high rates. This hybrid cell-free expression system allows the facile production of high-yield protein arrays suitable for downstream assays. © 2010 Zárate et al; licensee BioMed Central Ltd.
• Andrés, F., Galbraith, D. W., Talón, M., & Domingo, C. (2009). Analysis of Photoperiod Sensitivity5 sheds light on the role of phytochromes in photoperiodic flowering in rice. Plant Physiology, 151(2), 681-690.
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  PMID: 19675157;PMCID: PMC2754645;Abstract: A great number of plants synchronize flowering with day length. In rice (Oryza sativa), photoperiod is the primary environmental cue that triggers flowering. Here, we show that the s73 mutant, identified in a g-irradiated Bahia collection, displays early flowering and photoperiodic insensitivity due to a null mutation in the PHOTOPERIOD SENSITIVITY5 (SE5) gene, which encodes an enzyme implicated in phytochrome chromophore biosynthesis. s73 mutant plants show a number of alterations in the characteristic diurnal expression patterns of master genes involved in photoperiodic control of flowering, resulting in up-regulation of the floral integrator Heading date3a (Hd3a). Early heading date1 (Ehd1), an additional rice floral activator, was also highly expressed in the s73 mutant, suggesting that SE5 represses Ehd1 in wild-type plants. Silencing of Ehd1 in both Bahia and s73 backgrounds indicated that SE5 regulates Ehd1 expression. The data also indicate that SE5 confers photoperiodic sensitivity through regulation of Hd1. These results provide direct evidence that phytochromes inhibit flowering by affecting both Hd1 and Ehd1 flowering pathways. © 2009 American Society of Plant Biologists.
• Galbraith, D. W. (2009). Simultaneous flow cytometric quantification of plant nuclear DNA contents over the full range of described angiosperm 2C values. Cytometry Part A, 75(8), 692-698.
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  PMID: 19565637;Abstract: Flow cytometry provides a rapid, accurate, and simple means to determine nuclear DNA contents (C-value) within plant homogenates. This parameter is extremely useful in a number of applications in basic and applied plant biology; for example, it provides an important starting point for projects involving whole genome sequencing, it facilitates characterization of plant species within natural and agricultural settings, it allows facile identification of engineered plants that are euploid or that represent desired ploidy classes, it points toward studies concerning the role of C-value in plant growth and development and in response to the environment and in terms of evolutionary fitness, and, in uncovering new and unexpected phenomena (for example endoreduplication), it uncovers new avenues of scientific enquiry. Despite the ease of the method, C-values have been determined for only around 2% of the described angiosperm (flowering plant) species. Within this small subset, one of the most remarkable observations is the range of 2C values, which spans at least two orders of magnitude. In determining C-values for new species, technical issues are encountered which relate both to requirement for a method that can provide accurate measurements across this extended dynamic range, and that can accommodate the large amounts of debris which accompanies flow measurements of plant homogenates. In this study, the use of the Accuri C6 flow cytometer for the analysis of plant C-values is described. This work indicates that the unusually large dynamic range of the C6, a design feature, coupled to the linearity of fluorescence emission conferred by staining of nuclei using propidium iodide, allows simultaneous analysis of species whose C-values span that of almost the entire described angiosperms. © 2009 International Society for Advancement of Cytometry.
• Galbraith, D., & Galbraith, D. W. (2009). Simultaneous flow cytometric quantification of plant nuclear DNA contents over the full range of described angiosperm 2C values. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 75(8).
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  Flow cytometry provides a rapid, accurate, and simple means to determine nuclear DNA contents (C-value) within plant homogenates. This parameter is extremely useful in a number of applications in basic and applied plant biology; for example, it provides an important starting point for projects involving whole genome sequencing, it facilitates characterization of plant species within natural and agricultural settings, it allows facile identification of engineered plants that are euploid or that represent desired ploidy classes, it points toward studies concerning the role of C-value in plant growth and development and in response to the environment and in terms of evolutionary fitness, and, in uncovering new and unexpected phenomena (for example endoreduplication), it uncovers new avenues of scientific enquiry. Despite the ease of the method, C-values have been determined for only around 2% of the described angiosperm (flowering plant) species. Within this small subset, one of the most remarkable observations is the range of 2C values, which spans at least two orders of magnitude. In determining C-values for new species, technical issues are encountered which relate both to requirement for a method that can provide accurate measurements across this extended dynamic range, and that can accommodate the large amounts of debris which accompanies flow measurements of plant homogenates. In this study, the use of the Accuri C6 flow cytometer for the analysis of plant C-values is described. This work indicates that the unusually large dynamic range of the C6, a design feature, coupled to the linearity of fluorescence emission conferred by staining of nuclei using propidium iodide, allows simultaneous analysis of species whose C-values span that of almost the entire described angiosperms.
• Lee, T., Kim, Y., Thi, T., Song, S. I., Kim, J., Kang, K. Y., Gynheung, A. n., Jung, K., Galbraith, D. W., Kim, M., Yoon, U., & Nahm, B. H. (2009). RiceArrayNet: A database for correlating gene expression from transcriptome profiling, and its application to the analysis of coexpressed genes in rice. Plant Physiology, 151(1), 16-33.
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  PMID: 19605550;PMCID: PMC2735985;Abstract: Microarray data can be used to derive understanding of the relationships between the genes involved in various biological systems of an organism, given the availability of databases of gene expression measurements from the complete spectrum of experimental conditions and materials. However, there have been no reports, to date, of such a database being constructed for rice (Oryza sativa). Here, we describe the construction of such a database, called RiceArrayNet (RAN; http://www.ggbio.com/arraynet/), which provides information on coexpression between genes in terms of correlation coefficients (γ values). The average number of coexpressed genes is 214, with SD of 440 at γ ≥ 0.5. Given the correlation between genes in a gene pair, the degrees of closeness between genes can be visualized in a relational tree and a relational network. The distribution of correlated genes according to degree of stringency shows how each gene is related to other genes. As an application of RAN, the 16-member L7Ae ribosomal protein family was explored for coexpressed genes and gene expression values within and between rice and Arabidopsis (Arabidopsis thaliana), and common and unique features in coexpression partners and expression patterns were observed for these family members. We observed a correlation pattern between Os01g0968800, a drought-responsive element-binding transcription factor, Os02g0790500, a trehalose-6-phosphate synthase, and Os06g0219500, a small heat shock factor, reflecting the fact that genes responding to the same biological stresses are regulated together. The RAN database can be used as a tool to gain insight into a particular gene by examining its coexpression partners. © 2009 American Society of Plant Biologists.
• Luo, Q., Samanta, M. P., Köksal, F., Janda, J., Galbraith, D. W., Richardson, C. R., Ou-Yang, F., & Rock, C. D. (2009). Evidence for antisense transcription associated with microRNA target mRNAs in arabidopsis. PLoS Genetics, 5(4).
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  PMID: 19381263;PMCID: PMC2664332;Abstract: Antisense transcription is a pervasive phenomenon, but its source and functional significance is largely unknown. We took an expression-based approach to explore microRNA (miRNA)-related antisense transcription by computational analyses of published whole-genome tiling microarray transcriptome and deep sequencing small RNA (smRNA) data. Statistical support for greater abundance of antisense transcription signatures and smRNAs was observed for miRNA targets than for paralogous genes with no miRNA cleavage site. Antisense smRNAs were also found associated with MIRNA genes. This suggests that miRNA-associated "transitivity" (production of small interfering RNAs through antisense transcription) is more common than previously reported. High-resolution (3 nt) custom tiling microarray transcriptome analysis was performed with probes 400 bp 5' upstream and 3' downstream of the miRNA cleavage sites (direction relative to the mRNA) for 22 select miRNA target genes. We hybridized RNAs labeled from the smRNA pathway mutants, including hen1-1, dcl1-7, hyl1-2, rdr6-15, and sgs3-14. Results showed that antisense transcripts associated with miRNA targets were mainly elevated in hen1- 1 and sgs3-14 to a lesser extent, and somewhat reduced in dcl11-7, hyl11-2, or rdr6-15 mutants. This was corroborated by semi-quantitative reverse transcription PCR; however, a direct correlation of antisense transcript abundance in MIR164 gene knockouts was not observed. Our overall analysis reveals a more widespread role for miRNA-associated transitivity with implications for functions of antisense transcription in gene regulation. HEN1 and SGS3 may be links for miRNA target entry into different RNA processing pathways. © 2009.
• Lynch, R. M., Lynch, R. M., Lapresto, L., Galbraith, D. W., Cates, J., Brooks, H. L., & Barthelson, R. A. (2009). Nucleomics: Targeting and Genetic Analysis of Nutrient-Sensing Cells. The FASEB Journal, 23.
• Mathiason, K., Dong, H. e., Grimplet, J., Venkateswari, J., Galbraith, D. W., Etti, O. r., & Fennell, A. (2009). Transcript profiling in Vitis riparia during chilling requirement fulfillment reveals coordination of gene expression patterns with optimized bud break. Functional and Integrative Genomics, 9(1), 81-96.
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  PMID: 18633655;Abstract: Endodormant grapevine buds require a period of chilling before they break and begin to grow. Custom Vitis bud cDNA microarrays (9,216 features) were used to examine gene expression patterns in overwintering Vitis riparia buds during 2,000 h of 4°C chilling. Three-node cuttings collected concurrently with buds were monitored to determine dormancy status. Chilling requirement was fulfilled after 1,500 h of chilling; however, 2,000 h of chilling significantly increased the rate of bud break. Microarray analysis identified 1,469 significantly differentially expressed (p value < 0.05) array features when 1,000, 1,500, and 2,000 h of chilling were compared to 500 h of chilling. Functional classification revealed that the majority of genes were involved in metabolism, cell defense/stress response, and genetic information processing. The number of significantly differentially expressed genes increased with chilling hour accumulation. The expression of a group of 130 genes constantly decreased during the chilling period. Up-regulated genes were not detected until the later stages of chilling accumulation. Hierarchical clustering of non-redundant expressed sequence tags revealed inhibition of genes involved in carbohydrate and energy metabolism and activation of genes involved in signaling and cell growth. Clusters with expression patterns associated with increased chilling and bud break were identified, indicating several candidate genes that may serve as indicators of bud chilling requirement fulfillment. © Springer-Verlag 2008.
• Mustroph, A., Zanetti, M. E., J., C., Holtan, H. E., Repetti, P. P., Galbraith, D. W., Girke, T., & Bailey-Serres, J. (2009). Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America, 106(44), 18843-18848.
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  PMID: 19843695;PMCID: PMC2764735;Abstract: Multicellular organs are composed of distinct cell types with unique assemblages of translated mRNAs. Here, ribosome-associated mRNAs were immunopurified from specific cell populations of intact seedlings using Arabidopsis thaliana lines expressing a FLAG-epitope tagged ribosomal protein L18 (FLAG-RPL18) via developmentally regulated promoters. The profiling of mRNAs in ribosome complexes, referred to as the translatome, identified differentially expressed mRNAs in 21 cell populations defined by cell-specific expression of FLAG-RPL18. Phloem companion cells of the root and shoot had the most distinctive translatomes. When seedlings were exposed to a brief period of hypoxia, a pronounced reprioritization of mRNA enrichment in the cell-specific translatomes occurred, including a ubiquitous rise in 49 mRNAs encoding transcription factors, signaling proteins, anaerobic metabolism enzymes, and uncharacterized proteins. Translatome profiling also exposed an intricate molecular signature of transcription factor (TF) family member mRNAs that was markedly reconfigured by hypoxia at global and cell-specific levels. In addition to the demonstration of the complexity and plasticity of cell-specific populations of ribosome-associated mRNAs, this study provides an in silico dataset for recognition of differentially expressed genes at the cell-, region-, and organ-specific levels.
• Mustroph, A., Zanetti, M. E., Jang, C., Holtan, H. E., Repetti, P. P., Galbraith, D. W., Girke, T., & Bailey-Serres, J. (2009). Profiling translatomes of discrete cell populations resolves altered cellular priorities during hypoxia in Arabidopsis. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 106(44), 18843-18848.
• Ophir, R., Pang, X., Halaly, T., Venkateswari, J., Lavee, S., Galbraith, D., & Etti, O. r. (2009). Gene-expression profiling of grape bud response to two alternative dormancy-release stimuli expose possible links between impaired mitochondrial activity, hypoxia, ethylene-ABA interplay and cell enlargement. Plant Molecular Biology, 71(4-5), 403-423.
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  PMID: 19653104;Abstract: A grape-bud-oriented genomic platform was produced for a large-scale comparative analysis of bud responses to two stimuli of grape-bud dormancy release, hydrogen cyanamide (HC) and heat shock (HS). The results suggested considerable similarity in bud response to the stimuli, both in the repertoire of responding genes and in the temporary nature of the transcriptome reprogramming. Nevertheless, the bud response to HC was delayed, more condensed and stronger, as reflected by a higher number of regulated genes and a higher intensity of regulation compared to the response to HS. Integrating the changes occurring in response to both stimuli suggested perturbation of mitochondrial activity, development of oxidative stress and establishment of a situation that resembles hypoxia, which coincides with induction of glycolysis and fermentation, as well as changes in the interplay between ABA and ethylene metabolism. The latter is known to induce various growth responses in submerged plants and the possibility of a similar mechanism operating in the bud meristem during dormancy release is raised. The new link suggested between sub lethal stress, mitochondrial activity, hypoxic conditions, ethylene metabolism and cell enlargement during bud dormancy release may be instrumental in understanding the dormancy-release mechanism. Temporary increase of acetaldehyde, ethanol and ethylene in response to dormancy release stimuli demonstrated the predictive power of the working model, and its relevance to dormancy release was demonstrated by enhancement of bud break by exogenous ethylene and its inhibition by an ethylene signal inhibitor. © 2009 Springer Science+Business Media B.V.
• Sliwinska, E., Pisarczyk, I., Pawlik, A., & Galbraith, D. W. (2009). Measuring genome size of desert plants using dry seeds. Botany, 87(2), 127-135.
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  Abstract: Use of seeds instead of leaves for the flow cytometric measurement of DNA content is of particular interest to botanists and plant ecologists, since it allows estimation of genome sizes for species having reduced leaves or that accumulate staining inhibitors within leaves, and also for species growing in regions where cytometers are not readily available. The seeds of 24 desert species, including wildflowers, cacti, shrubs, and trees were analyzed by flow cytometry. Nuclei were used from either total seeds or seed tissues, following dissection to determine the seed parts that were most suitable for genome size measurement. In addition, the mass of 100 seeds was established. The seeds of 14 species contained only cells occupying a mitotic cell cycle. For 10 other species, endoreplicated nuclei (up to 32C) were detected. Using entire seeds or their parts, it was possible to estimate genome sizes for all of the species, which ranged from 0.79 pg per 2C in Parkinsonia aculeata L. to 26.96 pg per 2C in Agave parryi Engelm., thus this kind of plant material can be used for the cytometric measuring of nuclear DNA content. However, a detailed understanding of seed biology is needed to interpret the results correctly. The relationships among genome size, seed mass, and desert growing conditions are also discussed. © 2009 NRC Canada.
• Bohnert, H. J., Kawasaki, S., Michalowski, C. B., Wang, H., Ozturk, N. Z., Deyholos, M. K., Galbraith, D. W., Khush, G. S., Brar, D. S., & Hardy, B. (2008). Isolation of candidate genes for tolerance of abiotic stresses.. Rice Genetics Collection (Rice Genetics IV), 345-363. doi:https://doi.org/10.1142/9789812814296_0024
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  High-throughput analysis of expressed genes, achieved by cataloguing expressed sequence tags (ESTs) and monitoring hybridization patterns by microarrays, has recently become possible in rice. As the first results become available, the value of these technologies can be gauged. Through ESTs and microarrays, we can obtain a more complete view than in the past of plant gene complexity, tissue specificity, and developmental or externally affected expression patterns. In particular, EST and microarray analyses can have tremendous impact in plant breeding, based on accelerated identification of complex traits such as those controlling plant responses to abiotic stresses. Owing to the novelty and lack of refinement in the use of microarray technology, we discuss advantages and limitations. We demonstrate responses to salt stress in rice (Oryza sativa) monitored by microarray analysis.
• Edwards, J. D., Janda, J., Sweeney, M. T., Gaikwad, A. B., Liu, B., Leung, H., & Galbraith, D. W. (2008). Development and evaluation of a high-throughput, low-cost genotyping platform based on oligonucleotide microarrays in rice. Plant Methods, 4(1).
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  PMID: 18510771;PMCID: PMC2435114;Abstract: Background. We report the development of a microarray platform for rapid and cost-effective genetic mapping, and its evaluation using rice as a model. In contrast to methods employing whole-genome tiling microarrays for genotyping, our method is based on low-cost spotted microarray production, focusing only on known polymorphic features. Results. We have produced a genotyping microarray for rice, comprising 880 single feature polymorphism (SFP) elements derived from insertions/deletions identified by aligning genomic sequences of the japonica cultivar Nipponbare and the indica cultivar 93-11. The SFPs were experimentally verified by hybridization with labeled genomic DNA prepared from the two cultivars. Using the genotyping microarrays, we found high levels of polymorphism across diverse rice accessions, and were able to classify all five subpopulations of rice with high bootstrap support. The microarrays were used for mapping of a gene conferring resistance to Magnaporthe grisea, the causative organism of rice blast disease, by quantitative genotyping of samples from a recombinant inbred line population pooled by phenotype. Conclusion. We anticipate this microarray-based genotyping platform, based on its low cost-per-sample, to be particularly useful in applications requiring whole-genome molecular marker coverage across large numbers of individuals. © 2008 Edwards et al; licensee BioMed Central Ltd.
• Facella, P., Lopez, L., Carbone, F., Galbraith, D. W., Giuliano, G., & Perrotta, G. (2008). Diurnal and circadian rhythms in the tomato transcriptome and their modulation by cryptochrome photoreceptors. PLoS ONE, 3(7).
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  PMID: 18665253;PMCID: PMC2474677;Abstract: Background: Circadian clocks are internal molecular time-keeping mechanisms that provide living organisms with the ability to adjust their growth and physiology and to anticipate diurnal environmental changes. Circadian clocks, without exception, respond to light and, in plants, light is the most potent and best characterized entraining stimulus. The capacity of plants to respond to light is achieved through a number of photo-perceptive proteins including cryptochromes and phytochromes. There is considerable experimental evidence demonstrating the roles of photoreceptors in providing light input to the clock. Methodology: In order to identify genes regulated by diurnal and circadian rhythms, and to establish possible functional relations between photoreceptors and the circadian clock in tomato, we monitored the temporal transcription pattern in plants entrained to long-day conditions, either by large scale comparative profiling, or using a focused approach over a number of photosensory and clock-related genes by QRT-PCR. In parallel, focused transcription analyses were performed in cry1a- and in CRY2-OX tomato genotypes. Conclusions: We report a large series of transcript oscillations that shed light on the complex network of interactions among tomato photoreceptors and clock-related genes. Alteration of cryptochrome gene expression induced major changes in the rhythmic oscillations of several other gene transcripts. In particular, over-expression of CRY2 had an impact not only on day/night fluctuations but also on rhythmicity under constant light conditions. Evidence was found for widespread diurnal oscillations of transcripts encoding specific enzyme classes (e.g. carotenoid biosynthesis enzymes) as well as for post-transcriptional diurnal and circadian regulation of the CRY2 transcript. © Facella et al.
• Galbraith, D., Zhang, C., Barthelson, R. A., Lambert, G. M., & Galbraith, D. W. (2008). Global characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant physiology, 147(1).
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  We describe a simple and highly effective means for global identification of genes that are expressed within specific cell types within complex tissues. It involves transgenic expression of nuclear-targeted green fluorescent protein in a cell-type-specific manner. The fluorescent nuclei are then purified from homogenates by fluorescence-activated sorting, and the RNAs employed as targets for microarray hybridization. We demonstrate the validity of the approach through the identification of 12 genes that are selectively expressed in phloem.
• Noizet, M., Harrabi, F., Vijayalakshmi, M., Galbraith, D., Thomas, D., & Thomasset, B. (2008). Targeted protein accumulation promoted by autoassembly and its recovery from plant cells. Biotechnology Journal, 3(3), 392-402.
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  PMID: 18264977;Abstract: The expression of a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme β-glucuronidase (GUS) in plants promotes the formation of new organization of the endoplasmic reticulunn in tobacco plants. This unusual organization of the membranes, never present in nontransformed plants, has been explained by the oligomerization of the GUS domains of the IBVM-GUS fusion proteins. These specific organized membranes could have broad implications for biotechnology since their formation could be used as a mechanism for retaining and accumulating resident proteins in specific and discrete membrane compartments. In this study, we have shown that the unusual organization of native membranes due to overexpression of the IBVM-GUS fusion gene in tobacco transgenic plants and calli is present at higher levels in plant cell suspensions than in plant tissues. In these cell suspensions, IBVM-GUS protein was continuously synthesized and accumulated throughout the cell culture. An enrichment of the chimeric IBVM-GUS protein corresponding to a five-fold increase in the microsomal fractions was achieved and the GUS enzyme did not show any modification on enzyme kinetics. However, the GUS activity could be differentially distributed in the fractions eluted at different pH suggesting differences in the surface topography of histidine residues for this recombinant GUS. © 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.
• Yuan, J. S., Galbraith, D. W., Dai, S. Y., Griffin, P., & Stewart Jr., C. N. (2008). Plant systems biology comes of age. TRENDS IN PLANT SCIENCE, 13(4), 165-171.
• Yuan, J. S., Galbraith, D. W., Dai, S. Y., Griffin, P., & Stewart Jr., C. N. (2008). Plant systems biology comes of age. Trends in Plant Science, 13(4), 165-171.
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  PMID: 18329321;Abstract: 'Omics' research approaches have produced copious data for living systems, which have necessitated the development of systems biology to integrate multidimensional biological information into networks and models. Applications of systems biology to plant science have been rapid, and have increased our knowledge about circadian rhythms, multigenic traits, stress responses and plant defenses, and have advanced the virtual plant project. © 2008 Elsevier Ltd. All rights reserved.
• Zhang, C., Barthelson, R. A., Lambert, G. M., & Galbraith, D. W. (2008). Global characterization of cell-specific gene expression through fluorescence-activated sorting of nuclei. Plant Physiology, 147(1), 30-40.
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  PMID: 18354040;PMCID: PMC2330299;Abstract: We describe a simple and highly effective means for global identification of genes that are expressed within specific cell types within complex tissues. It involves transgenic expression of nuclear-targeted green fluorescent protein in a cell-type-specific manner. The fluorescent nuclei are then purified from homogenates by fluorescence-activated sorting, and the RNAs employed as targets for microarray hybridization. We demonstrate the validity of the approach through the identification of 12 genes that are selectively expressed in phloem. © 2008 American Society of Plant Biologists.
• Barthelson, R. A., Lambert, G. M., Vanier, C., Lynch, R. M., & Galbraith, D. W. (2007). Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells. BMC Genomics, 8.
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  PMID: 17894886;PMCID: PMC2048942;Abstract: Background: In the most general sense, studies involving global analysis of gene expression aim to provide a comprehensive catalog of the components involved in the production of recognizable cellular phenotypes. These studies are often limited by the available technologies. One technology, based on microarrays, categorizes gene expression in terms of the abundance of RNA transcripts, and typically employs RNA prepared from whole cells, where cytoplasmic RNA predominates. Results: Using microarrays comprising oligonucleotide probes that represent either protein-coding transcripts or microRNAs (miRNA), we have studied global transcript accumulation patterns for the HepG2 (human hepatoma) cell line. Through subdividing the total pool of RNA transcripts into samples from nuclei, the cytoplasm, and whole cells, we determined the degree of correlation of these patterns across these different subcellular locations. The transcript and miRNA abundance patterns for the three RNA fractions were largely similar, but with some exceptions: nuclear RNA samples were enriched with respect to the cytoplasm in transcripts encoding proteins associated with specific nuclear functions, such as the cell cycle, mitosis, and transcription. The cytoplasmic RNA fraction also was enriched, when compared to the nucleus, in transcripts for proteins related to specific nuclear functions, including the cell cycle, DNA replication, and DNA repair. Some transcripts related to the ubiquitin cycle, and transcripts for various membrane proteins were sorted into either the nuclear or cytoplasmic fractions. Conclusion: Enrichment or compartmentalization of cell cycle and ubiquitin cycle transcripts within the nucleus may be related to the regulation of their expression, by preventing their translation to proteins. In this way, these cellular functions may be tightly controlled by regulating the release of mRNA from the nucleus and thereby the expression of key rate limiting steps in these pathways. Many miRNA precursors were also enriched in the nuclear samples, with significantly fewer being enriched in the cytoplasm. Studies of mRNA localization will help to clarify the roles RNA processing and transport play in the regulation of cellular function. © 2007 Barthelson et al; licensee BioMed Central Ltd.
• Galbraith, D. W. (2007). Analysis of Plant Gene Expression Using Flow Cytometry and Sorting. Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes, 405-422.
• Galbraith, D. W. (2007). Nanobiotechnology: Silica breaks through in plants. Nature Nanotechnology, 2(5), 272-273.
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  PMID: 18654282;Abstract: Designer nanotubes based on mesoporous silica can now penetrate the thick cell walls of plants and deliver DNA and their activators. This opens the way to precisely manipulate gene expression in plants at the single-cell level. ©2007 Nature Publishing Group.
• Galbraith, D. W. (2007). Protoplast Analysis using Flow Cytometry and Sorting. Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes, 231-250.
• Galbraith, D., Barthelson, R. A., Lambert, G. M., Vanier, C., Lynch, R. M., & Galbraith, D. W. (2007). Comparison of the contributions of the nuclear and cytoplasmic compartments to global gene expression in human cells. BMC genomics, 8.
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  In the most general sense, studies involving global analysis of gene expression aim to provide a comprehensive catalog of the components involved in the production of recognizable cellular phenotypes. These studies are often limited by the available technologies. One technology, based on microarrays, categorizes gene expression in terms of the abundance of RNA transcripts, and typically employs RNA prepared from whole cells, where cytoplasmic RNA predominates.
• Keilin, T., Pang, X., Venkateswari, J., Halaly, T., Crane, O., Keren, A., Ogrodovitch, A., Ophir, R., Volpin, H., Galbraith, D., & Etti, O. r. (2007). Digital expression profiling of a grape-bud EST collection leads to new insight into molecular events during grape-bud dormancy release. Plant Science, 173(4), 446-457.
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  Abstract: The application of genomic approaches may serve as an initial step in broadening our understanding of the complex network of biochemical and cellular processes responsible for the regulation and execution of grape-bud dormancy release. However, bud tissue in general, and the dormant bud in particular, are under-represented in the public Vitis genomic resources. Here we describe a large-scale grape-bud EST collection representing a wide range of bud developmental stages. A collection of 5516 consensus sequences is presented, of which 59% were not included within the Vitis TIGR collection at the time of current analysis. About 22% of these transcripts bear no resemblance to any known plant transcript and thus corroborate the need for this targeted EST collection. The added value of the presented EST collection lies in the conferred ability to compare EST frequencies between the different cDNA libraries. Such comparison was implemented and allowed us to identify several genes/functions whose expression is altered in response to the dormancy-release treatment. Based on this analysis, it is suggested that oxidative stress, calcium signaling, intracellular vesicle trafficking and anaerobic mode of carbohydrate metabolism play a role in the regulation and execution of grape-bud dormancy release. © 2007 Elsevier Ireland Ltd. All rights reserved.
• Kris, R. M., Felder, S., Deyholos, M., Lambert, G. M., Hinton, J., Botros, I., Martel, R., Seligmann, B., & Galbraith, D. W. (2007). High-throughput, high-sensitivity analysis of gene expression in arabidopsis. Plant Physiology, 144(3), 1256-1266.
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  PMID: 17496109;PMCID: PMC1914134;Abstract: High-throughput gene expression analysis of genes expressed during salt stress was performed using a novel multiplexed quantitative nuclease protection assay that involves customized DNA microarrays printed within the individual wells of 96-well plates. The levels of expression of the transcripts from 16 different genes were quantified within crude homogenates prepared from Arabidopsis (Arabidopsis thaliana) plants also grown in a 96-well plate format. Examples are provided of the high degree of reproducibility of quantitative dose-response data and of the sensitivity of detection of changes in gene expression within limiting amounts of tissue. The lack of requirement for RNA purification renders the assay particularly suited for high-throughput gene expression analysis and for the discovery of novel chemical compounds that specifically modulate the expression of endogenous target genes. © 2007 American Society of Plant Biologists.
• Lynch, R. M., Lynch, R. M., Galbraith, D. W., Cates, J., Brooks, H. L., & Barthelson, R. A. (2007). Nucleomics of glucose sensing cells: methodology for cell-type specific analysis of gene expression. The FASEB Journal, 21(5). doi:10.1096/fasebj.21.5.a476-d
• Pang, X., Halaly, T., Crane, O., Keilin, T., Keren-Keiserman, A., Ogrodovitch, A., Galbraith, D., & Etti, O. r. (2007). Involvement of calcium signalling in dormancy release of grape buds. Journal of Experimental Botany, 58(12), 3249-3262.
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  PMID: 17977848;Abstract: Artificial induction of grape bud dormancy release by hydrogen cyanamide (HC) serves as a reliable model system to explore the events occurring shortly after the induction of dormancy release. Recently, a group of genes with remarkable differences in expression level between HC-treated and control buds was identified. The identification of several calcium signalling-related genes within that group raised the hypothesis of the involvement of Ca2+ signalling in grape bud dormancy release. Therefore, the effects of HC treatment on the expression profiles of several calcium sensors, the effect of the plasma membrane calcium channel blocker LaCl3 and the calcium chelator EGTA on HC-induced and chilling-induced bud-break, and the effect of HC application on calcium-dependent protein phosphorylation activities in the bud tissue were studied. Here the HC-induced expression of Ca2+-ATPase is described, indicating that this treatment might evoke an increase in [Ca2+]cyt. Similar induction was confirmed for calmodulin, calmodulin-binding protein, and calcium-dependent protein kinase (CDPK). Both LaCl3 and EGTA blocked the inducing effect of HC on bud-break, and their inhibitory effects were removed by supplying exogenous Ca2+. Calcium-dependent histone phosphorylation was up to 70% higher in HC-treated buds. Endogenous protein phosphorylation assays detected four proteins exhibiting increased phosphorylation following HC treatment, of which two were phosphorylated in a calcium-dependent manner. One of these, a 47 kDa protein, presented strong and Ca2+-dependent phosphorylation only in HC-treated buds. The potential role of CDPK in the phosphorylation of this protein was supported by an immunoprecipitation assay. The data suggest, for the first time, that calcium signalling is involved in the mechanism of bud dormancy release. © 2007 The Author(s).
• Weng, Z., Barthelson, R., Gowda, S., Hilf, M. E., Dawson, W. O., Galbraith, D. W., & Xiong, Z. (2007). Persistent infection and promiscuous recombination of multiple genotypes of an RNA virus within a single host generate extensive diversity. PLoS ONE, 2(9).
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  PMID: 17878952;PMCID: PMC1975466;Abstract: Recombination and reassortment of viral genomes are major processes contributing to the creation of new, emerging viruses. These processes are especially significant in long-term persistent infections where multiple viral genotypes co-replicate in a single host, generating abundant genotypic variants, some of which may possess novel host-colonizing and pathogenicity traits. In some plants, successive vegetative propagation of infected tissues and introduction of new genotypes of a virus by vector transmission allows for viral, populations to increase in complexity for hundreds of years allowing co-replication and subsequent recombination of the multiple viral genotypes. Using a resequencing microarray, we examined a persistent infection by a Citrus tristeza virus (CTV) complex in citrus, a vegetatively propagated, globally important fruit crop, and found that the complex comprised three major and a number of minor genotypes. Subsequent deep sequencing analysis of the viral population confirmed the presence of the three major CTV genotypes and, in addition, revealed that the minor genotypes consisted of an extraordinarily large number of genetic variants generated by promiscuous recombination between the major genotypes. Further analysis provided evidence that some of the recombinants underwent subsequent divergence, further increasing the genotypic complexity. These data demonstrate that persistent infection of multiple viral genotypes within a host organism is sufficient to drive the large-scale production of viral genetic variants that may evolve into new and emerging viruses. © 2007 Weng et al.
• Ammiraju, J. S., Luo, M., Goicoechea, J. L., Wang, W., Kudrna, D., Mueller, C., Talag, J., Kim, H., Sisneros, N. B., Blackmon, B., Fang, E., Tomkins, J. B., Brar, D., MacKill, D., McCouch, S., Kurata, N., Lambert, G., Galbraith, D. W., Arumuganathan, K., , Rao, K., et al. (2006). The Oryza bacterial artificial chromosome library resource: Construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza. Genome Research, 16(1), 140-147.
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  PMID: 16344555;PMCID: PMC1356138;Abstract: Rice (Oryza sativa L.) is the most important food crop in the world and a model system for plant biology. With the completion of a finished genome sequence we must now functionally characterize the rice genome by a variety of methods, including comparative genomic analysis between cereal species and within the genus Oryza. Oryza contains two cultivated and 22 wild species that represent 10 distinct genome types. The wild species contain an essentially untapped reservoir of agriculturally important genes that must be harnessed if we are to maintain a safe and secure food supply for the 21st century. As a first step to functionally characterize the rice genome from a comparative standpoint, we report the construction and analysis of a comprehensive set of 12 BAC libraries that represent the 10 genome types of Oryza. To estimate the number of clones required to generate 10 genome equivalent BAC libraries we determined the genome sizes of nine of the 12 species using flow cytometry. Each library represents a minimum of 10 genome equivalents, has an average insert size range between 123 and 161 kb, an average organellar content of 0.4%-4.1% and nonrecombinant content between 0% and 5%. Genome coverage was estimated mathematically and empirically by hybridization and extensive contig and BAC end sequence analysis. A preliminary analysis of BAC end sequences of clones from these libraries indicated that LTR retrotransposons are the predominant class of repeat elements in Oryza and a roughly linear relationship of these elements with genome size was observed. ©2006 by Cold Spring Harbor Laboratory Press.
• Ammiraju, J., Luo, M. Z., Goicoechea, J. L., Wang, W. M., Kudrna, D., Mueller, C., Talag, J., Kim, H., Sisneros, N. B., Blackmon, B., Fang, E., Tomkins, J. B., Brar, D., MacKill, D., McCouch, S., Kurata, N., Lambert, G., Galbraith, D. W., Arumuganathan, K., , Rao, K. R., et al. (2006). The Oryza bacterial artificial chromosome library resource: Construction and analysis of 12 deep-coverage large-insert BAC libraries that represent the 10 genome types of the genus Oryza. GENOME RESEARCH, 16(1), 140-147.
• Galbraith, D. W. (2006). DNA microarray analyses in higher plants. OMICS A Journal of Integrative Biology, 10(4), 455-473.
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  PMID: 17233557;Abstract: DNA microarrays were originally devised and described as a convenient technology for the global analysis of plant gene expression. Over the past decade, their use has expanded enormously to cover all kingdoms of living organisms. At the same time, the scope of applications of microarrays has increased beyond expression analyses, with plant genomics playing a leadership role in the on-going development of this technology. As the field has matured, the rate-limiting step has moved from that of the technical process of data generation to that of data analysis. We currently face major problems in dealing with the accumulating datasets, not simply with respect to how to archive, access, and process the huge amounts of data that have been and are being produced, but also in determining the relative quality of the different datasets. A major recognized concern is the appropriate use of statistical design in microarray experiments, without which the datasets are rendered useless. A vigorous area of current research involves the development of novel statistical tools specifically for microarray experiments. This article describes, in a necessarily selective manner, the types of platforms currently employed in microarray research and provides an overview of recent activities using these platforms in plant biology. © Mary Ann Liebert, Inc.
• Galbraith, D. W. (2006). The daunting process of MIAME [2]. Nature, 444(7115), 31-.
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  PMID: 17080064;
• Galbraith, D. W., & Birnbaum, K. (2006). Global studies of cell type-specific gene expression in plants. Annual Review of Plant Biology, 57, 451-475.
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  PMID: 16669770;Abstract: Technological advances in expression profiling and in the ability to collect minute quantities of tissues have come together to allow a growing number of global transcriptional studies at the cell level in plants. Microarray technology, with a choice of cDNA or oligo-based slides, is now well established, with commercial full-genome platforms for rice and Arabidopsis and extensive expressed sequence tag (EST)-based designs for many other species. Microdissection and cell sorting are two established methodologies that have been used in conjunction with microarrays to provide an early glimpse of the transcriptional landscape at the level of individual cell types. The results indicate that much of the transcriptome is compartmentalized. A minor but consistent percentage of transcripts appear to be unique to specific cell types. Functional analyses of cell-specific patterns of gene expression are providing important clues to cell-specific functions. The spatial dissection of the transcriptome has also yielded insights into the localized mediators of hormone inputs and promises to provide detail on cell-specific effects of microRNAs. Copyright © 2006 by Annual Reviews. All rights reserved.
• Galbraith, D., & Galbraith, D. W. (2006). DNA microarray analyses in higher plants. Omics : a journal of integrative biology, 10(4).
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  DNA microarrays were originally devised and described as a convenient technology for the global analysis of plant gene expression. Over the past decade, their use has expanded enormously to cover all kingdoms of living organisms. At the same time, the scope of applications of microarrays has increased beyond expression analyses, with plant genomics playing a leadership role in the on-going development of this technology. As the field has matured, the rate-limiting step has moved from that of the technical process of data generation to that of data analysis. We currently face major problems in dealing with the accumulating datasets, not simply with respect to how to archive, access, and process the huge amounts of data that have been and are being produced, but also in determining the relative quality of the different datasets. A major recognized concern is the appropriate use of statistical design in microarray experiments, without which the datasets are rendered useless. A vigorous area of current research involves the development of novel statistical tools specifically for microarray experiments. This article describes, in a necessarily selective manner, the types of platforms currently employed in microarray research and provides an overview of recent activities using these platforms in plant biology.
• Galbraith, D., Li, J., Li, X., Su, H., Chen, H., & Galbraith, D. W. (2006). A framework of integrating gene relations from heterogeneous data sources: an experiment on Arabidopsis thaliana. Bioinformatics (Oxford, England), 22(16).
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  One of the most important goals of biological investigation is to uncover gene functional relations. In this study we propose a framework for extraction and integration of gene functional relations from diverse biological data sources, including gene expression data, biological literature and genomic sequence information. We introduce a two-layered Bayesian network approach to integrate relations from multiple sources into a genome-wide functional network. An experimental study was conducted on a test-bed of Arabidopsis thaliana. Evaluation of the integrated network demonstrated that relation integration could improve the reliability of relations by combining evidence from different data sources. Domain expert judgments on the gene functional clusters in the network confirmed the validity of our approach for relation integration and network inference.
• Galbraith, D., Song, C., & Galbraith, D. W. (2006). AtSAP18, an orthologue of human SAP18, is involved in the regulation of salt stress and mediates transcriptional repression in Arabidopsis. Plant molecular biology, 60(2).
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  In yeast and mammalian systems, it is well established that transcriptional down-regulation by DNA-binding repressors involves core histone deacetylation, mediated by their interaction within a complex containing histone deacetylase (e.g. HDA1), as well as various other proteins (e.g. SIN3, SAP18, SAP30, and RbAp46). Here we identify that a Arabidopsis thaliana gene related in sequence to SAP18, designated AtSAP18, functions in transcription regulation in plants subjected to salt stress. The AtSAP18 loss- of-function mutant is more sensitive to NaCl, and is impaired in chlorophyll synthesis as compared to the wild-type. Using GST pull-down, two-hybrid, and transient transcription assays, we have characterized SAP18 and HDA1 orthologues and provide evidence that SAP18 and HDA1 function as transcriptional repressors. We further demonstrate that they associate with Ethylene-Responsive Element binding Factors (ERFs) to create a hormone-sensitive multimeric repressor complex under conditions of environmental stress. Our results indicate that AtSAP18 functions to link the HDA complex to transcriptional repressors that are bound to chromatin in a sequence-specific manner, thereby providing the specificity of signal transduction accompanying transcriptional repression under stress conditions.
• Ozkan, H., Tuna, M., & Galbraith, D. W. (2006). No DNA loss in autotetraploids of Arabidopsis thaliana. Plant Breeding, 125(3), 288-291.
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  Abstract: To address the issue of genome evolution in autopolyploids and particularly to investigate whether rapid sequence elimination also occurs in autopolyploids as in allopolyploids, amplified fragment length polymorphism (AFLP) fingerprinting was employed to examine a large number of genomic loci in F 1 hybrids between two different autotetraploids of Arabidopsis thaliana accessions, namely Ler and Col. Using this approach, perfect additivity in the F1 hybrids was found between the newly-formed autopolyploids when compared with their parental lines. Using flow cytometry, the study was extended in a quantitative manner, in which the nuclear DNA contents in one autotetraploid A. thaliana accession Ler, was determined. The increase in genome size of the autotetraploid line was additive. Taken together, no evidence was found for genome size reduction due to autopolyploidization of A. thaliana. The results indicating that there was no DNA loss in autotetraploid A. thaliana suggest that a different type of genome evolution may occur in autopolyploids during the initial stages of their formation when compared with allopolyploids. © 2006 Blackwell Verlag.
• Rangwala, S. H., Elumalai, R., Vanier, C., Ozkan, H., Galbraith, D. W., & Richards, E. J. (2006). Meiotically stable natural epialleles of Sadhu, a novel Arabidopsis retroposon.. PLoS genetics, 2(3), e36.
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  PMID: 16552445;PMCID: PMC1401498;Abstract: Epigenetic variation is a potential source of genomic and phenotypic variation among different individuals in a population, and among different varieties within a species. We used a two-tiered approach to identify naturally occurring epigenetic alleles in the flowering plant Arabidopsis: a primary screen for transcript level polymorphisms among three strains (Col, Cvi, Ler), followed by a secondary screen for epigenetic alleles. Here, we describe the identification of stable, meiotically transmissible epigenetic alleles that correspond to one member of a previously uncharacterized non-LTR retroposon family, which we have designated Sadhu. The pericentromeric At2g10410 element is highly expressed in strain Col, but silenced in Ler and 18 other strains surveyed. Transcription of this locus is inversely correlated with cytosine methylation and both the expression and DNA methylation states map in a Mendelian manner to stable cis-acting variation. The silent Ler allele can be converted by the epigenetic modifier mutation ddm1 to a meiotically stable expressing allele with an identical primary nucleotide sequence, demonstrating that the variation responsible for transcript level polymorphism among Arabidopsis strains is epigenetic. We extended our characterization of the Sadhu family members and show that different elements are subject to both genetic and epigenetic variation in natural populations. These findings support the view that an important component of natural variation in retroelements is epigenetic.
• Rangwala, S. H., Elumalai, R., Vanier, C., Ozkan, H., Galbraith, D. W., & Richards, E. J. (2006). Meiotically stable natural epialleles of Sadhu, a novel arabidopsis retroposon. PLoS Genetics, 2(3), 0270-0281.
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  Abstract: Epigenetic variation is a potential source of genomic and phenotypic variation among different individuals in a population, and among different varieties within a species. We used a two-tiered approach to identify naturally occurring epigenetic alleles in the flowering plant Arabidopsis: a primary screen for transcript level polymorphisms among three strains (Col, Cvi, Ler), followed by a secondary screen for epigenetic alleles. Here, we describe the identification of stable, meiotically transmissible epigenetic alleles that correspond to one member of a previously uncharacterized non-LTR retroposon family, which we have designated Sadhu. The pericentromeric At2g10410 element is highly expressed in strain Col, but silenced in Ler and 18 other strains surveyed. Transcription of this locus is inversely correlated with cytosine methylation and both the expression and DNA methylation states map in a Mendelian manner to stable cis-acting variation. The silent Ler allele can be converted by the epigenetic modifier mutation ddm1 to a meiotically stable expressing allele with an identical primary nucleotide sequence, demonstrating that the variation responsible for transcript level polymorphism among Arabidopsis strains is epigenetic. We extended our characterization of the Sadhu family members and show that different elements are subject to both genetic and epigenetic variation in natural populations. These findings support the view that an important component of natural variation in retroelements is epigenetic. Copyright: © 2006 Rangwala et al.
• Song, C., & Galbraith, D. W. (2006). AtSAP18, an orthologue of human SAP18, is involved in the regulation of salt stress and mediates transcriptional repression in Arabidopsis. Plant Molecular Biology, 60(2), 241-257.
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  PMID: 16429262;Abstract: In yeast and mammalian systems, it is well established that transcriptional down-regulation by DNA-binding repressors involves core histone deacetylation, mediated by their interaction within a complex containing histone deacetylase (e.g. HDA1), as well as various other proteins (e.g. SIN3, SAP18, SAP30, and RbAp46). Here we identify that a Arabidopsis thaliana gene related in sequence to SAP18, designated AtSAP18, functions in transcription regulation in plants subjected to salt stress. The AtSAP18 loss- of-function mutant is more sensitive to NaCl, and is impaired in chlorophyll synthesis as compared to the wild-type. Using GST pull-down, two-hybrid, and transient transcription assays, we have characterized SAP18 and HDA1 orthologues and provide evidence that SAP18 and HDA1 function as transcriptional repressors. We further demonstrate that they associate with Ethylene-Responsive Element binding Factors (ERFs) to create a hormone-sensitive multimeric repressor complex under conditions of environmental stress. Our results indicate that AtSAP18 functions to link the HDA complex to transcriptional repressors that are bound to chromatin in a sequence-specific manner, thereby providing the specificity of signal transduction accompanying transcriptional repression under stress conditions. © Springer 2006.
• Woosley, R. L., Woosley, R. L., Sundareshan, P., Galbraith, D. W., & Barthelson, R. A. (2006). Development of a comprehensive detection method for medicinal and toxic plant species.. American journal of botany, 93(4), 566-74. doi:10.3732/ajb.93.4.566
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  Pharmacologically active ingredients in plants can cause significant morbidity through their increasingly common use in herbal alternative medicines and dietary supplements. Monitoring consumer products for the presence of toxic plants is encumbered by the lack of rapid and specific assays. To create a sensitive, reliable, fast, and broad-spectrum assay for medicinal or toxic plant species, we tested multiplexed ligation-dependent probe amplification (MLPA), which requires partial genomic DNA sequences from species of plants that are not well represented in currently available genetic databases. Genomic DNA was obtained from 21 species of medicinal and/or toxic plants. The PCR products were amplified from these plants and cloned for sequencing. The MLPA method was successful with DNA samples from many different species. The use of a microarray to facilitate screening of potentially thousands of plants in a single assay also was successful. The combination of the specificity of the MLPA assay with the broad-scale capabilities of microarray technology should make this an especially useful tool in screening in foods and commercial herbal preparations to identify the plant compounds actually present. Other applications could potentially extend to the identification of any plant species in samples for academic botanical studies and for biodefense and forensics applications.
• Birnbaum, K., Jung, J. W., Wang, J. Y., Lambert, G. M., Hirst, J. A., Galbraith, D. W., & Benfey, P. N. (2005). Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines. Nature Methods, 2(8), 615-619.
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  PMID: 16170893;Abstract: To investigate the relationship between developmental events and gene expression, cell-specific resolution of gene activity is critical. Such high-resolution data have been difficult to obtain at a genomic level because cells first need to be isolated, and then sufficient amounts of mRNA must be collected, or subsequently amplified, for a large-scale profiling analysis. Genomics methods have tremendous potential to infer developmental circuits and, in combination with genetic tools, to discover the unknown downstream targets of known developmental regulators. We have developed a method that can be used to isolate up to hundreds of thousands of plant cells of a specific cell type, with very high purity, which can then be used for microarray analysis. The method makes use of reporter lines expressing green fluorescent protein (GFP) in histologically defined cell types, of which large collections are now available (Table 1). The GFP Line of interest is bulked and the tissue is collected and rapidly converted into protoplasts. GFP-positive cells are then isolated using a fluorescence-activated cell sorter (FACS). Total RNA is isolated, labeled using standard procedures and applied to microarrays (Fig. 1). The technique has been used to generate expression profiles of cell types and tissues in the Arabidopsis thaliana root, although it can be used for any tissue whose cell walls can be readily digested. The protocol presented here has been optimized for roots.
• Birnbaum, K., Jung, J. W., Wang, J. Y., Lambert, G. M., Hirst, J. A., Galbraith, D. W., & Benfey, P. N. (2005). Cell type-specific expression profiting in plants via cell sorting of protoplasts from fluorescent reporter lines. NATURE METHODS, 2(8), 615-619.
• Galbraith, D., Brown, J. K., Lambert, G. M., Ghanim, M., Czosnek, H., & Galbraith, D. W. (2005). Nuclear DNA content of the whitefly Bemisia tabaci (Aleyrodidae: Hemiptera) estimated by flow cytometry. Bulletin of entomological research, 95(4).
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  The nuclear DNA content of the whitefly Bemisia tabaci (Gennnadius) was estimated using flow cytometry. Male and female nuclei were stained with propidium iodide and their DNA content was estimated using chicken red blood cells and Arabidopsis thaliana L. (Brassicaceae) as external standards. The estimated nuclear DNA content of male and female B. tabaci was 1.04 and 2.06 pg, respectively. These results corroborated previous reports based on chromosome counting, which showed that B. tabaci males are haploid and females are diploid. Conversion between DNA content and genome size (1 pg DNA=980 Mbp) indicate that the haploid genome size of B. tabaci is 1020 Mbp, which is approximately five times the size of the genome of the fruitfly Drosophila melanogaster Meigen. These results provide an important baseline that will facilitate genomics-based research for the B. tabaci complex.
• Galbraith, D., Murthi, S., Sankaranarayanan, S., Xia, B., Lambert, G. M., Rodríguez, J. J., & Galbraith, D. W. (2005). Performance analysis of a dual-buffer architecture for digital flow cytometry. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 66(2).
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  Most current commercial flow cytometers employ analog circuitry to provide feature values describing the pulse waveforms produced from suspended cells and particles. This restricts the type of features that can be extracted (typically pulse height, width, and integral) and consequently places a limit on classification performance. In previous work, we described a first-generation digital data acquisition and processing system that was used to demonstrate the classification advantages provided by the extraction of additional waveform features. An improved version of the system is discussed in this paper, focusing on dual-buffering to ensure increased pulse capture. A mathematical model of the system is also presented for performance analysis.
• Galbraith, D., Zhang, C., Gong, F. C., Lambert, G. M., & Galbraith, D. W. (2005). Cell type-specific characterization of nuclear DNA contents within complex tissues and organs. Plant methods, 1(1).
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  Eukaryotic organisms are defined by the presence of a nucleus, which encloses the chromosomal DNA, and is characterized by its DNA content (C-value). Complex eukaryotic organisms contain organs and tissues that comprise interspersions of different cell types, within which polysomaty, endoreduplication, and cell cycle arrest is frequently observed. Little is known about the distribution of C-values across different cell types within these organs and tissues.
• Gardiner, J. M., Buell, C. R., Elumalai, R., Galbraith, D. W., Henderson, D. A., Iniguez, A. L., Kaeppler, S. M., Kim, J. J., Liu, J., Smith, A., Zheng, L., & Chandler, V. L. (2005). Design, production, and utilization of long oligonucleotide microarrays for expression analysis in maize. Maydica, 50(3-4), 425-435.
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  Abstract: Analysis of gene expression on a genome scale can provide useful insights into plant growth and development, and an understanding of the mechanisms used by plants to cope with biotic and abiotic stress. To facilitate analysis of genome-wide gene expression in maize, we have assembled a large collection of maize EST and genomic sequences, designed a set of 57,442 maize 70-mer oligonucleotides to represent these sequences, and printed a two-slide microarray set (MOA and MOB) which is available to the maize research community at minimal cost. To monitor array quality, we have developed a series of printing controls and procedures that when coupled with a 9-mer hybridization assay, allow tracking of spot morphology and printing pin carryover. An optimized hybridization protocol has been developed by testing a series of hybridization temperatures and performing detailed statistical analyses. To facilitate management of all long-oligonucleotide associated array data, Zeamage, a Sybase relational database has been developed and is available at www.maizearray.org. Zeamage contains the appropriate tables and fields for tracking the oligonucleotide sequences and associated annotation, array design, and biological information associated with the microarray hybridizations. The www.maizearray.org web-site provides additional information on the project, array content, and data analysis tools.
• Kristensen, C., Morant, M., Olsen, C. E., Ekstrom, C. T., Galbraith, D. W., Moller, B. L., & Bak, S. (2005). Metabolic engineering of dhurrin in transgenic Arabidopsis plants with marginal inadvertent effects on the metabolome and transcriptome. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 102(5), 1779-1784.
• Kristensen, C., Morant, M., Olsen, C. E., Ekstrøm, C. T., Galbraith, D. W., Møller, B. L., & Bak, S. (2005). Metabolic engineering of dhurrin in transgenic Arabidopsis plants with marginal inadvertent effects on the metabolome and transcriptome. Proceedings of the National Academy of Sciences of the United States of America, 102(5), 1779-1784.
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  PMID: 15665094;PMCID: PMC545087;Abstract: Focused and nontargeted approaches were used to assess the impact associated with introduction of new high-flux pathways in Arabidopsis thaliana by genetic engineering. Transgenic A. thaliana plants expressing the entire biosynthetic pathway for the tyrosine-derived cyanogenic glucoside dhurrin as accomplished by insertion of CYP79A1, CYP71E1, and UGT85B1 from Sorghum bicolor were shown to accumulate 4% dry-weight dhurrin with marginal inadvertent effects on plant morphology, free amino acid pools, transcriptome, and metabolome. In a similar manner, plants expressing only CYP79A1 accumulated 3% dry weight of the tyrosine-derived glucosinolate, p-hydroxybenzylglucosinolate with no morphological pleitropic effects. In contrast, insertion of CYP79A1 plus CYP71E1 resulted in stunted plants, transcriptome alterations, accumulation of numerous glucosides derived from detoxification of intermediates in the dhurrin pathway, and in loss of the brassicaceae-specific UV protectants sinapoyl glucose and sinapoyl malate and kaempferol glucosides. The accumulation of glucosides in the plants expressing CYP79A1 and CYP71E1 was not accompanied by induction of glycosyltransferases, demonstrating that plants are constantly prepared to detoxify xenobiotics. The pleiotrophic effects observed in plants expressing sorghum CYP79A1 and CYP71E1 were complemented by retransformation with S. bicolor UGT85B. These results demonstrate that insertion of high-flux pathways directing synthesis and intracellular storage of high amounts of a cyanogenic glucoside or a glucosinolate is achievable in transgenic A. thaliana plants with marginal inadvertent effects on the transcriptome and metabolome.
• LaJeunesse, T. C., Lambert, G., Andersen, R. A., Coffroth, M. A., & Galbraith, D. W. (2005). Symbiodinium (Pyrrhophyta) genome sizes (DNA content) are smallest among dinoflagellates. JOURNAL OF PHYCOLOGY, 41(4), 880-886.
• LaJeunesse, T. C., Lambert, G., Andersen, R. A., Coffroth, M. A., & Galbraith, D. W. (2005). Symbiodinium (Pyrrhophyta) genome sizes (DNA content) are smallest among dinoflagellates. Journal of Phycology, 41(4), 880-886.
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  Abstract: Using flow cytometric analysis of fluorescence, we measured the genome sizes of 18 cultured "free-living" species and 29 Symbiodinium spp. isolates cultured from stony corals, gorgonians, anemones, jellyfish, and giant clams. Genome size directly correlated with cell size, as documented previously for most eukaryotic cell lines. Among the smallest of dinoflagellates, Symbiodinium spp. (6-15 μm) possessed the lowest DNA content that we measured (1.5-4.8 pg · cell-1). Bloom-forming or potentially harmful species in the genera Alexandrium, Karenia, Pfiesteria, and Prorocentrum possessed genomes approximately 2 to 50 times larger in size. A phylogenetic analysis indicated that genome/cell size has apparently increased and decreased repeatedly during the evolution of dinoflagellates. In contrast, genome sizes were relatively consistent across distantly and closely related Symbiodinium spp. This may be the product of intracellular host habitats imposing strong selective pressures that have restricted symbiont size. © 2005 Phycological Society of America.
• Lambert, G. M., Lajeunesse, T. C., Galbraith, D. W., Coffroth, M. A., & Andersen, R. A. (2005). SYMBIODINIUM (PYRRHOPHYTA) GENOME SIZES (DNA CONTENT) ARE SMALLEST AMONG DINOFLAGELLATES. Journal of Phycology, 41(4), 880-886. doi:10.1111/j.0022-3646.2005.04231.x
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  Using flow cytometric analysis of fluorescence, we measured the genome sizes of 18 cultured "free-living" species and 29 Symbiodinium spp. isolates cultured from stony corals, gorgonians, anemones, jellyfish, and giant clams. Genome size directly correlated with cell size, as documented previously for most eukaryotic cell lines. Among the smallest of dinoflagellates, Symbiodinium spp. (6-15 μm) possessed the lowest DNA content that we measured (1.5-4.8 pg.cell - 1 ). Bloom-forming or potentially harmful species in the genera Alexandrium, Karenia, Pfiesteria, and Prorocentrum possessed genomes approximately 2 to 50 times larger in size. A phylogenetic analysis indicated that genome/cell size has apparently increased and decreased repeatedly during the evolution of dinoflagellates. In contrast, genome sizes were relatively consistent across distantly and closely related Symbiodinium spp. This may be the product of intracellular host habitats imposing strong selective pressures that have restricted symbiont size.
• Zanetti, M. E., Chang, I. F., Gong, F. C., Galbraith, D. W., & Bailey-Serres, J. (2005). Immunopurification of polyribosomal complexes of arabidopsis for global analysis of gene expression. PLANT PHYSIOLOGY, 138(2), 624-635.
• Zanetti, M. E., Chang, I., Gong, F., Galbraith, D. W., & Bailey-Serres, J. (2005). Immunopurification of polyribosomal complexes of Arabidopsis for global analysis of gene expression. Plant Physiology, 138(2), 624-635.
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  PMID: 15955926;PMCID: PMC1150383;Abstract: Immunoaffinity purification of polyribosomes (polysomes) from crude leaf extracts of Arabidopsis (Arabidopsis thaliana) was achieved with transgenic genotypes that overexpress a translational fusion of a ribosomal protein (RP) with a His6-FLAG dual epitope tag. In plants with a cauliflower mosaic virus 35S:HF-RPL18 transgene immunopurification with anti-FLAG agarose beads yielded 60-Svedberg ribosomal subunits, intact 80-Svedberg monosomes and polysomes. Sucrose density gradient fractionation of the purified complexes demonstrated that the distribution of polysome size was similar in crude cell extracts and the purified complexes. The immunopurified complexes included putative cytosolic KPs of Arabidopsis and ribosome-associated proteins, as well as full-length transcripts of high and low abundance. Whole-genome profiling using long DNA oligonucleotide-based microarrays provided a high level of reproducibility between polysomal mRNA samples immunopurified from two independent biological replicates (r approximately 0.90). Comparison of immunopurified and total cellular RNA samples revealed that for most of the genes, the mRNAs were associated with the epitope-tagged polysomal complexes, with an average relative level of association of 62.06% + 4.39%. The results demonstrate that the immunopurification of polysomes can be a valuable tool for the quantification of mRNAs present in translation complexes in plant cells. This technology can be extended to evaluation of mRNA populations at the cell- or tissue-specific level by regulation of the tagged RP with distinct promoters. © 2005 American Society of Plant Biologists.
• Zhang, C., Gong, F. C., Lambert, G. M., & Galbraith, D. W. (2005). Cell type-specific characterization of nuclear DNA contents within complex tissues and organs. Plant Methods, 1(1).
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  Abstract: Background: Eukaryotic organisms are defined by the presence of a nucleus, which encloses the chromosomal DNA, and is characterized by its DNA content (C-value). Complex eukaryotic organisms contain organs and tissues that comprise interspersions of different cell types, within which polysomaty, endoreduplication, and cell cycle arrest is frequently observed. Little is known about the distribution of C-values across different cell types within these organs and tissues.Results: We have developed, and describe here, a method to precisely define the C-value status within any specific cell type within complex organs and tissues of plants. We illustrate the application of this method to Arabidopsis thaliana, specifically focusing on the different cell types found within the root.Conclusion: The method accurately and conveniently charts C-value within specific cell types, and provides novel insight into developmental processes. The method is, in principle, applicable to any transformable organism, including mammals, within which cell type specificity of regulation of endoreduplication, of polysomaty, and of cell cycle arrest is suspected. © 2005 Zhang et al; licensee BioMed Central Ltd.
• Galbraith, D. W. (2004). Cytometry and Plant Sciences: A Personal Retrospective. Cytometry Part A, 58(1), 37-44.
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  PMID: 14994218;
• Galbraith, D. W. (2004). Nuclear dynamics in higher plants.. Symposia of the Society for Experimental Biology, 217-228.
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  PMID: 15565884;
• Galbraith, D. W. (2004). The Rainbow of Fluorescent Proteins. Methods in Cell Biology, 75, 153-169.
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  PMID: 15603425;
• Galbraith, D. W., Elumalai, R., & Gong, F. C. (2004). Integrative flow cytometric and microarray approaches for use in transcriptional profiling.. Methods in molecular biology (Clifton, N.J.), 263, 259-280.
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  PMID: 14976371;Abstract: Flow cytometry and cell sorting provides unparalleled means for the identification and purification of specific cell types. It is a mature technology having been in existence commercially for the last 25 yr. High-throughput transcriptional profiling methods have emerged relatively recently. These provide the means to characterize efficiently the genome-wide contribution of individual genes to gene expression. A combination of these methods offers the opportunity to explore the relationship between gene expression and the ways in which different cell types are formed and maintained. This chapter provides a review of published methods for analysis of global gene expression within different cell types in complex tissues and organs, and provides practical details concerning microarray fabrication and use based on presynthesized 70-mer oligonucleotide array elements.
• Galbraith, D., & Galbraith, D. W. (2004). Cytometry and plant sciences: a personal retrospective. Cytometry. Part A : the journal of the International Society for Analytical Cytology, 58(1).
• Galbraith, D., & Galbraith, D. W. (2004). Nuclear dynamics in higher plants. Symposia of the Society for Experimental Biology.
• Galbraith, D., & Galbraith, D. W. (2004). The rainbow of fluorescent proteins. Methods in cell biology, 75.
• Inan, G., Zhang, Q., Li, P. H., Wang, Z. L., Cao, Z. Y., Zhang, H., Zhang, C. Q., Quist, T. M., Goodwin, S. M., Zhu, J. H., Shi, H. H., Damsz, B., Charbaji, T., Gong, Q. Q., Ma, S. S., Fredricksen, M., Galbraith, D. W., Jenks, M. A., Rhodes, D., , Hasegawa, P. M., et al. (2004). Salt cress. A halophyte and cryophyte Arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles. PLANT PHYSIOLOGY, 135(3), 1718-1737.
• Inan, G., Zhang, Q., Pinghua, L. i., Wang, Z., Cao, Z., Zhang, H., Zhang, C., Quist, T. M., Goodwin, S. M., Zhu, J., Shi, H., Damsz, B., Charbaji, T., Gong, Q., Shisong, M. a., Fredricksen, M., Galbraith, D. W., Jenks, M. A., Rhodes, D., , Hasegawa, P. M., et al. (2004). Salt cress. A halophyte and cryophyte arabidopsis relative model system and its applicability to molecular genetic analyses of growth and development of extremophiles. Plant Physiology, 135(3), 1718-1737.
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  PMID: 15247369;PMCID: PMC519085;Abstract: Salt cress (Thellungiella halophila) is a small winter annual crucifer with a short life cycle. It has a small genome (about 2 x Arabidopsis) with high sequence identity (average 92%) with Arabidopsis, and can be genetically transformed by the simple floral dip procedure. It is capable of copious seed production. Salt cress is an extremophile native to harsh environments and can reproduce after exposure to extreme salinity (500 mM NaCl) or cold to -15°C. It is a typical halophyte that accumulates NaCl at controlled rates and also dramatic levels of Pro (>150 mM) during exposure to high salinity. Stomata of salt cress are distributed on the leaf surface at higher density, but are less open than the stomata of Arabidopsis and respond to salt stress by closing more tightly. Leaves of salt cress are more succulent-like, have a second layer of palisade mesophyll cells, and are frequently shed during extreme salt stress. Roots of salt cress develop both an extra endodermis and cortex cell layer compared to Arabidopsis. Salt cress, although salt and cold tolerant, is not exceptionally tolerant of soil desiccation. We have isolated several ethyl methanesulfonate mutants of salt cress that have reduced salinity tolerance, which provide evidence that salt tolerance in this halophyte can be significantly affected by individual genetic loci. Analysis of salt cress expressed sequence tags provides evidence for the presence of paralogs, missing in the Arabidopsis genome, and for genes with abiotic stress-relevant functions. Hybridizations of salt cress RNA targets to an Arabidopsis whole-genome oligonucleotide array indicate that commonly stress-associated transcripts are expressed at a noticeably higher level in unstressed salt cress plants and are induced rapidly under stress. Efficient transformation of salt cress allows for simple gene exchange between Arabidopsis and salt cress. In addition, the generation of T-DNA-tagged mutant collections of salt cress, already in progress, will open the door to a new era of forward and reverse genetic studies of extremophile plant biology.
• Meyers, B. C., Galbraith, D. W., Nelson, T., & Agrawal, V. (2004). Methods for transcriptional profiling in plants. Be fruitful and replicate. Plant Physiology, 135(2), 637-652.
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  PMID: 15173570;PMCID: PMC514100;
• Miyazaki, S., Fredricksen, M., Hollis, K. C., Poroyko, V., Shepley, D., Galbraith, D. W., Long, S. P., & Bohnert, H. J. (2004). Transcript expression profiles of Arabidopsis thaliana grown under controlled conditions and open-air elevated concentrations of CO ₂ and of O ₃. Field Crops Research, 90(1), 47-59.
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  Abstract: Microarrays have significantly enhanced our ability to track transcripts and changes in gene expression under different conditions and environments. In order to find determinants of plant performance influenced by atmospheric changes, we tracked transcript profiles in Arabidopsis thaliana (L.) Heynh. grown in a field within SoyFACE. This facility (http://www.soyface.uiuc.edu/ index.htm) consists of a series of open-air rings within which either [CO 2] or [O 3] are elevated above current atmospheric concentrations by 1.5× and 1.2×, respectively. A microarray platform including ∼26,000 DNA elements was used to monitor transcript abundance and changes due to exposure to elevated [CO 2] and [O 3]. Transcripts from plants in growth chambers were further compared with material from plants grown in Free Air Concentration Enrichment (FACE) rings under ambient conditions. Most changes in gene expression were observed between growth chamber and ambient field conditions. Two to four times the number of transcripts were either up- or down-regulated between controlled versus field ambient conditions compared with high versus low [CO 2] or [O 3] contrasts. The differences showed a preponderance of regulated transcripts in categories such as chaperones and general defense reactions, altered (secondary) metabolic functions, redox control, energy provision, protein turnover, signaling and transcription. We discuss the results obtained in a model non-crop plant for their possible relevance in studies with crop species. © 2004 Elsevier B.V. All rights reserved.
• Song, C. P., Guo, Y., Qiu, Q. S., Lambert, G., Galbraith, D. W., Jagendorf, A., & Zhu, J. K. (2004). A probable Na+(K+)/H+ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 101(27), 10211-10216.
• Song, C., Guo, Y., Qiu, Q., Lambert, G., Galbraith, D. W., Jagendorf, A., & Zhu, J. (2004). A probable Na⁺(K⁺)/H⁺ exchanger on the chloroplast envelope functions in pH homeostasis and chloroplast development in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America, 101(27), 10211-10216.
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  PMID: 15220473;PMCID: PMC454189;Abstract: Electroneutral monovalent cation/proton antiport across the chloroplast envelope has been shown previously to have an important regulatory effect on stromal pH and thereby on photosynthetic carbon reduction. Here we report that an Arabidopsis nuclear gene, AtCHX23, encodes a putative Na+(K +)/H+ exchanger and functions in the adjustment of pH in the cytosol and possibly in maintaining a high pH level in the chloroplast stroma. The AtCHX23 protein is localized in the chloroplast envelope. Plastids from chx23 mutants had straight thylakoids but lacked grana lamellae. chx23 mutant leaves were pale yellow and had a much reduced chlorophyll content. The chlorophyll content of chx23 was increased by growing in medium at low (4.0) pH and decreased by growing at high (7.0) pH. The cytosolic pH in the leaves of the mutant was significantly higher than that in the wild type. chx23 mutants displayed a high sensitivity to NaCl. Together, these data indicate that CHX23 is a probable chloroplast Na+(K+)/H+ exchanger important for pH homeostasis and chloroplast development and function.
• Birnbaum, K., Shasha, D. E., Wang, J. Y., Jung, J. W., Lambert, G. M., Galbraith, D. W., & Benfey, P. N. (2003). A Gene Expression Map of the Arabidopsis Root. Science, 302(5652), 1956-1960.
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  PMID: 14671301;Abstract: A global map of gene expression within an organ can identify genes with coordinated expression in localized domains, thereby relating gene activity to cell fate and tissue specialization. Here, we present localization of expression of more than 22,000 genes in the Arabidopsis root. Gene expression was mapped to 15 different zones of the root that correspond to cell types and tissues at progressive developmental stages. Patterns of gene expression traverse traditional anatomical boundaries and show cassettes of hormonal response. Chromosomal clustering defined some coregulated genes. This expression map correlates groups of genes to specific cell fates and should serve to guide reverse genetics.
• Birnbaum, K., Shasha, D. E., Wang, J. Y., Jung, J. W., Lambert, G. M., Galbraith, D. W., & Benfey, P. N. (2003). A gene expression map of the Arabidopsis root. SCIENCE, 302(5652), 1956-1960.
• Boyce, J. M., Knight, H., Deyholos, M., Openshaw, M. R., Galbraith, D. W., Warren, G., & Knight, M. R. (2003). The sfr6 mutant of Arabidopsis is defective in transcriptional activation via CBF/DREB1 and DREB2 and shows sensitivity to osmotic stress. Plant Journal, 34(4), 395-406.
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  PMID: 12753580;Abstract: The sfr6 mutant of Arabidopsis displays a deficit in freezing tolerance after cold acclimation. We previously observed that the transcripts of three cold-, ABA- and drought-inducible genes, each having a C-repeat motif or the drought-responsive element (CRT/DRE) in its promoter, failed to normally accumulate in this mutant. We now report that the effects of sfr6 upon transcript levels are reflected in the levels of the encoded proteins, confirming that the cold-inducible protein expression is affected by the sfr6 mutation. Using microarray analysis, we found not only that this effect may be general to cold-inducible genes with CRT/DRE promoter elements, but also that it extends to some other genes whose promoters lack a CRT/DRE element. The role of the CRT/DRE has been empirically tested by use of a synthetic promoter, confirming that the CRT/DRE is sufficient to confer the sfr6 effect upon expression. Tolerance of osmotic stress was also found to be reduced in sfr6, consistent with a role in osmotic stress tolerance for the cold-, ABA-and drought-inducible genes whose expression is affected by the sfr6 mutation.
• Galbraith, D. W. (2003). Global analysis of cell type-specific gene expression. Comparative and Functional Genomics, 4(2), 208-215.
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  PMID: 18629131;PMCID: PMC2447418;Abstract: The tissues and organs of multicellular eukaryotes are frequently observed to comprise complex three-dimensional interspersions of different cell types. It is a reasonable assumption that different global patterns of gene expression are found within these different cell types. This review outlines general experimental strategies designed to characterize these global gene expression patterns, based on a combination of methods of transgenic fluorescent protein (FP) expression and targeting, of flow cytometry and sorting and of high-throughput gene expression analysis. Copyright © 2003 John Wiley & Sons, Ltd.
• Galbraith, D., & Galbraith, D. W. (2003). Global analysis of cell type-specific gene expression. Comparative and functional genomics, 4(2).
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  The tissues and organs of multicellular eukaryotes are frequently observed to comprise complex three-dimensional interspersions of different cell types. It is a reasonable assumption that different global patterns of gene expression are found within these different cell types. This review outlines general experimental strategies designed to characterize these global gene expression patterns, based on a combination of methods of transgenic fluorescent protein (FP) expression and targeting, of flow cytometry and sorting and of high-throughput gene expression analysis.
• Postier, B. L., Wang, H., Singh, A., Impson, L., Andrews, H. L., Klahn, J., Hong, L. i., Risinger, G., Pesta, D., Deyholos, M., Galbraith, D. W., Sherman, L. A., & Burnap, R. L. (2003). The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy. BMC Genomics, 4.
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  Abstract: Background: DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein). We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. Results: Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. Conclusions: The technique detailed here minimizes the cost and effort to replicate a PCRgenerated DNA gene fragment library and facilitates several downstream processes (e.g. directional cloning of fragments and gene expression as affinity-tagged fusion proteins) beyond the primary objective of producing DNA microarrays for global gene expression profiling. © 2003 Postier et al; licensee BioMed Central Ltd.
• Postier, B. L., Wang, H., Singh, A., Impson, L., Andrews, H. L., Klahn, J., Hong, L. i., Risinger, G., Pesta, D., Deyholos, M., Galbraith, D. W., Sherman, L. A., & Burnap, R. L. (2003). The construction and use of bacterial DNA microarrays based on an optimized two-stage PCR strategy.. BMC genomics [electronic resource], 4(1), 23-.
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  PMID: 12803655;PMCID: PMC165429;Abstract: BACKGROUND: DNA microarrays are a powerful tool with important applications such as global gene expression profiling. Construction of bacterial DNA microarrays from genomic sequence data using a two-stage PCR amplification approach for the production of arrayed DNA is attractive because it allows, in principal, the continued re-amplification of DNA fragments and facilitates further utilization of the DNA fragments for additional uses (e.g. over-expression of protein). We describe the successful construction and use of DNA microarrays by the two-stage amplification approach and discuss the technical challenges that were met and resolved during the project. RESULTS: Chimeric primers that contained both gene-specific and shared, universal sequence allowed the two-stage amplification of the 3,168 genes identified on the genome of Synechocystis sp. PCC6803, an important prokaryotic model organism for the study of oxygenic photosynthesis. The gene-specific component of the primer was of variable length to maintain uniform annealing temperatures during the 1st round of PCR synthesis, and situated to preserve full-length ORFs. Genes were truncated at 2 kb for efficient amplification, so that about 92% of the PCR fragments were full-length genes. The two-stage amplification had the additional advantage of normalizing the yield of PCR products and this improved the uniformity of DNA features robotically deposited onto the microarray surface. We also describe the techniques utilized to optimize hybridization conditions and signal-to-noise ratio of the transcription profile. The inter-lab transportability was demonstrated by the virtual error-free amplification of the entire genome complement of 3,168 genes using the universal primers in partner labs. The printed slides have been successfully used to identify differentially expressed genes in response to a number of environmental conditions, including salt stress. CONCLUSIONS: The technique detailed here minimizes the cost and effort to replicate a PCR-generated DNA gene fragment library and facilitates several downstream processes (e.g. directional cloning of fragments and gene expression as affinity-tagged fusion proteins) beyond the primary objective of producing DNA microarrays for global gene expression profiling.
• Wang, H., Miyazaki, S., Kawai, K., Deyholos, M., Galbraith, D. W., & Bohnert, H. J. (2003). Temporal progression of gene expression responses to salt shock in maize roots. PLANT MOLECULAR BIOLOGY, 52(4), 873-891.
• Wang, H., Miyazaki, S., Kawai, K., Deyholos, M., Galbraith, D. W., & Bohnert, H. J. (2003). Temporal progression of gene expression responses to salt shock in maize roots. Plant Molecular Biology, 52(4), 873-891.
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  PMID: 13677474;Abstract: Using a cDNA microarray containing 7943 ESTs, the behavior of the maize root transcriptome has been monitored in a time course for 72 h after imposition of salinity stress (150 mM NaCl). Under these conditions, root sodium amounts increased faster than in leaves, and root potassium decreased significantly. Although the overall free amino acid concentration was not affected, amino acid composition was changed with proline and asparagine increasing. Microarray analysis identified 916 ESTs representing genes whose steady-state RNA levels were significantly altered at various time points, corresponding to 11% of the ESTs printed. The response of the transcriptome to sub-lethal salt stress was rapid and transient, leading to a burst of changes at the three-hour time point. The salt-regulated ESTs represented 472 tentatively unique genes (TUGs), which, based on functional category analysis, are involved in a broad range of cellular and biochemical activities, prominent amongst which were transport and signal transduction pathways. Clustering of regulated transcripts based on the timing and duration of changes suggests a structured succession of induction and repression for salt responsive genes in multiple signal and response cascades. Within this framework, 16 signaling molecules, including six protein kinases, two protein phosphatases and eight transcription factors, were regulated with distinct expression patterns by high salinity.
• Fernandes, J., Brendel, V., Gai, X. W., Lal, S., Chandler, V. L., Elumalai, P., Galbraith, D. W., Pierson, E. A., & Walbot, V. (2002). Comparison of RNA expression profiles based on maize expressed sequence tag frequency analysis and micro-array hybridization. PLANT PHYSIOLOGY, 128(3), 896-910.
• Fernandes, J., Brendel, V., Gai, X., Lal, S., Chandler, V. L., Elumalai, R. P., Galbraith, D. W., Pierson, E. A., & Walbot, V. (2002). Comparison of RNA expression profiles based on maize expressed sequence tag frequency analysis and micro-array hybridization. Plant Physiology, 128(3), 896-910.
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  PMID: 11891246;PMCID: PMC152203;Abstract: Assembly of 73,000 expressed sequence tags (ESTs) representing multiple organs and developmental stages of maize (Zea mays) identified approximately 22,000 tentative unique genes (TUGs) at the criterion of 95% identity. Based on sequence similarity, overlap between any two of nine libraries with more than 3,000 ESTs ranged from 4% to 20% of the constituent TUGs. The most abundant ESTs were recovered from only one or a minority of the libraries, and only 26 EST contigs had members from all nine EST sets (presumably representing ubiquitously expressed genes). For several examples, ESTs for different members of gene families were detected in distinct organs. To study this further, two types of micro-array slides were fabricated, one containing 5,534 ESTs from 10- to 14-d-old endosperm, and the other 4,844 ESTs from immature ear, estimated to represent about 2,800 and 2,500 unique genes, respectively. Each array type was hybridized with fluorescent cDNA targets prepared from endosperm and immature ear poly(A+) RNA. Although the 10- to 14-d-old postpollination endosperm TUGs showed only 12% overlap with immature ear TUGs, endosperm target hybridized with 94% of the ear TUGs, and ear target hybridized with 57% of the endosperm TUGs. Incomplete EST sampling of low-abundance transcripts contributes to an underestimate of shared gene expression profiles. Reassembly of ESTs at the criterion of 90% identity suggests how cross hybridization among gene family members can overestimate the overlap in genes expressed in micro-array hybridization experiments.
• Liwińska, E., Lambert, G. M., & Galbraith, D. W. (2002). Factors affecting nuclear dynamics and green fluorescent protein targeting to the nucleus in Arabidopsis thaliana roots. Plant Science, 163(3), 425-430.
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  Abstract: Nuclear movement and targeting of a green fluorescent protein (GFP)-nuclear localization sequence (NLS)-β-glucuronidase (GUS) chimeric protein to the roots of transformed Arabidopsis has been studied using time lapse analysis. To characterize the mechanism governing this movement, metabolic inhibitors, and chemicals that alter the concentrations of divalent cations in the cell were applied. Flow cytometry was used to examine the condition of nuclear membranes. The results showed that nuclear movement in Arabidopsis roots was energy-dependent. The concentration of magnesium ions seemed to play a crucial role in the movement of the nuclei and targeting of the chimeric GFP protein into the nucleoplasm. In contrast, alterations in concentration of calcium ions did not affect either nuclear dynamics or nucleoplasmic GFP accumulation. The results are discussed in terms of some of the physiological processes occurring within the cell. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
• Ozturk, Z. N., Talame, V., Deyholos, M., Michalowski, C. B., Galbraith, D. W., Gozukirmizi, N., Tuberosa, R., & Bohnert, H. J. (2002). Monitoring large-scale changes in transcript abundance in drought- and salt-stressed barley. PLANT MOLECULAR BIOLOGY, 48(5), 551-573.
• Ozturk, Z. N., Talamé, V., Deyholos, M., Michalowski, C. B., Galbraith, D. W., Gozukirmizi, N., Tuberosa, R., & Bohnert, H. J. (2002). Monitoring large-scale changes in transcript abundance in drought- and salt-stressed barley. Plant Molecular Biology, 48(5-6), 551-573.
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  PMID: 11999834;Abstract: Responses to drought and salinity in barley (Hordeum vulgare L. cv. Tokak) were monitored by microarray hybridization of 1463 DNA elements derived from cDNA libraries of 6 and 10 h drought-stressed plants. Functional identities indicated that many cDNAs in these libraries were associated with drought stress. About 38% of the transcripts were novel and functionally unknown. Hybridization experiments were analyzed for drought- and salinity-regulated sequences, with significant changes defined as a deviation from the control exceeding 2.5-fold. Responses of transcripts showed stress-dependent expression patterns and time courses. Nearly 15% of all transcripts were either up- or down-regulated under drought stress, while NaCl led to a change in 5% of the transcripts (24 h, 150 mM NaCl). Transcripts that showed significant up-regulation under drought stress are exemplified by jasmonate-responsive, metallothionein-like, late-embryogenesis-abundant (LEA) and ABA-responsive proteins. Most drastic down-regulation in a category was observed for photosynthesis-related functions. Up-regulation under both drought and salt stress was restricted to ESTs for metallothionein-like and LEA proteins, while increases in ubiquitin-related transcripts characterized salt stress. A number of functionally unknown transcripts from cDNA libraries of drought-stressed plants showed up-regulation by drought but down-regulation by salt stress, documenting how precisely transcript profiles report different growth conditions and environments.
• Bak, S., Tax, F. E., Feldmann, K. A., Galbraith, D. W., & Feyereisen, R. (2001). CYP83B1, a cytochrome P450 at the metabolic branch paint in auxin and indole glucosinolate biosynthesis in Arabidopsis. PLANT CELL, 13(1), 101-111.
• Bohnert, H. J., Ayoubi, P., Borchert, C., Bressan, R. A., Burnap, R. L., Cushman, J. C., Cushman, M. A., Deyholos, M., Fischer, R., Galbraith, D. W., Hasegawa, P. M., Jenks, M., Kawasaki, S., Koiwa, H., Kore-eda, S., Lee, B. -., Michalowski, C. B., Misawa, E., Nomura, M., , Ozturk, N., et al. (2001). A genomics approach towards salt stress tolerance. Plant Physiology and Biochemistry, 39(3-4), 295-311.
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  Abstract: Abiotic stresses reduce plant productivity. We focus on gene expression analysis following exposure of plants to high salinity, using salt-shock experiments to mimic stresses that affect hydration and ion homeostasis. The approach includes parallel molecular and genetic experimentation. Comparative analysis is employed to identify functional isoforms and genetic orthologs of stress-regulated genes common to cyanobacteria, fungi, algae and higher plants. We analyze global gene expression profiles monitored under salt stress conditions through abundance profiles in several species: in the cyanobacterium Synechocystis PCC6803, in unicellular (Saccharomyces cerevisiae) and multicellular (Aspergillus nidulans) fungi, the eukaryotic alga Dunaliella salina, the halophytic land plant Mesembryanthemum crystallinum, the glycophytic Oryza sativa and the genetic model Arabidopsis thaliana. Expanding the gene count, stress brings about a significant increase of transcripts for which no function is known. Also, we generate insertional mutants that affect stress tolerance in several organisms. More than 400 000 T-DNA tagged lines of A. thaliana have been generated, and lines with altered salt stress responses have been obtained. Integration of these approaches defines stress phenotypes, catalogs of transcripts and a global representation of gene expression induced by salt stress. Determining evolutionary relationships among these genes, mutants and transcription profiles will provide categories and gene clusters, which reveal ubiquitous cellular aspects of salinity tolerance and unique solutions in multicellular species. © 2001 Éditions scientifiques et médicales Elsevier SAS.
• Bohnert, H. J., Ayoubi, P., Borchert, C., Bressan, R. A., Burnap, R. L., Cushman, J. C., Cushman, M. A., Deyholos, M., Fischer, R., Galbraith, D. W., Hasegawa, P. M., Jenks, M., Kawasaki, S., Koiwa, H., Kore-eda, S., Lee, B. H., Michalowski, C. B., Misawa, E., Nomura, M., , Ozturk, N., et al. (2001). A genomics approach towards salt stress tolerance. PLANT PHYSIOLOGY AND BIOCHEMISTRY, 39(3-4), 295-311.
• Deyholos, M. K., & Galbraith, D. W. (2001). High-Density Microarrays for Gene Expression Analysis. Cytometry, 43(4), 229-238.
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  PMID: 11260590;
• Galbraith, D. W., Lambert, G. M., Macas, J., & Dolezel, J. (2001). Analysis of nuclear DNA content and ploidy in higher plants.. Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.], Chapter 7, Unit 7.6.
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  PMID: 18770733;Abstract: This is the first of a series of units discussing the application of cytometry to plant material. Techniques commonly used for mammalian nuclei evaluation need considerable modification to be successful with plant material. David Galbraith and his colleagues bring together many years of knowledge in plant cytometry. Their unit provides detailed protocols on measuring DNA content, ploidy, and cell cycle status of plant tissue using both conventional laser based instruments as well as arc lamp cytometers. This unit provides an excellent starting point for those interested in doing cytometry with plants.
• Galbraith, D. W., Macas, J., Pierson, E. A., Xu, W., & Nouzová, M. (2001). Printing DNA microarrays using the Biomek 2000 laboratory automation workstation.. Methods in molecular biology (Clifton, N.J.), 170, 131-140.
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  PMID: 11357677;
• Galbraith, D., Deyholos, M. K., & Galbraith, D. W. (2001). High-density microarrays for gene expression analysis. Cytometry, 43(4).
• Galbraith, D., Xu, W., Bak, S., Decker, A., Paquette, S. M., Feyereisen, R., & Galbraith, D. W. (2001). Microarray-based analysis of gene expression in very large gene families: the cytochrome P450 gene superfamily of Arabidopsis thaliana. Gene, 272(1-2).
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  Cytochrome P450 (P450s) are heme-thiolate protein products of a very large gene superfamily, present in all kingdoms and involved in a variety of metabolic reactions. P450s are classified according to the degree of amino acid sequence identity, with P450s of the same family defined as having >40% identity, and P450s of the same subfamily having >55% identity. Currently, 273 P450 genes distributed over 45 families have been identified in Arabidopsis, and its genome is estimated to contain as many as 286. Genome-wide DNA microarrays make it possible to broadly correlate P450 gene activity with alterations in physiological or developmental states. A potential problem with microarray research is that sequence similarity between and within these families of closely related genes may lead to cross-hybridization. We designed experiments to systematically evaluate the specificity of P450 microarrays, and showed that conditions could be optimized to provide a very high degree of hybridization specificity. Under these conditions, and employing a 20% intensity value of maximum hybridization intensity as a cut-off, labeled P450 genes exhibited essentially no cross-hybridization between families and within subfamilies. We also compared the gene transcription levels of microarray probes derived from EST clones and from genomic DNA sequences for which ESTs were not available, using cDNA produced from RNA from various Arabidopsis tissue as the target. Many of the P450 genes displayed tissue-specific expression, leading to hypotheses as to the function of individual genes and their regulation. We also observed that several of the genomic sequences reported high levels of expression, highlighting the limitations of expression analysis based on ESTs alone.
• Kawasaki, S., Borchert, C., Deyholos, M., Wang, H., Brazille, S., Kawai, K., Galbraith, D., & Bohnert, H. J. (2001). Gene expression profiles during the initial phase of salt stress in rice. Plant Cell, 13(4), 889-905.
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  PMID: 11283343;PMCID: PMC135538;Abstract: Transcript regulation in response to high salinity was investigated for salt-tolerant rice (var Pokkali) with microarrays including 1728 cDNAs from libraries of salt-stressed roots. NaCl at 150 mM reduced photosynthesis to one tenth of the prestress value within minutes. Hybridizations of RNA to microarray slides probed for changes in transcripts from 15 min to 1 week after salt shock. Beginning 15 min after the shock, Pokkali showed upregulation of transcripts. Approximately 10% of the transcripts in Pokkali were significantly upregulated or downregulated within 1 hr of salt stress. The initial differences between control and stressed plants continued for hours but became less pronounced as the plants adapted over time. The interpretation of an adaptive process was supported by the similar analysis of salinity-sensitive rice (var IR29), in which the immediate response exhibited by Pokkali was delayed and later resulted in downregulation of transcription and death. The upregulated functions observed with Pokkali at different time points during stress adaptation changed over time. Increased protein synthesis and protein turnover were observed at early time points, followed by the induction of known stress-responsive transcripts within hours, and the induction of transcripts for defense-related functions later. After 1 week, the nature of upregulated transcripts (e.g., aquaporins) indicated recovery.
• Kore-eda, S., Galbraith, D. W., Deyholos, M. K., Cushman, M. A., Cushman, J. C., & Agarie, S. (2001). Gene expression profiling of salinity stress responses using expressed sequence tag (EST)-based microarrays in the common ice plant, Mesembryanthemum crystallinum. Plant and Cell Physiology, 42.
• Nouzová, M., Neumann, P., Navrátilová, A., Galbraith, D. W., & Macas, J. (2001). Microarray-based survey of repetitive genomic sequences in Vicia spp.. Plant Molecular Biology, 45(2), 229-244.
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  PMID: 11289513;Abstract: A modified DNA microarray-based technique was devised for preliminary screening of short fragment genomic DNA libraries from three Vicia species (V. melanops, V. narbonensis, and V. sativa) to isolate representative highly abundant DNA sequences that show different distribution patterns among related legume species. The microarrays were sequentially hybridized with labeled genomic DNAs of thirteen Vicia and seven other Fabaceae species and scored for hybridization signals of individual clones. The clones were then assigned to one of the following groups characterized by hybridization to: (1) all tested species, (2)most of the Vicia and Pisum species, (3) only a few Vicia species, and (4) preferentially a single Vicia species. Several clones from each group, 65 in total, were sequenced. All Group I clones were identified as rDNA genes or fragments of chloroplast genome, whereas the majority of Group II clones showed significant homologies to retroelement sequences. Clones in Groups III and IV contained novel dispersed repeats with copy numbers 102-106/1C and two genus-specific tandem repeats. One of these belongs to the VicTR-B repeat family, and the other clone (S12) Contains an amplified portion of the rDNA intergenic spacer. In situ hybridization using V. sativa metaphase chromosomes revealed the presence of the S12 sequences not only within rDNA genes, but also at several additional loci. The newly identified repeats, as well as the retroelement-like sequences, were characterized with respect to their abundance within individual genomes. Correlations between the repeat distributions and the current taxonomic classification of these species are discussed.
• Wenying, X. u., Bak, S., Decker, A., Paquette, S. M., Feyereisen, R., & Galbraith, D. W. (2001). Microarray-based analysis of gene expression in very large gene families: The cytochrome P450 gene superfamily of Arabidopsis thaliana. Gene, 272(1-2), 61-74.
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  PMID: 11470511;Abstract: Cytochrome P450 (P450s) are heme-thiolate protein products of a very large gene superfamily, present in all kingdoms and involved in a variety of metabolic reactions. P450s are classified according to the degree of amino acid sequence identity, with P450s of the same family defined as having >40% identity, and P450s of the same subfamily having >55% identity. Currently, 273 P450 genes distributed over 45 families have been identified in Arabidopsis, and its genome is estimated to contain as many as 286. Genome-wide DNA microarrays make it possible to broadly correlate P450 gene activity with alterations in physiological or developmental states. A potential problem with microarray research is that sequence similarity between and within these families of closely related genes may lead to cross-hybridization. We designed experiments to systematically evaluate the specificity of P450 microarrays, and showed that conditions could be optimized to provide a very high degree of hybridization specificity. Under these conditions, and employing a 20% intensity value of maximum hybridization intensity as a cut-off, labeled P450 genes exhibited essentially no cross-hybridization between families and within subfamilies. We also compared the gene transcription levels of microarray probes derived from EST clones and from genomic DNA sequences for which ESTs were not available, using cDNA produced from RNA from various Arabidopsis tissue as the target. Many of the P450 genes displayed tissue-specific expression, leading to hypotheses as to the function of individual genes and their regulation. We also observed that several of the genomic sequences reported high levels of expression, highlighting the limitations of expression analysis based on ESTs alone. © 2001 Elsevier Science B.V. All rights reserved.
• Xiong, L., Gong, Z., Rock, C. D., Subramanian, S., Guo, Y., Wenying, X. u., Galbraith, D., & Zhu, J. (2001). Modulation of Abscisic Acid Signal Transduction and Biosynthesis by an Sm-like Protein in Arabidopsis. Developmental Cell, 1(6), 771-781.
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  PMID: 11740939;Abstract: The phytohormone abscisic acid (ABA) regulates plant growth and development as well as stress tolerance. The Arabidopsis sad1 (supersensitive to ABA and drought) mutation increases plant sensitivity to drought stress and ABA in seed germination, root growth, and the expression of some stress-responsive genes. sad1 plants are also defective in the positive feedback regulation of ABA biosynthesis genes by ABA and are impaired in drought stress induction of ABA biosynthesis. SAD1 encodes a polypeptide similar to multifunctional Sm-like snRNP proteins that are required for mRNA splicing, export, and degradation. These results suggest a critical role for mRNA metabolism in the control of ABA signaling as well as in the regulation of ABA homeostasis. © 2001 Cell Press.
• Xu, W. Y., Bak, S., Decker, A., Paquette, S. M., Feyereisen, R., & Galbraith, D. W. (2001). Microarray-based analysis of gene expression in very large gene families: the cytochrome P450 gene superfamily of Arabidopsis thaliana. GENE, 272(1-2), 61-74.
• Zhang, X., Zhang, L., Dong, F. C., Gao, J. F., Galbraith, D. W., & Song, C. P. (2001). Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. PLANT PHYSIOLOGY, 126(4), 1438-1448.
• Zhang, X., Zhang, L., Dong, F., Gao, J., Galbraith, D. W., & Song, C. -. (2001). Hydrogen peroxide is involved in abscisic acid-induced stomatal closure in Vicia faba. Plant Physiology, 126(4), 1438-1448.
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  PMID: 11500543;PMCID: PMC117144;Abstract: One of the most important functions of the plant hormone abscisic acid (ABA) is to induce stomatal closure by reducing the turgor of guard cells under water deficit. Under environmental stresses, hydrogen peroxide (H2O2), an active oxygen species, is widely generated in many biological systems. Here, using an epidermal strip bioassay and laser-scanning confocal microscopy, we provide evidence that H2O2 may function as an intermediate in ABA signaling in Vicia faba guard cells. H2O2 inhibited induced closure of stomata, and this effect was reversed by ascorbic acid at concentrations lower than 10-5 M. Further, ABA-induced stomatal closure also was abolished partly by addition of exogenous catalase (CAT) and diphenylene iodonium (DPI), which are an H2O2 scavenger and an NADPH oxidase inhibitor, respectively. Time course experiments of single-cell assays based on the fluorescent probe dichlorofluorescein showed that the generation of H2O2 was dependent on ABA concentration and an increase in the fluorescence intensity of the chloroplast occurred significantly earlier than within the other regions of guard cells. The ABA-induced change in fluorescence intensity in guard cells was abolished by the application of CAT and DPI. In addition, ABA microinjected into guard cells markedly induced H2O2 production, which preceded stomatal closure. These effects were abolished by CAT or DPI micro-injection. Our results suggest that guard cells treated with ABA may close the stomata via a pathway with H2O2 production involved, and H2O2 may be an intermediate in ABA signaling.
• Zhu, J., Wang, H., Prade, R. A., Nomura, M., Michalowski, C. B., Kore-eda, S., Koiwa, H., Kawasaki, S., Hasegawa, P. M., Galbraith, D. W., Deyholos, M. K., Cushman, M. A., Cushman, J. C., Clark, E., Burmap, R., Bressan, R. A., Bohnert, H. J., Ayoubi, P., & Agarie, S. (2001). GENOMICS-BASED UNDERSTANDING OF ABIOTIC STRESS RESPONSES IN PLANTS :. Plant and Cell Physiology, 42.
• Chytilova, E., Macas, J., Sliwinska, E., Rafelski, S. M., Lambert, G. M., & Galbraith, D. W. (2000). Nuclear dynamics in Arabidopsis thaliana. MOLECULAR BIOLOGY OF THE CELL, 11(8), 2733-2741.
• Chytilova, E., Macas, J., Sliwinska, E., Rafelski, S. M., Lambert, G. M., & Galbraith, D. W. (2000). Nuclear dynamics in Arabidopsis thaliana. Molecular Biology of the Cell, 11(8), 2733-2741.
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  PMID: 10930466;PMCID: PMC14952;Abstract: The nucleus is a definitive feature of eukaryotic cells, comprising twin bilamellar membranes, the inner and outer nuclear membranes, which separate the nucleoplasmic and cytoplasmic compartments. Nuclear pores, complex macromolecular assemblies that connect the two membranes, mediate communication between these compartments. To explore the morphology, topology, and dynamics of nuclei within living plant cells, we have developed a novel method of confocal laser scanning fluorescence microscopy under time-lapse conditions. This is used for the examination of the transgenic expression in Arabidopsis thaliana of a chimeric protein, comprising the GFP (Green-Fluorescent Protein of Aequorea victoria) translationally fused to an effective nuclear localization signal (NLS) and to β-glucuronidase (GUS) from E. coli. This large protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm. This article provides online access to movies that illustrate the remarkable and unusual properties displayed by the nuclei, including polymorphic shape changes and rapid, long-distance, intracellular movement. Movement is mediated by actin but not by tubulin; it therefore appears distinct from mechanisms of nuclear positioning and migration that have been reported for eukaryotes. The GFP-based assay is simple and of general applicability. It will be interesting to establish whether the novel type of dynamic behavior reported here, for higher plants, is observed in other eukaryotic organisms.
• Galbraith, D., Chytilova, E., Macas, J., Sliwinska, E., Rafelski, S. M., Lambert, G. M., & Galbraith, D. W. (2000). Nuclear dynamics in Arabidopsis thaliana. Molecular biology of the cell, 11(8).
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  The nucleus is a definitive feature of eukaryotic cells, comprising twin bilamellar membranes, the inner and outer nuclear membranes, which separate the nucleoplasmic and cytoplasmic compartments. Nuclear pores, complex macromolecular assemblies that connect the two membranes, mediate communication between these compartments. To explore the morphology, topology, and dynamics of nuclei within living plant cells, we have developed a novel method of confocal laser scanning fluorescence microscopy under time-lapse conditions. This is used for the examination of the transgenic expression in Arabidopsis thaliana of a chimeric protein, comprising the GFP (Green-Fluorescent Protein of Aequorea victoria) translationally fused to an effective nuclear localization signal (NLS) and to beta-glucuronidase (GUS) from E. coli. This large protein is targeted to the nucleus and accumulates exclusively within the nucleoplasm. This article provides online access to movies that illustrate the remarkable and unusual properties displayed by the nuclei, including polymorphic shape changes and rapid, long-distance, intracellular movement. Movement is mediated by actin but not by tubulin; it therefore appears distinct from mechanisms of nuclear positioning and migration that have been reported for eukaryotes. The GFP-based assay is simple and of general applicability. It will be interesting to establish whether the novel type of dynamic behavior reported here, for higher plants, is observed in other eukaryotic organisms.
• Chytilova, E., Macas, J., & Galbraith, D. W. (1999). Green fluorescent protein targeted to the nucleus, a transgenic phenotype useful for studies in plant biology. Annals of Botany, 83(6), 645-654.
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  Abstract: We present a characterization of transgenic Arabidopsis thaliana (L.) Heynh. plants expressing a chimeric gene comprising the Green Fluorescent Protein (GFP) and β-glucuronidase (GUS) coding sequences, fused to an efficient nuclear localization signal (NLS). The transgenic plants accumulate the fusion protein in their nuclei, and this provides a novel phenotype, that of green-fluorescent nuclei. The fluorescent nuclei are readily observed using conventional epifluorescence and laser scanning confocal microscopy. We describe the use of this phenotype for in vivo studies of nuclear shape and movement, cell division, and for analysis of the transcriptional activities of constitutive and tissue-specific promoters. We propose that the phenotype of fluorescent nuclei will prove particularly valuable in histological, physiological and developmental studies of higher plants that require the facile observation of nuclei within living cells and in the absence of fixation or external staining.
• Feldmann, K. A., Xu, W., Watts, G. S., Walbot, V., Pierson, E. A., Macas, J., Galbraith, D. W., Feyereisen, R., Feldmann, K. A., Deyholos, M. K., & Bohnert, H. J. (1999). Implementing microarrays for the study of plant genomics. Nature Genetics, 23(3), 45-45. doi:10.1038/14307
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  Microarrays provide the means for the large-scale analysis of gene expression patterns in living organisms. My laboratory is part of three federally funded projects that are directed toward an understanding of the regulation of gene expression in higher plants.
• Galbraith, D. W., Anderson, M. T., & Herzenberg, L. A. (1999). Flow cytometric analysis and FACS sorting of cells based on GFP accumulation. Methods in Cell Biology, 315-341.
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  PMID: 9891389;
• Galbraith, D. W., Herzenberg, L. A., & Anderson, M. T. (1999). Flow cytometric analysis of transgene expression in higher plants: Green fluorescent protein. Methods in Enzymology, 302, 296-315.
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  PMID: 12876781;
• Johnston, J. S., Bennett, M. D., Rayburn, A. L., Galbraith, D. W., & Price, H. J. (1999). Reference standards for determination of DNA content of plant nuclei. AMERICAN JOURNAL OF BOTANY, 86(5), 609-613.
• Johnston, J. S., Bennett, M. D., Rayburn, A. L., Galbraith, D. W., & Price, H. J. (1999). Reference standards for determination of DNA content of plant nuclei. American Journal of Botany, 86(5), 609-613.
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  PMID: 10330063;Abstract: Flow cytometry was used to compare 14 potential reference standards for plant DNA content determination. Both chicken and plant internal standards were used, as were propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) as fluorochromes. Means and standard errors of the means are presented for the 14 potential reference standards, and the means are compared to those obtained by Feulgen densitometry. Five species are recommended as an initial set of international standards for future plant DNA content determinations: Sorghum bicolor cv. Pioneer 8695 (2C = 1.74 pg), Pisum sativum cv. Minerva Maple (2C = 9.56 pg), Hordeum vulgare cv. Sultan (2C = 11.12 pg), Vicia faba (2C = 26.66 pg), and Allium cepa cv. Ailsa Craig (2C = 33.55 pg). It is recommended that the reference standard of choice be one with 2C and 4C nuclear DNA content peaks similar to, but not overlapping, the 2C and 4C peaks of the target species. We recommend PI as the fluorochrome of choice for flow cytometric determination of plant DNA content. DAPI should be used only if the estimated DNA value is corroborated by using a second stain that has no bias for AT- or GC-rich sequences within genomes.
• Schmelz, E. A., Grebenok, R. J., Galbraith, D. W., & Bowers, W. S. (1999). Insect-induced synthesis of phytoecdysteroids in spinach, Spinacia oleracea. Journal of Chemical Ecology, 25(8), 1739-1757.
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  Abstract: Spinach (Spinacia oleracea) foliage is known to synthesize and accumulate insect molting hormones, predominantly in the form of 20- hydroxyecdysone (20E). We previously demonstrated that root 20E accumulation is increased following root damage. We designed two further experiments to address root responses to both mechanical and insect damage. In plants grown hydroponically, removal of 35% or less of the root mass did not result in changes in root 20E levels. However, removal of 70% of the root mass stimulated 6.0- and 1.5-fold increases in the root and shoot 20E concentrations, respectively. The effects of insect damage on soil-grown plants were investigated by infesting plant roots with black vine weevil (BVW: Otiorhynchus sulcatus) larvae and allowing them to feed for seven days. Decreases in root mass occurred in young plants; however, no changes were detected in mature plants. In all cases, root herbivory resulted in at least a 3.0-fold increase in root 20E concentrations. Our previous experiments implicated jasmonic acid and the analog methyl jasmonate (MJ) in signaling the damage-induced accumulation of root 20E levels. We investigated the activity of other phytohormones and growth regulators (GRs) on the 20E accumulation patterns of young plants as a means of examining the significance of jasmonates in the induction response. Hydroponic additions of MJ (0.5 μM) and the synthetic auxin, 1-naphthaleneacetic acid (NAA; 0.5 μM), resulted in significant increases in root 20E levels. At the concentrations tested, indole-3-acetic acid (IAA), gibberellic acid (GA3), abscisic acid (ABA), and trans-zeatin (Z) had no effects on root 20E concentrations. However, both NAA (0.5-5.0 μM) and Z (5.0 μM) treatments caused increases in the root/shoot dry mass ratios, indicating shifts in resource allocation to the roots. Treatments involving ABA (5.0 μM) and Z (0.5-5.0 μM) caused significant increases in shoot 20E concentrations. No other hormone treatments altered shoot accumulation patterns. The mechanisms underlying the root 20E induction phenomena were investigated through the incorporation of [2-14C]mevalonic acid ([14C]MVA). Within one day, excised roots readily incorporated radioactivity into 20E from [14C]MVA. In intact plants, [14C]MVA absorbed by the roots was rapidly incorporated into root 20E pools following damage and MJ treatments. This implies that the wound-induced root 20E accumulation is the result of increased de novo 20E synthesis in the root.
• Galbraith, D., Macas, J., Lambert, G. M., Dolezel, D., & Galbraith, D. W. (1998). Nuclear expressed sequence Tag (NEST) analysis: a novel means to study transcription through amplification of nuclear RNA. Cytometry, 33(4).
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  We describe a novel concept and corresponding methods for the analysis of transcription in higher plant cells. The concept is that an examination of the presence of different polyadenylated transcripts within isolated nuclei reflects the state of gene expression at a given moment more precisely than do conventional techniques using total cellular mRNA. The methods involve isolation of polyadenylated nuclear transcripts from flow-sorted nuclei, reverse transcription, amplification using the polymerase chain reaction, and analysis of the products through gel electrophoresis and sequencing. By using specific primers, we have demonstrated detection of selected gene products in nuclei from transgenic plants. We also employed a technique for analysis of individual transcripts based on the length polymorphisms of restriction fragments derived from their 3' ends. Because the technique does not require a priori knowledge of the analyzed sequences, it is suitable for displaying the complete spectra of RNA transcripts present in nuclei at the moment of their isolation. These fragments can be easily isolated and sequenced and the sequence information used for assignment of putative function of corresponding genes. These techniques have been used to identify leaf-, root-, and cell cycle-specific transcripts. In principle, they should be applicable to the tissues of any eukaryotic species that contain transcriptionally active nuclei.
• Galbraith, D., Macas, J., Nouzová, M., & Galbraith, D. W. (1998). Adapting the Biomek 2000 Laboratory Automation Workstation for printing DNA microarrays. BioTechniques, 25(1).
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  The Biomek 2000 Laboratory Automation Workstation is used for liquid handling and other repetitive operations in many laboratories. Since it has very good spatial positioning capabilities, we have modified this workstation to deliver samples at high densities onto microscope slides to produce DNA microarrays. The workstation tool, originally designed for bacterial colony replication, was adapted to carry special printing pins and was further modified to improve its positional accuracy. Software written in the Tool Command Language was concurrently developed to control the movements of the workstation arm during the process of printing. With these modifications, the workstation can reliably deliver individual samples at a spacing of 0.5 mm, corresponding to a total of more than 3000 samples on a single slide. Arrays prepared in this way were successfully tested in hybridization experiments.
• Grebenok, R. J., Ohnmeiss, T. E., Yamamoto, A., Huntley, E. D., Galbraith, D. W., & Penna, D. D. (1998). Isolation and characterization of an Arabidopsis thaliana C-8,7 sterol isomerase: Functional and structural similarities to mammalian C-8,7 sterol isomerase/emopamil-binding protein. Plant Molecular Biology, 38(5), 807-815.
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  PMID: 9862498;Abstract: The yeast C-8,7 sterol isomerase contains a polyvalent high-affinity drug binding site similar to mammalian sigma receptors. Exogenously supplied sigma ligands inhibit sterol biosynthesis in yeast, demonstrating a pharmacological relationship between sigma ligand-binding and C-8,7 sterol isomerase activity. We report the isolation of an Arabidopsis thaliana C-8,7 sterol isomerase by functional complementation of the corresponding sterol mutant in yeast and its characterization by exposure to sigma ligands. The yeast erg2 mutant, which lacks the C-8,7 sterol isomerase gene and activity, was transformed with an Arabidopsis cDNA yeast expression library. Transformed colonies were selected for restoration of C-8,7 sterol isomerase activity (i.e. wild-type ergosterol production) by enhanced resistance to the antibiotic cycloheximide. Sterols produced in complemented lines were characterized by gas chromatography-mass spectroscopy (GC-MS). The full-length A. thaliana cDNA (pA.t.SI1) that complemented the erg2 mutation contains an open reading frame encoding a 21 kDa protein that shares 68% similarity and 35% amino acid identity to the recently isolated mouse C-8,7 sterol isomerase. The sigma ligands, haloperidol, ifenprodil and verapamil inhibited the production of ergosterol in wild-type Saccharomyces cerevisiae and in the erg2 mutant complemented with pA.t.SI1. Structural and biochemical similarities between the A. thaliana C-8,7 sterol isomerase and the mammalian emopamil-binding protein (EBP) are discussed.
• Macas, J., Lambert, G. M., Dolezel, D., & Galbraith, D. W. (1998). Nuclear expressed sequence tag (NEST) analysis: A novel means to study transcription through amplification of nuclear RNA. Cytometry, 33(4), 460-468.
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  PMID: 9845441;Abstract: We describe a novel concept and corresponding methods for the analysis of transcription in higher plant cells. The concept is that an examination of the presence of different polyadenylated transcripts within isolated nuclei reflects the state of gene expression at a given moment more precisely than do conventional techniques using total cellular mRNA. The methods involve isolation of polyadenylated nuclear transcripts from flow-sorted nuclei, reverse transcription, amplification using the polymerase chain reaction, and analysis of the products through gel electrophoresis and sequencing. By using specific primers, we have demonstrated detection of selected gene products in nuclei from transgenic plants. We also employed a technique for analysis of individual transcripts based on the length polymorphisms of restriction fragments derived from their 3' ends. Because the technique does not require a priori knowledge of the analyzed sequences, it is suitable for displaying the complete spectra of RNA transcripts present in nuclei at the moment of their isolation. These fragments can be easily isolated and sequenced and the sequence information used for assignment of putative function of corresponding genes. These techniques have been used to identify leaf-, root- , and cell cycle-specific transcripts. In principle, they should be applicable to the tissues of any eukaryotic species that contain transcriptionally active nuclei.
• Macas, J., Nouzová, M., & Galbraith, D. W. (1998). Adapting the Biomek® 2000 Laboratory Automation Workstation for printing DNA microarrays. BioTechniques, 25(1), 106-110.
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  PMID: 9668984;Abstract: The Biomek® 2000 Laboratory Automation Workstation is used for liquid handling and other repetitive operations in many laboratories. Since it has very good spatial positioning capabilities, we have modified this workstation to deliver samples at high densities onto microscope slides to produce DNA microarrays. The workstation tool, originally designed for bacterial colony replication, was adapted to carry special printing pins and was further modified to improve its positional accuracy. Software written in the Tool Command Language was concurrently developed to control the movements of the workstation arm during the process of printing. With these modifications, the workstation can reliably deliver individual samples at a spacing of 0.5 mm, corresponding to a total of more than 3000 samples on a single slide. Arrays prepared in this way were successfully tested in hybridization experiments.
• Schmelz, E. A., Grebenok, R. J., Galbraith, D. W., & Bowers, W. S. (1998). Damage-induced accumulation of phytoecdysteroids in spinach: A rapid root response involving the octadecanoic acid pathway. Journal of Chemical Ecology, 24(2), 339-360.
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  Abstract: Some plant defenses are known to be rapidly induced following attack by phytophagous insects. Plant-produced insect molting hormones, trained phytoecdysteroids, are believed to aid plant resistance; however, their dynamics are poorly understood. Using spinach (Spinacia oleracea) as a model system, we examined the inducibility of phytoecdysteroids, primarily 20-hydroxyecdysone (20E), in an effort to characterize potential interactions with herbivorous insects. Rapid phytochemical induction was investigated using damage treatments and applications of defense-related plant-signal analogs, specifically methyl jasmonate (MJ) and methyl salicylate (MSA). Within two days, mechanically damaged roots exhibited two to three fold increases in phytoecdysteroid concentrations. Four days after root damage, small increases in shoot levels were also detectable. Unlike roots, foliar 20E concentrations were unaltered over a range of shoot treatments including insect herbivory (Spodoptera exigua), mechanical damage, and MJ applications. Additions of MJ (12.5-50 μg/liter) to the root systems of hydroponically grown plants stimulated accumulations of root phytoecdysteroids in a dose-dependent manner, similar in magnitude to the response induced by root damage. Under identical conditions, MSA did not affect the accumulation of 20E when added to the hydroponic solutions of undamaged plants. Moreover, MSA inhibited the induction of 20E in wounded roots, but did not interfere with the action of applied MJ. In contrast to mechanical damage, roots did not induce 20E levels when challenged with two different fungal pathogens (Pythium aphanidermatum and Phytophthora capsici). We propose that wound- induced accumulations of 20E are generated in the roots, signaled via endogenous jasmonates, and may confer enhanced resistance against subterranean herbivorous insects.
• Winkler, R. G., Frank, M. R., Galbraith, D. W., Feyereisen, R., & Feldmann, K. A. (1998). Systematic reverse genetics of transfer-DNA-tagged lines of Arabidopsis - Isolation of mutations in the cytochrome P450 gene superfamily. PLANT PHYSIOLOGY, 118(3), 743-749.
• Winkler, R. G., Frank, M. R., Galbraith, D. W., Feyereisen, R., & Feldmann, K. A. (1998). Systematic reverse genetics of transfer-DNA-tagged lines of arabidopsis: Isolation of mutations in the cytochrome P450 gene superfamily. Plant Physiology, 118(3), 743-750.
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  PMID: 9808718;PMCID: PMC34784;Abstract: We have developed an efficient reverse-genetics protocol that uses expedient pooling and hybridization strategies to identify individual transfer-DNA insertion lines from a collection of 6000 independently transformed lines in as few as 36 polymerase chain reactions. We have used this protocol to systematically isolate Arabidopsis lines containing insertional mutations in individual cytochrome P450 genes. In higher plants P450 genes encode enzymes that perform an exceptionally wide range of functions, including the biosynthesis of primary metabolites necessary for normal growth and development, the biosynthesis of secondary products, and the catabolism of xenobiotics. Despite their importance, progress in assigning enzymatic function to individual P450 gene products has been slow. Here we report the isolation of the first 12 such lines, including one (CYP83B1-1) that displays a runt phenotype (small plants with hooked leaves), and three insertions in abundantly expressed genes. The DNAs used in this study are publicly available and can be used to systematically isolate mutants in Arabidopsis.
• Galbraith, D., Grebenok, R. J., Pierson, E., Lambert, G. M., Gong, F. C., Afonso, C. L., Haldeman-Cahill, R., Carrington, J. C., & Galbraith, D. W. (1997). Green-fluorescent protein fusions for efficient characterization of nuclear targeting. The Plant journal : for cell and molecular biology, 11(3).
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  The green-fluorescent protein (GFP) from Aequorea victoria has been shown to be a convenient and flexible reporter molecule within a variety of eukaryotic systems, including higher plants. It is particularly suited for applications in vivo, since the mechanism of fluorophore formation involves an intramolecular autoxidation and does not require exogenous co-factors. Unlike standard histochemical procedures of fixation and staining required for analysis of the cellular or tissue-specific expression of other popular reporter molecules, such as the beta-glucuronidase (GUS) marker, analysis of GFP can be done in living cells with no specific pretreatments. This implies that GFP might also be particularly suited for studies of intracellular protein targeting. In this paper, the use of GUS is compared with that of GFP for the analysis of nuclear targeting in tobacco. A novel oligopeptide motif from a tobacco protein is described which confers nuclear localization of GUS. The use of this oligopeptide and two from potyviral proteins to target GFP to the nucleus is examined. An essential modification of GFP is described, which specifically increases its molecular weight to eliminate its passive penetration into the nucleus. Three examples of the targeting of these enlarged GFP molecules to the nucleus are illustrated. GFP, in combination with confocal microscopy, offers significant advantages over traditional methods of studying nuclear targeting.
• Grebenok, R. J., Galbraith, D. W., & Penna, D. D. (1997). Characterization of Zea mays endosperm C-24 sterol methyltransferase: One of two types of sterol methyltransferase in higher plants. Plant Molecular Biology, 34(6), 891-896.
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  PMID: 9290641;Abstract: We report the characterization of a higher-plant C-24 sterol methyltransferase by yeast complementation. A Zea mays endosperm expressed sequence tag (EST) was identified which, upon complete sequencing, showed 46% identity to the yeast C-24 methyltransferase gene (ERG6) and 75% and 37% amino acid identity to recently isolated higher-plant sterol methyltransferases from soybean and Arabidopsis, respectively. When placed under GAL4 regulation, the Z. mays cDNA functionally complemented the erg6 mutation, restoring ergosterol production and conferring resistance to cycloheximide. Complementation was both plasmid-dependent and galactose-inducible. The Z. mays cDNA clone contains an open reading frame encoding a 40 kDa protein containing motifs common to a large number of S-adenosyl-L-methionine methyltransferases (SMTs). Sequence comparisons and functional studies of the maize, soybean and Arabidopsis cDNAs indicates two types of C-24 SMTs exist in higher plants.
• Grebenok, R. J., Lambert, G. M., & Galbraith, D. W. (1997). Characterization of the targeted nuclear accumulation of GFP within the cells of transgenic plants. Plant Journal, 12(3), 685-696.
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  Abstract: The soluble proteins of the nucleoplasm are synthesized on cytoplasmic ribosomes. Proteins larger than about 40 kDa are post-translationally targeted to the nucleus via energy-dependent processes, passing through the nuclear pore complex into the nucleoplasm. Targeting involves nuclear localization signals (NLSs) found within the primary sequences of the imported proteins. In higher plants, information has come primarily from study of proteins carrying 'classical' NLSs, comprising stretches of basic amino acids, and has required assays to measure nuclear uptake both in vitro and in vivo. In general, these assays are not entirely satisfactory; they are either technically demanding, are of limited accuracy and statistical rigor, or are unsuitable for in vivo applications. The green-fluorescent protein (GFP) of Aequorea victoria has recently emerged as a versatile marker for transgenic expression in vivo. Conditions under which GFP gene fusions can be employed for the analysis of nuclear targeting in plant protoplasts have been described. This study demonstrates for the first time the nuclear targeting of chimeric GFP molecules in transgenic tobacco. This is accompanied by a description and evaluation of novel analytical methods, involving flow and image cytometry, for the quantitative temporal and spatial analysis of nuclear targeting, and these unique methods are used to provide information concerning the targeting process. Finally, the way in which the chimeric GFP molecules might be employed for the study of various important problems in plant cell and developmental biology is discussed.
• Grebenok, R. J., Pierson, E., Lambert, G. M., Gong, F. C., Afonso, C. L., HaldemanCahill, R., Carrington, J. C., & Galbraith, D. W. (1997). Green-fluorescent protein fusions for efficient characterization of nuclear targeting. PLANT JOURNAL, 11(3), 573-586.
• Grebenok, R. J., Pierson, E., Lambert, G. M., Gong, F., Afonso, C. L., Haldeman-Cahill, R., Carrington, J. C., & Galbraith, D. W. (1997). Green-fluorescent protein fusions for efficient characterization of nuclear targeting. Plant Journal, 11(3), 573-586.
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  PMID: 9107043;Abstract: The green-fluorescent protein (GFP) from Aequorea victoria has been shown to be a convenient and flexible reporter molecule within a variety of eukaryotic systems, including higher plants. It is particularly suited for applications in vivo, since the mechanism of fluorophore formation involves an intramolecular autoxidation and does not require exogenous co-factors. Unlike standard histochemical procedures of fixation and staining required for analysis of the cellular or tissue-specific expression of other popular reporter molecules, such as the β-glucuronidase (GUS) marker, analysis of GFP can be done in living cells with no specific pretreatments. This implies that GFP might also be particularly suited for studies of intracellular protein targeting. In this paper, the use of GUS is compared with that of GFP for the analysis of nuclear targeting in tobacco. A novel oligopeptide motif from a tobacco protein is described which confers nuclear localization of GUS. The use of this oligopeptide and two from potyviral proteins to target GFP to the nucleus is examined. An essential modification of GFP is described, which specifically increases its molecular weight to eliminate its passive penetration into the nucleus. Three examples of the targeting of these enlarged GFP molecules to the nucleus are illustrated. GFP, in combination with confocal microscopy, offers significant advantages over traditional methods of studying nuclear targeting.
• Shen, Z., Corbin, D. R., Greenplate, J. T., Grebenok, R. J., Galbraith, D. W., & Purcell, J. P. (1997). Studies on the mode of action of cholesterol oxidase on insect midgut membranes. Archives of Insect Biochemistry and Physiology, 34(4), 429-442.
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  Abstract: Cholesterol oxidase (EC 1.1.3.6.) is an insecticidal protein known to have potent activity against the boll weevil, milder activity against a number of lepidopteran species, and virtually no activity against other insects. Several factors that could explain its species-dependent differential activity were examined. We compared cholesterol concentrations and rates of cholesterol oxidation in the midgut membranes from larvae of boll weevil (Anthonomus grandis grandis Boheman), southern corn rootworm (Diabrotica undecimpunctata Howardi Barber), tobacco budworm (Heliothis virescens Fabricius), and yellow mealworm (Tenebrio molitor Linnaeus). Results showed that cholesterol concentration alone could not account for the differences in insecticidal activity and that midgut brush-border membranes of all species tested were generally susceptible to oxidation by cholesterol oxidase in vitro. We also demonstrated that cholesterol oxidase stability in the midgut environment was similar for the species tested and thus could not account for the differential activity. However, comparison of the pH of the insect midgut fluids with the pH optimum of cholesterol oxidase indicated that the lower sensitivity of lepidopteran larvae to the enzyme may be partially due to the alkaline nature of their midgut environments. In some species, oxidation caused significant changes in the activities of brush-border membrane alkaline phosphatase, and these changes did correlate with the susceptibility of the insect to cholesterol oxidase. © 1997 Wiley-Liss, Inc.
• Winkler, R., Frank, M. R., Feldmann, K. A., Galbraith, D. W., & Feyereisen, R. (1997). Systematic reverse genetics of the P450 gene family in arabidopsis thaliana. FASEB Journal, 11(9), A811.
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  Abstract: The great diversity of P450 genes in higher eukaryotes makes it difficult to assign a function to each member of this gene family. In particular, little is known about the function of the very diverse P450 genes in plants. We have initiated a systematic reverse genetics approach to address this problem. A large number of Arabidopsis thaliana P450 expressed sequence tags (ESTs) are available in the EST database. These represent at least 60 independent P450 genes, perhaps close to half of all the A. thaliana P450 genes. Null mutations (gene knock outs) in P450 genes induced by the insertion of the T-DNA of Agrobacterium tumefaciens in A. thaliana transformants were screened by a systematic PCR-based approach. This screen used primers to the P450 EST sequences or degenerate primers to conserved P450 sequences, in combination with T-DNA border primers. DNA from over 6,000 transformed lines was screened and null mutant lines rapidly isolated by a multi-arrayed pooling strategy. Insertion of T-DNA in the P450 gene and the identity of the P450 gene thus tagged were confirmed by sequencing. We will present a detailed molecular and phenotypic description of the first 12 P450 gene mutants, which belong to at least 6 different CYP families. Supported by NSF/DOE/USDA Joint Program on Collaborative Research in Plant Biology.
• Galbraith, D., Godavarti, M., Rodriguez, J. J., Yopp, T. A., Lambert, G. M., & Galbraith, D. W. (1996). Automated particle classification based on digital acquisition and analysis of flow cytometric pulse waveforms. Cytometry, 24(4).
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  In flow cytometry, the typical use of front-end analog processing limits the pulse waveform features that can be measured to pulse integral, height, and width. Direct digitizing of the waveforms provides a means for the extraction of additional features, for example, pulse skewness and kurtosis, and Fourier properties. In this work, we have first demonstrated that the Fourier properties of the pulse can be employed usefully for discrimination between different types of cells that otherwise cannot be classified by using only time-domain features of the pulse. We then implemented and evaluated automatic procedures for cell classification based on neural networks. We established that neural networks could provide an efficient means of classification of cell types without the need for user interaction. The neural networks were also employed in an innovative manner for analysis of the digital flow cytometric data without feature extraction. The performance of the neural networks was compared with that of a more conventional means of classification, the K-means clustering algorithm. Neural networks can be realized in hardware, and this, in addition to their highly parallel architecture, makes them an important potential part of real-time analysis systems. These results are discussed in terms of the design of a real-time digital data acquisition system for flow cytometry.
• Galbraith, D., Gong, F. C., Giddings, T. H., Meehl, J. B., Staehelin, L. A., & Galbraith, D. W. (1996). Z-membranes: artificial organelles for overexpressing recombinant integral membrane proteins. Proceedings of the National Academy of Sciences of the United States of America, 93(5).
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  We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of beta-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 micrometers in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.
• Gong, F., Giddings, T. H., Meehl, J. B., Staehelin, L. A., & Galbraith, D. W. (1996). Z-membranes: Artificial organelles for overexpressing recombinant integral membrane proteins. Proceedings of the National Academy of Sciences of the United States of America, 93(5), 2219-2223.
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  PMID: 8700911;PMCID: PMC39938;Abstract: We have expressed a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme β-glucuronidase in transgenic tobacco cells. Electron microscope images of such cells demonstrate that overexpression of this fusion protein gives rise to a type of endoplasmic reticulum membrane domain in which adjacent membranes become zippered together apparently as a consequence of the oligomerizing action of β-glucuronidase. These zippered (Z-) membranes lack markers of the endoplasmic reticulum (NADH cytochrome c reductase and ribosomes) and accumulate in the cells in the form of multilayered scroll-like structures (up to 2 μm in diameter; 20-50 per cell) without affecting plant growth. The discovery of Z-membranes has broad implications for biology and biotechnology in that they provide a means for accumulating large quantities of recombinant membrane proteins within discrete domains of native membranes.
• Grebenok, R. J., Galbraith, D. W., Benveniste, I., & Feyereisen, R. (1996). Ecdysone 20-monooxygenase, a cytochrome P450 enzyme from spinach, Spinacia oleracea. Phytochemistry, 42(4), 927-933.
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  Abstract: A microsomal preparation isolated from first leaves of 25-day-old spinach catalysed the hydroxylation of ecdysone to produce the insect moulting hormone, 20-hydroxyecdysone. Hydroxylation was dependent on NADPH and molecular oxygen, and was inhibited by carbon monoxide. Carbon monoxide inhibition was partially reversible by white light. Polyclonal antibodies to the Jerusalem artichoke NADPH-cytochrome P450 reductase inhibited the hydroxylation reaction as well as the spinach microsomal NADPH cytochrome c reductase. These results taken together establish ecdysone hydroxylation as a cytochrome P450 dependent reaction in spinach, which is known to synthesize large amounts of phytoecdysteroids.
• Galbraith, D. W., Bohnert, H. J., & Bourque, D. P. (1995). Preface. Methods in Cell Biology, 49(C), xix-xx.
• Galbraith, D. W., Bourque, D. P., & Bohnert, H. J. (1995). Preface. Methods in Cell Biology, 50(C), xxi-xxii.
• Galbraith, D. W., Lambert, G. M., Grebenok, R. J., & Sheen, J. (1995). Chapter 1 Flow Cytometric Analysis of Transgene Expression in Higher Plants: Green-Fluorescent Protein. Methods in Cell Biology, 50(C), 3-14.
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  PMID: 8531802;
• Galbraith, D., Zilmer, N. A., Godavarti, M., Rodriguez, J. J., Yopp, T. A., Lambert, G. M., & Galbraith, D. W. (1995). Flow cytometric analysis using digital signal processing. Cytometry, 20(2).
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  Current commercial flow cytometers employ analog circuits to produce the feature values of the pulse waveforms that result from particle analysis. The use of analog pulse processing limits the features that can be measured to pulse integral, pulse height, and pulse width, and a large amount of potentially relevant information about the shape of the pulse waveform is lost. Direct digitizing of the waveform provides a means for the extraction of additional features, for example, pulse skewness and kurtosis, as well as the Fourier properties of the pulse. Here we describe a digital pulse waveform processing system that is compatible both with a commercial flow cytometer, and with a readily available computational platform. The performance of the digital and analog systems were compared through analysis of synthetic waveforms, and the waveforms produced by standard fluorescence microspheres and biological particles. The digital waveform processing system was found to be accurate and flexible, and the value of several of its unique attributes was demonstrated using biological cells. A protocol was designed in which digital pulse processing provided a means for the quantitative monitoring of the optical alignment of the flow cytometer. It was shown that digital pulse processing could be used to discriminate between particle classes which produce feature values indistinguishable through analog pulse processing, and to discriminate accurately single cells from doublets and larger aggregates.
• SHEEN, J., HWANG, S. B., NIWA, Y., KOBAYASHI, H., & GALBRAITH, D. W. (1995). GREEN-FLUORESCENT PROTEIN AS A NEW VITAL MARKER IN PLANT-CELLS. PLANT JOURNAL, 8(5), 777-784.
• Sheen, J., Hwang, S., Niwa, Y., Kobayashi, H., & Galbraith, D. W. (1995). Green-fluorescent protein as a new vital marker in plant cells. Plant Journal, 8(5), 777-784.
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  PMID: 8528289;Abstract: The green-fluorescent protein (GFP) from jellyfish Aequorea victoria has been used as a convenient new vital marker in various heterologous systems. However, it has been problematic to express GFP in higher eukaryotes, especially in higher plants. This paper reports that either a strong constitutive or a heat-shock promoter can direct the expression of GFP which is easily detectable in maize mesophyll protoplasts. In this single-cell system, bright green fluorescence emitted from GFP is visible when excited with UV or blue light even in the presence of blue fluorescence from the vacuole or the red chlorophyll autofluorescence from chloroplasts using a fluorescence microscope. No exogenous substrate, co-factor, or other gene product is required. GFP is very stable in plant cells and shows little photobleaching. Viable cells can be obtained after fluorescence-activated cell sorting based on GFP. The paper further reports that GFP can be detected in intact tissues after delivering the constructs into Arabidopsis leaf and root by microprojectile bombardment. The successful detection of GFP in plant cells relies on the use of a universal transcription enhancer from maize or the translation enhancer from tobacco mosaic virus (TMV) to boost the expression. This new reporter could be used to monitor gene expression, signal transduction, co-transfection, transformation, protein trafficking and localization, protein-protein interaction, cell separation and purification, and cell lineage in higher plants.
• Afonso, C. L., & Galbraith, D. W. (1994). The callus associated protein (CAP) gene of Nicotiana tabacum: Isolation, characterization, and evidence for possible function as a transcriptional factor. In Vitro Cellular & Developmental Biology - Plant, 30(1), 44-54.
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  Abstract: We have identified a callus associated protein (CAP) as a new molecular marker for proliferative growth and early cellular differentiation in higher plants. Antiserum directed against the Sorghum callus associated protein (CAP) was employed to isolate a cDNA clone (CAP-C2) from an expression library prepared from mRNA from actively dividing cell suspension cultures of Nicotiana tabacum L. cv. xanthi. The derived amino acid sequence contained structural features of eukaryotic transcriptional factors, including three putative zinc fingers, three activator domains, and a nuclear localization signal. A fusion protein formed between part of the CAP-C2 polypeptide and Escherichia coli beta-glucuronidase specifically accumulated within the nuclei of transfected protoplasts, suggesting a nuclear localization for the CAP protein. Increased levels of expression of CAP in vivo were observed in actively proliferating tobacco tissues, whereas expression in vitro was induced when mature, differentiated explants proliferated into callus or cell suspensions. A 6.2 kb unstable mRNA and two low-abundance nuclear proteins (p66 and p68) containing epitopes encoded by CAP-C2 accumulated in plant tissues undergoing proliferative growth but were absent in mature, differentiated tissues. The corresponding genes were also present in Nicotiana sylvestris and Petunia hybrida © 1994 Tissue Culture Association.
• Bharathan, G., Lambert, G., & Galbraith, D. W. (1994). Nuclear DNA content of monocotyledons and related taxa. American Journal of Botany, 81(3), 381-386.
• Galbraith, D. W. (1994). Chapter 31 Flow Cytometry and Sorting of Plant Protoplasts and Cells. Methods in Cell Biology, 42(C), 539-561.
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  PMID: 7877508;
• Galbraith, D., & Galbraith, D. W. (1994). Flow cytometry and sorting of plant protoplasts and cells. Methods in cell biology, 42 Pt B.
• Galbraith, D. W., Zeiher, C. A., Harkins, K. R., & Afonso, C. L. (1992). Biosynthesis, processing and targeting of the G-protein of vesicular stomatitis virus in tobacco protoplasts. Planta, 186(3), 324-336.
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  PMID: 24186728;Abstract: Leaf protoplasts of tobacco (Nicotlana tabacum L.) were employed for transfection of chimeric transcriptional gene fusions comprising the 35S promoter from cauliflower mosaic virus, the coding sequence of the G-protein from vesicular stomatitis virus (VSVG) and the transcriptional terminator from the Agrobacterium tumefaciens nopaline-synthetase gene. Transient expression of the chimeric gene was monitored through Northern analysis of total protoplast RNA using a labeled VSV cDNA probe, and through Western-blot analysis of protoplast proteins using a polyclonal and-VSV antiserum. Although a single species of mRNA was detected in the transfected protoplasts, two glycoproteins differing in mass by approx. 9 kDa were detected by the antiserum. Biosynthesis of the VSVG isoforms was not impeded by chemical inhibitors of cell-wall production or of proline hydroxylation. Transfection using mutant forms of the VSVG coding sequence in which either one or both consensus glycosylation sites were removed resulted in the production of progressively smaller VSVG proteins. Those proteins produced from the double mutant had mobilities on sodium dodecyl sulfate-polyacrylamide gel electrophoresis that were very similar to those produced from the wild-type construct in the presence of tunicamycin. Analysis of protoplast homogenates by differential centrifugation showed that the two VSVG isoforms were exclusively associated with cellular membranes. The larger protein co-localized with the plasma membrane and with the organelles of the endomembrane-secretory pathway leading to the plasma membrane. The smaller protein was associated with membranes of lower isopycnic densities which were not identical to the endoplasmic reticulum. The larger protein displayed greater sensitivity than did the smaller to degradation in vivo by exogenously added protease. Immunofluorescence microscopy demonstrated that the VSVG isoforms were present both within the protoplasts and at the surface of the plasma membrane. The intracellular distribution was either punctate or reticulate. These results are consistent with the progressive and accurate glycosylation of the newly synthesized VSVG polypeptide during its passage through the endomembrane-secretory pathway, the access of the larger isoform to the cell surface, and the conversion of the larger to the small isoform by selective proteolysis. © 1992 Springer-Verlag.
• GALBRAITH, D. W., HARKINS, K. R., & KNAPP, S. (1991). SYSTEMIC ENDOPOLYPLOIDY IN ARABIDOPSIS-THALIANA. PLANT PHYSIOLOGY, 96(3), 985-989.
• Galbraith, D. W., Harkins, K. R., & Knapp, S. (1991). Systemic endopolyploidy in Arabidopsis thaliana. Plant Physiology, 96(3), 985-989.
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  PMID: 16668285;PMCID: PMC1080875;Abstract: Microfluorometric analysis of the nuclear DNA contents of the somatic tissues of Arabidopsis thaliana has revealed extensive endoreduplication, resulting in tissues that comprise mixtures of polyploid cells. Endoreduplication was found in all tissues except those of the inflorescences and was developmentally regulated according to the age of the tissues and their position within the plant.
• DEROCHER, E. J., HARKINS, K. R., GALBRAITH, D. W., & BOHNERT, H. J. (1990). DEVELOPMENTALLY REGULATED SYSTEMIC ENDOPOLYPLOIDY IN SUCCULENTS WITH SMALL GENOMES. SCIENCE, 250(4977), 99-101.
• Galbraith, D. W. (1990). Chapter 47 Isolation and Flow Cytometric Characterization of Plant Protoplasts. Methods in Cell Biology, 33(C), 527-547.
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  PMID: 2084484;
• Galbraith, D. W. (1990). Chapter 48 Flow Cytometric Analysis of Plant Genomes. Methods in Cell Biology, 33(C), 549-562.
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  PMID: 2150685;
• Galbraith, D., & Galbraith, D. W. (1990). Flow cytometric analysis of plant genomes. Methods in cell biology, 33.
• Galbraith, D., & Galbraith, D. W. (1990). Isolation and flow cytometric characterization of plant protoplasts. Methods in cell biology, 33.
• Galbraith, D., Harkins, K. R., Jefferson, R. A., Kavanagh, T. A., Bevan, M. W., & Galbraith, D. W. (1990). Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts. Proceedings of the National Academy of Sciences of the United States of America, 87(2).
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  We have analyzed the expression of chimeric genes in populations of protoplasts isolated from the photosynthetic and nonphotosynthetic tissues within leaves of transgenic tobacco plants and separated by fluorescence-activated cell sorting. Expression of transcriptional gene fusions controlled by promoters from photosynthesis-associated genes showed a striking dependence on cell type. These patterns of expression were preserved when the gene fusions were transfected into normal (nontransgenic) tobacco leaf protoplasts.
• Harkins, K. R., Jefferson, R. A., Kavanagh, T. A., Bevan, M. W., & Galbraith, D. W. (1990). Expression of photosynthesis-related gene fusions is restricted by cell type in transgenic plants and in transfected protoplasts. Proceedings of the National Academy of Sciences of the United States of America, 87(2), 816-820.
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  PMID: 2300565;PMCID: PMC53357;Abstract: We have analyzed the expression of chimeric genes in populations of protoplasts isolated from the photosynthetic and nonphotosynthetic tissues within leaves of transgenic tobacco plants and separated by fluorescence-activated cell sorting. Expression of transcriptional gene fusions controlled by promoters from photosynthesis-associated genes showed a striking dependence on cell type. These patterns of expression were preserved when the gene fusions were transfected into normal (nontransgenic) tobacco leaf protoplasts.
• Jay, E., Harkins, K. R., Galbraith, D. W., & Bohnert, H. J. (1990). Developmentally regulated systemic endopolyploidy in succulents with small genomes. Science, 250(4977), 99-101.
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  Abstract: Nuclei from Mesembryanthemum crystallinum (ice plant) exhibit multiple levels of ploidy in every tissue as revealed by flow microfluorometric analysis of isolated nuclei stained with mithramycin. Multiples of the haploid nuclear genome complement (1C) corresponding to 2C, 4C, 8C, 16C, 32C, and 64C were observed. The distribution of nuclei among the different ploidy levels is tissue-specific and in leaves is characteristic of the stage of development. This type of genome organization has been identified in eight other succulent CAM (crassulacean acid metabolism) plant species with small genomes. Multiploidy may be a common property of this type of plant.
• Knapp, S. J., Harkins, K. R., & Galbraith, D. W. (1990). Systemic endopolyploidy in Arabidopsis thaliana.. Philippine Journal of Crop Science, 15(3), 985-989.
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  Microfluorometric analysis of the nuclear DNA contents of the somatic tissues of Arabidopsis thaliana has revealed extensive endoreduplication, resulting in tissues that comprise mixtures of polyploid cells. Endoreduplication was found in all tissues except those of the inflorescences and was developmentally regulated according to the age of the tissues and their position within the plant.
• Galbraith, D. W. (1989). Analysis of Higher Plants by Flow Cytometry and Cell Sorting. International Review of Cytology-a Survey of Cell Biology, 116, 165-228. doi:10.1016/s0074-7696(08)60640-3
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  Publisher Summary This chapter discusses the process of development of flow cytometry and cell sorting procedures for the analysis of higher plant cells. The development of the flow cytometry and cell sorting technologies provides the ways in which the acquisition of unique information about populations of cells and subcellular organelles, coupled to the manipulation of these populations in a manner not possible using other techniques, can significantly augment the understanding of the molecular organization of living cells. A fundamental aspect of instrumentation used in flow cytometry and cell sorting centers is based on the optical properties of populations of individual cells suspended within a high-velocity fluid stream. The principle type of measurement made with flow cytometers involves the detection and quantitation of fluorescence. The characteristics and attributes that are unique to plant systems are: (1) the direct isolation of rare mutant or variant types, including those possessing enhanced capabilities for embryogenesis and those exhibiting altered photosynthetic activities, (2) the isolation of mutant or variant gametophytes, such as pollen at the microspore level, and (3) the isolation of transformed protoplasts in vivo .
• Galbraith, D. W., Harkins, K. R., & Jefferson, R. A. (1988). Flow cytometric characterization of the chlorophyll contents and size distributions of plant protoplasts.. Cytometry, 9(1), 75-83.
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  PMID: 3409786;Abstract: We have employed flow cytometry for the characterization of populations of protoplasts prepared from tobacco (Nicotiana tabacum) leaf tissues. We first investigated the possibility of using flow cytometric analysis of the emission of chlorophyll autofluorescence for measurement of the chlorophyll contents of leaf protoplasts. Defined numbers of leaf protoplasts were sorted according to different, nonoverlapping windows placed on the one-dimensional histograms of chlorophyll autofluorescence emission. The amounts of cellular chlorophyll were measured in cell-free extracts of these sorted protoplasts using fluorometry. A high degree of correlation (r2 = 0.983) was observed between these two parameters. We then examined the distribution of protoplast diameters in these protoplast populations through the use of pulse-width time-of-flight (TOF) analysis. Through sorting of protoplasts using a series of narrow, nonoverlapping TOF windows, we were able to demonstrate that the TOF parameter was linearly correlated with protoplast diameter, over the range of 15-55 micron (r2 greater than 0.99). We also compared the use of fluorescein diacetate (FDA) fluorochromasia and chlorophyll autofluorescence as the source of fluorescent signals for TOF analysis. We found that the presence of chloroplasts introduced distortions into the measurement of apparent size afforded by TOF analysis of FDA fluorochromasia. These results are discussed in terms of the application of techniques of flow analysis and sorting for the measurement of gene expression within the various different cell types found in plant tissues and organs.
• Meyer, D. J., Afonso, C. L., & Galbraith, D. W. (1988). Isolation and characterization of monoclonal antibodies directed against plant plasma membrane and cell wall epitopes: Identification of a monoclonal antibody that recognizes extensin and analysis of the process of epitope biosynthesis in plant tissues and cell cultures. Journal of Cell Biology, 107(1), 163-175.
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  PMID: 2455722;PMCID: PMC2115190;Abstract: Membranes from tobacco cell suspension cultures were used as antigens for the preparation of monoclonal antibodies. Use of solid phase and indirect immunofluorescence assays led to the identification of hybridomas producing antibodies directed against cell surface epitopes. One of these monoclonal antibodies (11.D2) was found to recognize a molecular species which on two-dimensional analysis (using nonequilibrium pH-gradient electrophoresis and SDS-PAGE) was found to have a high and polydisperse molecular mass and a very basic isoelectric point. This component was conspicuously labeled by [3H]proline in vivo. The monoclonal antibody cross-reacted with authentic tomato extensin, but not with potato lectin nor larch arabinogalactan. Use of the monoclonal antibody as an immunoaffinity reagent allowed the purification of a tobacco glycoprotein which was identical in amino acid composition to extensin. Finally, immunocytological analyses revealed tissue-specific patterns of labeling by the monoclonal antibody that were identical to those observed with a polyclonal antibody raised against purified extensin. We have concluded that monoclonal antibody 11.D2 recognizes an epitope that is carried exclusively by extensin. Analysis of cellular homogenates through differential and isopycnic gradient centrifugation revealed that biosynthesis of the extensin epitope was found on or within the membranes of the endoplasmic reticulum, Golgi region and plasma membrane. This result is consistent with the progressive glycosylation of the newly-synthesized extensin polypeptide during its passage through a typical eukaryotic endomembrane pathway of secretion. The 11.D2 epitope was not found in protoplasts freshly isolated from leaf tissues. However, on incubation of these protoplasts in appropriate culture media, biosynthesis of the epitope was initiated. This process was not impeded by the presence of chemicals that are reported to be inhibitors of cell wall production or of proline hydroxylation.
• Harkins, K. R., & Galbraith, D. W. (1987). Factors governing the flow cytometric analysis and sorting of large biological particles. Cytometry, 8(1), 60-70.
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  PMID: 3803096;
• Shoemaker, R. C., Christofferson, S. E., & Galbraith, D. W. (1987). Storage protein accumulation patterns in somatic embryos of cotton (Gossypium hirsutum L.). Plant Cell Reports, 6(1), 12-15.
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  PMID: 24248439;Abstract: The storage protein content of somatic embryos of Gossypium hirsutum L. cv. Coker 201 was determined using extinction level, antigen/antibody association detection methods. Mature storage protein was first detected in early globular-stage somatic embryos at a total concentration of 0.36% of the embryo protein mass. Tulip-stage and mature somatic embryos were comprised of 3.0% and 1.3% mature storage protein, respectively. Maximum storage protein synthesis was found to occur during early globular- and early heart-stages. During this period of development, significant levels of protein precursors were found also to accumulate. The pattern of storage protein synthesis, processing and accumulation paralleled the pattern that has been reported for the zygotic system, although somatic embryos accumulate storage protein at much earlier stages and to a lesser degree. The possibility of using complex biochemical pathways to monitor embryogenic systems in vitro is discussed. © 1987 Springer-Verlag.
• Shoemaker, R. C., Couche, L. J., & Galbraith, D. W. (1986). Characterization of somatic embryogenesis and plant regeneration in cotton (Gossypium hirsutum L.). Plant Cell Reports, 5(3), 178-181.
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  PMID: 24248126;Abstract: Seventeen cultivars of cotton (Gossypium hirsutum L.) were evaluated for callus initiation and maintenance using 3 initiation media and 3 maintenance media. After a series of transfers of a 3% glucose media, calli were placed on a 3% sucrose medium. After several weeks calli were observed for the presence of embryo-like structures. Cultivars Coker 201 and Coker 315 were identified as embryogenic. Embryogenic callus has since been routinely obtained within 6 weeks by initiating callus on glucose media for 3-4 weeks followed by transfer to sucrose media. Histological examination has shown that embryos are derived from isodiametric, densely cytoplasmic cells and follow predictable patterns of development. Upon maturity, transfer to auxin-free media with reduced sucrose levels results in embryo germination. Regenerated plants can be transferred to greenhouse within 90 days of callus initiation. © 1986 Springer-Verlag.
• Thomas-compton, M. A., Krejci, A. E., Harkins, K. R., Galbraith, D. W., & Afonso, C. L. (1985). Selection of Somatic Hybrid Plants in Nicotiana Through Fluorescence-Activated Sorting of Protoplasts. Nature Biotechnology, 3(9), 811-816. doi:10.1038/nbt0985-811
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  Selection of Somatic Hybrid Plants in Nicotiana Through Fluorescence-Activated Sorting of Protoplasts
• Firoozabady, E., & Galbraith, D. W. (1984). Presence of a plant cell wall is not required for transformation of Nicotiana by Agrobacterium tumefaciens. Plant Cell, Tissue and Organ Culture, 3(2), 175-188.
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  Abstract: Agrobacterium has been used to transform zero to six-day-old cell wall nonregenerating (CWNR) and cell wall regenerating (CWR) leaf protoplasts of tobacco. Transformed cells were selected by phoytohormone autotrophic growth and were verified by detection of the presence of lysopine dehydrogenase. Transformation frequencies in CWNR protoplasts were at least as high as those in CWR protoplasts, indicating that a plant cell wall is not required for the process of crown gall tumorigenesis. Transformation frequencies were highest in two-day-old protoplasts. This age coincides with the onset of DNA synthesis and the first mitosis within the cell populations. We suggest that the initiation of cell cycle activity may be important for the transformation process. © 1984 Martinus Nijhoff/Dr W. Junk Publishers.
• Galbraith, D. W., Afonso, C. L., & Harkins, K. R. (1984). Flow sorting and culture of protoplasts: Conditions for high-frequency recovery, growth and morphogenesis from sorted protoplasts of suspension cultures of nicotiana. Plant Cell Reports, 3(4), 151-155.
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  PMID: 24253474;Abstract: Protoplasts were prepared from suspension cultures of Nicotiana tabacum cv Wisconsin 38 that had been prelabeled with FITC. The protoplasts were subjected to flow sorting based on fluorescence content using a Coulter EPICS V Flow Cytometer - Cell Sorter. Conditions were established that allowed the recovery after sorting of approximately 30% of the initial protoplasts in a viable state. These were subsequently regenerated into calli that underwent shoot morphogenesis. © 1984 Springer-Verlag.
• Harkins, K. R., & Galbraith, D. W. (1984). Flow sorting and culture of plant protoplasts. Physiologia Plantarum, 60(1), 43-52. doi:10.1111/j.1399-3054.1984.tb04247.x
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  Conditions have been established for the rapid flow analysis of leaf protoplasts of Nicotiana tabacum L. cv. Xanthi using a flow cytometer-cell sorter. A procedure based upon chlorophyll autofluorescence was devised to permit the systematic evaluation of flow conditions in order to identify those under which protoplast damage was minimized. These conditions were employed for the flow sorting of protoplasts, following which it was possible to regenerate the sorted protoplasts into complete plants. The application of flow sorting is discussed for the rapid identification and selection of somatic hybrids produced by protoplast fusion.
• Galbraith, D. W., Harkins, K. R., & Maddox, J. M. (1983). Rapid flow cytometric analysis of the cell cycle in intact plant tissues. Science, 220(4601), 1049-1051.
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  PMID: 17754551;Abstract: Mechanical chopping of plant tissues in the presence of mithramycin released intact nuclei representative of the cells within the tissues. The amount of nuclear DNA in the homogenates of monocotyledonous and dicotyledonous plants was accurately and rapidly determined by flow microfluorometry, and the distribution of nuclei involved in the cell cycle was charted for tissues selected from different physical locations or developmental stages.
• Sharma, D. P., Galbraith, D. W., Firoozabady, E., & Ayres, N. M. (1983). Improvement of Anther Culture in Nicotiana: Media, Cultural Conditions and Flow Cytometric Determination of Ploidy Levels. Zeitschrift für Pflanzenphysiologie, 111(5), 441-451. doi:10.1016/s0044-328x(83)80008-7
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  Summary Maximum embryogenesis was observed in N. paniculate when anthers, excised from flower buds with petals just emerging from out of the calyx, were inoculated on a double layer medium (Johansson et al., 1982) and chill-treated at 7°C for a week prior to culture. The resulting micropropagules were multiplied on a shoot proliferating medium containing charcoal before rooting and subsequent transfer to pots in the greenhouse. Similar results were obtained in N. sylvestris by using the components of A medium (Kasperbauer and Wilson, 1979) in a double layer. Three hundred lux of light proved optimum for embryogenesis. A flow cytometric procedure is presented for fast and accurate determination of ploidy status in anther derived plants.
• Shields, B. A., & Galbraith, D. W. (1982). The effects of inhibitors of cell wall synthesis on tobacco protoplast development. Physiologia Plantarum, 55(1), 25-30. doi:10.1111/j.1399-3054.1982.tb00279.x
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  The effect of 2,6-dichlorobenzonitrile (DB) and coumarin on tobacco protoplast development has been studied using a combination of flow microfluorimetry and quantitative fluorescence microscopy. The results indicated that, over the initial period of culture, DB largely inhibited cellulose production at the cell surface, but did not inhibit DNA synthesis or protein accumulation by the protoplasts. Continuous culture of protoplasts in DB resulted in the accumulation of an increased capacity for cellulose synthesis as expressed following the removal of inhibitor. The possible sites of the specific inhibitory action of DB are discussed.
• Galbraith, D. W. (1981). Microfluorimetric quantitation of cellulose biosynthesis by plant protoplasts using Calcofluor White. Physiologia Plantarum, 53(2), 111-116. doi:10.1111/j.1399-3054.1981.tb04119.x
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  A microfluorimetric procedure, using Calcofluor White, has been developed for the measurement of cellulose biosynthesis by cultured protoplasts of tobacco (Nicotiana tabacum L. cv. Xanthi nc). The procedure was compared to a conventional method for cellulose estimation, that employing the anthrone reagent following exhaustive extraction of the developing cell walls. The results indicate that the amount of fluorescence emitted following Calcofluor White treatment is a specific measurement of cell wall cellulose levels. The procedure possesses the twin advantages of ease of manipulation and of greatly enhanced sensitivity (in the picogram range) as compared to other methods.
• Shields, B. A., Mauch, T. J., & Galbraith, D. W. (1981). Analysis of the initial stages of plant protoplast development using 33258 Hoechst: reactivation of the cell cycle. Physiologia Plantarum, 51(4), 380-386. doi:10.1111/j.1399-3054.1981.tb05573.x
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  A microfluorimetric procedure, employing the fluorescent stain 33258 Hoechst, has been developed for the investigation of the process of DNA synthesis during the initial stages of culture of tobacco (N. tabacum cv. Xanthi) leaf protoplasts. In this system, the freshly-isolated protoplasts exhibited a unimodal distribution of nuclear DNA content characteristic of the diploid state. The almost immediate onset of DNA synthesis during culture resulted in a doubling of nuclear DNA levels prior to the first mitoses. Although the majority of the protoplasts subsequently entered into synchronous mitosis and cell division, a proportion of the remainder developed into large polyploid cells. Upon further culture, the polyploid cells became subdivided into clusters of small diploid cells. Measurement of total cell protein and cell volumes during culture indicated that a relationship existed between these parameters and the initiation of mitosis. The significance of these observations is discussed.
• Galbraith, D. W., & Northcote, D. H. (1977). The isolation of plasma membrane from protoplasts of soybean suspension cultures. Journal of Cell Science, Vol.24, 295-310.
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  PMID: 561089;

Proceedings Publications

• Rodriguez, J. J., Xia, B., Sankaranarayanan, S., Rodriguez, J. J., Murthi, S., & Galbraith, D. W. (2002). Improved data acquisition system for digital flow cytometry. In 2002 IEEE International Symposium on Circuits and Systems. Proceedings (Cat. No.02CH37353), 1, 669-672.
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  Digital flow cytometry offers the flexibility to explore novel feature extraction and classification schemes for efficient sorting of biological cells. A prototype of a second-generation digital data acquisition system (DDAPS-2) - a mixed-signal design operating at 40 MHz - was built to interface to a commercial flow cytometer. The DDAPS-2 intercepts the analog signal from the photomultiplier tube and preamp, performs analog-to-digital conversion, extracts various features and then feeds these extracted features into one of the several pattern classification algorithms. This paper describes the design and operation of the various sub-systems that constitute the DDAPS-2. The novelty of the DDAPS-2 is the use of dual-buffering FIFO memories to acquire digital samples of the pulse voltage signal. Experimental results demonstrate the improvement in the pulse capture performance of DDAPS-2 over DDAPS-1, which used a single-buffering FIFO memory.
• Rodriguez, J. J., Yopp, T. A., Rodriguez, J. J., Lambert, G. M., Godavarti, M., & Galbraith, D. W. (1995). Neural network analysis of digital flow cytometric data. In Proceedings of ICNN'95 - International Conference on Neural Networks, 5, 2211-2216.
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  In flow cytometry, the pulse waveform features measurable by current analog instruments are limited to the pulse integral, peak, and width. Digitalization of the waveforms provides a means for the extraction of additional features, such as skewness, kurtosis, and Fourier properties. The introduction of additional features requires automated procedures for classification of biological particles. In this work, we implemented and evaluated neural network classification algorithms using derived, complex features, as well as using the raw, sampled data without feature extraction. The performance of the neural networks was compared with that of a more conventional means of classification in flow cytometry, the K-means clustering algorithm.

Presentations

• Galbraith, D. W. (2021, December). Cytotaxonomy. UCD Conway Flow Cytometry eWinter School. Virtual (Zoom): University College Dublin -- Conway.
• Galbraith, D. W. (2021, January). Advanced technologies for investigating the complex regulatory mechanisms of eukaryotes at the level of single cells. Academic Forum of Interdisciplinary Biology, & Inaugural Ceremony of Exploration. Kaifeng, China (via Zoom): Henan University, Chinese Association of Nanobiology, Wiley, and Exploration.
• Galbraith, D. W. (2021, June). Biological Applications of Cytometry: Flow Cytometry and Sorting for monitoring Cell Fate in Maize. Annual Meeting of the Society for In Vitro Biology. On-line (Zoom): Society for In Vitro Biology.
• Galbraith, D. W. (2021, June). Grow with the Flow! -- Creative Changes in Advanced Flow Cytometry for the 21st Century. Society for In Vitro Biology Meeting 2021. On-line (Zoom): Society for In Vitro Biology.
• Galbraith, D. W. (2021, November). Non-mammalian cytometry. Annual Australasian Cytometry Society Conference 2021. On-line (Zoom): Australasian Cytometry Society.
• Galbraith, D. W. (2019, February). Analyzing Gene Expression, Agnostically, at the Level of Single Cells and Nuclei  . Gordon Research Conference on Quantitative Genetics and Genomics. Il Ciocco, Italy.: Gordon Research Conferences.
• Galbraith, D. W. (2019, February). Preparation of vaccines in plants. VacciniAMO Conference. University of Modena, Italy.: University of Modena.
• Galbraith, D. W. (2019, January). Gene Expression Analysis. XXVIIth Plant and Animal Genome Meeting. San Diego: PAG.
• Galbraith, D. W. (2019, June). Fluorescent Proteins in Flow and Image Cytometry. 42nd Annual Course in Flow Cytometry University of Wisconsin. Madison: University of Wisconsin.
• Galbraith, D. W. (2019, March). There is no Planet B. TEDxUofA: Abberance. University of Arizona: TEDxUofA.
• Galbraith, D. W. (2019, November). Future Flow: Where will Cytometry take Us?. First International Workshop on Exotic Flow Cytometry: Checking Small Things... Better! ExoFlowMetry. ENEA Casaccia, Rome, Italy (Keynote Speaker).. Rome, Italy: ENEA Casaccia.
• Galbraith, D. W., & Villalobos-Menuey, L. (2019, June). Workshop: Flow cytometry of plants: a practical introduction to angiosperm genome size measurement . Botany 2019 Conference in Tucson, Arizona. Tucson: Botanical Society of America.
• Galbraith, D. W., Sun, G., Song, C., Xia, M., Li, J., Guo, G., & Fang, S. (2019, June). Agnostic clustering of RNA-Seq transcripts from single sorted nuclei identifies novel cell types within maize organs. CYTO2019, Vancouver, Canada.. Vancouver: International Society for Advancement of Cytometry.
• Galbraith, D. W. (2018, May). Cell-type Discovery within Complex Tissues and Organs using Flow Cytometry and Sorting. Seminar: Max Planck Institute of Molecular Plant Physiology. Golm: Max Planck Institute.
• Galbraith, D. W. (2018, Spring). Advances in the cytometry of single cells and organelles . 2018 Korea University-University of Arizona Joint International Symposium in Biomedical Engineering. Bio5 Institute, University of Arizona, Tucson: Korea University and University of Arizona.
• Galbraith, D. W. (2018, Spring). Workshop on Gene Expression Analysis. International Plant and Animal Genomes XXVI. San Diego CA: International Plant and Animal Genomes.
• Galbraith, D. W., & Loureiro, J. (2018, Spring). Flow Cytometry and Sorting of Plants, of Large Particles, and of Organ Homogenates of Plants and Animals. CYTO 2018: Congress of the International Society for Advancement of Cytometry. Prague, Czech Republic: International Society for Advancement of Cytometry.
• Galbraith, D. W. (2017, Fall). Flow cytometry and sorting with plants, large particles, and homogenates. Departmental seminar: Department of Flow & Image Cytometry, Roswell Park Comprehensive Cancer Center. Roswell Park Comprehensive Cancer Center, Buffalo, New York.: Roswell Park Comprehensive Cancer Center.
• Galbraith, D. W. (2017, Spring). A Guide to Publishing your Science in English (2). Institute seminar, Henan University, China. Henan University, Kaifeng, China: Institute for Plant Stress Biology.
• Galbraith, D. W. (2017, Spring). A Guide to Publishing your Science in English (3). Institute seminar, Henan University, China. Henan University, Kaifeng, China: Institute for Plant Stress Biology.
• Galbraith, D. W. (2017, Spring). A Guide to Publishing your Science in English. Institute seminar, Henan University, China. Henan University, Kaifeng, China: Institute for Plant Stress Biology.
• Galbraith, D. W. (2017, Spring). Analysis and modulation of genome-wide transcription at the level of single cells and nuclei. USDA-ALARC Seminar Series. USDA-ALARC, Maricopa: USDA-ALARC.
• Galbraith, D. W. (2017, Spring). Analysis of the initiation and progression of cancer at the level of single cells. 2nd Annual A.B.R.C. Research Conference. University of Arizona, College of Medicine, Phoenix: A.B.R.C..
• Galbraith, D. W. (2017, Spring). Workshop on Gene Expression Analysis. International Plant and Animal Genomes XXV. San Diego: Intl. Plant and Animal Genomes.
• Galbraith, D. W. (2017, Summer). Accelerating research through the use of flow cytometry. Becton Dickinson/Biocompare Webinar: on-line presentation. Internet webinar presentation: Becton Dickinson/Biocompare.
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  Flow cytometry is a powerful and versatile analytical technique that boasts an ever-expanding number of applications. In this webinar, we will review two innovative uses of flow cytometry technology that are very different but are similar in that they highlight the use of flow technology to accelerate research and uncover novel insights. The first presentation will focus on immunophenotyping by flow cytometry to efficiently classify and monitor multiple innate and adaptive cells. Specifically, we will examine the use of flow with antibodies against well-defined immune cell markers to determine the distribution of T cells, B cells, monocytes and activation markers, memory markers and apoptosis-related changes.The second presentation will focus on RNA-seq experiments conducted using tissues and organs, but examining these at the level of single cells. In this approach, flow cytometry and sorting is used to identify and purify single cells, which are then utilized for gene expression profiling.
• Galbraith, D. W. (2017, Summer). Fluorescent Proteins in Flow and Image Cytometry. Annual International Workshop in Flow Cytometry, Albuquerque. Albuquerque: University of New Mexico, Program in Biomedical Engineering.
• Galbraith, D. W., DeLay, M., & Koh, B. (2017, Summer). Strategies, instrumentation, and best practices for Next-Generation characterization of single cells and nuclei flow sorted from complex tissues and organs. CYTO2017, Congress of the International Society for Advancement of Cytometry. Boston MA: International Society for Advancement of Cytometry.
• Galbraith, D. W., Yoon, J., Mandel, M. A., M, N. A., & Toth, T. D. (2017, Summer). In situ optical detection of pathogens using emulsion loop-mediated isothermal amplification. CYTO2017, Congress of the International Society for Advancement of Cytometry. Boston MA: International Society for Advancement of Cytometry.
• Galbraith, D. W. (2016, January). Workshop presentation: Gene Expression Analysis. International Plant and Animal Genomes XXIV Conference. San Diego: Scherago International.
• Nicolini, A. M., Harshman, D. K., Toth, T. D., Mandel, M. A., Galbraith, D. W., & Yoon, J. (2016, Apr.). DOTS qPCR: a handheld, rapid molecular diagnostic tool for Ebola. Annual Meeting of IBE. Greenville, SC: IBE.
• Galbraith, D. W. (2015, January). Workshop presentation: Gene Expression Analysis. International Plant and Animal Genomes XXIII Conference. San Diego: Scherago International.
• Galbraith, D. W. (2014, December). Seminar presentation: Global Strategies for Analyzing Cell Type-specific Expression in Development and Disease.. Department of Botany, Delhi University, New Delhi.. Department of Botany, Delhi University, New Delhi.: Department of Botany, Delhi University, New Delhi..
• Galbraith, D. W. (2014, December). Symposium & Workshop presentation: Flow Cytometry of Plants.. Delhi University, New Delhi.. Delhi University, New Delhi.: Delhi University, New Delhi..
• Galbraith, D. W. (2014, December). Symposium & workshop presentation: Flow Cytometry of Plants.. Calcutta University, India.. Calcutta University, India.: Calcutta University, India..
• Galbraith, D. W. (2014, December). Symposium & workshop presentation: Flow Cytometry of Plants.. IARI (Indian Agricultural Research Institute), New Delhi, India.. IARI (Indian Agricultural Research Institute), New Delhi, India.: IARI (Indian Agricultural Research Institute), New Delhi, India..
• Galbraith, D. W. (2014, December). Symposium presentation at the FACSAcademy: Flow Cytometry of Plants.. Jamia Hamdard University, New Delhi.. Jamia Hamdard University, New Delhi.: Jamia Hamdard University, New Delhi..
• Galbraith, D. W. (2014, December). Symposium presentation: Flow Cytometry of Plants.. Punjab Agricultural University, Ludhiana, India.. Punjab Agricultural University, Ludhiana, India.: Punjab Agricultural University, Ludhiana, India..
• Galbraith, D. W. (2014, December). Symposium presentation: Flow Cytometry of Plants. School of Life Sciences, University of Hyderabad, India.. School of Life Sciences, University of Hyderabad, India.: School of Life Sciences, University of Hyderabad, India..
• Galbraith, D. W. (2014, JANUARY). Workshop organizer: Gene Expression Analysis. XXII Plant and Animal Genome Meeting, San Diego. San Diego, California.: SCHERAGO.
• Galbraith, D. W. (2014, January). Seminar presentation: New methods for the production of protein microarrays based on cell-free expression. PepTalk 2014, Palm Springs, California. Palm Springs, California: Cambridge Healthtech Institute.
• Galbraith, D. W. (2014, June). Workshop organizer, Annual Conference: Cytometry of Non-Mammalian Species. International Society for Advancement of Cytometry CYTO 2014, Fort Lauderdale.. Fort Lauderdale, Florida.: International Society for Advancement of Cytometry.
• Galbraith, D. W. (2014, June). Workshop presentation and laboratory: Cytometry of Fluorescent Proteins. Biennial National Flow Cytometry Course, Bowdoin College, Maine. Bowdoin College, Maine: Biennial National Flow Cytometry Course.
• Galbraith, D. W. (2014, May). Seminar presentation: Strategies for analyzing cell type-specific expression in development and disease. Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel.. Department of Plant Sciences, Weizmann Institute of Science, Rehovot, Israel.: Weizmann Institute of Science.
• Galbraith, D. W. (2014, May). Seminar presentation: Strategies for analyzing cell type-specific expression in development and disease. Institute of Plant Sciences, Volcani Center, Beit Dagan, Israel.. Institute of Plant Sciences, Volcani Center, Beit Dagan, Israel.: Institute of Plant Sciences, Volcani Center, Beit Dagan, Israel..
• Galbraith, D. W. (2014, October). Seminar: Novel Technologies for Studying Functional Genomics at the Level of Single Cells. SELECTBIO Advances in Plant Genomics online conference.. On-line/internet: SELECTBIO.
• Galbraith, D. W. (2013, August). Invited seminar: Arizona Biological and Biomedical Sciences Program. University of Arizona. Tucson: ABBS.
• Galbraith, D. W. (2013, January). Keynote speaker. CellTech 2013, San Diego. San Diego: Cambridge Healthtech.
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  Platform presentation at conference
• Galbraith, D. W. (2013, January). Organize and present workshop at PAG XXI. International Plant and Animal Genomes Conference (PAG XXI). San Diego: Intl PAG/Scherago.
• Galbraith, D. W. (2013, July). Invited seminar, University of Queensland. University of Queensland. Brisbane, Australia: University of Queensland.
• Galbraith, D. W. (2013, July). Lecturer and Laboratory Instructor: (1) Fluorescent Proteins in Flow Cytometry, and (2) Plant Applications in Flow Cytometry. Queensland Flow Cytometry Workshop, Queensland Institute of Medical Research. Brisbane, Australia.: Queensland Institute of Medical Research.
• Galbraith, D. W. (2013, June). Workshop Lecturer and Lab Instructor: Fluorescent Proteins in Flow Cytometry and Sorting. Biennial National Flow Cytometry Course, Albuquerque, New Mexico. Albuquerque, New Mexico: University of New Mexico.
• Galbraith, D. W. (2013, March). Seminar presentation: Flow Cytometry and Cell Sorting: Investigating Single Cells, Cell Types, and Species. Departmental seminar, Plant Sciences, NCSU. Raleigh, NC: North Carolina State University.
• Galbraith, D. W. (2013, November). Invited seminar: Arizona Cancer Bioimaging Program. Bioimaging Program. Tucson: Arizona Cancer Bioimaging Program.
• Galbraith, D. W. (2012). Lecture at the Royal Botanical Gardens, Kew. London, England.
• Galbraith, D. W. (2012). Seminar at the BMCB retreat at Biosphere 2. Oracle, Arizona.
• Galbraith, D. W. (2012). Seminar at the Gregor Mendel Institute in Vienna. Vienna, Austria.
• Galbraith, D. W. (2012, Fall). Seminar. MARC monthly seminar series. Maricopa Agricultural Research Center: USDA-MARC.
• Galbraith, D. W. (2012, February). Webinar. Genetic Engineering News. Tucson, AZ: Genetic Engineering News.
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  Internet webinar
• Galbraith, D. W. (2012, January). Gene Expression Analysis. XVIIIth Plant and Animal Genome. San Diego, CA.
• Galbraith, D. W. (2012, June). Flow Cytometry. I participated in a one day workshop in flow cytometry at Regeneron, Inc. Elmsford, NY: Regeneron, Inc.
• Galbraith, D. W. (2012, June). National Flow Cytometry Course. Biennial National Flow Cytometry Course. Bowdoin ME: Bowdoin College.
• Galbraith, D. W. (2012, March). Webinar. Biotechniques. Tucson, AZ: Biotechniques.
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  Internet webinar
• Galbraith, D. W. (2011). I gave a one-hour seminar at the Institute for Experimental Botany in Olomouc, and met with scientists and students. Olomouc, Czech Republic.
• Galbraith, D. W. (2011). Lecture at Liverpool University. Liverpool, England.
• Galbraith, D. W. (2011). One hour seminar at the Chinese Agricultural University in Beijing, and met with faculty and students. Beijing, China.
• Galbraith, D. W. (2011). Plenary Speaker at SUMMIT2011. SUMMIT2011: the Inaugural meeting of the Southern California Flow Cytometry Association. Los Angeles: Southern California Flow Cytometry Association.
• Galbraith, D. W. (2011). Plenary Speaker in the Cyto Symposium at this Congress. (ISAC XXVI; Baltimore). Baltimore.
• Galbraith, D. W. (2011). Seminar at BGI Shenzhen. Shenzhen, China.
• Galbraith, D. W. (2011). Seminar in the UA Quantitative Biology Colloquium series. Tucson, AZ.
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  Video
• Galbraith, D. W. (2011). Seminar. Hong Kong, China.
• Galbraith, D. W. (2011, Fall). Lecture and met with faculty and students. University of Toronto. Toronto, Canada: University of Toronto.
• Galbraith, D. W. (2011, January). Gene Expression Analysis. XVIIIth Plant and Animal Genome meeting. San Diego, CA.
• Galbraith, D. W. (2011, July). Flow Cytometry. Western New York Flow Cytometry Group. Rochester.
• Galbraith, D. W. (2011, June). Flow Cytometry. Flow Cytometry. Irvine, California: Stem Cell Institute at UC Irvine.
• Galbraith, D. W. (2011, June). Genomics of Tropical Biodiversity. Genomics of Tropical Biodiversity. Xishuangbanna, China.
• Galbraith, D. W. (2011, June). National Flow Cytometry Course. Biennial National Flow Cytometry Course. Albuquerque NM.

Others

• Galbraith, D. W. (2013, July). Participate in Gates Foundation Workshop on African Agriculture. Lake Naivasha. Kenya.
• Galbraith, D. W. (2010, Fall). SPLS website.
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