Chris Frost

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Scholarly Contributions

Chapters

• Frost, C. (2012). Effects of Herbivores on Terrestrial Ecosystem Processes: The Role of Trait-Mediated Indirect Effects. In Trait-Mediated Indirect Effects in Ecology.

Journals/Publications

• Bagley, R. K., Hurst, M. N., Frederick, J., Wolfe, J., Terbot, J. W., Frost, C. J., & Linnen, C. R. (2023).

  Multiple mechanisms contribute to isolation-by-environment in the redheaded pine sawfly, Neodiprion lecontei

  . Evolution. doi:10.1093/evolut/qpad137
• Frost, C. J. (2023).

  Information potential of an ubiquitous phytochemical cue

  . New Phytologist, 238(5), 1749-1751. doi:10.1111/nph.18890
• Frost, C. J. (2023).

  Overlaps and trade‐offs in the diversity and inducibility of volatile chemical profiles among diverse sympatric neotropical canopy trees

  . Plant, Cell & Environment, 46(10), 3059-3071. doi:10.1111/pce.14594
• Frost, C. J., Ramirez-Mata, A., Khattri, R. B., Merritt, M. E., & Frost, S. C. (2023). Effects of β-caryophyllene and oxygen availability on cholesterol and fatty acids in breast cancer cells. PloS one, 18(3), e0281396.
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  Hypoxia is a common feature of most solid tumors, one that favors tumor progression and limits treatment effectiveness. Targeting hypoxia has long been a goal in cancer therapy, by identifying factors that reverse or ameliorate the effects of hypoxia on cancer cells. We, and others, have shown that β-caryophyllene (BCP) exhibits anti-proliferative properties in cancer cells. We have further shown that non-cytotoxic concentrations of BCP affect cholesterol and lipid biosynthesis in hypoxic hBrC cells at both transcriptional and translational levels. This led us to hypothesize that BCP may reverse the hypoxic phenotype in hBrC cells. To test this, we determined the effect of BCP on hypoxic sensitive pathways, including oxygen consumption, glycolysis, oxidative stress, cholesterol and fatty acid biosynthesis, and ERK activation. While each of these studies revealed new information on the regulation by hypoxia and BCP, only the lipidomic studies showed reversal of hypoxic-dependent effects by BCP. These later studies showed that hypoxia-treated samples lowered monounsaturated fatty acid levels, shifting the saturation ratios of the fatty acid pools. This signature was ameliorated by sub-lethal concentrations of BCP, possibly through an effect on the C:16 fatty acid saturation ratios. This is consistent with BCP-induced upregulation of the stearoyl-CoA desaturase (SCD) gene, observed previously. This suggests that BCP may interfere with the lipid signature modulated by hypoxia which could have consequences for membrane biosynthesis or composition, both of which are important for cell replication.
• Frost, C. (2022). A visual technique used by citizen scientists shows higher herbivory in understory vs. canopy leaves of a tropical forest. Ecology.
• Frost, C. (2022). Acid soil stress compromises sugar maple induced resistance against an invasive insect herbivore.
• Frost, C. (2022). Below ground regulation of stress response genes in hybrid poplar.
• Frost, C. (2022). Ozone pollution compromises plant defense responses to insect herbivory.
• Frost, C. (2022). Plant responses to airborne volatiles depend on signal source and specificity.
• Santangelo, J. S., Ness, R. W., Cohan, B., Fitzpatrick, C. R., Innes, S. G., Koch, S., Miles, L. S., Munim, S., Peres-Neto, P. R., Prashad, C., Tong, A. T., Aguirre, W. E., Akinwole, P. O., Alberti, M., Álvarez, J., Anderson, J. T., Anderson, J. J., Ando, Y., Andrew, N. R., , Angeoletto, F., et al. (2022). Global urban environmental change drives adaptation in white clover. Science (New York, N.Y.), 375(6586), 1275-1281.
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  Urbanization transforms environments in ways that alter biological evolution. We examined whether urban environmental change drives parallel evolution by sampling 110,019 white clover plants from 6169 populations in 160 cities globally. Plants were assayed for a Mendelian antiherbivore defense that also affects tolerance to abiotic stressors. Urban-rural gradients were associated with the evolution of clines in defense in 47% of cities throughout the world. Variation in the strength of clines was explained by environmental changes in drought stress and vegetation cover that varied among cities. Sequencing 2074 genomes from 26 cities revealed that the evolution of urban-rural clines was best explained by adaptive evolution, but the degree of parallel adaptation varied among cities. Our results demonstrate that urbanization leads to adaptation at a global scale.
• Frost, C. (2021). Dispensing a Synthetic Green Leaf Volatile to Two Plant Species in a Common Garden Differentially Alters Physiological Responses and Herbivory. Agronomy.
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  Herbivore-induced plant volatile (HIPV)-mediated eavesdropping by plants is a well-documented, inducible phenomenon that has practical agronomic applications for enhancing plant defense and pest management. However, as with any inducible phenomenon, responding to volatile cues may incur physiological and ecological costs that limit plant productivity. In a common garden experiment, we tested the hypothesis that exposure to a single HIPV would decrease herbivore damage at the cost of reduced plant growth and reproduction. Lima bean (Phaseolus lunatus) and pepper (Capsicum annuum) plants were exposed to a persistent, low dose (~10 ng/h) of the green leaf volatile cis-3-hexenyl acetate (z3HAC), which is a HIPV and damage-associated volatile. z3HAC-treated pepper plants were shorter, had less aboveground and belowground biomass, and produced fewer flowers and fruits relative to controls, while z3HAC-treated lima bean plants were taller and produced more leaves and flowers than did controls. Natural herbivory was reduced in z3HAC-exposed lima bean plants, but not in pepper. Cyanogenic potential, a putative direct defense mechanism in lima bean, was lower in young z3HAC-exposed leaves, suggesting a growth–defense tradeoff from z3HAC exposure alone. Plant species-specific responses to an identical volatile cue have important implications for agronomic costs and benefits of volatile-mediated interplant communication under field conditions.
• Frost, C. (2021). Effects of Azteca trigona alarm pheromones on heterospecific ant behavior. Insectes Sociaux.
• Frost, C. (2021). Poplar Allene Oxide Synthase 1 Gene Promoter Drives Rapid and Localized Expression by Wounding. Biotechnology Journal International.
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  Promoters play critical roles in controlling the transcription of genes and are important as tools to drive heterologous expression for biotechnological applications. In addition to core transcription factor-binding motifs that assist in the binding of RNA polymerases, there are specific nucleotide sequences in a promoter region to allow regulation of gene expression. The allene oxide synthase (AOS) gene family are cytochrome P450s that are responsive to a variety of environmental stress, making them good candidates for the discovery of inducible promoters. Populus AOS homologs separate phylogenetically into two clades. Based on the 19 promoter motifs with significant abundance differences between the two clades, Clade I AOS genes are likely more responsive to hormones, salt, and pathogen, whereas clade II homologs are likely inducible by water stress. In this study, an upstream promoter from a Clade I poplar AOS encoding gene (AOS1) was cloned and used to drive the expression of a ß-glucuronidase (GUS) gene in Arabidopsis. AOS is an essential enzyme in the lipoxygenase pathway that is responsible for the production of many non-volatile oxylipins in plants, including the jasmonates, which are regulatory phytohormones coordinating a variety of biological and stress response functions. Consistent with AOS transcript expression patterns, we found that the poplar AOS1 promoter drives rapid and localized expression by wounding. The study provides insight on the responsive elements in the poplar AOS promoters, but more importantly identifies a strong wound-inducible and localized promoter for future applications.
• Frost, C. J. (2021). A visual technique used by citizen scientists shows higher herbivory in understory vs. canopy leaves of a tropical forest. Ecology, 103(1). doi:10.1002/ecy.3539
• Frost, C. J. (2022). A visual technique used by citizen scientists shows higher herbivory in understory vs. canopy leaves of a tropical forest. Ecology, 103(1), e03539.
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  Citizen science (CS) initiatives can transform how some ecological data are collected. Herbivory is a fundamental ecological interaction, but herbivory rates in many natural systems are unknown due in part to lack of personnel for monitoring efforts. This limits our ability to understand broad ecological patterns relevant to herbivory. Fortunately, accurate and reliable visual estimation techniques for assessing herbivory could be amenable to CS approaches. In 2008, I developed a CS training initiative (the Million Leaf Project, MLP) to measure herbivory based on a seven-category visual assessment of leaf area removed (LAR). From 2010 to 2018, 394 citizen scientists assessed damage on 175,421 leaves to test the hypothesis that herbivory varies between understory and canopy leaves in a Peruvian tropical forest. In support of this hypothesis, the longitudinal CS data reveal that understory leaves consistently experience more herbivory than do canopy leaves on average (18.3% vs. 12.3%, P 
• Frost, C., Patel, R., & Maurya, A. (2020). Acute toxicity of the plant volatile indole depends on herbivore specialization. Journal of Pest Science. doi:10.1007/s10340-020-01218-6
• Adams, R. M., Wells, R. L., Yanoviak, S. P., Yanoviak, S. P., Frost, C. J., & Fox, E. G. (2020). Interspecific Eavesdropping on Ant Chemical Communication. Frontiers in Ecology and Evolution, 8. doi:10.3389/fevo.2020.00024
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  Chemical communication is a fundamental, highly complex component of social insect societies. Ants in particular employ a remarkable diversity of chemical signals to maintain social cohesion among nestmates, gain essential resources through coordinated foraging, and warn of danger. Although the chemicals used can be functionally specific, they are vulnerable to exploitation by eavesdropping natural enemies (e.g., parasitoids, predators, parasites) and other associates (e.g., myrmecophiles). Ant nests are nutrient hotspots due to their collection of resources warranting keen defense systems; yet the heavily-defended hideouts are frequently invaded. Many organisms exploit ant species, but how they locate hosts—including what host-derived cues are used—is still poorly understood. Here, we review current knowledge about how ant chemical communication systems can be exploited by unintended receivers. We take a case study approach and illustrate the diversity of ant associates and host traits that may predispose ants to exploitation. We identify knowledge gaps by reviewing host systems and listing: 1) the types of associates (e.g., fly, wasp, beetle) where eavesdropping is likely occurring, organized by the host communication system that is being exploited; 2) the ant parasites that exploit trail pheromones; and 3) the experimentally-determined chemicals (i.e., alarm/defensive pheromones), used by eavesdroppers. At least 25 families of arthropods (10 orders) potentially eavesdrop on ant communication systems and nearly 20 host ant species are vulnerable to trail parasite ant species. We also propose future research that will improve our understanding of community assembly by examining host traits (e.g., latitude, nest characteristics, trail system) that influence their susceptibility to eavesdropping associates.
• Frost, C. (2020). Acute toxicity of the plant volatile indole depends on herbivore specialization. Journal of Pest Science.
• Frost, C. (2020). Defoliation‐induced compensatory transpiration is compromised in SUT4 ‐RNAi Populus. Plant Direct.
• Harding, S. A., Frost, C. J., & Tsai, C. J. (2020). Defoliation-induced compensatory transpiration is compromised in -RNAi. Plant direct, 4(9), e00268.
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  The tonoplast sucrose transporter PtaSUT4 is well expressed in leaves of  ×  (INRA 717-IB4), and its inhibition by RNA-interference (RNAi) alters leaf sucrose homeostasis. Whether sucrose partitioning between the vacuole and the cytosol is modulated by PtaSUT4 for specific physiological outcomes in remains unexplored. In this study, partial defoliation was used to elicit compensatory increases in photosynthesis and transpiration by the remaining leaves in greenhouse-grown poplar. Water uptake, leaf gas exchange properties, growth and nonstructural carbohydrate abundance in source and sink organs were then compared between wild-type and -RNAi lines. Partial defoliation increased maximum photosynthesis rates similarly in all lines. There was no indication that source leaf sugar levels changed differently between wild-type and RNAi plants following partial defoliation. Sink levels of hexose (glucose and fructose) and starch decreased similarly in all lines. Interestingly, plant water uptake after partial defoliation was not as well sustained in RNAi as in wild-type plants. While the compensatory increase in photosynthesis was similar between genotypes, leaf transpiration increased less robustly in RNAi than wild-type plants. SUT4-RNAi and wild-type source leaves differed constitutively in their bulk modulus of elasticity, a measure of leaf turgor, and storage water capacitance. The data demonstrate that reduced sucrose partitioning due to -RNAi altered turgor control and compensatory transpiration capacity more strikingly than photosynthesis and sugar export. The results are consistent with the interpretation that SUT4 may control vacuolar turgor independently of sink carbon provisioning.
• Mboge, M. Y., Chen, Z., Khokhar, D., Wolff, A., Ai, L., Heldermon, C. D., Bozdag, M., Carta, F., Supuran, C. T., Brown, K. D., McKenna, R., Frost, C. J., & Frost, S. C. (2019). A non-catalytic function of carbonic anhydrase IX contributes to the glycolytic phenotype and pH regulation in human breast cancer cells. The Biochemical journal, 476(10), 1497-1513.
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  The most aggressive and invasive tumor cells often reside in hypoxic microenvironments and rely heavily on rapid anaerobic glycolysis for energy production. This switch from oxidative phosphorylation to glycolysis, along with up-regulation of the glucose transport system, significantly increases the release of lactic acid from cells into the tumor microenvironment. Excess lactate and proton excretion exacerbate extracellular acidification to which cancer cells, but not normal cells, adapt. We have hypothesized that carbonic anhydrases (CAs) play a role in stabilizing both intracellular and extracellular pH to favor cancer progression and metastasis. Here, we show that proton efflux (acidification) using the glycolytic rate assay is dependent on both extracellular pH (pH) and CA IX expression. Yet, isoform-selective sulfonamide-based inhibitors of CA IX did not alter proton flux, which suggests that the catalytic activity of CA IX is not necessary for this regulation. Other investigators have suggested the CA IX co-operates with the MCT transport family to excrete protons. To test this possibility, we examined the expression patterns of selected ion transporters and show that members of this family are differentially expressed within the molecular subtypes of breast cancer. The most aggressive form of breast cancer, triple-negative breast cancer, appears to co-ordinately express the monocarboxylate transporter 4 (MCT4) and carbonic anhydrase IX (CA IX). This supports a possible mechanism that utilizes the intramolecular H shuttle system in CA IX to facilitate proton efflux through MCT4.
• Mboge, M. Y., Ramirez-Mata, A., Bullock, A., O'Donnell, R., Mathias, J. V., Davila, J., Frost, C. J., & Frost, S. C. (2019). β-caryophyllene enhances the transcriptional upregulation of cholesterol biosynthesis in breast cancer cells. Current topics in biochemical research, 20, 1-16.
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  β-caryophyllene (BCP) exhibits anti-proliferative properties in cancer cells. Here, we examine the hypothesis that BCP induces membrane remodeling. Our data show that high concentrations of BCP increase membrane permeability of human breast cells (hBrC) causing detachment and cell death. At a sub-lethal concentration of BCP, we show that BCP induces a striking upregulation of genes involved in cholesterol biosynthesis, including the gene that encodes for HMGCoA reductase (HMGCR), the rate-determining step in cholesterol biosynthesis. In addition, stearoyl-CoA desaturase (SCD) is also upregulated which would lead to the enhanced formation of monounsaturated fatty acids, specifically oleate and palmitoleate from stearoyl CoA and palmitoyl CoA, respectively. These fatty acids are major components of membrane phospholipids and cholesterol esters. Together, these data suggest that cells respond to BCP by increasing the synthesis of components found in membranes. These responses could be viewed as a repair mechanism and/or as a mechanism to mount resistance to the cytotoxic effect of BCP. Blocking HMGCR activity enhances the cytotoxicity of BCP, suggesting that BCP may provide an additional therapeutic tool in controlling breast cancer cell growth.
• Chen, Z., Ai, L., Mboge, M. Y., McKenna, R., Frost, C. J., Heldermon, C. D., & Frost, S. C. (2018). UFH-001 cells: A novel triple negative, CAIX-positive, human breast cancer model system. Cancer biology & therapy, 19(7), 598-608.
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  Human cell lines are an important resource for research, and are often used as in vitro models of human diseases. In response to the mandate that all cells should be authenticated, we discovered that the MDA-MB-231 cells that were in use in our lab, did not validate based on the alleles of 9 different markers (STR Profile). We had been using this line as a model of triple negative breast cancer (TNBC) that has the ability to form tumors in immuno-compromised mice. Based on marker analysis, these cells most closely resembled the MCF10A line, which are a near diploid and normal mammary epithelial line. Yet, the original cells express carbonic anhydrase IX (CAIX) both constitutively and in response to hypoxia and are features that likely drive the aggressive nature of these cells. Thus, we sought to sub-purify CAIX-expressing cells using Fluorescence Activated Cell Sorting (FACS). These studies have revealed a new line of cells that we have name UFH-001, which have the TNBC phenotype, are positive for CAIX expression, both constitutively and in response to hypoxia, and behave aggressively in vivo. These cells may be useful for exploring mechanisms that underlie progression, migration, and metastasis of this phenotype. In addition, constitutive expression of CAIX allows its evaluation as a therapeutic target, both in vivo and in vitro.
• Mboge, M. Y., Bullock, A. P., O'dennell, R., Matthias, J. V., Davila, J. A., Frost, C. J., & Frost, S. C. (2018). Abstract 1436: Beta-caryophyllene regulates lipid biosynthesis in breast cancer cells. Cancer Research, 78, 1436-1436. doi:10.1158/1538-7445.am2018-1436
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  The search for therapeutic anti-cancer drugs has spanned both synthetic and natural products approaches, and notable success has been achieved from unique chemistries produced by plants. Early studies in E.coli showed that cyclic hydrocarbons, including terpenes, interact directly with biological membranes. Accumulation of hydrocarbons results in membrane swelling and increased membrane fluidity, both signs of cell stress. At biological temperatures, membrane fluidity is controlled by the saturation state of the acyl chains of fatty acids (primarily in phospholipids) and cholesterol content. Changes in either of these parameters leads to membrane remodeling which can affect membrane function. Terpenes are, themselves, precursors to complex sterols across all kingdoms of life. Beta-caryophylene (BCP), a bicyclic sesquiterpene, induces cell death across a variety of cancer cell types, although the mechanism(s) by which this occurs is not completely known. Our data show that BCP induces membrane permeability in breast cancer lines representing both ER-positive and triple negative phenotypes (TNBC), as measured by lactate dehydrogenase release from cells. We sought to understand this phenomenon by looking at changes in the transcriptome of treated cells compared to controls. We chose to examine cells exposed to hypoxia, rationalizing that this condition, in vivo, creates an aggressive phenotype, is associated with TNBC and drug-resistant (recurrent) breast cancers, and is an independent prognosticator for poor patient outcome. We isolated high quality RNA (RIN>9) at the University of Florida. The Genomics Core at the University of Louisville prepared libraries and performed the sequencing run (Illumina NextSeq 500). This generated over 144 million 75bp reads that aligned to the human genome (96.3% alignment rate), or approximately 24 million reads per sample. As a first approach in data analysis, we selected differential expression based on the FDR adjusted p-values (q-values) Citation Format: Mam Y. Mboge, Adam P. Bullock, Riley O9Dennell, John V. Matthias, Julie A. Davila, Christopher J. Frost, Susan C. Frost. Beta-caryophyllene regulates lipid biosynthesis in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1436.
• Mboge, M. Y., Chen, Z., Ai, L., Tu, C., Carta, F., Frost, C. J., Zhang, Z., Mckenna, R., Frost, S. C., Supuran, C. T., & Heldermon, C. D. (2018). Abstract 2102: UFH-001: A novel triple-negative, CAIX-positive cell line for small-molecule targeting of human breast cancer. Cancer Research, 78, 2102-2102. doi:10.1158/1538-7445.am2018-2102
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  Specific treatment options for patients with metastatic breast cancers, especially of the triple negative subtype (TNBC), are limited and most patients develop resistance to radiation and/or chemotherapy. Because of this, the mortality rate in women with TNBC remains high. Carbonic anhydrases (CAs), most specifically membrane-bound CAIX, has been shown to play an important role in tumor progression and as a result is validated as a therapeutic target in several aggressive cancers. In breast cancer, CAIX expression has been shown to be ubiquitous in hypoxic TNBC tissues and is considered a prognostic marker and a potential therapeutic target. Therefore targeting CAIX activity, using small molecule inhibitors, in these highly aggressive and metastatic TNBCs may serve to improve overall disease free survival and therapeutic outcome in clinics. Our goal for this study was to 1) Identify and/or develop a cell line that expresses CAIX in a hypoxia independent manner and is representative of TNBCs and 2) To target CAIX activity in this cell line and observe the effects of CAIX inhibition on its growth and metastasis. Our hypothesis is that CAIX inhibition, in the context of a hypoxic and/or acidic microenvironment, will dysregulate its ability to maintain the acidic pH preferred by cancer cells which favors their growth and migration. To achieve our aims, we newly developed and characterized an endogenous CAIX-expressing cell line named UFH-001. Our results show that CAIX expression and activity in this cell line, although endogenous under normoxic conditions, is increased more than 2 fold under hypoxic conditions. This cell line is also representative of TNBCs (ER/PR and HER2 negative) with a more epithelial-like morphology and phenotype. UFH-001 cells grow aggressively and have the ability to migrate/invade and form tumors in vivo. In these studies we have also shown how inhibition of CAIX activity, using sulfonamide-based inhibitors, affects UFH-001 growth in an apoptosis independent manner and decrease the migratory/inhibitory capacity of these cells. Taken together, our observations indicate that CAIX is a viable small molecular drug target for the treatment of patients with TNBCs. Citation Format: Mam Y. Mboge, Zhijuan Chen, Lingbao Ai, Chingkuang Tu, Fabrizio Carta, Claudiu Supuran, Christopher J. Frost, Zaihui Zhang, Robert McKenna, Coy Heldermon, Susan C. Frost. UFH-001: A novel triple-negative, CAIX-positive cell line for small-molecule targeting of human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2102.
• Frost, C. (2017). Reduction of Motion Artifacts and Noise Using Independent Component Analysis in Task-Based Functional MRI for Preoperative Planning in Patients with Brain Tumor. American Journal of Neuroradiology.
• Xue, L. J., Frost, C. J., Tsai, C. J., & Harding, S. A. (2016). Drought response transcriptomes are altered in poplar with reduced tonoplast sucrose transporter expression. Scientific reports, 6, 33655.
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  Transgenic Populus tremula x alba (717-1B4) plants with reduced expression of a tonoplast sucrose efflux transporter, PtaSUT4, exhibit reduced shoot growth compared to wild type (WT) under sustained mild drought. The present study was undertaken to determine whether SUT4-RNAi directly or indirectly altered poplar predisposition and/or response to changes in soil water availability. While sucrose and hexose levels were constitutively elevated in shoot organs, expression responses to drought were most altered in the root tips of SUT4-RNAi plants. Prior to any drought treatment, constitutively elevated transcript levels of abscisic acid biosynthetic genes and bark/vegetative storage proteins suggested altered metabolism in root tips of RNAi plants. Stronger drought-stimulation of stress-inducible genes encoding late-embryogenesis-abundant proteins in transgenic roots was consistent with increased vulnerability to soil drying. Transcript evidence suggested an RNAi effect on intercellular water trafficking by aquaporins in stem xylem during soil drying and recovery. Co-expression network analysis predicted altered integration of abscisic acid sensing/signaling with ethylene and jasmonate sensing/signaling in RNAi compared to WT roots. The overall conclusion is that steepened shoot-root sugar gradient in RNAi plants increased sensitivity of root tips to decreasing soil water availability.
• Frost, C. (2015). Acute Invasive Fungal Rhinosinusitis: A Comprehensive Update of CT Findings and Design of an Effective Diagnostic Imaging Model. American Journal of Neuroradiology.
• Frost, C. (2013). Constitutively elevated salicylic acid levels alter photosynthesis and oxidative state but not growth in transgenic populus. The Plant Cell.
• Xue, L. J., Guo, W., Yuan, Y., Anino, E. O., Nyamdari, B., Wilson, M. C., Frost, C. J., Chen, H. Y., Babst, B. A., Harding, S. A., & Tsai, C. J. (2013). Constitutively elevated salicylic acid levels alter photosynthesis and oxidative state but not growth in transgenic populus. The Plant cell, 25(7), 2714-30.
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  Salicylic acid (SA) has long been implicated in plant responses to oxidative stress. SA overproduction in Arabidopsis thaliana leads to dwarfism, making in planta assessment of SA effects difficult in this model system. We report that transgenic Populus tremula × alba expressing a bacterial SA synthase hyperaccumulated SA and SA conjugates without negative growth consequences. In the absence of stress, endogenously elevated SA elicited widespread metabolic and transcriptional changes that resembled those of wild-type plants exposed to oxidative stress-promoting heat treatments. Potential signaling and oxidative stress markers azelaic and gluconic acids as well as antioxidant chlorogenic acids were strongly coregulated with SA, while soluble sugars and other phenylpropanoids were inversely correlated. Photosynthetic responses to heat were attenuated in SA-overproducing plants. Network analysis identified potential drivers of SA-mediated transcriptome rewiring, including receptor-like kinases and WRKY transcription factors. Orthologs of Arabidopsis SA signaling components NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 and thioredoxins were not represented. However, all members of the expanded Populus nucleoredoxin-1 family exhibited increased expression and increased network connectivity in SA-overproducing Populus, suggesting a previously undescribed role in SA-mediated redox regulation. The SA response in Populus involved a reprogramming of carbon uptake and partitioning during stress that is compatible with constitutive chemical defense and sustained growth, contrasting with the SA response in Arabidopsis, which is transient and compromises growth if sustained.
• Frost, C. (2012). A petiole-galling insect herbivore decelerates leaf lamina litter decomposition rates. Functional ecology.
• Frost, C. (2012). The tonoplast-localized sucrose transporter in Populus (PtaSUT4) regulates whole-plant water relations, responses to water stress, and photosynthesis. PLoS One.
• Frost, C. (2012). ecosystem processes: the role of. Trait-Mediated Indirect Interactions: Ecological and Evolutionary Perspectives.
• Frost, C. J., Nyamdari, B., Tsai, C. J., & Harding, S. A. (2012). The tonoplast-localized sucrose transporter in Populus (PtaSUT4) regulates whole-plant water relations, responses to water stress, and photosynthesis. PloS one, 7(8), e44467.
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  The Populus sucrose (Suc) transporter 4 (PtaSUT4), like its orthologs in other plant taxa, is tonoplast localized and thought to mediate Suc export from the vacuole into the cytosol. In source leaves of Populus, SUT4 is the predominantly expressed gene family member, with transcript levels several times higher than those of plasma membrane SUTs. A hypothesis is advanced that SUT4-mediated tonoplast sucrose fluxes contribute to the regulation of osmotic gradients between cellular compartments, with the potential to mediate both sink provisioning and drought tolerance in Populus. Here, we describe the effects of PtaSUT4-RNA interference (RNAi) on sucrose levels and raffinose family oligosaccharides (RFO) induction, photosynthesis, and water uptake, retention and loss during acute and chronic drought stresses. Under normal water-replete growing conditions, SUT4-RNAi plants had generally higher shoot water contents than wild-type plants. In response to soil drying during a short-term, acute drought, RNAi plants exhibited reduced rates of water uptake and delayed wilting relative to wild-type plants. SUT4-RNAi plants had larger leaf areas and lower photosynthesis rates than wild-type plants under well-watered, but not under chronic water-limiting conditions. Moreover, the magnitude of shoot water content, height growth, and photosynthesis responses to contrasting soil moisture regimes was greater in RNAi than wild-type plants. The concentrations of stress-responsive RFOs increased in wild-type plants but were unaffected in SUT4-RNAi plants under chronically dry conditions. We discuss a model in which the subcellular compartmentalization of sucrose mediated by PtaSUT4 is regulated in response to both sink demand and plant water status in Populus.
• Frost, C. (2011). Tracing the history of plant traits under domestication in cranberries: potential consequences on anti-herbivore defences. Journal of Experimental Botany.
• Rodriguez-Saona, C., Vorsa, N., Singh, A. P., Johnson-Cicalese, J., Szendrei, Z., Mescher, M. C., & Frost, C. J. (2011). Tracing the history of plant traits under domestication in cranberries: potential consequences on anti-herbivore defences. Journal of experimental botany, 62(8), 2633-44.
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  The process of selecting certain desirable traits for plant breeding may compromise other potentially important traits, such as defences against pests; however, specific phenotypic changes occurring over the course of domestication are unknown for most domesticated plants. Cranberry (Vaccinium macrocarpon) offers a unique opportunity to study such changes: its domestication occurred recently, and we have access to the wild ancestors and intermediate varieties used in past crosses. In order to investigate whether breeding for increased yield and fruit quality traits may indirectly affect anti-herbivore defences, the chemical defences have been examined of five related cranberry varieties that span the history of domestication against a common folivore, the gypsy moth (Lymantria dispar). Direct defences were assessed by measuring the performance of gypsy moth caterpillars and levels of phenolic compounds in leaves, and indirect defences by assaying induced leaf volatile emissions. Our results suggest that breeding in cranberry has compromised plant defences: caterpillars performed best on the derived NJS98-23 (the highest-yielding variety) and its parent Ben Lear. Moreover, NJS98-23 showed reduced induction of volatile sesquiterpenes, and had lower concentrations of the defence-related hormone cis-jasmonic acid (JA) than ancestral varieties. However, induced direct defences were not obviously affected by breeding, as exogenous JA applications reduced caterpillar growth and increased the amounts of phenolics independent of variety. Our results suggest that compromised chemical defences in high-yielding cranberry varieties may lead to greater herbivore damage which, in turn, may require more intensive pesticide control measures. This finding should inform the direction of future breeding programmes.
• Barakat, A., Bagniewska-Zadworna, A., Frost, C. J., & Carlson, J. E. (2010). Phylogeny and expression profiling of CAD and CAD-like genes in hybrid Populus (P. deltoides x P. nigra): evidence from herbivore damage for subfunctionalization and functional divergence. BMC plant biology, 10, 100.
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  Cinnamyl Alcohol Dehydrogenase (CAD) proteins function in lignin biosynthesis and play a critical role in wood development and plant defense against stresses. Previous phylogenetic studies did not include genes from seedless plants and did not reflect the deep evolutionary history of this gene family. We reanalyzed the phylogeny of CAD and CAD-like genes using a representative dataset including lycophyte and bryophyte sequences. Many CAD/CAD-like genes do not seem to be associated with wood development under normal growth conditions. To gain insight into the functional evolution of CAD/CAD-like genes, we analyzed their expression in Populus plant tissues in response to feeding damage by gypsy moth larvae (Lymantria dispar L.). Expression of CAD/CAD-like genes in Populus tissues (xylem, leaves, and barks) was analyzed in herbivore-treated and non-treated plants by real time quantitative RT-PCR.
• Frost, C. (2010). Cytokinin primes plant responses to wounding and reduces insect performance. Journal of Plant Growth Regulation.
• Frost, C. (2010). Phylogeny and expression profiling of CAD and CAD-like genes in hybrid Populus (P. deltoides$\times$ P. nigra): evidence from herbivore damage for subfunctionalization and functional divergence. BMC plant biology.
• Frost, C. (2010). Red oak responses to nitrogen addition depend on herbivory type, tree family, and site. Forest Ecology and Management.
• Frost, C. (2010). Red oak responses to nitrogen addition depend on herbivory type, tree family, and site. Forest ecology and management.
• Rodriguez-Saona, C. R., & Frost, C. J. (2010). New evidence for a multi-functional role of herbivore-induced plant volatiles in defense against herbivores. Plant signaling & behavior, 5(1), 58-60.
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  A diverse, often species-specific, array of herbivore-induced plant volatiles (HIPVs) are commonly emitted from plants after herbivore attack. Although research in the last 3 decades indicates a multi-functional role of these HIPVs, the evolutionary rationale underpinning HIPV emissions remains an open question. Many studies have documented that HIPVs can attract natural enemies, and some studies indicate that neighboring plants may eavesdrop their undamaged neighbors and induce or prime their own defenses prior to herbivore attack. Both of these ecological roles for HIPVs are risky strategies for the emitting plant. In a recent paper, we reported that most branches within a blueberry bush share limited vascular connectivity, which restricts the systemic movement of internal signals. Blueberry branches circumvent this limitation by responding to HIPVs emitted from neighboring branches of the same plant: exposure to HIPVs increases levels of defensive signaling hormones, changes their defensive status, and makes undamaged branches more resistant to herbivores. Similar findings have been reported recently for sagebrush, poplar and lima beans, where intra-plant communication played a role in activating or priming defenses against herbivores. Thus, there is increasing evidence that intra-plant communication occurs in a wide range of taxonomically unrelated plant species. While the degree to which this phenomenon increases a plant's fitness remains to be determined in most cases, we here argue that within-plant signaling provides more adaptive benefit for HIPV emissions than does between-plant signaling or attraction of predators. That is, the emission of HIPVs might have evolved primarily to protect undamaged parts of the plant against potential enemies, and neighboring plants and predators of herbivores later co-opted such HIPV signals for their own benefit.
• Rodriguez-Saona, C. R., Rodriguez-Saona, L. E., & Frost, C. J. (2009). Herbivore-induced volatiles in the perennial shrub, Vaccinium corymbosum, and their role in inter-branch signaling. Journal of chemical ecology, 35(2), 163-75.
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  Herbivore feeding activates plant defenses at the site of damage as well as systemically. Systemic defenses can be induced internally by signals transported via phloem or xylem, or externally transmitted by volatiles emitted from the damaged tissues. We investigated the role of herbivore-induced plant volatiles (HIPVs) in activating a defense response between branches in blueberry plants. Blueberries are perennial shrubs that grow by initiating adventitious shoots from a basal crown, which produce new lateral branches. This type of growth constrains vascular connections between shoots and branches within plants. While we found that leaves within a branch were highly connected, vascular connectivity was limited between branches within shoots and absent between branches from different shoots. Larval feeding by gypsy moth, exogenous methyl jasmonate, and mechanical damage differentially induced volatile emissions in blueberry plants, and there was a positive correlation between amount of insect damage and volatile emission rates. Herbivore damage did not affect systemic defense induction when we isolated systemic branches from external exposure to HIPVs. Thus, internal signals were not capable of triggering systemic defenses among branches. However, exposure of branches to HIPVs from an adjacent branch decreased larval consumption by 70% compared to those exposed to volatiles from undamaged branches. This reduction in leaf consumption did not result in decreased volatile emissions, indicating that leaves became more responsive to herbivory (or "primed") after being exposed to HIPVs. Chemical profiles of leaves damaged by gypsy moth caterpillars, exposed to HIPVs, or non-damaged controls revealed that HIPV-exposed leaves had greater chemical similarities to damaged leaves than to control leaves. Insect-damaged leaves and young HIPV-exposed leaves had higher amounts of endogenous cis-jasmonic acid compared to undamaged and non-exposed leaves, respectively. Our results show that exposure to HIPVs triggered systemic induction of direct defenses against gypsy moth and primed volatile emissions, which can be an indirect defense. Blueberry plants appear to rely on HIPVs as external signals for inter-branch communication.
• Frost, C. (2008). Improved Sugar Release from Lignocellulosic Material by Introducing a Tyrosine-rich Cell Wall Peptide Gene in Poplar. CLEAN--Soil, Air, Water.
• Frost, C. (2008). Insect herbivores and their frass affect Quercus rubra leaf quality and initial stages of subsequent litter decomposition. Oikos.
• Frost, C. (2008). Priming defense genes and metabolites in hybrid poplar by the green leaf volatile cis-3-hexenyl acetate. New Phytologist.
• Frost, C. (2008). Priming defense genes and metabolites in hybrid poplar by the green leaf volatilecis-3-hexenyl acetate. New Phytologist.
• Frost, C. (2008). Slow-cycle effects of foliar herbivory alter the nitrogen acquisition and population size of Collembola. Soil Biology and Biochemistry.
• Frost, C. (2008). Why do distance limitations exist on plant-plant signaling via airborne volatiles?. Plant signaling & behavior.
• Frost, C. J., & Hunter, M. D. (2008). Herbivore-induced shifts in carbon and nitrogen allocation in red oak seedlings. The New phytologist, 178(4), 835-845.
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  * A dual-isotope, microcosm experiment was conducted with Quercus rubra (red oak) seedlings to test the hypothesis that foliar herbivory would increase belowground carbon allocation (BCA), carbon (C) rhizodeposition and nitrogen (N) uptake. Plant BCA links soil ecosystems to aboveground processes and can be affected by insect herbivores, though the extent of herbivore influences on BCA is not well understood in woody plants. * Microcosms containing 2-yr-old Q. rubra seedlings and soil collected from the Coweeta Hydrologic Laboratory (NC, USA) were subjected to herbivory or left as undamaged controls. All microcosms were then injected with 15N-glycine and pulsed with 13CO2. * Contrary to our hypothesis, herbivore damage reduced BCA to fine roots by 63% and correspondingly increased allocation of new C to foliage. However, 13C recoveries in soil pools were similar between treatments, suggesting that exudation of C from roots is an actively regulated component of BCA. Herbivore damage also reduced N allocation to fine roots by 39%, apparently in favor of storage in taproot and stem tissues. * Oak seedlings respond to moderate insect herbivore damage with a complex suite of allocation shifts that may simultaneously increase foliar C, maintain C rhizodeposition and N assimilation, and shift N resources to storage.
• Frost, C. J., Mescher, M. C., Carlson, J. E., & De Moraes, C. M. (2008). Plant defense priming against herbivores: getting ready for a different battle. Plant physiology, 146(3), 818-24.
• Frost, C. J., Mescher, M. C., Carlson, J. E., & De Moraes, C. M. (2008). Why do distance limitations exist on plant-plant signaling via airborne volatiles?. Plant signaling & behavior, 3(7), 466-8.
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  Plant volatiles are known to mediate many important ecological interactions between plants and insects. Plants themselves have also been shown to perceive volatile signals, but the short transmission distances documented thus far in nature raise questions about the ecological significance of plant-to-plant signaling. Recently, we reported that herbivore-induced plant volatiles (HIPVs) can function within an individual plant to overcome vascular constraints on systemic wound signaling. Within-plant signaling is consistent with the limited distances over which HIPVs have been shown to be perceived by plants. However, it remains unclear why these distance limitations should exist. Such limitations cannot be explained by volatile transport distance alone, since parasitoids respond to HIPVs over much greater distances. Thus, we suggest that the apparent distance limitations on plant-to-plant volatile signaling may arise from the mechanisms by which volatile signals are received by plants. These limitations may reflect physiological constraints on plants' ability to perceive volatiles or an adaptive mechanism to avoid responding to signals from other plants. Distinguishing between these possibilities will require additional research into the mechanisms of signal reception, about which little is currently known. Deciphering the ecological significance of HIPVs as phytohormones depends on understanding the mechanisms of HIPV reception.
• Frost, C. J., Mescher, M. C., Dervinis, C., Davis, J. M., Carlson, J. E., & De Moraes, C. M. (2008). Priming defense genes and metabolites in hybrid poplar by the green leaf volatile cis-3-hexenyl acetate. The New phytologist, 180(3), 722-734.
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  * Herbivore-induced plant volatiles (HIPVs), in addition to attracting natural enemies of herbivores, can serve a signaling function within plants to induce or prime defenses. However, it is largely unknown, particularly in woody plants, which volatile compounds within HIPV blends can act as signaling molecules. * Leaves of hybrid poplar saplings were exposed in vivo to naturally wound-emitted concentrations of the green leaf volatile (GLV) cis-3-hexenyl acetate (z3HAC) and then subsequently fed upon by gypsy moth larvae. Volatiles were collected throughout the experiments, and leaf tissue was collected to measure phytohormone concentrations and expression of defense-related genes. * Relative to controls, z3HAC-exposed leaves had higher concentrations of jasmonic acid and linolenic acid following gypsy moth feeding. Furthermore, z3HAC primed transcripts of genes that mediate oxylipin signaling and direct defenses, as determined by both qRT-PCR and microarray analysis using the AspenDB 7 K expressed sequence tags (EST) microarray containing c. 5400 unique gene models. Moreover, z3HAC primed the release of terpene volatiles. * The widespread priming response suggests an adaptive benefit to detecting z3HAC as a wound signal. Thus, woody plants can detect and use z3HAC as a signal to prime defenses before actually experiencing damage. GLVs may therefore have important ecological functions in arboreal ecosystems.
• Frost, C. (2007). COS 115-10: Induced airborne volatiles coordinate systemic defense responses to insect herbivores.
• Frost, C. (2007). Within-plant signalling via volatiles overcomes vascular constraints on systemic signalling and primes responses against herbivores. Ecology Letters.
• Frost, C. J., & Hunter, M. D. (2007). Recycling of nitrogen in herbivore feces: plant recovery, herbivore assimilation, soil retention, and leaching losses. Oecologia, 151(1), 42-53.
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  Herbivores directly and indirectly affect ecosystem functioning in forests. Feces deposition is a direct effect that supplies ephemeral N pulses to soils. Herbivore-mediated changes in plant N allocation and uptake are indirect effects that can also influence soil N availability. These effects may interact if defoliation influences the ability of plants to recover fecal N, and this may affect subsequent generations of herbivores. We added (15)N-enriched insect feces (frass) to a series of replicated red oak, Quercus rubra, mesocosms that had been damaged experimentally and then followed the frass N over the course of 2 years. In the first season, some frass N was mineralized in the soil and leached in organic form from the mesocosms within 1 week of deposition. Within 1 month, frass N had been acquired by the oaks and enriched the foliage; late-season herbivores assimilated the frass N within the same growing season. In the second season, herbivore damage from the previous year lowered total leaf N contents and (15)N recovered in the foliage. A subsequent cohort of early-season herbivores fed on this foliage consequently derived less of their N from the previous year's frass, and feral leaf rollers colonized fewer of these saplings. The 0- to 5-cm soil fraction was the largest N sink measured, and 42% of the frass N was recovered in the soil. The results demonstrate that: (1) some frass N can be recycled rapidly into foliage and assimilated by successive cohorts of herbivore within the same season; (2) damage can affect N allocation in the following year's foliage, influencing N availability to and host selection by herbivores; and (3) leaching losses occur soon after deposition but are buffered by soil pools, which are the largest sinks for frass N.
• Frost, C. J., Appel, H. M., Carlson, J. E., De Moraes, C. M., Mescher, M. C., & Schultz, J. C. (2007). Within-plant signalling via volatiles overcomes vascular constraints on systemic signalling and primes responses against herbivores. Ecology letters, 10(6), 490-8.
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  Plant volatiles play important roles in signalling between plants and insects, but their role in communication among plants remains controversial. Previous research on plant-plant communication has focused on interactions between neighbouring plants, largely overlooking the possibility that volatiles function as signals within plants. Here, we show that volatiles released by herbivore-wounded leaves of hybrid poplar (Populus deltoides x nigra) prime defences in adjacent leaves with little or no vascular connection to the wounded leaves. Undamaged leaves exposed to volatiles from wounded leaves on the same stem had elevated defensive responses to feeding by gypsy moth larvae (Lymantria dispar L.) compared with leaves that did not receive volatiles. Volatile signals may facilitate systemic responses to localized herbivory even when the transmission of internal signals is constrained by vascular connectivity. Self-signalling via volatiles is consistent with the short distances over which plant response to airborne cues has been observed to occur and has apparent benefits for emitting plants, suggesting that within-plant signalling may have equal or greater ecological significance than signalling between plants.
• Frost, C. (2004). INSECT CANOPY HERBIVORY AND FRASS DEPOSITION AFFECT SOIL NUTRIENT DYNAMICS AND EXPORT IN OAK MESOCOSMS. Ecology.
• Frost, C. (2004). Insect canopy herbivory and frass deposition affect soil nutrient dynamics and export in oak mesocosms. Ecology.

Proceedings Publications

• Frost, C. (2012). Tubulin Manipulations Alter Wood Properties and Drought Tolerance Characteristics in Populus. In IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL.
• Frost, C. (2011). Partial suppression of a strongly expressed tonoplast sucrose transporter affects water use and carbon partitioning in Populus. In BMC proceedings.
• Frost, C. (2010). COS 32-8: A gall-inducing caterpillar species differentially alters decomposition dynamics of leaf litter from two Populus host plant species. In The 95th ESA Annual Meeting.
• Frost, C. (2010). OOS 47-4: Ozone pollution compromises plant defense responses to insect herbivory. In The 95th ESA Annual Meeting.
• Frost, C. (2010). OOS 52-8: Plant responses to airborne volatiles depend on signal source and specificity. In The 95th ESA Annual Meeting.
• Frost, C. (2008). COS 71-3: Defense priming against herbivores by plants using volatile cues as indicators of potential attack. In The 93rd ESA Annual Meeting.

Presentations

• Frost, C. (2024, October). Plants Stink! The Nuance and Importance of Odor in Modulating Plant Chemical Ecology: Mechanisms and Potential Applications. Invited Seminar at the Odum School of Ecology, University of Georgia. Athens, GA: Odum School of Ecology, University of Georgia.
• Frost, C. (2023, June). Plants Can Smell and Remember – Really. But Why?  And How?. KEYS Summer Internship Seminar Series. Tucson, AZ: BIO5, University of Arizona.
• Frost, C. (2021). Mechanisms and Applications of Plant Smells in Modulating Innate Plant Immunity. School of Plant Sciences Seminar. University of Arizona: School of Plant Sciences.
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  Plants sense and react to reliable information cues in their environment. While some plants like the venus fly trap or the sensitive plant are notable examples of how dynamic such responses can be, almost all plants are responsive in ways that are not visibly apparent. For example, plants have evolved sophisticated chemical defense strategies to resist or tolerate environmental stresses including insect herbivory, and these defenses are inducible and therefore change in real time. A prominent chemical response to herbivory is the synthesis and emission of distinct bouquets of volatile organic compounds (VOCs) that provide ecologically relevant information used by a variety of animals including herbivores’ natural enemies. Remarkably, plants can also eavesdrop these volatile cues and “prime” themselves against future stress. Priming is therefore an inducible phenomenon requiring changes within a plant to modify its physiological condition to more rapidly mount defense against a future foe. This talk will explore this remarkable feature of plant biology and some ways in which it provides an opportunity to enhance agricultural sustainability.

Poster Presentations

• Waggoner, E., Frost, C., & Duhamel, S. (2022, September). Organophosphate Esters alon the Amazon River Plume and into the North Atlantic Ocean. BioRetreat 2022. Biosphere 2: University of Arizona.

Others

• Frost, C. (2020, January). Defoliation-induced compensatory transpiration is compromised in SUT4-RNAi Populus. https://doi.org/10.1101/2020.01.13.905406
• Frost, C. (2019, January). Plant seeds are primed by herbivore-induced plant volatiles. https://doi.org/10.1101/522839
• Frost, C. (2019, September). Acute toxicity of the plant volatile indole depends on herbivore specialization. https://doi.org/10.1101/784165
• Frost, C. (2018, July). Dispensing a synthetic green leaf volatile to two plant species in a common garden differentially alters physiological responses and herbivory. https://doi.org/10.1101/370692
• Frost, C. (2005). Direct and indirect effects of insect herbivores on terrestrial ecosystem processes.