Progress 06/15/20 to 06/14/23
Outputs
Target Audience:This reporting period consisted of a continuation of research on the impact of dopamine on auxin signaling and plant physiology, and the publication of my results gathered on this topic during the duration of this fellowship. The target audience of this publication is the plant physiology research community, and will hopefully stimulate more interest in the role of catecholamines in plants and shed light on the impact of dopamine on plant physiology. Changes/Problems:One major problem was encounted during the completion of Research objective 2. The parental line used in the bulk segrigant analysis was the WS ecotype, which was obtained from lehle seeds. Unfortunitly, this ecotype was likely outcrossed with Col-0 at some point (determined due to high sequence alignment with the TAIR10 database, and comparitivly poorer alignment with the publically available WS genome). This problem with seedstocks at lehle seeds has been documented in the literature, and the company and community are now aware that some batches of seeds were effected. While this delayed the analysis of the datasets, it stimulated me to write my own analytical pipeline which can handle messier bulk-segrigant datasets. What opportunities for training and professional development has the project provided?During this funding period, my abilities in quantitative genomics and data analysis have expanded, predominantly as a result of setback described in the change/problems section of this report. Due to problems analyzing my bulk segrigant datasets resulting from apparent impurities in the genetic material used as the parental line, I developed an analytical pipeline using shell and python which, beyond the typical ratio-metric analysis of allele depth between the datasets, calculates g-statistics for every SNP and considers undocumented background polymorphisms if you have sequenced multiple bulk segrigant populations backcrossed into the same parental line. This experience was invaluable, and despite delaying some project goals has left me with a skillset that is highly useful in several agricultural research efforts. How have the results been disseminated to communities of interest?The research in this funding period as pertains to Research Objective 1 was published in the MDPI journal Stresses under the title Dopamine Inhibits "Arabidopsis Growth through Increased Oxidative Stress and Auxin Activity". What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Research objective 1, We proceeded to examine if the increase in auxin signaling described in the last reporting period was the result of an alteration of auxin transport. To test this, we employed two different strategies: To test if DA-induced IAA hypersensitivity is the result of altered auxin transport, we compared the impact of DA on the sensitivity of plants to n-naphthalene acetic acid (NAA) to IAA. NAA is not actively transported. We found that DA-induced auxin hypersensitivity was absent when with NAA, suggesting that the auxin-hypersensitive response induced by DA is possibly the result of altered auxin transport. We used the auxin transport deficient mutant aux1-7, comparing its response to combinational treatments of DA and the natural auxin IAA. We found that in the mutant, DA-induced IAA hypersensitivity is absent. We concluded that DA causes auxin hypersensitivity through alteration of auxin transport. We identified two potential mechanisms for this activity: GSH metabolism and oxidative stress: Previously, we had noted that one of the highest upregulated genes in our RNA-seq analysis was the GSH transferase GSTU11, and that there is an established link between GSH content in roots and auxin regulation. We found that in addition to GSTU11, 11 other GSH transferases and as well as the gene encoding the GSH synthetase enzyme GSH2 were upregulated in response to DA. Arabidopsis plants grown for 14 days on 0.2 mM DA treated media had ~52% larger rosettes than the DA-only treated control, suggesting the GSH may have a protective effect against DA-induced oxidative stress. We incubated plants grown on 0 or 0.25 mM DA and 0 or 100 µM GSH for 7 days in H2DCF-DA, which upon oxidation produces the fluorescent DCF. Plants grown on DA-treated media accumulate more DCF in their roots compared to the control, indicating that DA leads to excess ROS. Plants grown on DA treated media supplemented with 100 µM GSH also showed an ~34% decrease in fluorescence compared to the DA-only control. Considering this, we tested the link between GSH metabolism and DA-induced auxin hypersensitivity using the γ-ECS inhibitor BSO. We grew plants on media containing 0, 0.5 or 0.75 mM BSO with or without 10 µM DA and 10 nM IAA and assessed the growth of roots after 11 days. The mean primary root length plants grown on 0.5 and 0.75 mM BSO alone were ~80% and ~65% of the untreated control plants. Confirming our results suggesting that GSH production is stimulated by DA exposure, we found that supplementation of BSO-grown plants with 10 µM DA reversed BSO-induced mean primary root length decreases. As expected, plants grown on 10 nM IAA and 10 µM DA alone did not have altered mean primary root growth, and plants grown on 10 nM IAA and 10 µM DA combinational treatments had mean primary root length of ~33% of the control. Surprisingly, we found that addition of 0.5 and 0.75 mM BSO to IAA/DA cotreated plants resulted in an ~102% increase in primary root length compared to the IAA/DA cotreated plants alone, suggesting that an aspect of DA-induced IAA hypersensitivity is DA-stimulated GSH production. DA and iron homeostasis: The iron homeostasis regulator encoding gene BHLH100 had a fold-change value of 9.44 at 2 hours of DA treatment in our RNAseq dataset. Due to an established link between auxin transport and iron availability, we tested how iron influences DA-induced IAA sensitivity. We grew Col-0 seedlings on 0 or 0.5 mM DA and analyzed the accumulation of iron in seedling tissue using ICP-MS. Our results indicate that plants grown on DA accumulate significantly lower levels of iron than control plants - DA-grown seedlings contained ~33% less iron than the control. Using SDS-PAGE we found that plants grown on 0.25 and 0.5 mM DA had ~0.45 and ~0.14 fold-change lower mean levels of FIT1, a positive regulator of iron homeostatic processes, suggesting the plants grown on DA take up less iron due to a downregulation of iron uptake mechanisms. Finally, we tested the secretion of ferric chelate reductases (FCRs) using ferrozine. Our analysis showed that DA-grown plants secrete significantly less FCRs, confirming that DA-grown plants have downregulated iron uptake mechanisms. We reasoned that since iron deficiency can have a dramatic impact on plants growth, decreased iron uptake in DA-treated plants may contribute to DA-induced growth repression. To test this, we grew plants on iron-depleted media supplemented with 5 µM (low), 50 µM (optimal) and 150 µM (high) Fe-EDTA, with or without 0.1 mM DA for 14 days. As expected, plants grown on optimal levels of iron had normal rosette development upon additional supplementation of media with 0.1 mM DA. Plants grown on low levels of iron were chlorotic and exhibited mean rosette diameters ~69% of the control. Surprisingly, the response of plants on low and high iron to additional DA treatment was the opposite of our expected result. Plants grown on low levels of iron were significantly stimulated by the addition of 0.1 mM DA, leading to rosettes insignificantly different from the optimal-iron grown control. Upon addition of DA to the high-iron grown plants, mean rosette size decreased to ~50% of the high-iron only control, suggesting that high levels of iron in media exacerbates DA-induced growth repression. We concluded that the combined pro-oxidant effects of DA and iron, which has been established in mammalian literature, is likely a major component of DA-induced growth reduction seen in aseptically grown plants. Research objective 2, "Isolate and characterize DA-resistant Arabidopsis mutants." To fulfill objective 2, 8 mutants exhibiting DA-resistant phenotypes were backcrossed into the WS ecotype, and the F2 populations were segregated based on the DA resistant phenotype, pools of ~25 seedlings were sequenced. The resulting datasets were analyzed using the delta-SNP approach, in which the allelic frequencies of the "reference genotype" of SNPs found in each bulk were subtracted from each other, yielding a linkage map. Dopamine-resistant #1 (dar1), bears a lesion in the PTAC12 gene, introducing an early stop codon at amino acid position 480. Dar1 plants are highly chlorotic upon germination and growth under continuous light and appear to resume normal chlorophyll accumulation upon transfer to soil and a long-day light regime. Dar1 plants are resistant to DA compared to WS; mean rosette size of control plants grown on 0.5 mM DA after 14 days are ~50% the size of dar1. Additionally, dar1 accumulates chlorophyll in response to being grown on DA-supplemented media; dar1 and WS grown on control media have a mean of ~15.65 and ~387.95 mg/m^2 chlorophyll in developed leaves, respectively. However, on 0.3 mM DA supplemented media, both lines accumulate ~ 285 mg/m^2 chlorophyll in developed leaves, suggesting that in wild-type plants, DA slightly decreases chlorophyll production, but in dar1 plants, DA dramatically stimulates chlorophyll production. Dopamine-resistant #38 (dar38) appears to bear a lesion in the XYL1 gene, introducing an early stop codon at amino acid position 560. when grown on 0.2 mM DA, dar38 rosettes are ~3 times the size of the control to assess the if the dar38 mutation was correctly identified, we compared the phenotype of dar38 to the phenotype of the documented XYL1 mutation xyl1-1, which is characterized by so-called "club-like" siliques, which are shorter and wider than the wild-type. We find that in a comparison between dar38 and xyl1-1 siliques, the length and width are insignificantly different, indicating that the mutation was correctly identified. We hypothesized that the resistance of dar38 to DA treatment. We hypothesized that the resistance of dar38 could be due to a decrease in DA-induced repression of cell expansion.
Publications
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2023
Citation:
Shull, T.E.; Kurepa, J.; Smalle, J.A. Dopamine Inhibits Arabidopsis Growth through Increased Oxidative Stress and Auxin Activity. Stresses 2023, 3, 351-371. https://doi.org/10.3390/stresses3010026
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Progress 06/15/21 to 06/14/22
Outputs
Target Audience:This reporting period consisted of a continuation of research on the impact of dopamine on auxin signaling and plant physiology. The results gathered during this period was presented at thew University of Kentucky Integrated Plant and Soil Sciences Mini symposium at the beginning of 2022, and a publication of the results gathered throughout the course of the past two funding periods will be submitted by the end of 2022, reaching the Plant Physiology community to support further research and interest in the function of catecholamines in plants. Finally, during this reporting period I was able to engage in some educational outreachby volunteering as a judge of the microbiology section at the Fayette Country District Science fair. Changes/Problems:1) Aspects of this research project were delayed during the Covid-19 pandemic, predominantly in the way of access to opportunities to present research in person early in the pandemic, and delayed shipping of some relevant materials. As such, a no-cost extension was filed and accepted to extend the funding period by 6 months. 2) Some difficulties have arisen from using WS-0 for the mapping by sequencing experiments, as the resources for this genome are not nearly as robust as the resources available for Col-0. What opportunities for training and professional development has the project provided?During the current funding period, I have had the opportunity undergotrainingin bioinformatics workflows byboth attending the MIT open courseware course "Introduction to computer science and programming in python" and through independent study using the text "Bioinformatics algorithms: Design and implementation in python". This experience has been invaluable in sharpening my computational biology skills, which I'm hoping will make me better able to lead research efforts in an increasingly complex research environment.In terms ofprofessional development, this project has given me valuable insight into project management and the process of obtaining grants anddelivering results, giving me asubstantial competitive edge in the academic job market. How have the results been disseminated to communities of interest?We plan to publish the results of our analysis on the impact of dopamine on auxin signaling, iron homeostasis and glutathione metabolism by the end of 2022. Moreover, preliminary results generated during this project were presented at the Integrated Plant and Soil Science Mini symposium at the University of Kentucky early this year, where it was awarded first place in a competition among my peers. What do you plan to do during the next reporting period to accomplish the goals?Primary objectives in the next reporting period include: 1) Publish the results of our analysis on dopamine's impact on auxin signaling and plant physiology 2) complementation analysis of thedar1anddar38mutants, and continued analysis of the remaining dopamine-resistant mutants.
Impacts
What was accomplished under these goals?
Allelopathy is an evolutionarily selected mechanism by which plants advantageously alter their immediate environment by producing and releasing compounds (i.e., allelochemicals) that negatively affect the growth of surrounding plants. The neurotransmitter dopamine has many of the properties of an allelochemical, in that it is produced by many plant species and severely inhibits growth when applied to growth medium. Understanding how dopamine influences plant growth and development has the potential to lead to novel forms of sustainable weed control by leveraging already existing biological mechanisms which repress the growth of competing plants. To date, we have confirmed previous reports that dopamine causes hypersensitivity to the plant hormone auxin and have established that dopamine-induced auxin hypersensitivity is likely a result of altered auxin transport. We have also found that dopamine increases oxidative stress in an iron dependent manner, and that antioxidant glutathione mitigates dopamine-induced stress. Finally, we find that plants grown on dopamine supplemented media accumulate ~33% less iron, a key element for plant growth and development. Our results during this funding period indicate that dopamine exerts its effect by hypersensitizing plants to the plant hormone auxin through increased auxin transport, inducing oxidative stress and inhibiting iron uptake. Research objective 1, "Analyze the interaction between cytokinin signaling and dopamine-induced allelopathy". Considering findings reported in the previous funding period that plants grown on DA do not hyperaccumulate the primary auxin indole-3-acetic acid (IAA), we conducted experiments to understand the cause of DA-induced IAA hypersensitivity. We hypothesized that DA exerts its effect on auxin signaling by altering transport of IAA. To test this hypothesis, we employed the synthetic auxin 1-naphthalenacetic acid (NAA), which diffuses passively into roots, rather than through active transport. This difference has been used to identify responses to IAA which depend on auxin transport. As previously reported, plants grown on media supplemented with both 10 µM DA and 10 nM IAA exhibit an ~68% decrease in primary root length. However, additional supplementation of DA-grown plants with NAA led to no detectable morphological changes compared to the NAA only control, suggesting that DA likely exerts its IAA-hypersensitizing effect on plants by altering the transport of IAA. To further test if DA-induced IAA hypersensitivity is indeed reliant on altered IAA transport, we next used the aux1 mutant background, which is deficient in auxin transport. Indeed, aux1-7 Arabidopsis plants do not exhibit decreased root length when grown on media supplemented with 10 µM DA and 10 nM IAA, confirming that DA induced auxin hypersensitivity is a downstream effect of the impact of DA on auxin transport. To gain a general overview of how the plant transcriptome response to DA treatment, we sequenced the mRNA of pooled seedlings treated with 0 or 50 mM DA for 2 or 8 hours and analyzed the resulting RNAseq dataset. In brief, we uncovered that the transcriptomic response to DA involves an upregulation of IAA metabolic genes which are aimed at the production of phytoalexins, differential regulation of genes central to iron homeostasis, and a marked increase in the expression of glutathione (GSH) transferases. Iron homeostasis and GSH biosynthesis are two well-documented physiological processes which are tightly linked to altered auxin transport. We hypothesized that the influence of DA on auxin transport may be the result of the influence of DA on one or both mechanisms. Therefore, we aimed to obtain a more detailed understanding of how DA influences iron homeostasis, GSH biosynthesis and plant redox status. To examine the influence of DA on GSH metabolism, we grew the Arabidopsis plants on MS/2 supplemented with 0 or 0.25 mM DA and subjected them to SDS-PAGE analysis using GSH1 antibodies, which detect the enzyme responsible for the first committed step in GSH biosynthesis. We found that seedlings grown on 0.25 mM DA accumulate ~2.7 fold change more GSH1 protein than the untreated control, confirming that plants grown on DA do indeed have altered GSH metabolism. To test the influence of GSH on the DA growth response, we supplemented MS/2 media with 0 or 100 µM GSH and 0, 0.1, 0.2 or 0.3 mM DA. Indeed, supplementation with 100 µM GSH rescues plants from DA-induced rosette size decreases, with an ~52.71% increase in mean rosette diameter at 0.3 mM DA and ~87.16% increase in mean rosette diameter at 0.2 mM DA compared to the DA-only controls. Using the ROS probe H2DCF-DA, we also find that DA-induced oxidative stress is ameliorated by GSH. We concluded that GSH metabolism is upregulated in response to DA to adjust plant redox status. We next examined the link between DA and iron homeostasis. First, we tested if plants grown on DA-supplemented media differentially accumulate the transcription factor FIT, which is a central positive regulator of iron uptake. Upon subjecting DA-grown plants to SDS-PAGE and anti-FIT antibodies, we found that FIT accumulates ~0.6 and ~0.1 fold change less in Col-0 seedlings grown on MS/2 supplemented with 0.25 and 0.5 mM DA compared to plants not exposed to DA, suggesting that DA downregulates mechanisms of iron uptake. Using ICP-MS, we confirmed this, and found that plants grown on 0.5 mM DA accumulate ~33% less iron than plants grown on control media. Moreover, we find that DA alleviates iron deficiency, likely due to the DA-dependent upregulation of GSH1 and that plants grown on higher than optimal levels of iron are hypersensitive to DA due to an increase in ROS accumulation. With a framework for how DA interacts with iron homeostasis and GSH metabolism, in the next reporting period we will assess the interaction of DA-induced IAA hypersensitivity with these mechanisms. Research objective 2, "Isolate and characterize dopamine-resistant Arabidopsis mutants" To achieve objective 2, we initially isolated 5 mutants in the WS-0 ecotype background which are resistant to DA-induced rosette growth inhibition. During the current funding period, 3 more mutants were isolated, and all 8 mutants were subjected to mapping-by-sequencing analysis by subjecting pooled wild-type and mutant phenotypic segregants to high-throughput illumina DNA sequencing. Sequencing datasets were aligned to a publicly available WS-0 genome and linked regions were identified using the SIMPLE pipeline, which uses background EMS-induced polymorphisms to create linkage maps, which subsequently allow the identification of casual mutations. To date, two putative casual mutations have been identified in the lines DAR1 (dopamine-resistant 1) and dar38. dar1, which appears to bear a point mutation on chromosome 2 in the gene plastid transcriptionally active 12, or PTAC12, is under normal growth conditions dwarfed and highly chlorotic, containing ~10 mg/m2 of chlorophyl in its rosettes. However, upon growth on media supplemented with 0.3 mM DA, PTAC12 accumulates ~ 300 mg/m2 chlorophyll, suggesting that DA may have a profound influence on the regulation of chloroplast localized processes. Moreover, dar1 plants are resistant to DA treatment, with plants ~1.8 fold change larger than WS-0 wild-type plants when grown on media containing 0.3 mM DA. dar38, which appears to bear a point mutation on chromosome 1, putatively in the gene XYL1, which is involved in the modification of xyloglucans, which are involved in cell-wall expansion, bears club-shaped siliques and slightly thickened cotyledons. Complementation analysis and further experimentation on all dar mutants, including dar1 and dar38, are to continue in the coming months, and are expected to yield novel information on the impact of DA on plant growth and development.
Publications
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Progress 06/15/20 to 06/14/21
Outputs
Target Audience:This reporting period primarily involved experimentation and evidence gathering, the results of which were presented at the University of Kentucky Integrated Plant and Soil Sciences Mini symposium at the beginning of 2021. Changes/Problems:Currently, the only twodeviations from the experiments outline in the project initiation are as follows: 1)Due to the observed dramatic impact of dopamine on auxin signaling, much of the research in this funding period was focused on uncovering how dopamine influences auxin signaling. Therefore, we opted to subject samples grown on dopamine treated media to a metabolomic analysis targeted atquantifying the levels of 26 metabolites involved in auxin biosynthesis and catabolism, the results of which are outlined under the "Accomplishments" section of this report. 2) Due to the results of the metabolomicanalysis, we decided that project funds were best spent on obtaininga broaderunderstanding of the physiological impact of dopamine through an analysis of thetranscriptomic changes induced by dopamine treatment. To that end, we subjected 7-day old Arabidopsis seedlings treated with 0- or 50-mM dopamine for 2or 8 hours to RNAseq analysis. The data analysis for this experiment is currently ongoing. What opportunities for training and professional development has the project provided?Managing a research timeline and planning the allocation of funds have beenthe most fruitfulprofessional developmentopportunities provided by thisproject. Indeed, gaining competence in mapping a research plan, orchestrating the research required to meet goals, and adapting experimentsto meet long-term goals in the face of unexpected outcomes, have all been invaluable experiences.Furthermore, interacting with industry services and researchers while pursuing thegoals of this project has provided insight into the research infrastructure that exists outside ofAcademia, and what is necessary to run and operate a service-based biotechnology business. In relation to specifictrainingactivities this project has provided, this year I used project funds toattend the American Society of Plant Biologists virtual conference, where I was able to attend a workshop in which I received information and training to use the Department of Energy's predictive biology platform, KBase. Additionally, I attended aworkshop focused on improving interview skills, in which I engaged in a conversation with an expert about what modern hiring managers tend to look for in job candidates. How have the results been disseminated to communities of interest?To date, the research conducted in pursuit of the fellowship goals has been presented at a symposium at the University of Kentucky. Currently, a manuscript is being produced outlining the findings of the research conducted in the first period of the project, which will hopefully be published during the second funding period of this project. Hopefully in the coming year I will get the opportunity to present my researchto a broader audience. What do you plan to do during the next reporting period to accomplish the goals?High priority goals forthe next reporting period are: 1) Disseminate the results of my research on the influence of dopamine on plant physiology through the publication of a research article as well as present my research personally at a conference or symposium. 2) Identify the casual mutations underlying the dopamine-resistant germplasm generated throughout the course of the previous year. 2) Engage in at least one education and outreach opportunity. 3) Attend a professional development workshop focused on the development of advanced bioinformatics skills.
Impacts
What was accomplished under these goals?
Allelopathy is an evolutionarily selected mechanism by which plants advantageously alter their immediate environment by producing and releasing compounds (i.e., allelochemicals) that negatively affect the growth of surrounding plants. The neurotransmitter dopaminehas many of the properties of an allelochemical, in that it is produced by many plant species and induces severe growth retardation in sensitive plants. While dopamine is widespread in the plant kingdom and many other branches of life, the mechanisms by which dopamine influences plant growth and development remains unknown. Understanding how dopamine influences plant growth and development has the potential to lead to novel forms of sustainable weed control by leveragingbiological mechanisms that have been evolutionarily honed to repress the growth of competing plants. To date, the funded research has addressed a knowledge gap in how plants respond to dopamine exposure. Specifically, it has been uncovered that dopamine is likely perceived as a warning signal forbiological threats, inducing profound changes in plant signaling and behavior. Research objective 1,"Analyze the interaction between cytokinin signaling and dopamine-induced allelopathy".To make progress on this objective,we studiedthe link between cytokinin signaling and dopamine on plants using Arabidopsis seedlings bearing genetic abnormalities which make them hypo- or hyper-sensitive to cytokinin. Considering our preliminary data showing that plants treated with low doses of cytokinin are more resistant to dopamine-induced rosette size decreases, we were not surprised to observe that some cytokinin hypersensitive lines are indeed resistant to dopamine. However, cytokinin hyposensitivity did not lead to dopamine hypersensitivity,leading us to conclude that while cytokinin plays a role in the dopamine response, other biological mechanisms are likely involved in dopamine-induced changes in plant growth and development. This led us to expand our analysis to include the impact ofdopamineon the sensitivity of plants toauxin, a hormone which acts synergistically and antagonistically with cytokinin to shape plant growth and development. To this end, Arabidopsis seedlings deficient in auxin signaling were grown on growth media supplemented with dopamine. We observed a profound resistance to dopamine-induced morphological changes. Specifically, dopamine does not further dwarf the rosettesor influence root growth of auxin-resistant plants. Furthermore, we found that supplementation of growth media withdopamine at concentrations as low as10 µM caused plants to be dramatically more sensitive to the preeminentnaturally occurringauxin, indole-3-acetic acid(IAA). This sensitivity presents the strongest in roots. Whileplants grown on media supplementedwith only10 µM dopamine or10 µM IAA had root lengths insignificantly different from the untreated control plants, supplementation of mediawith both compounds simultaneously led to an ~65% and ~68%decrease in mean root length compared to the dopamine andIAAcontrols, respectively.Due to the strength of the increase in auxin sensitivity by low levels of dopamine, and the previously established link between the repression of cytokinin signaling and increased auxin signaling, we concluded thatdopamine-induced cytokinin insensitivityis possibly the downstreamresult ofdopamine-inducedhypersensitivity toauxin and opted to quantify the impact of dopamine on both auxin and cytokinin signaling. To measure the impact of dopamine on both auxin and cytokinin signaling mechanisms, we produced and grew Arabidopsis seedlingscontaining transgenes which allow the quantification of auxin (DR5rev::erRFP ) and cytokinin (TCSn::eGFP) signaling, through the measurement of the expression levels of fluorescent proteinsdriven by promotors activated by hormone signaling. Supporting the hypothesis that exposure to dopamine enhances auxin signaling, we observed a significantincrease (~3.5 fold) in DR5rev driven RFP accumulation in Arabidopsis seedlings grown on plates containing 1 mM dopamine had a small butinsignificant increase in TCSndriveneGFP expression. Due to the significant increase in auxin signaling seen in response to dopamine, as well as previous research claimingthat dopamine inhibits the activity of auxin oxidases, we analyzed the accumulation of 26 different metabolites involved in auxin biosynthesis and catabolism in Arabidopsis seedlings grown on solid media supplemented with 0- or 0.5-mM dopamine for 7 days. Our analysis revealed that baseline levels of IAA were not significantly different between plants grown ontreated and untreated media, nor was there a differential accumulation ofoxidized IAA, the known product of IAA oxidases.However, there was a marked increase in the biosynthesis of the camalexin-related compounds indole-3-carboxcylic acid (~2.4 fold increase) and indole-3-carboxyaldehyde (~7.2 fold increase) in plants grown on dopamine treated media. These two compounds are associated with the plantresponse topathogens, and their accumulation suggests that plants perceive dopamine as a xenobiotic compound and in turn upregulate defense related metabolic pathways. Considering the impressive increase in both auxin signaling and sensitivity to auxin in response to dopamine, it is perplexing that there is no organism-wideaccumulation of IAA in plants grown on dopamine treated media. However,it remainspossible that changes in the metabolism and production of indole-containing compounds in response to dopamine exposure is sufficient to severely alter the orchestration oflocal auxin minima and maxima, which isa key featureof auxin signaling and its downstreaminfluence on plant growth and development. To understand why there isa markedincrease in auxin sensitivity without the organism-wide hyperaccumulation of IAA, more information about the organismal response to short- and long-term dopamine treatments is needed. To gain further insight into the plant response to dopamine, we treated 7-day old Arabidopsis seedlings with 0- or 50-mM dopamine for 2 and 8 hours and subjected the collected whole seedlings to RNA sequencing. Theanalysis of the resulting datasets is still ongoing and is expected to yield insight into the organismal response to dopamine and the downstream processes which ultimately lead to auxin hypersensitivity. Collectively, the data we have gathered to satisfy research objective 1 in thisfirst project period suggest that the repression ofdopamine-induced rosette size decreases by cytokinin is likely a downstream effect of dopamine's capacityto stronglyinduce the auxin signaling pathway and increase auxin sensitivity. Additionally, we find that plants respond to dopamine defensively, upregulating auxin-associated metabolitestypically associated with plant defense from pathogens and herbivory. Finally, our metabolomic analysis revealed that while it remains possible that dopamine has an influence on auxin catabolism, it currently seems unlikely that the inhibition of auxin oxidases is the only factor in dopamine-induced changes toplant physiology. Research objective 2,"Isolate and characterize dopamine-resistant Arabidopsis mutants".To fulfill objective 2, we isolated several hundred lines with apparent resistance todopamine treatment from aprimary EMS screen. 5 mutantswith phenotypes of particular interest (phenotypes include one line presenting a phenotype in which dopamine inducesrelief of severe baseline chlorosis, and the other 4 arehighly resistant todopamine) have been selected for further analysis. To date, we have generated segregating F2 populations of the high interest mutants,and in the coming weeks tissue samples from the wild-type and mutant populationswill be subjected to sequencing-by-mapping experiments designed to identify underlying casual mutations of the dopamine resistant phenotypes.
Publications
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