Consumption of food crops with heavy metal and metalloid content may be associated with harmful health risks. After exposure to heavy metals, plants induce significant gene expression changes, but the mechanisms underlying this transcriptional response are unknown. Forward genetic screens for mutants displaying altered heavy metal-induced gene expression were performed to address this issue. Subsequent mapping of the causal genes provides an approach to illuminate the underlying mechanisms. “Luciferase reporter lines” were created by merging the promoter of cadmium-induced SULTR1;2 gene to the bioluminescent firefly luciferase gene within Arabidopsis thaliana (Jobe et al., 2012). Mutant groups were created by subjecting “luciferase reporter lines” to ethyl methanesulfonate (Jobe et al., 2012). Forward genetic luciferase screens of mutagenized lines showed a phenotypic response when exposed to cadmium. Three mutant subgroups were defined as “constitutive response to cadmium (crc1)”, “super response to cadmium (src1)”, and “non-response & reduced response to cadmium (nrc1,2 were characterized)” (Jobe et al., 2012). Evaluation of bulk segregation analysis led to the rough mapping of the cadmium-induced luciferase luminescence mutants crc1 and src1. Genes in candidate regions were mapped, and their respective T-DNA insertion lines were ordered. ICP-OES studies displayed no significant distinction in cadmium accumulation between wild-type, crc1, and src1 mutants. Using RT-qPCR, src1 mutants and candidate T-DNA insertion lines displayed similar transcriptional gene expression responses to cadmium. These results may expand our knowledge of plant genes involved in the heavy metal response.

Toxic heavy metals such as arsenic (As) and cadmium (Cd) contaminate agricultural soils through natural and industrial causes which can lead to hyperaccumulation of toxic metalloids in crops grown on such soils. Phytochelatins, peptides produced by plants in response to heavy metal exposure, have been shown to detoxify toxic heavy metals and arsenic in planta by binding and forming complexes that are then sequestered into vacuoles. This thesis investigates whether Phytochelatin synthase 1 (PCS1) can be overexpressed under a root specific promoter to confine toxic metalloids to underground root systems and prevent hyperaccumulation in aerial and edible tissues of Ozyra Sativa (rice) plants. ICP-MS analysis was used to determine Cd and As content in transgenic and wildtype rice plants. The results showed that root specific overexpression of PCS-1 led to an over 3- fold decrease in shoot accumulation of Cd compared to wild type plants. Additionally, these transgenic plants displayed a root accumulation of Cd that was 6-fold greater per dry weight than wild type roots. Accumulation of As in roots and shoots of transgenic plants followed a similar trend to that of Cd, with roots storing significantly greater amounts of As while shoots stored dramatically less when compared to wild type plants. Altogether, the data suggests that root specific overexpression of PCS1 is effective in redistributing heavy metal accumulation to favor root storage. These findings warrant further research into root specific overexpression of heavy metal tolerance genes to reduce crop accumulation, and thus human consumption of toxic heavy metals.

As the climate changes, plants– which cannot relocate– must adapt genetically. We can exploit these adaptations in an effort to combat and adapt to global changes in climate. However, to do so, we must develop new methods of investigation to increase our overall understanding of plant systems. One such method has been to create inducible genetic systems. This allows for the study of genes that would have deleterious or lethal effects if studied through traditional methods, such as constitutively expressed trans-genes. Another method of experimentation has been to perform cell specific studies. This yields a range of approaches to investigate multicellular organisms. In this paper, the two methods will be combined to attempt creation of a cell specific inducible system in Arabidopsis thaliana, with a focus on the guard cells. Guard cells play a crucial role in gas exchange, transpiration, and reducing the risk of high temperature stress, making them an ideal candidate. Using the guard cell preferential promoter (pGC1) in Arabidopsis thaliana, one can conduct studies with guard cell preferential genetics. Genetic components from the ethanol utilization operon from Aspergillus nidulans were investigated in this project to attempt creation of and test feasibility of an inducible system. Due to unforeseen circumstances in the experimental design, it could not be concluded if a guard cell preferential inducible system could be successfully constructed. As such, this paper will discuss the results obtained, troubleshooting methods used, and recommendations for future attempts of developing this system.

Heavy metals and metalloids are a prominent threat to human health worldwide. Unfortunately, plants expose people to heavy metals and metalloids through diet. To develop plants capable of protecting people from heavy metal and metalloid exposure, the molecular systems of plants in their response to heavy metals and metalloids must be elucidated. In the first chapter of this study, a luciferase reporter construct containing a promotor region for the SULTR1;2 gene was inserted into Arabidopsis thaliana lines. These lines were then mutated with Ethane methosulfornate (EMS), and a forward genetic screen was conducted that searched for shifts in luciferase luminescence in response to cadmium. In these screens, 3 types of classes of shifts in luminescence were procured from the mutant lines, and were classified as having a constitutive (crc1), super (src1), or non-response (nrc1, 2) to cadmium. The nrc1, 2 mutants have since been characterized. Using bulk-segregation analysis, mutated genomic regions that separated with the src1 and crc1 luciferase phenotypes were determined. In addition, the crc1 mutants expressed a root-growth phenotype in sulfur- free media supplemented with cadmium and selenium. Src1 mutants showed a greater SULTR 1;2 expression in response to cadmium. T-DNA knockout lines for all candidate genes were ordered, genotyped, and propagated, and were used to determine potential causative genes for the src1 and crc1 phenotypes. In the second chapter, inductively-coupled electron optical electron spectroscopy was conducted to determine the heavy metal and arsenic concentrations of edible crop tissues grown in Campus Community Gardens around UCSD.

Drought is a major stress which reduces crop yields, and which will continue to be an increasing problem in the coming years as climate change and limited fresh water supplies lead to higher temperatures, desertification, and increased soil salinity. These environmental stresses can significantly impact both the seed yield and quality of crops. There are several strategies which plants utilize to mitigate the effects of water deficit, making the identification of specific traits which convey drought tolerance difficult. As drought tolerance is a complex trait, accurate phenotyping to select for resilient genotypes is needed to improve our understanding of plant drought responses.

In this study, stable carbon isotope screening (δ13C) of a diversity set of the crop plant Brassica napus grown in the field was used to identify accessions with traits linked with extremes in water use efficiency (WUE). We investigated physiological characteristics of the selected accessions to identify how these characteristics translate to differences in WUE. Using gas exchange techniques, we identified an interesting spring-type accession (G302, Mozart), which exhibited the highest WUE in the field, based on δ13C measurements. This line displayed high CO2 assimilation rates coupled with an increased electron transport capacity (Jmax) under lab conditions. We also analyzed stomatal conductance response to exogenous abscisic acid (ABA) in the selected accessions. While little variation was observed in the response rates of spring-type accessions, one semi-winter accession demonstrated a significantly more rapid response to exogenous ABA, which was in line with a higher WUE derived from δ13C measurements. This research supports the genetic data showing distinct genetic lineages for spring and semi-winter accessions. It also illustrates the importance of examining natural variation at a physiological level for understanding the underlying mechanisms of drought responses.

Abscisic acid (ABA) is an important phytohormone whose major functions include regulation of seed germination, stomatal movement, and other plant responses to biotic and abiotic stress. Recent research has revealed that ABA signaling exhibits cross-talk with effector triggered immunity, a pathway in pathogen defense signaling. However, the exact mechanisms of this crosstalk remain unknown. Previously, our lab discovered that a new small molecule DFPM ([5-(3,4-dichlorophenyl)furan-2-yl]- piperidine-1-ylmethanethione) induces ETI-linked signaling and inhibits ABA signaling, providing us with a method to screen for genes involved in the crosstalk between ETI signaling and ABA signaling. Using this screening method, the novel mutant rda3 (resistant to dfpm induced aba inhibition 3) was isolated here. rda3 exhibits resistance to DFPM induced ABA signaling inhibition in the leaves of Arabidopsis thaliana. Its genetic locus is yet to be determined. In addition, the lab previously identified another gene involved in DFPM/ABA crosstalk called VICTR, (VARATION IN COMPOUND TRIGGERED ROOT), which codes for a Nucleotide Binding – Leucine Rich Repeat (NB-LRR) protein involved in early ETI signaling. It has a highly homologous tandem gene VICTL1, and since NB-LRR genes are often functionally redundant, generating a victr victl1 double mutant will allow us to more accurately characterize both genes. The close proximity of VICTR and VICTL1 makes a higher order mutant by cross-pollination inefficient, so we used CRISPR/Cas9 to mutate VICTR in victl1 mutant plants. Although we isolated plants harboring victr-like phenotypes, eliminating the CRISPR/Cas9 construct from the mutant plants and creating stable mutations remain a challenge.

With atmospheric CO2 levels steadily increasing, it is important for humans to understand how plants utilize CO2 and release O2 in the air we breathe. Several CO2 signaling components have been characterized, but there are still many unknown components of the CO2 signaling pathway. This thesis characterizes candidate mutant plants from a comprehensive artificial microRNA (amiRNA) forward genetic screen designed to isolate putative mutants involved in CO2 signaling. Each mutant carried an amiRNA specifically designed to downregulate a few genes in Arabidopsis thaliana. 39 putative mutants were isolated and confirmed at the T3 generation. This project aims to elucidate the role of these genes in CO2-mediated stomatal responses and stomatal development. Gas exchange assays under defined changes in CO2 concentrations were performed to quantify the stomatal conductance and kinetic responses of the candidates. Stomatal density assays were performed to quantify the number of stomata of the candidates. The database ePlant and a Python script were utilized to determine potential interactions between the targeted amiRNA loci and known CO2-mediated stomatal signaling components. The results highlight 15 candidate mutants with unique responses to imposed shifts in CO2 concentration and varying stomatal densities compared to HsMYO (wild-type control) plants. 6 of the putative mutants show an inhibited response to defined changes in CO2 concentration or are affected in stomatal development and warrant further investigation. The 6 putative mutants have been retransformed in wild-type plants and will be examined to verify the robustness of the mutant phenotypes seen in this thesis.

Drought-induced crop reduction challenges food security among the most vulnerable human populations. One of the strategies to address this challenge is to develop drought-resistant crops. It requires a comprehensive understanding of the genes and molecular and cellular mechanisms activated by drought. Dehydrated plants produce the stress hormone abscisic acid (ABA). Accumulation of ABA in guard cells triggers stomatal closure that is mediated by the activation of sucrose non-fermenting 1-related kinases 2 (SnRK2s). Activated SnRK2-type kinases cause ABA-induced gene expression mediated by basic-domain leucine-zipper (bZIP)-type transcription factors. In this study, we applied TurboID to label and purify Arabidopsis thaliana guard cell nuclear proteomes, as well as nuclear proteins that interact specifically with this class of transcription factors. Here, we report that TurboID is indeed suitable for labeling and purifying guard cell nuclear proteomes, including protein-specific complex members. Another goal was to confirm the findings reported by a former member of our laboratory. We retransformed the Arabidopsis wildtype line with an artificial microRNA (amiRNA) to silence two basic helix-loop-helix (bHLH) transcription factors, candidates for regulating ABA responses in roots. Root growth assays demonstrated significantly reduced primary root growth in amiRNA mutants compared to Col-0, which is consistent with previous findings. Further investigations of which genes are regulated by these ABA-responsive transcription factors will be an important step toward a better understanding of the natural resistance mechanisms of plants in response to abiotic stressors and ultimately bring us closer to the development of drought-resistant crops.

The industrial and urban evolution of the modern world has contributed to the innovative developments and economic growth of many countries. This growth has also resulted in elevated levels of heavy metal pollutants in the biological systems of these nations, such as their cropland and bodies of water. This becomes a predicament, as some of these nations are leading producers of agricultural products worldwide. Since the exposure of heavy metal toxicants via ingestion of contaminated crops can cause many serious illnesses to humans, this dilemma has received global attention from health organizations and science communities. To contribute to these efforts, this thesis research project aims to evaluate a heavy metal phytoremediation technique in rice plants known as “phytostabilization”. Phytostabilization refers to the immobilization of heavy metal pollutants in the roots of plants to prevent their uptake into the shoots, leaves, and seeds. This was tested with transgenic rice lines with root-targeted overexpression of the following transgenes: Phytochelatin Synthase Gene from T. aestivum (TaPCS1) and Heavy Metals Associate3 gene from A. thaliana (AtHMA3). TaPCS1 overexpressing rice lines were exposed to different concentrations of both cadmium (CdCl2) and arsenite (As(III)), while AtHMA3 overexpressing lines were only exposed to different concentrations of cadmium (CdCl2). ICP-MS analysis was utilized to measure heavy metal contents in the root vs. shoot tissues of the aforementioned rice lines upon exposure to their respective heavy metal conditions. The preliminary results of this study suggested that rice lines with root-targeted overexpression of AtHMA3 were able to achieve lower Cd accumulation in the shoot tissues when compared to the wildtype controls. Furthermore, there was promising evidence that rice lines with root-targeted overexpression of TaPCS1 exhibited lower Cd and As(III) accumulation in the shoot tissues than the wildtype controls; however, further experimentation must be done on multiple independent transgenic lines and several exposure regimens.

In nature, plants are continuously subjected to various abiotic stresses, such as drought, cold or high salinity. In agriculture, one of the most destructive abiotic stresses is drought, which is becoming an increasingly important problem due to climate change and increasing water scarcity. In agriculture, drought stress has a heavy impact on crop yields and overall crop health, representing a problem with major economic ramifications. As such, development of novel methods for drought-tolerant crops becomes a foremost solution for farming in increasingly arid conditions.

In this study, we looked into knocking down known negative regulators of the drought signaling pathway to generate more drought tolerant plants for both Arabidopsis thaliana and Brassica napus. We generated constructs allowing stress inducible and constitutive knockdowns A.thaliana and stress inducible knockdowns of PP2Cs in B.napus. We found that for both stress inducible and constitutive A. thaliana knockdown lines allowed enhanced ABA responsiveness in germination. By developing a well-controlled low moisture assay, we were able to demonstrate thatstress inducible and ubiquitous knockdown lines in A.thaliana had increased drought tolerance. In B.napus stress inducible lines, we found some variability in the insertional strength, with only some lines showing enhanced ABA responsiveness, but enhanced drought responsiveness is still uncertain. This research demonstrates the use of knocking down specific negative regulators in the drought signaling pathway in order to generate more drought tolerant plants.