Browsing by Subject "Plant sciences"

The main objective of this dissertation research is to develop measurement and data-analysis tools for improving the quantitative accuracy of radiotracer studies of small plants, e.g., grasses in their early growth stages and tree seedlings. Improved accuracy is needed due to the thin nature of plant organs, e.g., leafs and stem. In addition, the methods developed in this thesis are applied to study the plant-environment interface of barley. Some of the approaches explored have potential to increase the statistical accuracy of counting data using PET imaging techniques. Improving the statistical precision of radionuclide tracking data will add to the analysis options. Another important goal is to measure the absolute photosynthetic rate. The standard approach in plant radiotracer experiments is to perform measurements of the relative distribution of radioactivity in various parts of the plant being studied. A limitation of this approach is that it does not take into account differences in the amount of radioisotope assimilated that are available for transport and allocation to the various sinks, that is, absolute CO2 uptake and photosynthetic rates are important factors in understanding the holistic physiological responses of plants to external conditions. For example, monitoring the movement of carbon-11 (11C) tagged carbohydrates in a plant requires an estimate of the average photosynthetic rate to determine the actual quantity of carbohydrates in each plant region (e.g. leaf, shoot, and root).

Radiotracing provides a method for real-time measurements of substance absorption, allocation and metabolic consumption and production in living organisms. Application of radioactive labelling in plants enables measurements associated with core physiological processes, e.g., photosynthesis, water uptake and nitrogen absorption and utilization. Plant uptake of radiotracers allows for tracking spatial and temporal distribution of substances, which enables studies of the plant-environment interface and the mechanisms involved in the allocation of resources (e.g., sugars, nutrients, and water). As such, these techniques are increasingly becoming an important tool for investigating the processes involved in the physiological responses of plants to changes in their local environmental conditions.

This dissertation has two major components: (1) development of experiment techniques for absolute photosynthetic rate measurements in plants using radio-isotope labeling, and (2) application of radioisotope tracing techniques to study the plant-environment interface in barley. The first component is covered in chapters one through three. The second component is presented in chapter four. An introduction into radio-tracing techniques is provided in chapter one. Chapter two describes radio-isotope production, radio-labelled compound preparation and delivery of labels to plant measurements. Chapter three outlines methods that can be employed to measure the absolute photosynthetic rate (µmol/m2/s) for a closed-loop system with [CO2] monitoring capabilities. Chapter four describes the background and results of our study on changing environmental conditions on a model system, barley seedlings. Chapter 5 will introduce the use of Monte-Carlo modeling for scaling the collected data to adjust the detected coincidence counts for losses due to positron escape from plant tissue. Chapter 6 describes the development of a novel imaging technique using direct positron detection that takes advantage of the high fraction of positrons escaping thin plant tissue.

In this dissertation, we have performed the most extensive measurements of carbohydrate allocation and translocation in a plant species using radio-isotope tracing techniques. A major practical limitation of studies based on radio-isotope labeling is the number of samples that can be measured in a single project. Our study on barley (Hordeum distichum) includes measurements on more than 30 plants. The short-lived radionuclide, 11C, was used to determine the real-time response to metabolite transport in barley. Sugars are photosynthesized and tagged with a positron-emitting radioisotope by flowing carbon dioxide (11CO2) tagged air over an active leaf. Data analysis of measurements taken in this dissertation indicates that the fraction of carbohydrates allocated to below ground sinks decreased, by 31% ± 9% in ambient [CO2] and by 37% ± 14% in elevated [CO2], when the nutrient conditions were rapidly changed from high to low nutrient.

Cells from all levels of life secrete vesicles, which are nanoscale proteoliposomes packaged with a variety of proteins, lipids, and small molecule cargo. Depending on their origin, these extracellular vesicles are termed exosomes, microvesicles, exomeres, and membrane vesicles, to list a few. Vesicles released from Gram-negative bacteria bud from the outer membrane and are, therefore, referred to as outer membrane vesicles (OMVs). In mammalian systems, OMVs facilitate bacterial survival by alleviating membrane stress, serving as a decoy for bacteriophage and antibiotics, and providing a fast membrane remodeling mechanism. OMVs also contribute to virulence by delivering toxins and other soluble and insoluble cargo to the host cell. The role OMVs play in plant systems remains unknown.

Previous studies revealed that plant pathogenic bacterial vesicles contain virulence factors, type III secretion system effectors, plant cell wall-degrading enzymes, and more, suggesting that vesicles may play similar roles to those from mammalian pathogens in host-pathogen interactions. Further, OMVs elicit several markers for pathogen-associated molecular pattern triggered immunity in plants. These responses include increased transcription of defense markers such as FRK1 and production of reactive oxygen species. Building on these findings, here we show that OMVs from the plant pathogen Pseudomonas syringae and the plant beneficial Pseudomonas fluorescens elicit plant immune responses in Arabidopsis thaliana that protect against future pathogen challenge. Intriguingly, protection is independent of salicylic acid plant defense pathways and bacterial type III secretion. OMVs also inhibit seedling growth, another indication of plant immune activation.

Our initial biochemical studies suggested that the immunogenic OMV cargo was larger than 10 kDa and differed between the pathogen and beneficial species despite similar plant immunity outcomes. Interestingly, protective OMV-mediated responses were protein-independent, while the seedling growth inhibition phenotype was entirely protein dependent. Proteomics analysis confirmed that OMV protein cargo differed between P. syringae and P. fluorescens. While media culture conditions did not dramatically impact the immunogenic activity of isolated OMVs from either species, proteomics analysis revealed a significant shift in P. syringae OMV cargo between complete and minimal media conditions. P. fluorescens OMV cargo was largely the same in the two media conditions, with no significantly enriched proteins in minimal or complete media. Further analysis of the proteins enriched in the P. syringae minimal OMV condition identified one set of proteins with the same baseline abundance in P. syringae and P. fluorescens complete OMVs and another set with a lower baseline abundance compared to P. fluorescens OMVs. These two subsets could contribute to virulence and stress tolerance, respectively. Enrichment analysis uncovered particularly interesting protein categories in the subset with the same baseline abundance. Of interest, several lipoprotein and lipid binding categories were enriched, and proteins involved in synthesis of the phytotoxin coronatine were also enriched in this same-baseline subset. These results support our hypothesis that proteins enriched in P. syringae minimal OMVs with the same baseline abundance in P. fluorescens complete OMVs may contribute to OMV-mediated bacterial virulence in plants. Our findings also suggest that our forthcoming OMV metabolomic analyses may reveal non-proteinaceous cargo that is critical for OMV-mediated plant immune activation.

The work presented here lays the groundwork for future exploration of OMV-plant interactions and adds a new layer of complexity to plant-bacteria interactions. Further, these results reveal that OMVs elicit complex plant immune responses that would be difficult for pathogens to adapt to and overcome, supporting a role for bacterial OMVs in agricultural applications to promote durable resistance and revealing a new potential avenue for disease prevention and management.

As habitat fragmentation and changing climatic conditions continues to pose threats to species persistence, it is important to study the traits that may ensure survival of individual plants and the population-level effects of those traits. Previous studies have highlighted the importance of one such trait, dispersal, for population persistence under changing conditions. Dispersal allows plants to track spatial and seasonal changes in the environment. Plants can disperse through space, i.e., pollen and seed dispersal, and through time, i.e., delaying germination within seasons (seed dormancy) or between years (seed banking). Spatial sorting of good dispersers has been highlighted as a mechanism to facilitate spatial habitat tracking, but few studies have evaluated how interactions between spatial sorting and plasticity of dispersal-related traits may interact to impact dispersal dynamics. In addition, theory predicts that both spatial dispersal as a form of bet-hedging, and seasonal seed dormancy as a form of habitat selection, may stabilize population demography across years, thereby reducing population extinction risk. Few studies have experimentally manipulated spatial and temporal dispersal in the field to test these theoretical predictions. My dissertation quantifies spatial dispersal at a small scale, tests for the genetic basis and plasticity of dispersal-related traits, tests the metapopulation consequences of local spatial dispersal versus spatial isolation, and quantifies the population-level consequences of seasonal seed dormancy. I combined field, greenhouse, and quantitative genetic approaches to assess the demographic effects of spatial and temporal dispersal in the model plant species Arabidopsis thaliana. In my first chapter I show that limited dispersal results in predictable variation in post-dispersal density across space. Further, I show that the traits that enhance dispersal ability in the field have a genetic basis, exhibit plasticity to density and season length, and genetic variation in that plasticity. Plasticity and genotypic differences in plasticity can alter the effects of spatial sorting on dispersal ability across a species range. Specifically, plasticity can augment the effects of spatial sorting of genotypes, by enhancing dispersal at low-post-dispersal density. Genetic differences in plasticity of good versus poor dispersers can mask genetic differences in dispersal ability and thereby slow spatial sorting of genotypes at high density but augment genetic differences and spatial sorting at low density. In my second chapter I show that, compared to isolated populations, populations open to dispersal had smaller between-year population size fluctuations, increased survival of individuals within years, and less between-population differentiation in morphological traits. Those demographic effects of dispersal may have increased the effective population size of populations open to dispersal, facilitating a recovery from the effects of harsh environmental conditions. Finally, my third chapter demonstrates that seasonal seed dormancy may allow populations to better take advantage of favorable conditions by increasing population size and stabilizing population demography over time in more permissive environments. However, contrary to expectations, dormancy did not reduce the effects of environmental variation due to its inability to counter the effects of population bottlenecks induced by harsh environmental conditions. The findings of my dissertation highlight the importance of considering the interaction between spatial sorting, phenotypic plasticity, and genetic variation in plasticity when projecting range expansion dynamics. My dissertation also provides some of the first experimental validation of the theory that predicts that spatial and temporal dispersal can stabilize population demography and facilitate population persistence. Therefore, when predicting how species may respond to anthropogenic changes it is important to not only consider the effects of dispersal on environmental tracking, but also the effects of dispersal on population demography. Finally, given the limited dispersal of Arabidopsis thaliana and many annual species like it, it is important to measure population dynamics at a micro-scale, otherwise researchers risk underestimating regional-level extinction risk.

This research spans multiple scales—from the entire organism, down to the genes that created it.The first project, “Gene Dynamics in Tissue Development”, explores how stem cell differentiation depends on the dynamics of gene networks. In the Arabidopsis thaliana root, the SCARECROW (SCR) transcription factor is required for an asymmetric cell division of a stem cell, resulting in two daughter cells that acquire different fates and tissue identities. Although much research has developed the network topology for this division, the dynamics of this process remain unknown. A core feature of the GRN controlling this stem cell asymmetric division is the SCR positive feedback loop. This research develops a synthetic biology approach to systematically and precisely tune various dynamics of SCR protein accumulation. Thus, one can explore the role and function of this positive feedback loop in the developmental process of asymmetric division in the Arabidopsis root. The following project, “Organ Form for Function” details how organ function depends on cellular form and hormonal signals. As sessile organisms, plants must establish a firm foundation into the terrain wherever the seed lands. Roots, especially the primary root (a seed’s first root), are the only anchor into the terrain. With a multiscale investigation, we identified a molecular pathway required for circumnutation, the circular growth of the root tip. We found the cellular physiology and key hormonal cell signaling events driving this behavior.

This dissertation examined the response to termination of CO2 enrichment of a forest ecosystem exposed to long-term elevated atmospheric CO2 condition, and aimed at investigating responses and their underlying mechanisms of two important factors of carbon cycle in the ecosystem, stomatal conductance and soil respiration. Because the contribution of understory vegetation to the entire ecosystem grew with time, we first investigated the effect of elevated CO2 on understory vegetation. Potential growth enhancing effect of elevated CO2 were not observed, and light seemed to be a limiting factor. Secondly, we examined the importance of aerodynamic conductance to determine canopy conductance, and found that its effect can be negligible. Responses of stomatal conductance and soil respiration were assessed using Bayesian state space model. In two years after the termination of CO2 enrichment, stomatal conductance in formerly elevated CO2 returned to ambient level, while soil respiration became smaller than ambient level and did not recovered to ambient in two years.

Nectar production is one of several traits that are reduced in flowering plants that display the selfing syndrome, a suite of trait reductions often associated with the transition from outcross-fertilization to self-fertilization. However, the evolutionary mechanisms that contribute to reduced nectar has not been explored. In this dissertation, I use a pair of sister morning glories, Ipomoea lacunosa, a highly selfing species that displays the selfing syndrome, and I. cordatotriloba, a mixed mating species, to address the question: how did reduced nectar evolve in the selfing syndrome? Through a combination of approaches from quantitative genetics, population genomics, and transcriptomics, I describe the genetic architecture of nectar production and attempt to identify candidate genes that could lead to reduced nectar production – nectar volume and nectar sugar concentration – in I. lacunosa. QTL analyses indicate that nectar traits are polygenic and evolved independently from floral size traits, likely due to direct selection. Transcriptomic analyses reveal several sets of genes that are unique to each nectar trait, but both nectar volume and nectar sugar concentration also share some genes in common. Finally, through incorporating population genomic analyses, a short list of candidate genes was obtained that may explain how reduced nectar evolved in I. lacunosa and how nectar traits remain diverged between the two morning glory species even in regions of sympatry.

Accurate species descriptions are critical for understanding evolutionary relationships. Ipomoea “austinii” was proposed to be a new species found in the US, but conflicting evidence suggested that it was instead I. grandifolia. By examining cross-compatibility between these individuals and through genetic analyses, I find no cross-incompatibility and no genetic differentiation between I. “austinii” and I. grandifolia, thus suggesting that I. “austinii” should be reclassified as I. grandifolia.

Plants commonly produce multiple, seemingly redundant defenses, but the reasons for this are poorly understood. The specificity of defenses to particular herbivores could drive investment in multiple defenses. Alternatively, genetic correlations between defenses could lead to their joint expression, even if possessing both defenses is non-adaptive. Plants may produce multiple defenses if putative resistance traits do not reduce damage, forcing plants to rely on tolerance of damage instead. Furthermore, resource shortages caused by herbivore damage could lead to compensatory changes in expression and selection on non-defense traits, such as floral traits. Natural selection could favor producing multiple defenses if synergism between defenses increases the benefits or decrease the costs of producing multiple defenses. Non-linear relationships between the costs and benefits of defense trait investment could also favor multiple defenses.

Passiflora incarnata (`maypop') is a perennial vine native to the southeast United States that produces both direct, physical traits (leaf toughness and trichomes) and rewards thought to function in indirect defense (extrafloral nectar in a defense mutualism with ants), along with tolerance of herbivore damage. I performed two year-long common garden experiments with clonal replicates of plants originating from two populations. I measured plant fitness, herbivore damage, and defense traits. I ran a genotypic selection analysis to determine if manipulating herbivore damage through a pesticide exclusion treatment presence mediated selection on floral traits, and if herbivore damage led to plastic changes in floral trait expression. To evaluate the role of selection in maintaining multiple defenses, I estimated fitness surfaces for pairwise combinations of defense traits and evaluated where the fitness optima were on each surface.

I found that resistance traits did not reduce herbivore damage, but plants demonstrated specific tolerance to different classes of herbivore damage. Tolerance was negatively correlated with resistance, raising the possibility that tolerance of herbivore damage instead of resistance may be the key defense in this plant, and that production of the two type of defense is constrained by underlying genetic architecture. Plants with higher levels of generalist beetle damage flowered earlier and produced proportionally more male flowers. I found linear selection for both earlier flowering and a lower proportion of male flowers in the herbivore exclusion treatment. I found that selection favored investment in multiple resistance traits. However, for two tolerance traits or one resistance and one tolerance trait, investment in only one trait was favored.

These results highlight the possibility of several mechanisms selecting for the expression of multiple traits, including non-defense traits. Resistance traits may have a non-defensive primary function in this plant, and tolerance may instead be a key defense strategy. These results also emphasize the need to consider the type of trait--resistance or tolerance--when making broad predictions about their joint expression.

Phytochromes are red and far-red light receptors that initiate photomorphogenesis by reprogramming both nuclear and plastidial genomes. Early light signaling events include translocation of photoactivated phytochromes from the cytoplasm to subnuclear photobodies and phytochrome-mediated degradation of a group of transcription factors, PHYTOCHROME INTERACTING FACTORs (PIFs). The degradation of PIFs not only inhibits the elongation of hypocotyl but also promotes chloroplast development by activating photosynthetic genes. However, the mechanisms by which phytochrome signaling initiates chloroplast development remain elusive. The main challenge in determining these mechanisms has been that previous genetic screens have been unable to distinguish mutants involved in plastidial gene regulation from mutants of essential components of chloroplast functionality (Chen et al., 2010; Chen and Chory, 2011). We have previously reported a new phytochrome signaling component, HEMERA (HMR), which is a transcriptional coactivator required for both phytochrome signaling and chloroplast development. The hmr mutant has a combination of long-hypocotyl and albino phenotypes, representing the founding member of a new class of photomorphogenetic mutants that has been overlooked by previous genetic screens (Chen et al., 2010; Chen and Chory, 2011). We hypothesized that these tall-and-albino mutants define uncharacterized components of phytochrome signaling required for chloroplast development. To investigate this hypothesis, we conducted a forward genetic screen for tall-and-albino mutants, which identified a novel phytochrome signaling component named Regulator-for-Chloroplast-Biogenesis-by-Light (RCBL). To determine the evolutionary history of RCBL, I acquired the homologous sequences of RCBL and its paralog, REGULATOR-FOR-CHLOROPLAST-BIOGENESIS (RCB), from the available genomes and transcriptomes of a wide range of land plants. Phylogenetic analyses of these sequences demonstrate that RCBL and RCB diverged after the emergence of seed plants, but their mutant phenotypes show they are not functionally redundant. Characterization of rcbl mutants show that RCBL is required for both red and far-red light signaling, and it acts genetically downstream of phytochrome A and B. Similar to HMR, RCBL is also essential for photobody assembly, PIF1 and PIF3 degradation, and the expression of PIF-dependent light-responsive genes. Knocking out four PIFs (pifq, pif1/pif3/pif4/pif5) in the rcbl mutant background largely rescued the elongated hypocotyl phenotype of rcbl, indicating that the phytochrome-mediated phenotype of rcbl is dependent on PIFs. In chloroplasts, RCBL is required for transcription of plastid-encoded photosynthesis genes. However, this defective chloroplast phenotype of rcbl cannot be rescued by knocking out PIFs, suggesting RCBL plays a PIF-independent role in chloroplast development.

Since RCBL is involved in both phy signal transduction and chloroplast biogenesis, I examined whether RCBL is dual-localized to the nucleus and chloroplasts. Fluorescently-tagged RCBL shows dual-localization to chloroplasts and nuclei in both tobacco and Arabidopsis. Additionally, RCBL protein can be detected in protein fractions isolated from nuclei and plastids. I therefore conclude that RCBL is a dual-localized protein, which suggests that RCBL might be directly involved in both nuclear and plastidial events of photomorphogenesis.

Further investigation of the structure of RCBL revealed that the C-terminus of RCBL contains a domain similar to E. coli thioredoxin but without the canonical catalytic CxxC motif. Biochemical analyses confirmed that RCBL lacks thioredoxin reductase activity. Instead, in vitro experiments suggest that RCBL directly interacts with RCB through its C-terminal thioredoxin-like domain.

Taken together, this study revealed a previously uncharacterized early phytochrome signaling component which plays a critical role in chloroplast development, and demonstrated a mechanistic link between the nucleus and plastids during the initiation of photomorphogenesis.

When a plant emerges from the soil, it faces a critical developmental transition from utilizing stored energy to grow rapidly toward the light, to developing chloroplasts and beginning photosynthesis. While it is known that this process involves massive transcriptional reprogramming of the nuclear and plastidial genomes, the connections between chloroplast development and nuclear light signaling events are not well understood. One very promising target for investigating these connections is HEMERA (HMR), a dual-localized regulatory protein that is found in both nuclei and chloroplasts. HMR was previously identified as pTAC12, an essential component of the plastid-encoded RNA polymerase complex responsible for transcription of chloroplast photosynthetic genes. In the nucleus, HMR acts within the phytochrome signaling pathway as a transcriptional co-activator of a subset of growth-relevant genes in response to light, to regulate the elongation of the embryonic stem, or hypocotyl. HMR’s combination of roles in the nucleus and chloroplasts are dramatically demonstrated by the phenotypes of the hmr mutant, with a long hypocotyl and albino leaves when grown in the light.

While the functions of HMR in each compartment have been studied separately, the mechanisms by which the HMR protein is targeted to each compartment have not yet been determined. To address this, I characterized the localization signals of HMR with a combination of in vitro approaches and characterization of transgenic Arabidopsis lines. These experiments revealed that HMR has a cleavable N-terminal chloroplast transit peptide within its first 50 amino acids, while two predicted nuclear localization signals proved not to be highly functional. Surprisingly, HMR in the chloroplasts and nucleus appeared to both be the same cleaved form of the protein. We thus identified the mature form of HMR by mass spectrometry, finding that it begins from lysine as the result of transit peptide cleavage and possibly additional N-terminal processing. Through GST pull-down assays, we determined that this mature form of HMR was fully capable of interacting light signaling components. However, analysis of transgenic lines showed that expression of mature HMR alone could not complement the long-hypocotyl phenotype of the hmr mutant. Analysis of the transcription of HMR nuclear target genes confirmed that mature HMR lacked nuclear functionality.

Further investigation revealed that mature HMR does not accumulate within the nucleus, most likely as a result of its nonfunctional nuclear localization signals. However, addition of the transit peptide from the small subunit of Rubisco fully restored nuclear accumulation and function of mature HMR in Arabidopsis. Additional experiments testing the localization of a simple model of dual-targeted proteins with two types of localization signal showed that transit peptides might take priority over nuclear localization signals. These results together suggest an unexpected model of localization where HMR is first targeted to the chloroplasts, and then it is subsequently re-localized to the nucleus, thus connecting its nuclear and plastidial functions. Further investigation of this proposed retrograde plastid-to-nucleus translocation pathway promises to shed additional light on the link between nuclear light signaling events and chloroplast development.

A cell’s trajectory from stem cell to differentiation, while often portrayed as a linear progression, is best described as a network that produces a mature state through several pathways acting together. There are few examples that describe gene regulatory network changes during the entire trajectory of cell differentiation. The goal of my project was to define the gene regulatory network required for a stem cell to become a differentiated cell in the Arabidopsis thaliana root. The root is a powerful model for identifying basic principles of differentiation. Plant cells do not migrate therefore entire lineages from stem cell to mature progeny are spatially confined. Furthermore, the root displays indeterminate growth, facilitating the study of many different developmental stages at a single time. One cell type of the root, the endodermis, is particularly suitable for study because the molecular components required for its formation and terminal differentiation are established. In order to understand the path from stem cell to differentiated cell in the endodermis, we asked what transcription factors are sufficient to program a non-native cell-type into endodermis. Our results show the transcription factors SHORTROOT and MYB36 have limited ability to reprogram a non-native cell-type (the epidermis) and that this reprogramming is reversible in the absence of additional cues. The stele-derived signaling peptide CIF2 stabilizes SHORTROOT-induced reprogramming. The outcome is a partially impermeable barrier deposited in the sub-epidermal cell layer that has a transcriptional signature similar to endodermis. The induction mechanism depends on MYB36 and CIF2’s receptor, but may be independent of the transcription factor SCARECROW. These results highlight a non cell-autonomous induction mechanism for endodermis that resembles differentiation in many animal systems.

Nitrogen is an essential element for all organisms as a crucial component of nucleic and amino acids. The NIN-LIKE PROTEINS (NLPs) are the central transcription factors (TFs) regulating the primary nitrate response (PNR) in plants. In Arabidopsis thaliana, NLP7 is the master regulator of the PNR, regulating the expression of thousands of nitrate responsive genes. The mechanisms governing Here I investigate the role of PB1 domain interactions in the biological function of NLP7 and how nucleocytoplasmic partitioning of NLP7 contributes to the plant nitrate response. Additionally, I compare the structure and mechanisms regulating NLP7 to the AUXIN RESPONSE FACTORS (ARFs) which are also DNA-binding transcription factors with PB1 domains and large IDRs, that undergo nucleocytoplasmic partitioning.

The evolutionary transition from outcrossing to self-pollination occurs frequently in flowering plants and has direct and indirect effects on genomics, life history and floral morphology. The life history and floral traits common to selfing plants are collectively called the "selfing syndrome." This dissertation uses the highly selfing morning glory Ipomoea lacunosa to address three major questions in the evolution of highly selfing plants: what are the genomic consequences of selfing, do the morphological changes associated with the transition to self-pollination result from natural selection or genetic drift, and how does the genetic architecture of those morphological traits affect their evolution? In the first chapter, we analyze genetic data from I. lacunosa and its outcrossing sister species I. cordatotriloba to compare the genomic consequences of selfing in I. lacunosa to theoretical predictions. We find that the reduction in genetic diversity is greater than that predicted by theory, suggesting a population bottleneck in I. lacunosa's history. There is also evidence for the relaxation of natural selection. The second chapter combines these genetic data with phenotypic measurements in a Qst-Fst comparison to determine whether natural selection is responsible for life history and floral morphology differences between I. lacunosa and its outcrossing relative I. cordatotriloba. Our analyses reveal that several component traits in the selfing syndrome diverged in response to natural selection. Chapter Three uses a quantitative trait locus (QTL) mapping approach to characterize the genetic architecture of the selfing syndrome and investigate how genetic correlations between traits affected its evolution. We find generally lower levels of genetic correlation between selfing syndrome traits than previous QTL studies of the selfing syndrome. The low level of genetic correlation indicates that independent selection on selfing syndrome traits is responsible for the evolution of the syndrome as a whole.

In the plant innate immune system, diverse signals from a wide range of pathogens converge on the same output, effector triggered immunity (ETI) and the associated programmed cell death (PCD). Past genetic studies have succeeded in uncovering the role of R-genes in recognizing the presence of pathogen effectors, and in identifying a number of downstream executors of the immune response. However, the gap between effector recognition and phenotype regulation remains poorly understood, with each signaling component only contributing a minor quantitative effect to the phenotype of ETI-PCD. In this dissertation, my goal is to fill in a portion of that gap.

I demonstrate that there is a prolonged nuclear increase of calcium ions during ETI, and that that nuclear calcium signal is essential for PCD. I also utilize cpr5, a point mutant identified for its constitutive defense response and programmed cell death lesions, to identify a new role for cell cycle regulators in regulating ETI-PCD. I show that phosphorylation of the cell cycle regulator Retinoblastoma-Related 1 (RBR1) is responsive to ETI. The RBR1 target transcription factors E2Fa, E2Fb, and E2Fc have an additive role regulating ETI, and a triple e2fabc mutant is susceptible to pathogens. Using a reverse genetics approach in e2fabc, I identify repression of nonphotochemical quenching in the chloroplasts as a key step in ETI-PCD regulation.

Together, these studies emphasize the role of organelles in PCD regulation, with the nucleus serving as a hub of second messenger signaling and transcription and the chloroplasts responding to ETI by remodeling to serve a new role as a platform for ROS production. In addition, they define a new pathway of ETI regulation that contributes quantitatively to ETI-PCD.

Theoretical and empirical studies have consistently shown that the optimal timing of seed germination reduces exposure to physical stress and minimizes competitive interactions with neighbors. However, this research has not accounted for facilitative (positive) interactions among plants, which become more pronounced as environmental stress increases. Facilitation is more likely to occur early in a plant's life when it is more susceptible to stress. In seasonal environments, the stress a given individual experiences can change throughout the year, and some years are more stressful than others. These sources of temporal variation in stress will dictate the facilitation-competition balance that individuals experience. However, it remains unclear how this balance affects the optimal timing of germination. My dissertation research asks how the timing of germination responds to neighbors, how those responses affect the facilitation-competition balance individuals experience, and how that balance in turn affects fitness and demography. More generally, it asks how the timing of germination and other types of emergence affect the facilitation and competition that individuals experience throughout their lives.

I used laboratory, greenhouse, and field experiments to examine how the timing of germination in the winter annual Arabidopsis thaliana (Brassicaceae) responds to cues of neighbors and how those responses affect interactions with neighbors. I then developed a mathematical model of population growth in an annual plant to examine how intraspecific facilitation and competition over ontogeny affect the optimal degree of investment in dormancy (i.e., delayed germination) in variable environments.

My experiments revealed that seeds of A. thaliana typically delay germination in response to neighbors and that these responses can promote facilitative interactions and reduce competitive ones with neighbors. Selection against delayed germination, which occurs because of stress later in the season, can be mitigated by facilitation. Further, delaying germination can be beneficial by increasing the difference in sizes between seedlings and their neighbors, which may promote resource partitioning. In the theoretical study, I found that increasing the degree of investment in the fraction of dormant seeds (i.e., delaying germination) can promote the persistence of populations that experience both facilitation and competition in variable environments. This occurs because increased dormancy prevents high juvenile densities that promote facilitation and consequently limit reproduction in large populations. The findings of this research indicate that plant-plant interactions depend strongly on temporal context, and they reveal that the facilitation-competition balance determined by temporal variation in stress plays a key role in how germination and dormancy traits will evolve in variable environments.

Angiosperms have evolved multiple mating systems that allow reproductive success under varied conditions. Striking among these are cleistogamous mating systems, where individuals can produce alternative flower types specialized for distinct mating strategies. Cleistogamy is thought to be environmentally-dependent, but little is known about environmental triggers that induce cleistogamous flower or the gene regulatory networks that determine the final floral phenotypes. If production of alternate flowers is environmentally induced, populations may evolve locally adapted responses. Mimulus douglasii, exhibits a cleistogamous mating system, and ranges across temperature and day length gradients, providing an ideal system to investigate environmental parameters that control cleistogamy and the gene regulatory networks responsible for the different floral forms. In these studies, we compared flowering responses across M. douglasii population accessions that produce phenotypically distinct outcrossing, and self-pollinating flower morphs. Under controlled conditions, we determined time to flower, and number and type of flowers produced under different temperatures and day lengths. We also compared gene expression profiles between chasmogamous and cleistogamous flowers using RNA-seq. We find that temperature and day length both effect onset of flowering. Long days shift flower type from predominantly chasmogamous to cleistogamous. The strength of the response to day length varies across accessions whether temperature varies or is held constant. We also find that gene expression patterns differ between the early development chasmogamous and cleistogamous flower buds. Cleistogamy is an environmentally sensitive polyphenism in Mimulus douglasii, allowing transition from one mating strategy to another. Longer days induce flowering and production of cleistogamous flowers. Shorter days induce chasmogamous flowers. Population origin has a small effect on response to environmental cues. Subtle shifts in the expression of cell division, cell expansion, and metabolic process related transcripts lead to the massive size difference observed between chasmogamous and cleistogamous flowers.

Plants have evolved the circadian clock to anticipate environmental changes and coordinate internal biological processes. Recent studies unveiled the circadian regulation on plant immune responses as well as a reciprocal effect of immune activation on the clock activity. However, it is still largely unknown how the circadian clock interacts with specific immune signals. Plant hormone salicylic acid (SA) is a key immune signal. Its accumulation is sufficient to trigger immune responses and establish broad-spectrum resistance, known as systemic acquired resistance (SAR). My dissertation work studied whether SA could interact with the circadian clock and what potential mechanisms and the biological significance are.

I first found that SA could reinforce the circadian clock through the modulation of redox state in an NONEXPRESSER OF PR 1 (NPR1)-dependent manner. The basal redox state manifested by the NADPH abundance is shown to display a circadian rhythm. Perturbation in this cellular redox rhythm caused by the immune signal SA is sensed by the master immune regulator NPR1. NPR1 then triggers defense genes expression to generate SAR as well as transcriptionally activates several clock genes to reinforce the circadian clock. Since the basal redox state, which reflects the cellular metabolic activities, is under the circadian control, the reinforced circadian clock may negate the SA-triggered redox perturbation to restore the normal redox rhythm. One of NPR1-regulated clock components is TIMMING OF CAB2 EXPRESSION 1 (TOC1). SA/NPR1-mediated increase in TOC1 expression alone could lead to dampening of SAR through direct transcriptional repression on defense genes. Since maintenance of the immune responses is an energy-costly process, the strength and duration of SAR, a preventative defense strategy, need to be fine-tuned to reduce unnecessary energy expenditure. Therefore, both SA-dependent circadian clock reinforcement and the specific clock component TOC1 induction help to ensure a proper immune induction and a balanced energy allocation between defense and normal metabolic activities.

Besides the SA effects on the circadian clock, the circadian clock is found to reciprocally regulate SA biosynthesis. The clock gene, CCA1 HIKING EXPEDITION (CHE), and the major SA synthesis gene, ISOCHORISMATE SYNTHASE 1 (ICS1), show in-phase oscillatory rhythms, indicating that CHE may contribute to generation of the circadian rhythm of the basal SA level. I found that CHE, as a transcription factor, directly binds to the promoter of ICS1 to positively regulate its expression. After pathogen infection, CHE promotes endogenous SA biosynthesis and acts as a positive regulator of SAR. The function of the clock component CHE in activating ICS1 not only reveals a novel transcriptional regulatory mechanism of SA accumulation but also provides a new molecular link between the circadian clock and plant immunity.

In summary, my dissertation studies identified previously unknown molecular mechanisms of how the circadian clock mediates SA biosynthesis and SA-triggered immune responses. The interplay between the circadian clock and SA achieves a balance between activation of immune responses and maintenance of normal metabolic activities. Further studies may explore how other plant immune signals affect the circadian clock as well as how different clock components coordinately regulate the plant immunity. These future directions will broaden our understanding about the clock-immunity crosstalk.

Salicylic acid (SA) is a central plant defense signal. It is not only required for closing the stomata upon infection to prevent pathogens from entering into the plant apoplast, but also mediates defense responses activated by pathogen-originated microbe-associated molecular patterns (MAMPs) and effectors in the infected tissues. In addition, SA is a necessary and sufficient signal for systemic acquired resistance (SAR). In Arabidopsis thaliana, SA level increases in response to pathogen attack, which is essential for activating defense responses. This SA accumulation involves transcriptional activation of several genes including ICS1 (ISOCHORISMATE SYNTHASE 1), EDS5 (ENHANCED DISEASE SUSCEPTIBILITY 5), EDS1 (ENHANCED DISEASE SUSCEPTIBILITY 1), PAD4 (PHYTOALEXIN-DEFICIENT 4) and PBS3 (avrPphB SUSCEPTIBLE 3). However, it is not well understood how pathogenic signals induce these SA accumulation genes. Interestingly, our time-course transcriptome analysis showed that these five genes share a similar pathogen-induced expression pattern, suggesting the existence of common transcription factors (TFs). Through yeast-one-hybrid screening, a TF NTL9 was identified for its interactions with the promoters of the SA accumulation genes. Preferentially expressed in guard cells, NTL9 activates the expression of SA accumulation genes in guard cells. The ntl9 mutant is defective in pathogen-induced stomatal closure mediated by a well-characterized MAMP, flg22. Consistent with the stomatal closure defect, the ntl9 mutant exhibits elevated susceptibility to surface-inoculated pathogens. The stomatal closure defect of the ntl9 mutant can be rescued by exogenous application of SA, demonstrating that NTL9 acts upstream of SA in stomatal closure response. These results suggest that NTL9-mediated activation of SA accumulation genes is essential for MAMP-triggered stomatal closure.

While plants induce SA to activate defense responses, pathogens can also produce virulence factors to counteract the effects of SA. Coronatine is one such virulence factor produced by Pseudomonas syringae. Coronatine is known to promote opening of stomata for bacterial entry, bacterial growth in the apoplast, systemic susceptibility and development of disease symptoms such as chlorosis. In the process of examining the mechanisms underlying coronatine-mediated virulence, three homologous TFs, ANAC019, ANAC055 and ANAC072, were found to be activated by coronatine directly through the TF, MYC2. Genetic characterization of these three TF mutants revealed that these TFs mediate multiple virulence effects of coronatine by inhibiting SA accumulation. To exert this inhibitory effect, these TFs repress ICS1 and activate BSMT1, genes involved in SA biosynthesis and inactivation modification, respectively. Thus, a signaling cascade downstream of coronatine was illustrated to dampen SA-mediated defense responses through differential transcriptional regulation of genes related to SA level.

Taken together, my dissertation studies revealed novel transcriptional regulation of SA production and demonstrated that this transcriptional regulation is a vital point not only for plant defense activation but also for pathogen manipulation to counteract defense responses. Further studies on the interplay of this transcriptional regulation by different TFs would broaden our understanding about the dynamics of plant-pathogen interaction.

Translational reprogramming allows organisms to adapt to changing conditions. In regulating translation, upstream start codons (uAUGs), prevalently present in mRNAs, play crucial roles by providing alternative translation start sites. However, what determines this selective translation initiation between conditions remains a fundamental question. By integrating transcriptome-wide translational and structural analyses during Arabidopsis pattern-triggered immunity, I found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Under normal conditions, these uORFs are selectively translated due to highly structured regions immediately downstream of uAUGs by slowing and engaging the scanning preinitiation complex. Deep learning modelling provides unbiased support for recognizable double-stranded RNA structures downstream of uAUGs (“uAUG-ds”) being responsible for the selective translation of uAUGs. I showed that uAUG-ds regulation is generalizable in human cells. Moreover, I found that uAUG-ds-mediated start codon selection is dynamically regulated. Upon immune challenge in plants, induced Ded1p/DDX3X-homologous RNA helicases resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defensce proteins. This study demonstrates that mRNA structures, rather than primary sequences, dynamically regulate start codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest the generality of mRNA structural remodelling in mediating translational reprogramming.