Transcription factors are a vital class of genes in eukaryotes that regulate almost every aspect of organism development, function, and physiology. Characterizing the function of transcription factors in plants, and the regulatory networks that they make up, becomes crucial as we search for ways to adapt our food sources to the rapidly changing climate. In this thesis, we start by creating a resource of transgenic lines to aid in the identification and characterization of Arabidopsis thaliana transcription factors. We then develop several methods of testing stress-response phenotype in A. thaliana, and use the initial state of this resource to identify transcription factors that are likely to regulate stress response. The resource and methods outlined in this thesis can be developed over time to become a useful pipeline for the identification of transcription factors involved in the regulation of any plant function of interest.

The circadian clock in plants coordinates internal biological processes to exhibit daily oscillations that are in synchrony with environmental rhythms. The roles of core clock genes CCA1, LHY, and TOC1 that regulate the molecular mechanism have already been well established in Arabidopsis thaliana and the mechanism by which TOC1 regulates CCA1 and LHY appears to be mediated by transcription factors of the TCP family. However, the role of cis-elements in the promoters of these clock genes has not been fully addressed. In this thesis, we utilized CRISPR/Cas9 technology to create mutations within the CCA1, LHY and TOC1 promoter regions. Through this, we hope to better understand the role of cis-elements in the promoters of core clock genes and how they influence the clock feedback regulatory mechanism. We propose a procedure to efficiently identify genome editing when Cas9 is expressed in early gametogenesis stages. Using this procedure, we generated indel mutations in the CCA1 and LHY promoters and found that one of them resulted in a short hypocotyl phenotype. However, the visual observation of mutant plants suggested that off-target mutations may be present, thus backcrossing is required before further analysis. As such, our results suggest that cis-elements in the promoters of core clock genes may play an important role in modulating the clock function and therefore, clock-controlled processes.

Many plant physiological and behavioral responses exhibit 24h rhythms that anticipate daily changes in the environment. This key adaptive response is regulated by the circadian clock, which provides an endogenous timekeeping mechanism that coordinates biological activities. Multiple environmental cues, including the infection of biotrophic plant pathogen Pseudomonas Syringae and pathogen induced phytohormone salicylic acid (SA) were shown to affect the clock function. However, the highly dynamic and variable nature of pathogen infection progression and related SA kinetics make it difficult to dissect the clock response to a practical infection. Here, we developed methods to perform local Pseudomonas Syringae infection and to apply transient SA treatment. These methods more closely resembled stimuli in a practical infection and allowed us to explore the circadian clock response to these stimuli. Our results indicated that a local infection lengthens the period and reduces the amplitude of circadian rhythms. Transient SA treatment recapitulated the amplitude phenotype and resulted in a phase delay with strongest phase effect at the beginning of the day. Apoplastic ROS production induced by transient SA treatment and Pseudomonas Syringae infection was shown to partly mediate these circadian clock responses. Moreover, NPR1, a master regulator of plant defense, counteracted the amplitude decrease by both infection and SA treatment. Furthermore, we discovered that localized Pseudomonas Syringae infection predominantly affected the circadian rhythms in the shoot apex and resulted in a delay in plant development. Some TCP transcription factors have been shown to regulate clock function and DNA binding activity of TCPs is regulated by ROS. We therefore characterized TCP transcription factors in order to explore the possible link between defense related ROS production and the regulation of circadian clock function. We performed yeast one hybrid analysis with a newly developed Y1H reporter system on class I TCPs and found that most of them interact with CCA1 promoter. Also, we developed a novel gene-centered Y2H screen system and discovered that class I TCP transcription factors were highly enriched for TCP21 interactors. These results uncovered the complex nature in which TCP transcription factors interact to regulate circadian clock function.

Biotic stresses such as crop disease are one of the major threats to food production worldwide. Global losses due to pathogens and pests associated with wheat, rice, maize, potato and soybean reduced the output of these five major crops, which generate 50% of the global human calorie intake, by 10-40% (Savary, 2019). It has been projected that crop disease and pest incidences are expanding in a poleward direction (2.7 km annually) (Bebber, 2014). As evidence of the negative impacts of environmental shifts accumulates in combination with a market preference for pesticide-free produce, it is expected that the main strategies to combat food insecurity will come from science and technology (Cole, 2018). Thus, scientists and policy makers alike consider increasing the pathogen tolerance of crop plants through molecular and plant breeding approaches as the most attractive and viable option to sustain food production (Cole, 2018). In this regard, disease resistant crops designed to constitutively overexpress NONEXPRESSER OF PR GENES1 (NPR1)- a master immune regulator protein homologous to many plant species- have gained popularity given their phenotypic ability to resist pathogenic attacks (Silva, 2018). However, this approach has led to undesirable side effects that contest whether NPR1 overexpressing (NOX) plants are a viable solution to improve crop fitness (Silva, 2018). given their tendency towards growth retardation. The finer details governing the signaling cascades involved in NPR1-associated defense responses remain elusive, calling for further plant defense studies. The Arabidopsis thaliana-Pseudomonas syringae pathosystem has been broadly employed to understand plant-pathogen interactions (Volko, 1998). Through morning/evening infection experiments and bioluminescence imaging of NPR1 luciferase reporter lines, we discovered that NPR1 mediates the circadian regulation of defense responses, but said regulation does not involve changes in NPR1 transcription. Given P. syringae’s hemibiotrophic approach (Thaler, 2004) we investigated changes in NPR1 expression following salicylic acid (SA) and jasmonic acid (JA) treatments, tissue wounding, and P. syr infection. Remarkably, we found that while NPR1 expression increased after any of the aforementioned treatments, it expressed rhythmically after JA treatments, tissue wounding, and P. syr infection. From this we concluded that wounding triggers increase in NPR1 expression and expression oscillations.

Plants, like many organisms, possess the ability to synchronize their biological processes with the diurnal conditions of the surrounding environment. This ability is largely controlled by an internal time-keeping mechanism known as the circadian clock. Clock function is intrinsic to each cell in the plant organism and at the same time is compartmentalized in the form of tissue-specific clocks. In addition, previous studies have shown that these tissue-specific clocks are further organized in a hierarchical structure. Still, the tissue-specific role of core clock components is largely unknown and is the focus of ongoing research. In this thesis, we tested a previously constructed tissue-specific estradiol-inducible system in Arabidopsis thaliana, and we discovered results that suggested a need for modifying this system to yield more effective conditional tissue-specific gene expression. In addition, phenotyping experiments were conducted to test this systems capacity to overexpress core clock elements and it was observed that for certain clock genes, the timing of induction is an important factor that should be taken into consideration for future experiments utilizing similar conditional systems. Altogether, the approach outlined in this study poses a useful tool for not only elucidating the tissue-specific role of clock genes in plants, but also for investigating tissue-specific functions of any gene of interest.

An internal time keeping mechanism known as the circadian clock allows organisms to anticipate changes in the environment (Michael et al., 2003; Dodd, 2005). Environmental stressors have been shown to disrupt the circadian clock so it cannot accurately predict those changes. For example, a previous study revealed that a Pseudomonas syringae infection altered the clock, leading to long period and dampened circadian rhythms (Li et al., 2018). These clock phenotypes were triggered by host and pathogen responses (Li et al., 2018). In this thesis, we explored the molecular mechanisms that regulate the observed clock phenotypes upon infection. We found that pathogen-triggered responses directly target clock components, which likely results in the observed clock phenotypes. Such results suggest that, at least in plants, the outcome of host-pathogen interactions relies on the control of the circadian clock function.

The circadian clock is an internal time-keeping mechanism that allows an organism to anticipate external changes by synchronizing its biological processes with the environment. In plants, previous evidence showed that the circadian clock function, although intrinsic to each cell, is compartmentalized in different tissues and that these tissue-specific clocks are organized in a hierarchical fashion. However, the function of cell-intrinsic clock components in each tissue is not well known. In this thesis, we developed a tissue-specific estradiol-inducible system for Arabidopsis thaliana aiming to better understand the role of clock proteins in a tissue-specific manner. We identified gene promoters that are active only at the shoot apex, root, or leaf tissues. We also experimentally confirmed the tissue-specific activities of these promoters and created conditional overexpression constructs for core clock genes. Additionally, we discussed possible phenotypes that could result from clock genes overexpression in each tissue. The strategy developed in this study provides a useful tool that could be universally implemented to study tissue-specific functions of any gene of interest.

The circadian clock is an internal timing mechanism that allows organisms to anticipate and respond to predictable changes in their environment throughout the day and across the year. Circadian processes are particularly important for plants because they are sessile organisms that must adjust their physiology to accommodate the changing world around them. This requires precise coordination of rhythmic gene expression across cells, tissues, and organs of the plant. However, more research is needed to understand the complexity of spatial clock regulation in plants. Here, we present a survey of the circadian transcriptomes of two major organs of the Arabidopsis shoot system: the shoot apex and the leaves (Chapter 2). Our comparative analysis revealed that there are unique rhythmic gene sets in each organ, and that rhythmic genes tend to reach their peak expression earlier in the shoot apex. The notion of organ-specific phase regulation was bolstered by the identification of a shoot apex-enriched transcription factor that regulates the timing of expression of the core clock component CCA1. Ultimately, we formulated a model of the circadian clock architecture in which organ-specific mechanisms modulate the phase of rhythmic genes through upstream regulation of the core clock, while the amplitude of rhythmic expression is controlled independently in each organ downstream of the core clock. Our model was further supported by the results of an organ-level transcriptome survey of the shoot system following a single leaf bacterial infection (Chapter 3). We found that the phase of clock components and of rhythmic gene expression more broadly was altered by the distal infection. This, combined with the observation that infection alters the makeup of organ-specific rhythmic gene sets, reinforces our assertion that phase is controlled upstream of the clock, and amplitude is controlled downstream of the clock, in an organ-specific manner. Organ-specific control of rhythmic gene expression may allow for precision tuning of clock-regulated processes in response to intra- and inter-organ signals, integrating microenvironmental information with internal rhythms in order to balance the needs of growth and reproduction with responses to stressors like pathogen attacks.

Kallman’s syndrome is an anosmic subtype of Isolated Gonadotropin releasing hormone Deficiency (IGD) and causes abnormal puberty and infertility. The etiology of Kallman’s syndrome is still not well understood; recently, a genetic approach has been taken towards elucidating how Kallman’s syndrome alters gonadotropin-releasing hormone (GnRH) neuron migration and hormone secretion patterns. An exome-wide association study has identified a single nucleotide polymorphism in DLX1 to be associated with IGD. To characterize functional consequences a mutation of DLX1 in vitro, we developed a patient mutation model in mature and developing immortalized GnRH neuron cell lines. We observed that the IGD patient mutation of an insertion of an adenine in the 380th base pair of the DLX1 gene leads to impaired homeodomain binding site activation and lower GnRH expression.