Characterisation of the metacaspase gene family in Arabidopsis thaliana

Caspases are known to be involved in animal programmed cell death (PCD). The objective of this thesis was to use gene expression analysis and reverse genetics to determine if Arabidopsis metacaspase (AtMC) genes play a role in plant PCD. The majority of AtMC genes were found to be expressed nearly constitutively in various tissues, developmental stages, and under various inductive treatments. Transgenic Arabidopsis plants generated with AtMCpromoter::AtMCgene::GUS fusions showed expression of the reporter gene in leaves, vasculature, trichomes, siliques, anthers, and during embryo development. Preliminary phenotypic characterization of single and double Arabidopsis AtMC loss-of-function mutants suggested that the expression of the AtMC genes are highly functionally redundant. Nevertheless, our results suggest that AtMC1, 2, 4, 6 and 9 may be directly involved in rosette and/or stem development. Although this study does not provide a definitive role of MCs in plant PCD, it lays the foundation for their further in-depth analysis.

Effects of Ethylene on Secondary Xylem Formation in Arabidopsis thaliana

Ethylene has myriad roles as a plant hormone, ranging from senescence and defending against pathogen attacks to fruit ripening and interactions with other hormones. It has been shown to increase cambial activity in poplar, but the effect on wood formation in Arabidopsis hypocotyl has not previously been studied. The Auxin-Regulated Gene involved in Organ Size (ARGOS), which increases organ size by lengthening the time for cell division, was found to be upregulated by ethylene. We tested the effect of ethylene treatment at 10 and 100 µM ACC on three genotypes of Arabidopsis, Col0 (wild-type), an ARGOS deficient mutant (argos), and ein3-1, an ethylene insensitive mutant. ARGOS expression analysis with qPCR indicated that ACC does induce ARGOS and ARGOS-LIKE (ARL) in the hypocotyl. As seen in poplar, ethylene also decreases stem elongation.Histochemical staining, showed that ethylene changes the way secondary xylem lignifies, causing gaps in lignification around the outer edge of secondary xylem. Our results also implied that ethylene treatment changes the proportion of secondary to total xylem, resulting in less secondary, whereas in poplar, ethylene treatment caused an increase.

Functional analysis of rice bidirectional promoters

Bidirectional promoters regulate adjacent genes organized in a divergent fashion (head to head orientation). Several Reports pertaining to bidirectional promoters on a genomic scale exists in mammals. This work provides the essential background on theoretical and experimental work to carry out a genomic scale analysis of bidirectional promoters in plants. A computational study was performed to identify putative bidirectional promoters and the over-represented cis-regulatory motifs from three sequenced plant genomes: rice (Oryza sativa), Arabidopsis thaliana, and Populus trichocarpa using the Plant Cis-acting Regulatory DNA Elements (PLACE) and PLANT CARE databases. Over-represented motifs along with their possible function were described with the help of a few conserved representative putative bidirectional promoters from the three model plants. By doing so a foundation was laid for the experimental evaluation of bidirectional promoters in plants. A novel Agrobacterium tumefaciens mediated transient expression assay (AmTEA) was developed for young plants of different cereal species and the model dicot Arabidopsis thaliana. AmTEA was evaluated using five promoters (six constructs) and two reporter genes, gus and egfp. Efficacy and stability of AmTEA was compared with stable transgenics using the Arabidopsis DEAD-box RNA helicase family gene promoter. AmTEA was primarily developed to overcome the many problems associated with the development of transgenics and expression studies in plants. Finally a possible mechanism for the bidirectional activity of bidirectional promoters was highlighted. Deletion analysis using promoter-reporter gene constructs identified three rice promoters to be bidirectional. Regulatory elements located in the 5’- untranslated regions (UTR) of one of the genes of the divergent gene pair were found to be responsible for their bidirectional ctivity

Regulation of woody plant development by gibberellin catabolic and signaling genes

Gibberellin (GA) is a growth promoting hormone implicated in regulating a diversity of plant processes. This dissertation examines the role of GA metabolic and signaling genes in woody plant growth and development. Transgenic modifications, expression analysis, physiological/biochemical assays, biometric measurements and histological analysis were used to understand the regulatory roles these genes play in the model woody plant, Populus. Our results highlight the importance of GA regulatory genes in woody perennial growth, including: phenology, wood formation, phenotypic plasticity, and growth/survival under field conditions. We characterize two putative Populus orthologs of the SHORT INTERNODES (SHI) gene from Arabidopsis, a negative regulator of GA signaling. RNAi-mediated suppression of Populus SHI-like genes increased several growth-related traits, including extent of xylem proliferation, in a dose-dependent manner. Three Populus genes, sharing sequence homology to the positive regulator of GA signaling gene PHOTOPERIOD-RESPONSIVE 1 (PHOR1) from Solanum, are up-regulated in GA-deficient and insensitive plants suggesting a conserved role in GA signaling. We demonstrate that Populus PHOR1-like genes have overlapping and divergent function(s). Two PHOR1-like genes are highly expressed in roots, predominantly affect root growth (e.g., morphology, starch quantity and gravitropism), and induced by short-days (SD). The other PHOR1-like gene is ubiquitously expressed with a generalized function in root and shoot development. The effects of GA catabolic and signaling genes on important traits (e.g., adaptive and productivity traits) were studied in a multi-year field trial. Transgenics overexpressing GA 2-oxidase (GA2ox) and DELLA genes showed tremendous variation in growth, form, foliage, and phenology (i.e., vegetative and reproductive). Observed gradients in trait modifications were correlated to transgene expression levels, in a manner suggesting a dose-dependent relationship. We explore GA2ox and DELLA genes involvement in mediating growth responses to immediate short-term drought stress, and SD photoperiods, signaling prolonged periods of stress (e.g., winter bud dormancy). GA2ox and DELLA genes show substantial up-regulation in response to drought and SDs. Transgenics overexpressing homologs of these genes subjected to drought and SD photoperiods show hypersensitive growth restraint and increased stress resistances. These results suggest growth cessation (i.e., dormancy) in response to adverse conditions is mediated by GA regulatory genes.

DISSECTION OF STRESS RESPONSE NETWORKS REGULATING MULTIPLE STRESSES IN RICE

Important food crops like rice are constantly exposed to various stresses that can have devastating effect on their survival and productivity. Being sessile, these highly evolved organisms have developed elaborate molecular machineries to sense a mixture of stress signals and elicit a precise response to minimize the damage. However, recent discoveries revealed that the interplay of these stress regulatory and signaling molecules is highly complex and remains largely unknown. In this work, we conducted large scale analysis of differential gene expression using advanced computational methods to dissect regulation of stress response which is at the heart of all molecular changes leading to the observed phenotypic susceptibility. One of the most important stress conditions in terms of loss of productivity is drought. We performed genomic and proteomic analysis of epigenetic and miRNA mechanisms in regulation of drought responsive genes in rice and found subsets of genes with striking properties. Overexpressed genesets included higher number of epigenetic marks, miRNA targets and transcription factors which regulate drought tolerance. On the other hand, underexpressed genesets were poor in above features but were rich in number of metabolic genes with multiple co-expression partners contributing majorly towards drought resistance. Identification and characterization of the patterns exhibited by differentially expressed genes hold key to uncover the synergistic and antagonistic components of the cross talk between stress response mechanisms. We performed meta-analysis on drought and bacterial stresses in rice and Arabidopsis, and identified hundreds of shared genes. We found high level of conservation of gene expression between these stresses. Weighted co-expression network analysis detected two tight clusters of genes made up of master transcription factors and signaling genes showing strikingly opposite expression status. To comprehensively identify the shared stress responsive genes between multiple abiotic and biotic stresses in rice, we performed meta-analyses of microarray studies from seven different abiotic and six biotic stresses separately and found more than thirteen hundred shared stress responsive genes. Various machine learning techniques utilizing these genes classified the stresses into two major classes' namely abiotic and biotic stresses and multiple classes of individual stresses with high accuracy and identified the top genes showing distinct patterns of expression. Functional enrichment and co-expression network analysis revealed the different roles of plant hormones, transcription factors in conserved and non-conserved genesets in regulation of stress response.