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

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.

Regulation of woody plant development by auxin and polar auxin transport

Auxin is a key regulator in plant growth and development. This dissertation examines the role of auxin and polar auxin transport in woody growth and development. Strategies of promoter reporter system, microarray expression analysis, transgenic modification, physiological assays, anatomical analysis, and histochemical/biochemical assays were employed to improve our understanding of auxin study in Populus. The results demonstrate various aspects of auxin regulation on shoot growth, root development, wood formation, and gravitropism in woody tissues. We describe the behavior of the DR5 reporter system for measuring auxin concentrations and response in stably transformed Populus trees. Our study shows that DR5 reporter system can be efficiently used in Populus to study auxin biology at a cellular resolution. We investigated the global gene expression in responding to auxin in Populus root. The results revealed groups of IBA up- and down- regulated genes involved in various biological processes including cell wall modification, root growth and lateral root formation, transporter activity and hormone crosstalk. We also verify two of the identified genes' function by transgenic modification in Populus, which encode auxin efflux carrier PtPIN9 and transcription factor PtERF72. We investigated the role of PtPIN9 in woody growth and development, especially in wood formation and gravitropic response in woody stem. We found that overexpressing PtPIN9 enhanced several growth parameters while suppression of PtPIN9 has inhibited tension wood formation. Our results show that PIN9 and other members from PIN family could be possible useful tools for increasing biomass productivity, wood quality, or in modifying plant form.