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Genetic analysis of At4CL gene regulation and AtMyb subfamily 14 functional characterization in Arabidopsis thaliana Soltani, Bahram M.

Abstract

Lignin is an important biopolymer that is developmentally deposited in secondary cell walls of specialized plant cells (e.g. tracheary elements and fiber cells [i.e. fibre cells]) and also in response to stresses such as wounding. Lignin biosynthesis occurs via the phenylpropanoid pathway, in which the enzyme 4-coumarate:CoA ligase (4CL) plays a key role by catalyzing the formation of hydroxycinnamoyl-CoA esters. These esters are subsequently reduced to the corresponding monolignols. Four At4CL genes have been identified in Arabidopsis thaliana (At4CL1-At4CL4). At4CL1 and At4CL2 genes are developmentally up regulated and co-expressed with other genes involved in lignin biosynthesis. Also, they are co-expressed in response to stresses such as wounding. This co-expression is probably through the engagement of common regulatory elements and cognate transcription factors such as Mybs and their recognition sites. In this thesis, I undertook three projects with the goals to identify components of the signaling [i.e. signalling] pathway(s) regulating developmental expression and wound responsiveness of the At4CL genes, to localize cis regulatory elements controlling developmental and wound responsiveness of At4CL1 and At4CL2 genes, and to investigate the functions of a subfamily of Arabidopsis Myb transcription factors. First, Arabidopsis transgenec lines containing At4CL1::GUS or At4CL2::GUS transgenes were mutagenized in order to find and map At4CL signaling pathway mutants. Several lines with reproducible patterns of reduced GUS-expression were identified. However, the GUS-expression phenotype segregated in a non-Mendelian manner in all of the identified lines. Also, GUS expression was restored by 5-azacytidine treatment suggesting DNA methylation of the transgene. Southern analysis confirmed DNA methylation of the proximal promoter sequences of the transgene only in the mutant lines. In addition, retransformation of At4CL::GUS lines with further At4CL promoter constructs resulted in a comparable GUS-silencing phenotype with higher frequency. Taken together, these results suggest that the isolated mutants are epimutants. Apparently, two specific modes of silencing were engaged in At4CL1::GUS and At4CL2::GUS (trans)genes silencing. While silencing in the seedlings of the At4CL1::GUS line was root-specific, it was global in the At4CL2::GUS line. Also, At4CL1::GUS transgene silencing was confined to the transgene but At4CL2::GUS silencing was extended to the endogenous At4CL2 gene. In the second project, we generated a series of transgenic Arabidopsis plants containing promoter fragments and parts of the transcribed region of the At4CL2 gene fused to the GUS reporter gene, in order to localize cis regulatory elements which are involved in developmental and wound responsiveness of this gene. We found that positive and negative regulatory elements effective in modulating developmental expression or wound responsiveness of the gene are located both in the promoter and transcribed regions of the At4CL2 gene. Also, histochemical GUS assays and molecular studies indicated a biphasic wounding response of the At4CL2 gene, attributing early or late response to distinct cis-regulatory elements involved in the response, suggesting that different signaling pathways may be involved in these different responses. In the third project, I initiated strategies to knock down/out multiple members of AtMyb subfamily #14 genes in Arabidopsis in an attempt to find phenotypes related to loss of function of these genes, since functional redundancy within the subfamily appears to have hampered previous studies. Single AtMyb gene knock down or knock out lines did not reveal any mutant phenotypes but RNAi generated AtMyb84 knockdown lines in the AtMyb68 knock out background showed small rosettes and a delay in shoot development.

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