''Elucidation of the expression patterns and funtions of the SET- domain genes ATX3 and ATX5 in Arabidopsis thaliana''

Abstract

Covalent modifications of the DNA and histones through acetylation, phosphorylation, methylation, etc are central to gene regulation, recombination, DNA replication and many other processes in the cell. With respect to gene regulation, modifications of histones and DNA have immense influence on the expression potential of genes within the modified regions of chromatin structure, mainly on the level of DNA packaging and recruitment of the transcriptional machinery. Histone methyl transferases form a part of this complex system of genes that carry such modifications. They catalyze the methylation of the N-terminal tails of histones. This function is known to reside in an evolutionarily conserved domain, SET which is found in all the kingdoms of life. Several hundreds of the SET-domain genes have been identified in several organisms. About 30 of these SET-domain genes are thought to exist in Arabidopsis genome with relatively little or no experimental information on some of them. Many genetic analyses in several organisms show that the SET-domain genes regulate the transcription of very important genes including homeotic genes. Two of such genes are the Arabidopsis Trithorax 3 (ATX3) and ATX5 on which data generated experimentally is too scanty. This project was therefore aimed at obtaining functional information on ATX3 and ATX5. This began with phenotypic analysis by generating ‘Knockdown lines’ of ATX5, using the RNA interference mechanism. Level of silencing was low. However lines where silencing is considered to have occurred exhibited phenotypic defects which gives an important hint which suggest ATX5 is involved in a number very vital processes in Arabidopsis development. Fertility and silique development, which is fertility-related, were adversely affected. The ATX5 RNAi lines also possessed abnormal phyllotaxy and enlarged inflorescence meristems. When SALK lines for T-DNA insertion for the ATX5 were later screened phenotypically, there was one isolated instance of enlarged inflorescence meristem which matched that obtained on one RNAi line. Phenotypic analysis was followed by in situ hybridization and RT-PCR, both of which gave consistent results. RT-PCR results showed a relatively higher level of ATX5 mRNA in the flowers. In situ hybridization analysis also showed that ATX5 is expressed in very vital tissues and organs of Arabidopsis, namely the shoot apical meristems of both seedlings and embryo. ATX5 transcript was also detected in the tapetum of stamens and the gynoecia of Arabidopsis flowers. Results from both experiments suggest that the phenotypes observed on the RNAi lines were due to down regulation of the ATX5 gene, although this was not an easy experiment to perform convincely, but does not explain the cause of the low level of silencing. Experiments are underway to determine the interacting partners of the ATX5 protein using the glutathione Sepharose tagged (GST)- pull down assay with the interacting partners found for the structurally similar ATX4, as well as test the possibility of histone methyl transferase activity. Preliminary results from RT-PCR analysis detected ATX3 mRNA in several tissues and organs. The mRNA level in seedlings was however higher in seedlings than in many other tissues and could be a reflection of a higher activity of ATX3 in seedlings. In situ hybridization analysis also showed that ATX3 is necessary for normal development of the pollen wall or lipid-rich exin coating, which is the function of tapetum of which the ATX3 trasncript was present. Preliminary screening of SALK lines for the ATX3 gene did not produce any obvious phenotypes.