The molecular basis of maternal control in seed development : genetic and molecular analysis of maternal effects in seed development: molecular mapping of cap2 and expression and functional analyses of AtLDC and AtHD2C

Abstract

The female gametophyte of higher plants gives rise to the diploid embryo and the triploid endosperm which develop to produce the mature seed. Seed development is a concerted interplay of the embryo, endosperm and the surrounding diploid maternal tissue. In addition, it is highly dependent on the contribution from genetic programs executed in the gametophytic generations. What role the gametophytic maternal factors play in this process is still largely unknown. This thesis describes two approaches to identify novel genes involved in seed development. A forward genetic approach addresses the molecular nature of the maternal effect mutant capulet2 (cap2) by molecular mapping and a reverse genetics approach analyze the role in seed development of candidate genes from a promoter trap screen. The capulet2 gametophytic maternal-effect mutant was found in a linkage based screen preformed to identify gametophytic mutants in Arabidopsis (Grini et al., 1999). cap2 embryo and endosperm development is blocked at a very early stages, and heterozygous plants display a 50% reduced seed set. To investigate the molecular nature of the CAP2 gene, a map-based cloning approach was performed. Using PCR-based molecular markers the cap2 mutation was mapped to a genetic interval of 4238 basepairs, on the tip of the right arm of chromosome 1. This interval spanned parts of two genes, one involved in monoterpenoid biosynthesis and the other putatively involved in triterpenoid biosynthesis. Neither of these two genes could be verified to be responsible for the cap2 phenotype by complementation analysis. However the mapping interval of cap2 was reduced from more than 1 Mb to less than 100 kb. In a reverse genetic approach two candidate genes (AtHD2C and AtLDC) selected from a collection of promoter trap lines were analyzed to elucidate their role in seed development. Reporter gene expression studies, expression analysis, and the analysis of T-DNA insertion lines revealed that the candidate genes were expressed in the seed, but also in other organs. The promoter reporter line of AtLDC was found to have a similar but in some respects also different expression patterns in the seed than the original promoter trap line. The AtHD2C gene was found to be redundant as no phenotype could be observed in knock out alleles of the gene