Molecular genetic approaches to understanding and manipulating seed development in Arabidopsis thaliana

Abstract

One of the major problems facing the world in the 21st century is feeding a growing population on ever-decreasing arable land. The majority of the world’s calorific intake comes from seeds, primarily those of wheat, rice and maize which are principally composed of endosperm tissue. Achieving a greater understanding of endosperm development, and applying this knowledge to improve seed yield is an important step towards increased food productivity. This project aims to add to the pool of knowledge associated with endosperm development in the model plant Arabidopsis thaliana. Our first goal was to generate a method by which we could rapidly assay seed size.We achieved this by placing seeds on a consumer-level scanner and processing the images with digital imaging software. This method was able to accurately identify differences in seed size caused by altered plant growth and mutations. We applied this method to perform a quantitative trait loci (QTL) analysis and a mutant screen, identifying 8 QTL and a novel seed size mutant, respectively. Rational alteration of seed size through the endosperm was achieved by taking a strong endosperm-specific promoter and fusing it to a number of genes associated with increased plant growth. Using transcriptome data we identified a number of putative endosperm-specific promoters and tested their activity using GFP fusion proteins. The most suitable promoter was fused to seven genes of interest and seed size was determined. Overexpression of DWARF4, a gene involved in brassinosteroid biosynthesis, resulted in a significant increase in seed size. A mutant screen of endosperm-preferred early-seed-specific genes identified previously by our laboratory was performed. We tested homozygous lines for a seed size effect (see above) and heterozygous lines for seed abortion phenotypes. This yielded a number of putative seed-lethal mutants, one of which was selected for further analysis. The MCM5 gene encodes a subunit of the replicative helicase complex, consisting of six homologous subunits. Analysis of mutants of each subunit revealed that different phenotypes occurred in the developing seed, suggesting unique individual functions. In addition, a pool of MCM5 and MCM7 protein was identified in the central cell which was capable of supporting seed development when de novo endosperm transcription was abolished with a microRNA. We have taken a multi-faceted approach to seed development and significantly contributed to the pool of knowledge associated with endosperm development in Arabidopsis thaliana. Future work can be directed at determining the mechanism by which seed size is altered in our rationally altered seed size mutants, and the causes of the different phenotypes seen in MCM2-7 mutants can be more fully characterized.