The food supply to a growing world population is based on grain crops, which are harvested for their seeds. Therefore, understanding the process of seed development and its regulation has been important to increase production. This has become even more relevant and important as grain production reaches a plateau post green revolution and food security for an ever-increasing population becomes more pressing. This study focuses on altering seed size via manipulation of the endosperm, an important component of the seed that not only nourishes the developing embryo and provides the majority of our food. A classic study done by crossing different ploidies of Arabidopsis thaliana (A. thaliana) results in changes to endosperm-led growth as a consequence of altered parental genome ratios in the endosperm specifically. Increased paternal contribution results in an enlarged endosperm, and a heavier seed, whereas increased maternal contribution has the opposite effect.
Whole genome transcript profiling using microarray analysis of siliques generated by interploidy crosses identified A. thaliana genes namely: PHE1, PHE2, AGL40, AGL62, AGL28, AGL45, CKX2, MAPK10, E2L2, GA1, CYCD4;1, CYCD4;2, GA20OX5, GA-regulated and AT5G46950, that are positively associated with endosperm overgrowth. In order to verify the role of these genes in endosperm proliferation, knock in (KI) which causes gene over-expression and knock out (KO) which results in gene inactivation, strategies were used. Constitutive over-expression of CKX2, MAPK10, and E2L2 showed abnormal vegetative and floral phenotypes, whereas targeted endosperm-specific over-expression of PHE2 and GA1 showed an increase in seed size and/or fertility. KI plants of AGL40, AGL62, AGL45, CYCD4;1, CYCD4;2, GA20OX5, GA-regulated and AT5G46950 did not result in any obvious phenotypic effects under normal growth conditions. KO mutant plants namely: phe1, phe2, agl40, agl62, agl28 and agl45, as single individual mutants were also indistinguishable to wild type plants in non-seed phenotypes. Embryo sac area, individual seed weight and total seed yield data obtained from a phe1 line showed a smaller embryo sac area, lighter seed and reduced total seed yield. Loss of function of agl62 showed precocious endosperm cellularisation and seed lethality. It is nearly impossible to generate phe1/phe2 double mutants due to a very tight linkage between these genes (on the chromosome physically the two genes lie next to each other) and hence doubles, triples and quadruple mutants where appropriate were made with either phe1 or phe2 in combination with other mutant lines of the MADS family of transcription factors. None of the double mutant combination tested had obvious developmental defects. However, the triple mutant phe1/phe1::agl40/agl40 ::agl45/agl45 and the quadruple mutant phe2/phe2::agl40/agl40::agl45/agl45 ::agl28/agl28 showed abnormal embryo and endosperm development which resulted to seed death. This indicates a functional redundancy among these MADS box genes. However, it is unclear how PHE1, PHE2, AGL40, AGL45 and AGL28 act to affect embryo and endosperm development. This result confirms that these genes are positively associated with endosperm over-proliferation as growth promoters and function in some cases as singly or as participants in a complex to control seed development. Thus manipulating expression of genes involved in endosperm development in A. thaliana and the potential use of this knowledge in crop plants provides us with a route to improvement of crop yields.
|Date of Award||1 Sep 2010|
|Supervisor||Roderick Scott (Supervisor)|