Proteome analysis of male gametophyte development in rice anthers

Abstract

In this thesis, two-dimensional electrophoresis (2-DE)-based comparative proteomics were applied to monitor the global changes in protein expression during the male gametophyte development in anthers of the Australian rice cultivar Doongara, with the aim of providing a protein-level insight into the molecular mechanism underlying this important reproductive developmental process. At the same time, this thesis also evaluates the potential application of 2-DE based proteomics to other aspects of plant developmental biology. In order to collect sufficient amount of homogenous anther populations which represent a number of discrete cellular events encompassing the process of microsporegenesis, the cytological examination of developing anthers was done to establish the allometric relationships between a number of growth parameters and anther developmental stages. This provided a base for the quick and nondestructive assessment of microspore developmental stages. From this study, a strategy for the collection of rice anthers for six developmental stages was established using a combination of anther length, auricle distance and days before heading. The findings of the cytological analysis opened up the possibility of establishing rice plants as a new model system for male gametophyte research of plants. Anther proteome maps were established for six microspore developmental stages within the pH ranges of 4 to 7 and 6 to 11. Over 3,500 protein spots were reproducibly resolved in the combined pH range of 4 to 11. Comparison of proteome maps of six developmental stages resulted in the detection of 150 differentially displayed protein spots at various stages. Putative identities were predicted for 49 out of 155 protein spots which were subjected to peptide mass fingerprinting (PMF) analysis. Eight low molecular weight protein spots were matched to putative translation products of rice expressed sequence tags (EST) by N-terminal terminal sequencing followed by homology searches. This verified the translation of these small open reading frames (ORF) and revealed the presence of some post translational modifications of these proteins. By integrating the information about the functions of identified proteins and their temporal regulation patterns, three developmentally regulated metabolic pathways Xl were identified and the significance of these pathways in relation to male gametophyte development was discussed. Based on the N-terminal sequencing data, three isoforms of rice homologues of grass group II pollen allergens (Ory s 2) were identified and further characterized using bioinformatics and immunochemical techniques. Polyclonal antibodies were produced against Ory s 2 isoforms using gel-separated proteins as the antigen. Immunoblot analysis revealed that Ory s 2 proteins are pollen specific and accumulated to high abundance at mature pollen, indicating their possible involvement in fertilization process. Immunochemical analysis also showed that rice group II allergens do not possess cross-reactivity with group II allergens of other grasses. This study produced valuable molecular data to provide some insight into the global changes of protein expression accompanying pollen development, and identified some developmentally regulated protein markers which have potential practical application to other research projects. From the promising results of this proteomic study it can be expected that our understanding of complex biological processes in plant development will be enhanced with the availability of a fully annotated rice genome and the application of integrated systems biology research approaches.