A Study on two ABC transporters that regulate the postmeiotic development of male gametophyte in Arabidopsis

DoctorThe male gametophytic cells of terrestrial plants, the pollen grains, travel far before fertilization, and thus require strong protective layers, which take the form of a pollen coat and pollen wall. The components of pollen wall, namely sporopollenin, is highly complex chemical mixture of lipids and phenolic molecules. The sporopollenin precursors are synthesized in the nutritive tapetal cells, the tissue surrounding the developing gametophytes in anther and exported to the developing microspores. However, the transport mechanism and the corresponding transporters for the sporopollenin precursors are largely unknown. To date, many ABC transporters have been shown to play essential roles in the formation of the protective layers. AtABCG11, 12, 13, and 32 transporters are reported for their function in the surface cuticular layer formation in various tissues. AtABCG26 and AtABCG9/31 are required for the pollen wall and pollen coat formation, respectively. Among the many ABC transporters involved in the surface layer formation, AtABCG26 is the only transporter reported to be involved in pollen wall formation. Therefore it is necessary to find out whether there are any additional transporters required for the pollen wall formation. In this study, I aimed to identify the transporters required for the formation of complex pollen wall structures. To achieve this goal, I hypothesized that the transporter is under the regulation of the master regulator of pollen wall development, ABORTED MICROSPORES (AMS) which directly binds to the promoter region of AtABCG26 and induce the gene expression. Therefore, I established the following criteria to screen out the candidate ABC transporters using in silico database; (i) The transporter would have altered gene expression in the knockout mutant of ams; (ii) The transporter would possess the putative binding motif of AMS; (iii) The transporter which belongs to ABCG sub-family. AtABCG1 satisfied all criteria. However, no visible phenotype was observed in the single knockout mutant of AtABCG1, suggesting a functional redundancy. Based on the amino acid sequence identity, I found out that AtABCG16 might have a function similar to AtABCG1, and generated the double knockout mutant. Whereas the microspores of atabcg1 or atabcg16 single knockout mutants developed into normal pollen grains, those of the atabcg1 atabcg16 double knockout mutant were eventually aborted and failed to deposit the normal exine and intine layers. To gain insight into the functional roles of AtABCG1 and AtABCG16 in pollen development, I investigated the spatiotemporal expression pattern of those proteins. AtABCG1 and AtABCG16 are localized at the plasma membrane of tapetal cells, specifically after postmeiotic microspore release, and this expression pattern implied that those transporters play critical roles in the postmeiotic stages of male gametophyte development. Consistent with this stage-specific expression, the atabcg1 atabcg16 double knockout mutant exhibited defects in pollen development after postmeiotic microspore release; their microspores lacked intact nexine layers, exhibited defects in pollen mitosis I, displayed ectopic deposits of arabinogalactan proteins, failed to complete cytokinesis, and lacked sperm cells. Interestingly, the double mutant exhibited abnormalities in the internal structures of tapetal cells, too; the storage organelles of tapetal cells, tapetosomes and elaioplasts, were morphologically altered. This study reveals that the lack of AtABCG1 and AtABCG16 at the tapetal cell plasma membrane causes a broad range of defects in pollen, as well as in tapetal cells themselves during post-meiotic developmental stages. Furthermore, this study demonstrates that the normal formation of pollen surface layer is critical for normal pollen development