A study on Arabidopsis ABC transporters that participate on the deposition of pollen surface lipids

DoctorPollen surface is covered with pollen wall and coat, comprising of various lipophilic materials. These lipidic structures are important for pollen development as well as the protection of the pollen against external stresses, which are necessary for successful plant reproduction. The synthesis of their precursors in the tapetum, which is nourishing cells for pollen development, was unraveled by recent reports on the identification of the enzymes involved in that process. However, the mechanisms underlying the transport of these lipid materials from the tapetum onto pollen surface remain elusive. I hypothesized that ABCG transporters are good candidates for such function, as they are well known with their involvement in the transport of surface lipids. Here, I identified 3 Arabidopsis ABCG transporters, ABCG26, ABCG9 and ABCG31, which function in this process.ABCG26 was first isolated by drastically reduced seed production of two independent knockout mutant plants, which was complemented by expression of ABCG26 driven by its native promoter. The severely reduced fertility of the abcg26 mutants was caused by a failure to produce mature pollens. Defective pollen development in the mutant was initially observed at pollen wall developmental stage, i.e. the reticulate pattern of the exine of wild-type microspores was absent in abcg26 microspores at the vacuolate stage, and the vast majority of the mutant pollen degenerated thereafter. ABCG26 was expressed specifically in tapetal cells at the early vacuolate stage of pollen development. It was also highly co-expressed with genes encoding enzymes required for sporopollenin precursor synthesis, i.e. CYP704B1, ACOS5, MS2 and CYP703A2. The tapetal cells of abcg26 accumulated numerous vesicles and granules, which was also observed in two other mutants with defects in pollen wall deposition (nef1 and dex1). ABCG26 was localized to the plasma membrane. Taken together, these results suggest that ABCG26 plays a crucial role in the transfer of sporopollenin lipid precursors from tapetal cells to anther locules, facilitating exine formation on the pollen surface.Next, I searched for ABCG transporter genes that are highly expressed in anther to find candidate transporters involved in transport of pollen surface lipids. Using in silico microarray database, together with quantitative reverse transcriptase-PCR and promoter-GUS assays, I found that ABCG9 and ABCG31 were highly expressed in anthers and that the two genes exhibited a strong co-expression. Vital staining of pollens of abcg9 abcg31 double knockout plants revealed that half of them were non-viable. When exposed to air, many of the mutant pollens were shriveled and collapsed. When exposed to a cold shock during the flowering period, abcg9 abcg31 plants could not produce as many seeds as the wild type or the single knockout mutants. Electron microscopic observation of the pollen coat revealed that the pollen coat of the mutant was not fully-filled, and contained many vesicular or linear, electron-translucent structures. The extensive analyses of lipid composition of the pollen revealed that steryl glucosidesglycosides were reduced to about half in the abcg9 abcg31 pollen without any changes in the steryl ester and free sterol contents. Pollens of ugt80A2 ugt80B1, a mutant deficient in the steryl glucosidesglycosides synthesis, were similarly reduced in their viability, and often collapsed when exposed to air. Together, these results indicate that steryl glucosidesglycosides are one of the important materials for pollen fitness and that the two ABC transporters contribute to the accumulation of this class of sterols on pollen surface.Despite the huge number of ABC transporters of plants, there are few researches on their function in the pollen yet. On the hypothesis that ABCG transporters may be involved in the pollen surface lipid transport, similarly as several other members already studied for their functions in the cuticular lipid formation, I screened all ABCG transporters by observing the mutant phenotypes and using in silico microarray databases to find candidates for the transport of pollen surface lipids. By this screening, I isolated 3 ABCG transporters working in this process, and elucidated the function of ABCG26 in pollen wall, and that of ABCG9 and ABCG31 in pollen coat formation. In addition to unraveling the transport mechanisms for the deposition of pollen surface lipids, my study introduced a new aspect of functions of ABC transporters in pollen development and its proper function. Furthermore, I showed the analogy of the functions of the ABCG transporters for the formation of the surface lipids in different tissues. This analogy will help to identify more ABCG proteins with similar activities, and also synergistically improve our understanding of the pollen surface lipids and the cuticular lipids