关键词: 水稻, OsPKS1, OsPKS2, 孢粉素, 花粉外壁, 雄性不育

Abstract:

【Background】 Plant pollen is surrounded by pollen wall which acts as a natural protective barrier for male gametes and plays a pivotal role in plant reproductive development. The main component of pollen wall is sporopollenin, which is mainly composed of lipidic and phenolic substances. Therefore, the metabolism of these two substances is a key step for anther wall and pollen wall formation. PKS1/PKSA/LAP6 and PKS2/PKSB/LAP5 show conserved biochemical functions in sporopollenin biosynthesis pathways among different species. 【Objective】 The role of OsPKS1 and OsPKS2 in rice anther wall and pollen wall development was studied to provide a new understanding for the mechanism of this process. 【Method】 A gene co-expressed network, AntherNet predicted a gene OsPKS1 that might be involved in sporopollenin biosynthesis, using the CRISPR/Cas9 genome editing system to generate ospks1 and ospks1 ospks2 in Japonica subspecies 9522 background and ospks2 background, respectively. Under the same growth condition, the vegetative growth and floral organ development of the mutant plants were analyzed by comparing the phenotypes of the wild type and the mutants. I₂-KI staining was utilized to analyze the pollen viability of ospks1 and ospks1 ospks2. Semi-thin section was performed to observe four cell layers and microspore development in the wild type and the mutants at different stages. Scanning electron microscope (SEM) was used to observe the fine structures of anther wall outer and inner surface as well as pollen wall surface both in the wild type and the mutants. And transmission electron microscopy (TEM) was performed to observe the fine structures of anther wall cell, Ubisch body and pollen wall of the wild type and the mutants.【Result】Four ospks1 and four ospks1 ospks2 were obtained by CRISPR/Cas9 approach, among which ospks1-3 and ospks1-4 ospks2 were homozygous mutants. Both ospks1-3 and ospks1-4 ospks2 were male sterile. ospks1-3 and ospks2 displayed abnormal pollen wall and Ubisch body, however, the detailed morphology was different between two mutants. ospks1-3 could form convex wall structure on the surface of pollen wall and the tapetal layer could be normally degraded; a large number of tiny cavities were formed in the inner structure of pollen wall and the bacula became shorter, which might cause invalid connection between tectum and nexine; the bottom structure of Ubisch bodies was decreased while the top structure was increased and Ubisch bodies were sharper than those of the wild type. By observing ospks1-4 ospks2, it was found that the cuticle was reduced and the tapetal layer could not be degraded normally. Besides, there was no obvious pollen wall structure on the surface of microspores and the microspores were degraded at stage 11; Ubisch bodies were less formed with abnormal structure at stage 9 and were detached from the anther wall at stage 11. 【Conclusion】 The function of PKS1/PKSA/LAP6 and PKS2/PKSB/LAP5 are conserved among various species and could affect the biosynthesis and accumulation of sporopollenin. However, these two genes show different function for the formation of the inner structure of pollen wall and Ubisch bodies in rice: OsPKS1 is more important for the formation of bacula and the bottom structure of Ubisch bodies; OsPKS2 is more important for the formation of tectum and the top structure of Ubisch bodies. They regulate the formation of pollen wall, anther wall and the degradation of tapetum.

Key words: Oryza sativa, OsPKS1, OsPKS2, sporopollenin, pollen wall, male sterile