OsMYB80 Regulates Anther Development and Pollen Fertility by Targeting Multiple Biological Pathways.

Pollen development is critical to the reproductive success of flowering plants, but how it is regulated is not well understood. Here, we isolated two allelic male-sterile mutants of OsMYB80 and investigated how OsMYB80 regulates male fertility in rice. OsMYB80 was barely expressed in tissues other than anthers, where it initiated the expression during meiosis, reached the peak at the tetrad-releasing stage and then quickly declined afterward. The osmyb80 mutants exhibited premature tapetum cell death, lack of Ubisch bodies, no exine and microspore degeneration. To understand how OsMYB80 regulates anther development, RNA-seq analysis was conducted to identify genes differentially regulated by OsMYB80 in rice anthers. In addition, DNA affinity purification sequencing (DAP-seq) analysis was performed to identify DNA fragments interacting with OsMYB80 in vitro. Overlap of the genes identified by RNA-seq and DAP-seq revealed 188 genes that were differentially regulated by OsMYB80 and also carried an OsMYB80-interacting DNA element in the promoter. Ten of these promoter elements were randomly selected for gel shift assay and yeast one-hybrid assay, and all showed OsMYB80 binding. The 10 promoters also showed OsMYB80-dependent induction when co-expressed in rice protoplast. Functional annotation of the 188 genes suggested that OsMYB80 regulates male fertility by directly targeting multiple biological processes. The identification of these genes significantly enriched the gene networks governing anther development and provided much new information for the understanding of pollen development and male fertility.

GDSL esterase/lipases OsGELP34 and OsGELP110/OsGELP115 are essential for rice pollen development.

Pollen exine contains complex biopolymers of aliphatic lipids and phenolics. Abnormal development of pollen exine often leads to plant sterility. Molecular mechanisms regulating exine formation have been studied extensively but remain ambiguous. Here we report the analyses of three GDSL esterase/lipase protein genes, OsGELP34, OsGELP110, and OsGELP115, for rice exine formation. OsGELP34 was identified by cloning of a male sterile mutant gene. OsGELP34 encodes an endoplasmic reticulum protein and was mainly expressed in anthers during pollen exine formation. osgelp34 mutant displayed abnormal exine and altered expression of a number of key genes required for pollen development. OsGELP110 was previously identified as a gene differentially expressed in meiotic anthers. OsGELP110 was most homologous to OsGELP115, and the two genes showed similar gene expression patterns. Both OsGELP110 and OsGELP115 proteins were localized in peroxisomes. Individual knockout of OsGELP110 and OsGELP115 did not affect the plant fertility, but double knockout of both genes altered the exine structure and rendered the plant male sterile. OsGELP34 is distant from OsGELP110 and OsGELP115 in sequence, and osgelp34 and osgelp110/osgelp115 mutants were different in anther morphology despite both were male sterile. These results suggested that OsGELP34 and OsGELP110/OsGELP115 catalyze different compounds for pollen exine development.

Identification of late-stage pollen-specific promoters for construction of pollen-inactivation system in rice.

Large-scale production of male sterile seeds can be achieved by introducing a fertility-restoration gene linked with a pollen-killer gene into a recessive male sterile mutant. We attempted to construct this system in rice by using a late-stage pollen-specific (LSP) promoter driving the expression of maize α-amylase gene ZM-AA1. To obtain such promoters in rice, we conducted comparative RNA-seq analysis of mature pollen with meiosis anther, and compared this with the transcriptomic data of various tissues in the Rice Expression Database, resulting in 269 candidate LSP genes. Initial test of nine LSP genes showed that only the most active OsLSP3 promoter could drive ZM-AA1 to disrupt pollen. We then analyzed an additional 22 LSP genes and found 12 genes stronger than OsLSP3 in late-stage anthers. The promoters of OsLSP5 and OsLSP6 showing higher expression than OsLSP3 at stages 11 and 12 could drive ZM-AA1 to inactivate pollen, while the promoter of OsLSP4 showing higher expression at stage 12 only could not drive ZM-AA1 to disrupt pollen, suggesting that strong promoter activity at stage 11 was critical for pollen inactivation. The strong pollen-specific promoters identified in this study provided valuable tools for genetic engineering of rice male sterile system for hybrid rice production.

Lectin receptor kinase OsLecRK-S.7 is required for pollen development and male fertility.

Pollen grains are covered by exine that protects the pollen from stress and facilitates pollination. Here we isolated a male sterile mutant s13283 in rice exhibiting aborted pollen with abnormal exine and defective aperture. The mutant gene encodes a novel plasma membrane-localized legume-lectin receptor kinase that we named OsLecRK-S.7. OsLecRK-S.7 was expressed at different levels in all tested tissues and throughout anther development. In vitro kinase assay showed OsLecRK-S.7 capable of autophosporylation. Mutation in s13283 (E560K) and mutation of the conserved ATP binding site (K418E) both knocked out the kinase activity. Mass spectrometry showed Thr376 , Ser378 , Thr386 , Thr403 , and Thr657 to be the autophosphorylation sites. Mutation of individual autophosphorylation site affected the in vitro kinase activity to different degrees, but did not abolish the gene function in fertility complementation. oslecrk-s.7 mutant plant overexpressing OsLecRK-S.7 recovered male fertility but showed severe growth retardation with reduced number of tillers, and these phenotypes were abolished by E560K or K418E mutation. The results indicated that OsLecRK-S.7 was a key regulator of pollen development.