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.

The pollen wall, which protects male gametophyte against various stresses and facilitates pollination, is essential for successful reproduction in flowering plants. The pollen wall consists of gametophyte-derived intine and sporophyte-derived exine. From outside to inside of exine are tectum, bacula, nexine I and nexine II layers. How these structural layers are formed has been under extensive studies, but the molecular mechanisms remain obscure. Here we identified two osabcg3 allelic mutants and demonstrated that OsABCG3 was required for pollen development in rice. OsABCG3 encodes a half-size ABCG transporter localized on the plasma membrane. It was mainly expressed in anther when exine started to form. Loss-function of OsABCG3 caused abnormal degradation of the tapetum. The mutant pollen lacked the nexine II and intine layers, and shriveled without cytoplasm. The expression of some genes required for pollen wall formation was examined in osabcg3 mutants. The mutation did not alter the expression of the regulatory genes and lipid metabolism genes, but altered the expression of lipid transport genes. Base on the genetic and cytological analyses, OsABCG3 was proposed to transport the tapetum-produced materials essential for pollen wall formation. This study provided a new perspective to the genetic regulation of pollen wall development.

Salinity stress significantly impairs plant growth and leads to substantial yield losses in rice and other crops. WRKY transcription factors (TFs) are well-documented regulators of plant stress responses, yet their specific roles in rice salt tolerance remain largely unexplored. This study investigates the function of OsWRKY18, a rice WRKY TF with transcriptional activation activity, in salt stress adaptation. Bioinformatics analysis revealed that OsWRKY18 contains conserved motifs and domains shared with other salt-tolerant WRKY TFs, indicating its potential involvement in stress response. Expression analysis showed that OsWRKY18 is predominantly expressed in roots, with significant upregulation under salt stress. Subcellular localization in rice protoplasts revealed that OsWRKY18 is primarily localized in the nucleus. Immunostaining assays confirmed its widespread expression in root tissues, particularly in the stele, but not in epidermal cells. Using CRISPR/Cas9, we generated Oswrky18 knockout mutants, which displayed increased salt sensitivity, marked by elevated Na+ accumulation in shoots and stunted growth compared to wild-type plants. Transcriptome sequencing and quantitative real-time PCR analysis revealed that OsWRKY18 regulates key genes involved in ABA signaling, osmotic adjustment, and ion homeostasis, including OsHOX22, OsSALP1, OsNHX4, and OsHKT1;5. Yeast one-hybrid assays further confirmed the direct binding of OsWRKY18 to the OsHKT1;5 promoter. These results highlight OsWRKY18's critical role in enhancing rice salt tolerance by maintaining ion balance and activating stress-responsive gene networks. This study advances our understanding of the molecular mechanisms underlying OsWRKY18-mediated salt-stress responses and the functional roles of WRKY TFs in rice.

Meiotic recombination plays a central role in maintaining genome stability and increasing genetic diversity. Although meiotic progression and core components are widely conserved across kingdoms, significant differences remain among species. Here we identify a rice gene ABERRANT GAMETOGENESIS 1 (AGG1) that controls both male and female gametogenesis. Cytological and immunostaining analysis showed that in the osagg1 mutant the early recombination processes and synapsis occurred normally, but the chiasma number was dramatically reduced. Moreover, OsAGG1 was found to interact with ZMM proteins OsHEI10, OsZIP4, and OsMSH5. These results suggested that OsAGG1 plays an important role in crossover formation. Phylogenetic analysis showed that OsAGG1 is a plant-specific protein with a highly conserved N-terminal region. Further genetic and protein interaction analyses revealed that the conserved N-terminus was essential for the function of the OsAGG1 protein. Overall, our work demonstrates that OsAGG1 is a novel and critical component in rice meiotic crossover formation, expanding our understanding of meiotic progression.This study identified a plant-specific gene ABERRANT GAMETOGENESIS 1 that is required for meiotic crossover formation in rice. The conserved N-terminus of the AGG1 protein was found to be essential for its function.

Phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P₂) is known to be an instrumental anionic phospholipid in governing pollen germination and pollen tube growth. However, the precise functions and regulatory mechanisms of PI(4,5)P₂ in pollen polarity establishment and germination remain poorly understood. Our previous studies demonstrated the pivotal involvement of Arabidopsis formin homology 5 (AtFH5)‐dependent vesicle trafficking in polarity establishment of pollen. Here, we observed that PI(4,5)P₂ accumulated and oscillated at the prospective germination site, a process closely associated with the rotational movement of AtFH5‐labeled vesicles. Disruption of the mobility of AtFH5‐labeled vesicles, either through AtFH5 mutation or pharmacological treatment, significantly perturbed the accumulation of PI(4,5)P₂ at the plasma membrane. Subcellular localization and genetic analysis revealed that two phosphatidylinositol 4‐phosphate 5‐kinases, AtPIP5K1 and AtPIP5K4, are essential for PI(4,5)P₂ oscillation at the germination site prior to pollen germination. Furthermore, we found that the dynamics of AtPIP5K4 depended on the mobility of AtFH5‐labeled vesicles and reduced PI(4,5)P₂ in turn disturbed the attachment of AtFH5‐labeled secretory vesicles to the plasma membrane. In conclusion, these findings collectively highlight the reciprocal regulation of AtFH5‐labeled secretory vesicles and PI(4,5)P₂ oscillations at the plasma membrane, providing critical insights into the molecular mechanism underlying polarity establishment during pollen germination.