Defective Pollen Wall 3 (DPW3), a novel alpha integrin-like protein, is required for pollen wall formation in rice.

In flowering plants, pollen wall is a specialized extracellular cell-wall matrix surrounding male gametophytes and acts as a natural protector of pollen grains against various environmental and biological stresses. The formation of pollen wall is a complex but well-regulated process, which involves the action of many different genes. However, the genetic and molecular mechanisms underlying this process remain largely unknown. In this study, we isolated and characterized a novel rice male sterile mutant, defective pollen wall3 (dpw3), which displays smaller and paler anthers with aborted pollen grains. DPW3 encodes a novel membrane-associated alpha integrin-like protein conserved in land plants. DPW3 is ubiquitously expressed in anther developmental stages and its protein is localized to the plasma membrane, endoplasmic reticulum (ER) and Golgi. Anthers of dpw3 plants exhibited unbalanced anther cuticular profile, abnormal Ubisch bodies, disrupted callose deposition, defective pollen wall formation such as abnormal microspore plasma membrane undulation and defective primexine formation, resulting in pollen abortion and complete male sterility. Our findings revealed a novel and vital role of alpha integrin-like proteins in plant male reproduction.

NERD1 is required for primexine formation and plasma membrane undulation during microsporogenesis in Arabidopsis thaliana.

The primexine formation and plasma membrane undulation are the crucial steps of pollen wall formation in many angiosperms. However, the molecular mechanism underlining these processes is largely unknown. In Arabidopsis, NEW ENHANCER OF ROOT DWARFISM1 (NERD1), a transmembrane protein, was reported to play pleiotropic roles in plant development including male fertility control; while, how NERD1 disruption impacts male reproduction is yet unclear. Here, we revealed that the male sterility of nerd1 mutants is attributed to defects in early steps of pollen wall formation. We found that nerd1-2 is void of primexine formation and microspore plasma membrane undulation, defective in callose deposition. Consequently, sporopollenin precursors are unable to deposit and assemble on the microspore surface, but instead accumulated in the anther locule and tapetal cells, and ultimately leading to microspore abortion. NERD1 is localized in the Golgi and is expressed in both vegetative and reproductive organs, with the highest expression in reproductive tissues, including the tapetum, male meiocytes, tetrads and mature pollen grains. Our results suggest that NERD1 is required for the primexine deposition and microspore plasma membrane undulation, thus essential for sporopollenin assembly and pollen exine formation.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2-3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.