Tapetum-specific NEF1 is essential for primexine AGP and polysaccharide formation during pollen wall pattern establishment.

The establishment of a specific pollen wall pattern is essential for pollen development in flowering plants. This process largely depends on the formation of a temporary wall surrounding microspores in tetrads, known as the primexine, which is primarily composed of Arabinogalactan proteins (AGPs) and polysaccharides. NEF1, a gene identified two decades ago in Arabidopsis thaliana, is required for primexine formation; however, its precise functional mechanism remains unclear. In this study, we generated a nef1 knockout mutant (nef1-2) that exhibits a complete absence of primexine and male sterility. Immuno-fluorescence analysis revealed severe defects in primexine components, including pectin, xylan, and AGPs, in the nef1-2 mutant. Notably, AGPs accumulated abnormally in the tapetum, where material transport was severely disrupted, suggesting that defective AGP accumulation may underlie the primexine deficiency. Fluorescence observations demonstrated that NEF1 is specifically expressed in the tapetum and localizes to the endoplasmic reticulum and Golgi apparatus. Furthermore, when GFP was fused to the N terminus of NEF1, the protein failed to localize to the Golgi apparatus. Consequently, transgenic plants expressing pNEF1:GFP-NEF1 displayed impaired deposition of both primexine polysaccharides and AGPs, further confirming that proper NEF1 localization is essential for its function. This study improves our understanding of NEF1's role in primexine formation and highlights the contribution of AGPs in pollen wall pattern establishment.