Regulation of Pollen Viability, Pollen Tube Growth and Seed Development in Maize by Application of Cysteine Protease ZmPCP

In the process of maize production, extreme meteorological conditions such as drought and high temperature are often the main environmental stress factors affecting pollination efficiency. Previous studies have shown that, under adversity, the germination rate of pollen grains on the filaments of female spikes directly affects the success rate of reproduction and ultimately determines the grain yield. This study focuses on a cysteine protease named ZmPCP. The expression of this protease in maize pollen is significantly higher than in other tissues, and its specific function has not been clearly defined. Its localization in the cell membrane or apoplast was further confirmed by transient transfection experiments and plasmolysis. The interaction between ZmPCP and ZmSNAP33 was verified by yeast two-hybrid technology and a GST pull-down experiment, indicating that ZmPCP may affect pollen germination and stress resistance by regulating vesicle transport. Secondly, by analyzing the pollen germination rate of maize inbred lines B104, <i>ZmPCP</i>-KO and <i>ZmPCP</i>-OE transgenic maize plants, we found that <i>ZmPCP</i> overexpression could significantly enhance pollen viability and pollen tube growth under drought stress. After 1 h of short-term drying treatment, the pollen germination rate of the <i>ZmPCP</i>-OE line was maintained at 44%, which was significantly higher than that of the other lines. In addition, the observation of pollen tube growth showed that <i>ZmPCP</i> overexpression could promote the extension of pollen tubes in the filament. Moreover, a transcriptome sequencing analysis revealed the regulatory effects of <i>ZmPCP</i> on pollen in multiple biological processes, including stress response, carbohydrate metabolism, growth and development, cell wall material metabolism, signal transduction, etc. The involved pathways of these differential genes indicate that <i>ZmPCP</i> enhances pollen drought tolerance and promotes pollen tube growth through a "metabolism signal structure". In the germination experiment on the seventh day, the germination rate of <i>ZmPCP</i>-OE maize seeds was the lowest, indicating that its overexpression inhibited seed germination. At the same time, <i>ZmPCP</i>-overexpressing Arabidopsis showed a significant advantage in taproot growth under high-concentration ABA stress. ZmPCP provides an important theoretical basis for regulating the pollination process and improving the pollination efficiency of maize varieties through interaction with ZmSNAP33.