Dual-Layer Transcriptional-Protein Regulation by HvAP2-12 Represses HvAP2-18 Activity to Fine-Tune Barley Starch Synthesis

Starch content and composition are key factors determining yield and quality of barley, but the molecular mechanisms regulating barley starch synthesis remain unclear. In this study, we identified an APETALA2/Ethylene-Responsive factor (AP2/ERF) family gene HvAP2-12, which has typical transcription factor characteristics. Overexpression of HvAP2-12had no effect in total starch and amylose content, whereas the gene-edited mutant lines showed increased total starch and significantly decreased amylose content. Further analysis revealed that HvAP2-12 does not directly bind to the promoter of major starch synthesis genes. However, it binds to another AP2/ERF family member, the HvAP2-18promoter and interacts with the HvAP2-18 protein.HvAP2-18also affects starch synthesis in barley grains, and its overexpression increases total starch content while significantly reducing amylose content.HvAP2-18binds directly to the promoters of key starch synthesis genes HvAGP-S1 and HvSBE1, activating their expression. Additionally,HvAP2-12 inhibits HvAP2-18 from binding to HvAGP-S1and HvSBE1 promoters. Based on these findings, we propose that HvAP2-12 and HvAP2-18 play critical roles in the regulation of starch genes and in determining barley grain quality.