ZmCNX2 regulates maize pre-harvest sprouting through ABA synthesis and energy Metabolism

• A novel pre-harvest sprouting mutant vpm in maize was created, and the gene ZmCNX2 related to pre-harvest sprouting in vpm was identified. • Overexpression of ZmCNX2 in Arabidopsis can delay seed germination, and overexpression of ZmCNX2 in wheat can improve resistance to pre-harvest sprouting, laying a foundation for breeding crop varieties resistant to pre-harvest sprouting. • Loss of ZmCNX2 function leads to defects in ABA synthesis, disorders in ABA signal transduction, and active cellular energy metabolism, which jointly result in the appearance of the pre-harvest sprouting phenotype. Abscisic acid (ABA) plays a crucial role in regulating seed dormancy and germination, and its disruption is the primary cause of preharvest sprouting (PHS) in crops. In this study, we generated a maize viviparous mutant, vpm , via EMS mutagenesis. Bulked segregant analysis identified ZmCNX2 with a stop-gained mutation (c.816T>A|p.Y272*), which was confirmed as the causative gene. Allelic validation for ZmCNX2 was performed in maize using two mutants, vpm and cnx2-1 , which demonstrated that they were alleles of the ZmCNX2 locus, and its ectopic expression in Arabidopsis and wheat resulted in delayed seed germination. ZmCNX2 encodes a GTP 3′,8-cyclase catalyzing the first step in the biosynthesis of molybdenum cofactor (MoCo), which accompanies the aldehyde oxidase (AO3) to catalyze the last step in ABA biosynthesis. In the vpm mutant, AO3 was downregulated, and there was a significant reduction in ABA-related metabolites. Further experiments revealed that ZmCNX2 interacts with ZmRPT3 and ZmO18. The interaction between ZmCNX2 and ZmRPT3 may be involved in the regulation of ABA signaling, while the interaction between ZmCNX2 and ZmO18 may affect energy metabolism. Therefore, ZmCNX2 coordinates ABA synthesis, signal transduction, and energy metabolism, collectively contributing to the regulation of pre-harvest sprouting.