The plant cuticle is essential in plant defense against biotic and abiotic stresses. To systematically elucidate the genetic architecture of maize (Zea mays L.) cuticular wax metabolism, 2 cuticular wax–related traits, the chlorophyll extraction rate (CER) and water loss rate (WLR) of 389 maize inbred lines, were investigated and a genome-wide association study (GWAS) was performed using 1.25 million single nucleotide polymorphisms (SNPs). In total, 57 nonredundant quantitative trait loci (QTL) explaining 5.57% to 15.07% of the phenotypic variation for each QTL were identified. These QTLs contained 183 genes, among which 21 strong candidates were identified based on functional annotations and previous publications. Remarkably, 3 candidate genes that express differentially during cuticle development encode β-ketoacyl-CoA synthase (KCS). While ZmKCS19 was known to be involved in cuticle wax metabolism, ZmKCS12 and ZmKCS3 functions were not reported. The association between ZmKCS12 and WLR was confirmed by resequencing 106 inbred lines, and the variation of WLR was significant between different haplotypes of ZmKCS12. In this study, the loss-of-function mutant of ZmKCS12 exhibited wrinkled leaf morphology, altered wax crystal morphology, and decreased C32 wax monomer levels, causing an increased WLR and sensitivity to drought. These results confirm that ZmKCS12 plays a vital role in maize C32 wax monomer synthesis and is critical for drought tolerance. In sum, through GWAS of 2 cuticular wax–associated traits, this study reveals comprehensively the genetic architecture in maize cuticular wax metabolism and provides a valuable reference for the genetic improvement of stress tolerance in maize.

Summary: A fast evolution within mitochondria genome(s) often generates discords between nuclear and mitochondria, which is manifested as cytoplasmic male sterility (CMS) and fertility restoration (Rf) system. The maize CMS‐C trait is regulated by the chimeric mitochondrial gene, atp6c, and can be recovered by the restorer gene ZmRf5. Through positional cloning in this study, we identified the nuclear restorer gene, ZmRf5, which encodes a P‐type pentatricopeptide repeat (PPR) family protein. The over‐expression of ZmRf5 brought back the fertility to CMS‐C plants, whereas its genomic editing by CRISPR/Cas9 induced abortive pollens in the restorer line. ZmRF5 is sorted to mitochondria, and recruited RS31A, a splicing factor, through MORF8 to form a cleaving/restoring complex, which promoted the cleaving of the CMS‐associated transcripts atp6c by shifting the major cleavage site from 480th nt to 344 th nt for fast degradation, and preserved just right amount of atp6c RNA for protein translation, providing adequate ATP6C to assembly complex V, thus restoring male fertility. Interestingly, ATP6C in the sterile line CMo17A, with similar cytology and physiology changes to YU87‐1A, was accumulated much less than it in NMo17B, exhibiting a contrary trend in the YU87‐1 nuclear genome previously reported, and was restored to normal level in the presence of ZmRF5. Collectively these findings unveil a new molecular mechanism underlying fertility restoration by which ZmRF5 cooperates with MORF8 and RS31A to restore CMS‐C fertility in maize, complemented and perfected the sterility mechanism, and enrich the perspectives on communications between nucleus and mitochondria.

The maize C system of cytoplasmic male sterility (CMS) and its fertility restoration gene Rf4 have been widely used for maize hybrid production; however, the underlying mechanism is still uncertain. The sterility factor functions in mitochondria, where it interacts directly or indirectly with the restorer. Mitoproteomics can capture all participants involved in CMS and restoration at the organelle level. In the present study, we identified and quantified anther mitochondrial proteins from CMS, maintainer and restorer lines. We obtained 14,528 unique peptides belonging to 3,369 proteins. Comparative analysis of 1840 high‐confidence proteins revealed 68 were differentially accumulated proteins likely involved in CMS or its restoration within mitochondria. These proteins were mainly associated with fatty acid metabolism, amino acid metabolism and protein‐processing pathways. These results suggest that an energy deficiency caused by the sterility factor hinders other proteins or protein complexes required for pollen development through nuclear‐mitochondrial interaction. The restorer factor may boost the energy generation by activating alternative metabolic pathways and by improving the post‐translation processing efficiency of proteins in energy‐producing complexes to restore pollen fertility. Our findings may aid detailed molecular analysis and contribute to a better understanding of maize CMS‐C restoration and sterility.