The DIR gene family, which encodes Dirigent proteins, plays a crucial role in plant development and stress responses. However, the functions and mechanisms of this family in maize remained underexplored. This study identified ZmDIR11, a member of the maize Dirigent protein family, and explored its role in drought tolerance. ZmDIR11 shared conserved regions with homologous proteins in wheat, rice, and Arabidopsis. RT-qPCR analysis revealed that ZmDIR11 expression is upregulated in leaves under drought and PEG stress, while subcellular localization confirmed its presence in the endoplasmic reticulum. Virus-induced gene silencing (VIGS) and EMS mutagenesis demonstrated that ZmDIR11 silencing or mutation significantly reduces drought tolerance in maize seedlings, indicating its positive regulatory role. Silencing or mutation of ZmDIR11 led to decreased growth parameters (plant height, root length, fresh weight, dry weight, and chlorophyll content) under drought stress, alongside a reduced antioxidant capacity, as evidenced by increased levels of MDA and ROS and decreased activities of SOD, CAT, and POD. Furthermore, ZmDIR11 mutation impaired the synthesis of ABA and zeatin, downregulating key genes in the ABA (ZmABA1, ZmNCED3, ZmSnRK2.6) and zeatin (ZmIPT4, ZmCKO5, ZmCKO4b) pathways. Drought-responsive genes (ZmRD20, ZmRD22, ZmDREB2A) and lignan biosynthesis genes (Zm4CL, ZmC3H, ZmCAD1) are also downregulated in ZmDIR11 mutants. In conclusion, ZmDIR11 enhances drought tolerance in maize by regulating antioxidant capacity, ABA and zeatin synthesis, and lignan metabolism. This study provides new insights into the role of DIR genes in drought tolerance and offers a potential genetic resource for breeding drought-resistant maize varieties.

The DIR (Dirigent) gene family plays a multifaceted role in plant growth, development, and stress responses, making it one of the key gene families for plant adaptation to environmental changes. However, research on ZmDIRs in maize remains limited. In this study, we identified a member of the maize DIR gene family, ZmDIR5, whose promoter region contains numerous elements associated with responses to abiotic stresses. ZmDIR5 is upregulated in response to waterlogging, salt, and drought stresses, and its protein is localized in the endoplasmic reticulum. Subsequent studies revealed that ZmDIR5-EMS (ethyl methane sulfonate) mutant lines exhibited reduced growth compared to WT (wild-type) plants under waterlogging, salt, and drought stress conditions. The mutant lines also demonstrated a relatively higher accumulation of malondialdehyde and reactive oxygen species, lower synthesis of proline and total lignans, and decreased antioxidant enzyme activity under these stress conditions. Additionally, the mutant lines displayed impaired sodium and potassium ion transport capabilities, reduced synthesis of abscisic acid and zeatin, and decreased expression of related genes. The mutation of ZmDIR5 also inhibited the phenylpropanoid biosynthesis pathway in maize. These results indicate that ZmDIR5 serves as a positive regulator of maize tolerance to waterlogging, salt, and drought stresses.

Drought and salt stresses are major abiotic factors limiting maize yield. Lignin, a key cell wall component, plays a crucial role in boosting plant stress resistance. Cinnamyl alcohol dehydrogenase (CAD) is a vital enzyme at the late stage of lignin biosynthesis; however, a systematic study of its functions in abiotic stress responses and its potential for genetic improvement in maize remains lacking. In this study, we conducted the first comprehensive, multi-dimensional analysis of the maize ZmCAD gene family, including gene identification, evolutionary relationships, protein interaction networks, and stress-responsive expression patterns. We identified 9 ZmCAD members that showed significant functional divergence in evolution, structure, and expression patterns. Expression analysis revealed complex, tissue-specific responses of ZmCAD genes to drought and salt stress, with ZmCAD6 strongly induced by drought. Importantly, through haplotype analysis of 157 waxy maize inbred lines, we successfully identified an elite haplotype (H3) of ZmCAD6 that is significantly associated with improved drought tolerance in maize. This study not only clarifies the functional differentiation mechanisms of the ZmCAD gene family but also provides the identified elite ZmCAD6-H3 haplotype as a valuable genetic resource and precise target for molecular breeding aimed at enhancing drought tolerance in maize.