Long-chain acyl-CoA synthetases (LACSs) are essential enzymes that activate free fatty acids to fatty acyl-CoA thioesters, playing key roles in fatty acid (FA) catabolism, lipid synthesis and storage, epidermal wax synthesis, and stress tolerance. Despite their importance, comprehensive information about LACS genes in maize, a primary food crop, remains scarce. In the present work, eleven maize LACS genes were identified and mapped across five chromosomes. Three pairs of segmentally duplicated genes were detected in the maize LACS gene family, which underwent significant purifying selection (Ka/Ks < 1). Subsequently, phylogenetic analysis indicated that ZmLACS genes were divided into four subclasses, as supported by highly conserved motifs and gene structures. On the basis of the PlantCARE database, analysis of the ZmLACS promoter regions revealed various cis-regulatory elements related to tissue-specific expression, hormonal regulation, and abiotic stress response. RT-qPCR analysis showed that ZmLACS genes exhibit tissue-specific expression patterns and respond to diverse abiotic stresses including drought and salt, as well as phytohormone abscisic acid. Furthermore, using the STRING database, several proteins involved in fatty acid and complex lipid synthesis were identified to be the potential interaction partners of ZmLACS proteins, which was also confirmed by the yeast two-hybrid (Y2H) assay, enhancing our understanding of wax biosynthesis and regulatory mechanisms in response to abiotic stresses in maize. These findings provide a comprehensive understanding of ZmLACS genes and offer a theoretical foundation for future research on the biological functions of LACS genes in maize environmental adaptability.

Drought stress globally poses a significant threat to maize (Zea mays L.) productivity and the underlying molecular mechanisms of drought tolerance remain elusive. In this study, we characterized ZmbHLH47, a basic helix–loop–helix (bHLH) transcription factor, as a positive regulator of drought tolerance in maize. ZmbHLH47 expression was notably induced by both drought stress and abscisic acid (ABA). Transgenic plants overexpressing ZmbHLH47 displayed elevated drought tolerance and ABA responsiveness, while the zmbhlh47 mutant exhibited increased drought sensitivity and reduced ABA sensitivity. Mechanistically, it was revealed that ZmbHLH47 could directly bind to the promoter of ZmSnRK2.9 gene, a member of the subgroup III SnRK2 kinases, activating its expression. Furthermore, ZmSnRK2.9-overexpressing plants exhibited enhanced ABA sensitivity and drought tolerance, whereas the zmsnrk2.9 mutant displayed a decreased sensitivity to both. Notably, overexpressing ZmbHLH47 in the zmsnrk2.9 mutant closely resembled the zmsnrk2.9 mutant, indicating the importance of the ZmbHLH47-ZmSnRK2.9 module in ABA response and drought tolerance. These findings provided valuable insights and a potential genetic resource for enhancing the environmental adaptability of maize.

Expansins contribute to maize tolerance to salt stress, but the molecular mechanisms by which they function under high-salinity conditions remain poorly understood. In this research, the α-expansin gene ZmEXPA3 was characterized. We obtained overexpression transgenic lines in maize and determined physiological and biochemical indices to elucidate its molecular role in salt stress. Our results confirmed that ZmEXPA3 functioned as a positive salt tolerance regulator and was potentially regulated by abscisic acid (ABA) and methyl jasmonate (MeJA). ZmEXPA3 located to the cytoplasm and cell wall. Overexpression of ZmEXPA3 achieved thicker cell wall and bigger cell size and thereby promoted biomass accumulation. The ZmEXPA3-OE lines showed a marked reduction in malondialdehyde (MDA) and H₂O₂ accumulation compared to the WT under salt stress. Overexpression of ZmEXPA3 elevated the enzyme activity of peroxidase (POD) and superoxide dismutase (SOD) and proline accumulation and decreased the Na⁺/K⁺ ratio in roots. Transcriptome and Gene Ontology (GO) enrichment analysis of ZmEXPA3-OE lines and WT showed that many differentially expressed genes (DEGs) were enriched in cell-wall-related terms, plant hormone response, osmotic stress response, salt stress response, oxidoreductase activity, etc. Changes in these processes may be the primary reasons why ZmEXPA3 overexpression promotes growth and salt tolerance.

ERF56, a member of the APETALA2/ETHYLENE-RESPONSIVE FACTOR (AP2/ERF) transcription factor (TF) family, was reported to be an early nitrate-responsive TF in Arabidopsis. But the function of ERF56 in nitrate signaling remains not entirely clear. This study aimed to investigate the role of ERF56 in nitrate-dependent plant growth and nitrate signaling. We confirmed with reverse transcription quantitative PCR (RT-qPCR) that the transcription of ERF56 is quickly induced by nitrate. ERF56 overexpressors displayed decreased nitrate-dependent plant growth, while erf56 mutants exhibited increased plant growth. Confocal imaging demonstrated that ERF56 is localized into nuclei. Assays with the glucuronidase (GUS) reporter showed that ERF56 is mainly expressed at the region of maturation of roots and in anthers. The dual-luciferase assay manifested that the transcription of ERF56 is not directly regulated by NIN-LIKE PROTEIN 7 (NLP7). The transcriptome analysis identified 1038 candidate genes regulated by ERF56 directly. A gene ontology (GO) over-representation analysis showed that ERF56 is involved in the processes of water transport, inorganic molecule transmembrane transport, secondary metabolite biosynthesis, and cell wall organization. We revealed that ERF56 represses nitrate-dependent growth through regulating the processes of inorganic molecule transmembrane transport, the secondary metabolite biosynthesis, and cell wall organization.

Maize is a globally vital crop for both grain and forage production. Its cultivation and growth are significantly restricted by salt stress. Expansins are non-enzymatic plant cell wall proteins that play pivotal roles in growth, development, and stress responses by mediating cell wall loosening. We identified ZmEXPA6, an α-expansin gene, as exhibiting high expression levels in maize roots under salt stress. In this study, the ZmEXPA6 gene was cloned and functionally characterized. Heterologous overexpression of ZmEXPA6 promoted root elongation and enhanced salt tolerance of Arabidopsis thaliana. Under salt stress, the ZmEXPA6 overexpression lines exhibited elevated levels of anthocyanin (61.70%, 59.70%), proline (16.39%, 15.11%), soluble sugars (11.97%, 8.68%), and soluble proteins (14.83%, 13.74%) compared to the WT. Concurrently, the expression of genes associated with anthocyanin and proline biosynthesis was markedly up-regulated in these overexpression lines. The ZmEXPA6 overexpression lines exhibited elevated activities of SOD (23.81%, 23.51%), CAT (13.86%, 10.93%), and POD (4.27%, 1.39%) compared to the WT, along with significantly reduced accumulation of MDA (23.47%, 24.48%), O₂⁻ (21.9%, 19.8%), and H₂O₂ (27.61%, 18.07%). These results indicate that ZmEXPA6 is involved in the growth and development of Arabidopsis thaliana and improves its salt tolerance through enhanced osmotic adjustment and elevated antioxidant capacity.

Improving nitrogen use efficiency (NUE) is one of the major objectives for crop breeding. As nitrate signaling plays pivotal roles in nitrogen use of plants, factors in this pathway might be valuable for improving the NUE of maize. In this research, we performed Gene Set Enrichment Analysis (GSEA) of maize transcriptomes in response to nitrate and found that the ethylene action pathway might participate in nitrate signaling. Through a modified reciprocal best hit approach, we obtained 16 maize aminocyclopropane-1-carboxylic acid (ACC) oxidase (ACO) and four ACC synthase (ACS) homologs in maize genome. In silico analyses and the reverse transcription quantitative PCR assays demonstrated that ZmACCO7, ZmACCO5, ZmACCO15, ZmACCO35, and ZmACCO31 are the top five highly expressed ACO genes, and ZmACS1 is the most highly expressed ACS gene in the primary and seminal roots of maize. We discovered that ACO and ACS genes have different regulatory modes in response to nitrate provision. Some ACO genes, which are mainly expressed in root regions far from the root tip like ZmACCO7, are repressed by nitrate, while the others, which are mainly expressed in root regions near the root tip like ZmACCO5, are induced by nitrate. ZmACS1, which has more uniform expression across maize roots, is induced in root regions near the root tip and repressed in regions far from the root tip. A phenotypic analysis indicated that upregulation of ACO and ACS genes by nitrate is linked to repression of axial root elongation by nitrate while the downregulation of these genes is associated with the promotion of growth of lateral roots of the axial roots. In addition, differences in regulation of ACO and ACS genes by nitrate were observed between genotypes, which is related to the differences in the responses of their primary root growth to nitrate. These results suggested that the ethylene synthesis pathway is involved in the responses of maize roots to nitrate, which is associated with the remodeling of maize root architecture by nitrate.