Maize (Zea mays L.) is sensitive to salt stress, especially during seed germination and seedling morphogenesis, which limits maize growth and productivity formation. As a novel recognized plant hormone, melatonin (MT) participates in multiple growth and developmental processes and mediates biotic/abiotic stress responses, yet the effects of salt stress on maize seedlings remain unclear. Herein, we investigated the effects of 150 μM exogenous MT on multiple phenotypes and physiologic metabolisms in three-leaf seedlings across eight maize inbred lines under 180 mM NaCl salt stress, including growth parameters, stomatal morphology, photosynthetic metabolisms, antioxidant enzyme activities, and reactive oxygen species (ROS). Meanwhile, the six gene expression levels controlling antioxidant enzyme activities and photosynthetic pigment biosynthesis in two materials with contrasting salt resistance were examined for all treatments to explore the possible molecular mechanism of exogenous MT alleviating salt injury in maize. The results showed that 150 μM exogenous MT application protected membrane integrity and reduced ROS accumulation by activating the antioxidant system in leaves of maize seedlings under salt stress, their relative conductivity and H₂O₂ level average reduced by 20.91% and 17.22%, while the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) averaged increased by 13.90%, 17.02%, 22.00%, and 14.24% relative to salt stress alone. The improvement of stomatal size and the deposition of photosynthetic pigments were more favorable to enhancing photosynthesis in leaves when these seedlings treated with MT application under salt stress, their stomatal size, chlorophyll content, and net photosynthetic rate averaged increased by 11.60%, 19.64%, and 27.62%. Additionally, Gene expression analysis showed that MT stimulation significantly increased the expression of antioxidant enzyme genes (Zm00001d009990, Zm00001d047479, Zm00001d014848, and Zm00001d007234) and photosynthetic pigment biosynthesis genes (Zm00001d011819 and Zm00001d017766) under salt stress. At the same time, 150 μM MT significantly promoted seedling growth and biomass accumulation. In conclusion, our study may unravel crucial evidence of the role of MT in maize seedlings against salt stress, which can provide a novel strategy for improving maize salt stress resistance.

Low-temperature (LT) is one of the major abiotic stresses that restrict the growth and development of maize seedlings. Brassinolides (BRs) have been shown to enhance LT tolerance in several plant species; the physiological and molecular mechanisms by which BRs enhance maize tolerance are still unclear. Here, we characterized changes in the physiology and transcriptome of N192 and Ji853 seedlings at the three-leaf stage with or without 2 μM 2,4-epibrassinolide (EBR) application at 25 and 15 °C environments via high-performance liquid chromatography and RNA-Sequencing. Physiological analyses revealed that EBR increased the antioxidant enzyme activities, enhanced the cell membrane stability, decreased the malondialdehyde formation, and inhibited the reactive oxygen species (ROS) accumulation in maize seedlings under 15 °C stress; meanwhile, EBR also maintained hormone balance by increasing indole-3-acetic acid and gibberellin 3 contents and decreasing the abscisic acid level under stress. Transcriptome analysis revealed 332 differentially expressed genes (DEGs) enriched in ROS homeostasis, plant hormone signal transduction, and the mitogen-activated protein kinase (MAPK) cascade. These DEGs exhibited synergistic and antagonistic interactions, forming a complex LT tolerance network in maize. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that 109 hub genes involved in LT stress regulation pathways were discovered from the four modules with the highest correlation with target traits. In conclusion, our findings provide new insights into the molecular mechanisms of exogenous BRs in enhancing LT tolerance of maize at the seedling stage, thus opening up possibilities for a breeding program of maize tolerance to LT stress.

Serotonin (5-HT), an indoleamine compound, has been known to mediate many physiological responses of plants under environmental stress. The deep-seeding (≥20 cm) of maize seeds is an important cultivation strategy to ensure seedling emergence and survival under drought stress. However, the role of 5-HT in maize deep-seeding tolerance remains unexplored. Understanding the mechanisms and evaluating the optimal concentration of 5-HT in alleviating deep-seeding stress could benefit maize production. In this study, two maize inbred lines were treated with or without 5-HT at both sowing depths of 20 cm and 3 cm, respectively. The effects of different concentrations of 5-HT on the growth phenotypes, physiological metabolism, and gene expression of two maize inbred lines were examined at the sowing depths of 20 cm and 3 cm. Compared to the normal seedling depth of 3 cm, the elongation of the mesocotyl (average elongation 3.70 cm) and coleoptile (average elongation 0.58 cm), secretion of indole-3-acetic acid (IAA; average increased 3.73 and 0.63 ng g⁻¹ FW), and hydrogen peroxide (H₂O₂; average increased 1.95 and 0.63 μM g⁻¹ FW) in the mesocotyl and coleoptile were increased under 20 cm stress, with a concomitant decrease in lignin synthesis (average decreased 0.48 and 0.53 A₂₈₀ g⁻¹). Under 20 cm deep-seeding stress, the addition of 5-HT activated the expression of multiple genes of IAA biosynthesis and signal transduction, including Zm00001d049601, Zm00001d039346, Zm00001d026530, and Zm00001d049659, and it also stimulated IAA production in both the mesocotyl and coleoptile of maize seedlings. On the contrary, 5-HT suppressed the expression of genes for lignin biosynthesis (Zm00001d016471, Zm00001d005998, Zm00001d032152, and Zm00001d053554) and retarded the accumulation of H₂O₂ and lignin, resulting in the elongation of the mesocotyl and coleoptile of maize seedlings. A comprehensive evaluation analysis showed that the optimum concentration of 5-HT in relieving deep-seeding stress was 2.5 mg/L for both inbred lines, and 5-HT therefore could improve the seedling emergence rate and alleviate deep-seeding stress in maize seedlings. These findings could provide a novel strategy for improving maize deep-seeding tolerance, thus enhancing yield potential under drought and water stress.

Low-temperature (LT) stress seriously affects the distribution, seedling survival, and grain yield of maize. At the seedling emergence stage, maize’s coleoptile is one of the most sensitive organs in sensing LT signaling and, in general, it can envelop young leaves to protect them from LT damage. In addition, brassinolides (BRs) have been shown to enhance LT tolerance from various species, but the effects of BRs on coleoptiles in maize seedlings under LT stress are unclear. Therefore, in this study, the pre-cultured coleoptiles of Zheng58 seedlings were treated with or without 2.0 μM 24-epibrassinolide (EBR) at 25 °C and 10 °C environments for five days to analyze their physiological and transcriptomic changes. Physiological analysis showed that a 10°C LT stress increased the content of glucose (0.43 mg g⁻¹ FW), sucrose (0.45 mg g⁻¹ FW), and starch (0.76 mg g⁻¹ FW) of Zheng58 coleoptiles compared to a 25°C environment. After the coleoptiles were exposed to a 2.0 μM EBR application under 10°C temperature for five days, the contents of these three sugars continued to increase, and reached 2.68 mg g⁻¹ FW, 4.64 mg g⁻¹ FW, and 9.27 mg g⁻¹ FW, respectively, indicating that sugar signaling and metabolism played key roles in regulating LT tolerance in the coleoptiles of maize seedlings. Meanwhile, a transcriptome analysis showed that 84 and 15 differentially expressed genes (DEGs) were enriched in the sucrose and starch metabolism and photosynthesis pathways, respectively, and multiple DEGs involved in these pathways were significantly up-regulated under LT stress and EBR stimulation. Further analysis speculated that the four DEGs responsible for sucrose-phosphate synthetase (SPS, i.e., Zm00001d048979, probable sucrose-phosphate synthase 5 and Zm00001d012036, sucrose-phosphate synthase 1), sucrose synthase (SUS, Zm00001d029091, sucrose synthase 2 and Zm00001d029087, sucrose synthase 4) were crucial nodes that could potentially link photosynthesis and other unknown pathways to form the complex interaction networks of maize LT tolerance. In conclusion, our findings provide new insights into the molecular mechanisms of exogenous EBR in enhancing LT tolerance of maize seedlings and identified potential candidate genes to be used for LT tolerance breeding in maize.

Benzoxazinoids (BXs) are unique bioactive metabolites with protective and allelopathic properties in maize in response to diverse stresses. The production of BXs involves the fine regulations of BXs biosynthetic gene cluster (BGC). However, little is known about whether and how the expression pattern of BGC members is impacted by biotic and abiotic stresses. Here, maize BGC was systemically investigated and 26 BGC gene members were identified on seven chromosomes, for which Bin 4.00–4.01/4.03–4.04/7.02 were the most enriched regions. All BX proteins were clearly divided into three classes and seven subclasses, and ten conserved motifs were further identified among these proteins. These proteins were localized in the subcellular compartments of chloroplast, endoplasmic reticulum, or cytoplasmic, where their catalytic activities were specifically executed. Three independent RNA-sequencing (RNA-Seq) analyses revealed that the expression profiles of the majority of BGC gene members were distinctly affected by multiple treatments, including light spectral quality, low-temperature, 24-epibrassinolide induction, and Asian corn borer infestation. Thirteen differentially expressed genes (DEGs) with high and specific expression levels were commonly detected among three RNA-Seq, as core conserved BGC members for regulating BXs biosynthesis under multiple abiotic/biotic stimulates. Moreover, the quantitative real-time PCR (qRT-PCR) verified that six core conserved genes in BGC were significantly differentially expressed in leaves of seedlings upon four treatments, which caused significant increases in 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) content under darkness and wound treatments, whereas a clear decrease in DIMBOA content was observed under low-temperature treatment. In conclusion, the changes in BX metabolites in maize were regulated by BGC gene members in multiple stress presences. Therefore, the identification of key genes associated with BX accumulation under biotic/abiotic stresses will provide valuable gene resources for breeding maize varieties with enhanced capability to adapt to environmental stresses.

Salt stress can seriously affect the growth and development of maize (Zea mays L.), resulting in a great yield loss. Melatonin (MT), an indole hormone, is a potential enhancer of plant tolerance against salt stress. However, the complex mechanisms of MT application in enhancing maize salt tolerance are still unclear. Herein, three-leaf seedlings of salt-susceptible P138 and its salt-resistant ethyl methane sulfonate (EMS)-104 mutant were cultured with or without 150 μM MT application under 0 and 100 mM Na₂CO₃ treatments for seven days, to systematically explore the response mechanisms of exogenous MT in improving the salt tolerance of maize. The results showed that salt stress triggered an escalation in reactive oxygen species production, enhanced multiple antioxidant enzymes’ activities, impaired cellular membrane permeability, inhibited photosynthetic pigment accumulation, and ultimately undermined the vigor and photosynthetic prowess of the seedlings. While suitable MT application counteracted the detrimental impacts of Na₂CO₃ on seedlings’ growth and photosynthetic capacity, the seedling length and net photosynthetic rate of P138 and EMS-104 were increased by 5.5% and 18.7%, and 12.7% and 54.5%, respectively. Quantitative real-time PCR (qRT-PCR) analysis further showed that MT application activated the expression levels of antioxidant enzyme-related genes (Zm00001d025106, Zm00001d031908, Zm00001d027511, and Zm00001d040364) and pigment biosynthesis-related genes (Zm00001d011819 and Zm00001d017766) in both maize seedlings under Na₂CO₃ stress; they then formed a complex interaction network of gene expression, multiple physiological metabolisms, and phenotype changes to influence the salt tolerance of maize seedlings under MT or Na₂CO₃ stress. To sum up, these observations underscore that 150 μM MT can alleviate salt injury of maize seedlings, which may provide new insights for further investigating MT regulation mechanisms to enhance maize seedlings’ salt resistance.