Maize is a cold-sensitive crop, and it exhibits severe retardation of growth and development when exposed to cold snaps during and right after seedling emergence. Although different agronomic, physiological, and molecular approaches have been tried to overcome the problems related to cold stress in recent years, the mechanisms causing cold resistance in maize are still unclear. Screening and breeding of varieties for cold resistance may be a sustainable option to boost maize production under low-temperature environments. Herein, seedlings of 39 different maize genotypes were treated under both 10 °C low temperature and 22 °C normal temperature conditions for 7 days, to assess the changes in seven growth parameters, two membrane characteristics, two reactive oxygen species (ROS) levels, and four antioxidant enzymes activities. The changes in ten photosynthetic performances, one osmotic substance accumulation, and three polyamines (PAs) metabolisms were also measured. Results indicated that significant differences among genotypes, temperature treatments, and their interactions were found in 29 studied traits, and cold–stressed seedlings were capable to enhance their cold resistance by maintaining high levels of membrane stability index (66.07%); antioxidant enzymes activities including the activity of superoxide dismutase (2.44 Unit g⁻¹ protein), peroxidase (1.65 Unit g⁻¹ protein), catalase (0.65 μM min⁻¹ g⁻¹ protein), and ascorbate peroxidase (5.45 μM min⁻¹ g⁻¹ protein); chlorophyll (Chl) content, i.e., Chl a (0.36 mg g⁻¹ FW) and Chl b (0.40 mg g⁻¹ FW); photosynthetic capacity such as net photosynthetic rate (5.52 μM m⁻² s⁻¹) and ribulose 1,5–biphosphate carboxylase activity (6.57 M m⁻² s⁻¹); PAs concentration, mainly putrescine (274.89 nM g⁻¹ FW), spermidine (52.69 nM g⁻¹ FW), and spermine (45.81 nM g⁻¹ FW), particularly under extended cold stress. Importantly, 16 traits can be good indicators for screening of cold–resistant genotypes of maize. Gene expression analysis showed that GRMZM2G059991, GRMZM2G089982, GRMZM2G088212, GRMZM2G396553, GRMZM2G120578, and GRMZM2G396856 involved in antioxidant enzymes activity and PAs metabolism, and these genes may be used for genetic modification to improve maize cold resistance. Moreover, seven strong cold–resistant genotypes were identified, and they can be used as parents in maize breeding programs to develop new varieties.

Lignin is an important factor affecting agricultural traits. The mechanism of lignin metabolism in maize (Zea mays) mesocotyl elongation was investigated during seed germination. Maize seeds were treated with 24-epibrassinolide (EBR) and brassinazole stimulation under 3 and 20 cm deep-seeding stress. Mesocotyl transcriptome sequencing together with targeted metabolomics analysis and physiological measurements were employed in two contrasting genotypes. Our results revealed differentially expressed genes (DEGs) were significantly enriched in phenylpropanoid biosynthesis, plant hormone signal transduction, flavonoid biosynthesis, and alpha-linolenic acid metabolism. There were 153 DEGs for lignin biosynthesis pathway, 70 DEGs for peroxisome pathway, and 325 differentially expressed transcription factors (TFs) of MYB, NAC, WRKY, and LIM were identified in all comparisons, and highly interconnected network maps were generated among multiple TFs (MYB and WRKY) and DEGs for lignin biosynthesis and peroxisome biogenesis. This caused p-coumaraldehyde, p-coumaryl alcohol, and sinapaldehyde down-accumulation, however, caffeyl aldehyde and caffeyl alcohol up-accumulation. The sum/ratios of H-, S-, and G-lignin monomers was also altered, which decreased total lignin formation and accumulation, resulting in cell wall rigidity decreasing. As a result, a significant elongation of maize mesocotyl was detected under deep-seeding stress and EBR signaling. These findings provide information on the molecular mechanisms controlling maize seedling emergence under deep-seeding stress and will aid in the breeding of deep-seeding maize cultivars.

Maize seedlings contain high amounts of 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), and the effect of DIMBOA is directly associated with multiple insect-resistance against insect pests such as Asian corn borer and corn leaf aphids. Although numerous genetic loci for multiple insect-resistant traits have been identified, little is known about genetic controls regarding DIMBOA content. In this study, the best linear unbiased prediction (BLUP) values of DIMBOA content in two ecological environments across 310 maize inbred lines were calculated; and their phenotypic data and BLUP values were used for marker-trait association analysis. We identified nine SSRs that were significantly associated with DIMBOA content, which explained 4.30–20.04% of the phenotypic variation. Combined with 47 original genetic loci from previous studies, we detected 19 hot loci and approximately 11 hot loci (in Bin 1.04, Bin 2.00–2.01, Bin 2.03–2.04, Bin 4.00–4.03, Bin 5.03, Bin 5.05–5.07, Bin 8.01–8.03, Bin 8.04–8.05, Bin 8.06, Bin 9.01, and Bin 10.04 regions) supported pleiotropy for their association with two or more insect-resistant traits. Within the 19 hot loci, we identified 49 candidate genes, including 12 controlling DIMBOA biosynthesis, 6 involved in sugar metabolism/homeostasis, 2 regulating peroxidases activity, 21 associated with growth and development [(auxin-upregulated RNAs (SAUR) family member and v-myb avian myeloblastosis viral oncogene homolog (MYB)], and 7 involved in several key enzyme activities (lipoxygenase, cysteine protease, restriction endonuclease, and ubiquitin-conjugating enzyme). The synergy and antagonism interactions among these genes formed the complex defense mechanisms induced by multiple insect pests. Moreover, sufficient genetic variation was reported for DIMBOA performance and SSR markers in the 310 tested maize inbred lines, and 3 highly (DIMBOA content was 402.74–528.88 μg g⁻¹ FW) and 15 moderate (DIMBOA content was 312.92–426.56 μg g⁻¹ FW) insect-resistant genotypes were major enriched in the Reid group. These insect-resistant inbred lines can be used as parents in maize breeding programs to develop new varieties.

Synergetic elongation of mesocotyl and coleoptile are crucial in governing maize seedlings emergence, especially for the maize sown in deep soil. Studying the genomic regions controlling maize deep-sowing tolerance would aid the development of new varieties that are resistant to harsh conditions, such as drought and low temperature during seed germination. Using 346 F_2:3 maize population families from W64A × K12 cross at three sowing depths, we identified 33 quantitative trait loci (QTLs) for the emergence rate, mesocotyl, coleoptile, and seedling lengths via composite interval mapping (CIM). These loci explained 2.89% to 14.17% of phenotypic variation in a single environment, while 12 of 13 major QTLs were identified at two or more sowing environments. Among those, four major QTLs in Bin 1.09, Bin 4.08, Bin 6.01, and Bin 7.02 supported pleiotropy for multiple deep-sowing tolerant traits. Meta-analysis identified 17 meta-QTLs (MQTLs) based on 130 original QTLs from present and previous studies. RNA-Sequencing of mesocotyl and coleoptile in both parents (W64A and K12) at 3 cm and 20 cm sowing environments identified 50 candidate genes expressed differentially in all major QTLs and MQTLs regions: six involved in the circadian clock, 27 associated with phytohormones biosynthesis and signal transduction, seven controlled lignin biosynthesis, five regulated cell wall organization formation and stabilization, three were responsible for sucrose and starch metabolism, and two in the antioxidant enzyme system. These genes with highly interconnected networks may form a complex molecular mechanism of maize deep-sowing tolerance. Findings of this study will facilitate the construction of molecular modules for deep-sowing tolerance in maize. The major QTLs and MQTLs identified could be used in marker-assisted breeding to develop elite maize varieties.

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.