Genetic Variation, DIMBOA Accumulation, and Candidate Gene Identification in Maize Multiple Insect-Resistance.

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.

Integrated QTL Mapping, Meta-Analysis, and RNA-Sequencing Reveal Candidate Genes for Maize Deep-Sowing Tolerance.

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 F2:3 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.

Exogenous Serotonin (5-HT) Promotes Mesocotyl and Coleoptile Elongation in Maize Seedlings under Deep-Seeding Stress through Enhancing Auxin Accumulation and Inhibiting Lignin Formation.

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.

New insights into light spectral quality inhibits the plasticity elongation of maize mesocotyl and coleoptile during seed germination.

The plastic elongation of mesocotyl (MES) and coleoptile (COL), which can be repressed by light exposure, plays a vital role in maize seedling emergence and establishment under adverse environmental conditions. Understanding the molecular mechanisms of light-mediated repression of MES and COL elongation in maize will allow us to develop new strategies for genetic improvement of these two crucial traits in maize. A maize variety, Zheng58, was used to monitor the transcriptome and physiological changes in MES and COL in response to darkness, as well as red, blue, and white light. The elongation of MES and COL was significantly inhibited by light spectral quality in this order: blue light > red light > white light. Physiological analyses revealed that light-mediated inhibition of maize MES and COL elongation was closely related to the dynamics of phytohormones accumulation and lignin deposition in these tissues. In response to light exposure, the levels of indole-3-acetic acid, trans-zeatin, gibberellin 3, and abscisic acid levels significantly decreased in MES and COL; by contrast, the levels of jasmonic acid, salicylic acid, lignin, phenylalanine ammonia-lyase, and peroxidase enzyme activity significantly increased. Transcriptome analysis revealed multiple differentially expressed genes (DEGs) involved in circadian rhythm, phytohormone biosynthesis and signal transduction, cytoskeleton and cell wall organization, lignin biosynthesis, and starch and sucrose metabolism. These DEGs exhibited synergistic and antagonistic interactions, forming a complex network that regulated the light-mediated inhibition of MES and COL elongation. Additionally, gene co-expression network analysis revealed that 49 hub genes in one and 19 hub genes in two modules were significantly associated with the elongation plasticity of COL and MES, respectively. These findings enhance our knowledge of the light-regulated elongation mechanisms of MES and COL, and provide a theoretical foundation for developing elite maize varieties with improved abiotic stress resistance.

Transcriptome sequencing and single-cell sequencing analysis identify GARS1 as a potential prognostic and immunotherapeutic biomarker for multiple cancers, including bladder cancer.

Background: Glycyl-tRNA synthetase 1 (GARS1) belongs to the aminoacyl-tRNA synthetase family, playing a crucial role in protein synthesis. Previous studies have reported a close association between GARS1 and various tumors. However, the role of GARS1 in human cancer prognosis and its impact on immunology remain largely unexplored. Methods: In this study, we comprehensively analyzed GARS1 expression at the mRNA and protein levels, examined genetic alterations, and assessed its prognostic implications in pan-cancer, with a specific emphasis on the immune landscape. Furthermore, we investigated the functional enrichment of genes related to GARS1 and explored its biological functions using single-cell data. Finally, we conducted cellular experiments to validate the biological significance of GARS1 in bladder cancer cells. Results: In general, GARS1 expression was significantly upregulated across multiple cancer types, and it demonstrated prognostic value in various cancers. Gene Set Enrichment Analysis (GSEA) revealed the association of GARS1 expression with multiple immune regulatory pathways. Moreover, GARS1 exhibited significant correlations with immune infiltrating cells (such as DC, CD8+T cells, Neutrophils, and Macrophages), immune checkpoint genes (CD274, CD276), and immune regulatory factors in tumors. Additionally, we observed that GARS1 could effectively predict the response to anti-PD-L1 therapy. Notably, Ifosfamide, auranofin, DMAPT, and A-1331852 emerged as potential therapeutic agents for GARS1-upregulated tumors. Our experimental findings strongly suggest that GARS1 promotes the proliferation and migration of bladder cancer cells. Conclusion: GARS1 holds promise as a potential prognostic marker and therapeutic target for pan-cancer immunotherapy, offering valuable insights for the development of more precise and personalized approaches to tumor treatment in the future.

Transcriptomic and Metabolic Profiling Reveals a Lignin Metabolism Network Involved in Mesocotyl Elongation during Maize Seed Germination.

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.

Understanding and Comprehensive Evaluation of Cold Resistance in the Seedlings of Multiple Maize Genotypes.

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.