The Teosinte-branched 1/Cycloidea/Proliferating (TCP) plant-specific transcription factors (TFs) have been demonstrated to play a fundamental role in plant development and organ patterning. However, it remains unknown whether or not the TCP gene family plays a role in conferring a tolerance to drought stress in maize, which is a major constraint to maize production. In this study, we identified 46 ZmTCP genes in the maize genome and systematically analyzed their phylogenetic relationships and synteny with rice, sorghum, and Arabidopsis TCP genes. Expression analysis of the 46 ZmTCP genes in different tissues and under drought conditions, suggests their involvement in maize response to drought stress. Importantly, genetic variations in ZmTCP32 and ZmTCP42 are significantly associated with drought tolerance at the seedling stage. RT-qPCR results suggest that ZmTCP32 and ZmTCP42 RNA levels are both induced by ABA, drought, and polyethylene glycol treatments. Based on the significant association between the genetic variation of ZmTCP42 and drought tolerance, and the inducible expression of ZmTCP42 by drought stress, we selected ZmTCP42, to investigate its function in drought response. We found that overexpression of ZmTCP42 in Arabidopsis led to a hypersensitivity to ABA in seed germination and enhanced drought tolerance, validating its function in drought tolerance. These results suggested that ZmTCP42 functions as an important TCP TF in maize, which plays a positive role in drought tolerance.

Waterlogging has increasingly become one of the major constraints to maize productivity in some maize production zones because it causes serious yield loss. Bulked segregant RNA-seq (BSR-seq) has been widely applied to profile candidate genes and map associated Single Nucleotide Polymorphism (SNP) markers in many species. In this study, ten waterlogging sensitive and eight tolerant inbred lines were selected from 60 maize inbred lines with waterlogging response determined and preselected by the International Maize and Wheat Improvement Center (CIMMYT) from over 400 tropical maize inbred lines. BSR-seq was performed to identify differentially expressed genes (DEGs) and SNPs associated with waterlogging tolerance. Upon waterlogging stress, 354 and 1094 genes were differentially expressed in the tolerant and sensitive pools, respectively, compared to untreated controls. When tolerant and sensitive pools were compared, 593 genes were differentially expressed under untreated and 431 genes under waterlogged conditions, of which 122 genes overlapped. To validate the BSR-seq results, the expression levels of six genes were determined by qRT-PCR. The qRT-PCR results were consistent with BSR-seq results. Comparison of allelic polymorphism in mRNA sequences between tolerant and sensitive pools revealed 165 (normal condition) and 128 (waterlogged condition) high-probability SNPs. We found 18 overlapping SNPs with genomic positions mapped. Eighteen SNPs were contained in 18 genes, and eight and nine of 18 genes were responsive to waterlogging stress in tolerant and sensitive lines, respectively. Six alleles of the 18 originated from tolerant pool were significantly up-regulated under waterlogging, but not those from sensitive pool. Importantly, one allele (GRMZM2G055704) of the six genes was mapped between umc1619 and umc1948 on chromosome 1 where a QTL associated with waterlogging tolerance was identified in a previous research, strongly indicating that GRMZM2G055704 is a candidate gene responsive to waterlogging. Our research contributes to the knowledge of the molecular mechanism for waterlogging tolerance in maize.

Vitreoscilla hemoglobin (VHb) is a type of hemoglobin found in the Gram-negative aerobic bacterium Vitreoscilla that has been shown to contribute to the tolerance of anaerobic stress in multiple plant species. Maize (Zea mays L.) is susceptible to waterlogging, causing significant yield loss. In this study, we approached this problem with the introduction of an exogenous VHb gene. We overexpressed the VHb gene in Arabidopsis and maize under the control of the CaMV35S promoter. After 14 days of waterlogging treatment, the transgenic VHb Arabidopsis plants remained green, while the controls died. Under waterlogging, important plant growth traits of VHb plants, including seedling height, primary root length, lateral root number, and shoot dry weight were significantly improved relative to those of the controls. The VHb gene was also introduced into a maize line through particle bombardment and was then transferred to two elite maize inbred lines through marker-assisted backcrossing. The introduction of VHb significantly enhanced plant growth under waterlogging stress on traits, including seedling height, primary root length, lateral root number, root dry weight, and shoot dry weight, in both Zheng58 and CML50 maize backgrounds. Under the waterlogging condition, transgenic VHb maize seedlings exhibited elevated expression of alcohol dehydrogenase (ADH1) and higher peroxidase (POD) enzyme activity. The two VHb-containing lines, Zheng58 (VHb) and CML50 (VHb), exhibited higher tolerance to waterlogging than their negative control lines (Zheng58 and CML50). These results demonstrate that the exogenous VHb gene confers waterlogging tolerance to the transgenic maize line. In Maize in the place of to the transgenic maize line, the VHb gene is a useful molecular tool for the improvement of waterlogging and submergence-tolerance.

Stearoyl-acyl carrier protein desaturase (SAD) is a key enzyme that catalyses the conversion of stearoyl-acyl carrier protein (ACP) to oleoyl-ACP, a precursor for the biosynthesis of polyunsaturated fatty acids. ZmSAD1 (GenBank: KU949326) is a major QTL for stearic acid content in maize seeds. To investigate the biological function and the application potential of maize ZmSAD1 in oil biosynthesis, we isolated the full-length ZmSAD1 cDNA from maize B73 and overexpressed it in Arabidopsis and maize. Under seed-specific overexpression of ZmSAD1 in Arabidopsis, the stearic acid content and the ratio of saturated to unsaturated fatty acids in the seeds were significantly decreased relative to those in the control. Conversely, in transgenic ZmSAD1 RNAi Arabidopsis seeds, the contents of stearic acid and long-chain saturated acids and the ratio of saturated to unsaturated fatty acids were significantly increased; in addition, the oleic acid content was significantly decreased. More importantly, transgenic ZmSAD1 maize that expressed high levels of ZmSAD1 in its mature seeds showed reduced stearic acid content (1.57 %) and a lower saturated to unsaturated fatty acid ratio (20.40 %) relative to those (1.64 % and 20.61 %, respectively) of the control. Conversely, down-regulation of ZmSAD1 in maize resulted in increased levels of stearic acid (1.78 %), long-chain saturated acids (0.85 %) and the ratio of saturated to unsaturated fatty acids (21.54 %) relative to those (1.64 %, 0.74 %, and 20.61 %, respectively) of the control, whereas the oleic acid (32.01 %) level was significantly decreased relative to that (32.68 %) of the control. Our work demonstrates that the contents of stearic acid, oleic acid, and long-chain saturated acids, and the ratio of saturated to unsaturated fatty acids, are modified in maize seeds by seed-specific overexpression or down-regulation of ZmSAD1. Therefore, the ZmSAD1 gene is a useful tool for engineering the seed oil composition in maize and other crops.