Regulatory mechanisms used by ZmMYB39 to enhance drought tolerance in maize (Zea mays) seedlings.

Drought is a significant abiotic stressor that limits maize (Zea mays L.) growth and development. Thus, enhancing drought tolerance is critical for promoting maize production. Our findings demonstrated that ZmMYB39 is an MYB transcription factor with transcriptional activation activity. Drought stress experiments involving ZmMYB39 overexpression and knockout lines indicated that ZmMYB39 positively regulated drought stress tolerance in maize. DAP-Seq, EMSA, dual-LUC, and RT-qPCR provided initial insights into the molecular regulatory mechanisms by which ZmMYB39 enhances drought tolerance in maize. ZmMYB39 directly promoted the expression of ZmP5CS1, ZmPOX1, ZmSOD2, ZmRD22, ZmNAC49, and ZmDREB2A, which are involved in stress resistance. ZmMYB39 enhanced drought tolerance by interacting with and promoting the expression of ZmFNR1, ZmHSP20, and ZmDOF6. Our study offers a theoretical basis for understanding the molecular regulatory networks involved in maize drought stress response. Furthermore, ZmMYB39 serves as a valuable genetic resource for breeding drought-resistant maize.

ZmABF4-ZmVIL2/ZmFIP37 module enhances drought tolerance in maize seedlings.

Drought, as a primary environmental factor, imposes significant constraints on developmental processes and productivity of plants. PHDs were identified as stress-responsive genes in a wide range of eukaryotes. However, the regulatory mechanisms governing PHD genes in maize under abiotic stress conditions are still largely unknown and require further investigation. Here, we identified a mutant, zmvil2, in the EMS mutant library with a C to T mutation in the exon of the Zm00001d053875 (VIN3-like protein 2, ZmVIL2), resulting in premature termination of protein coding. ZmVIL2 belongs to PHD protein family. Compared to WT, zmvil2 mutant exhibited increased sensitivity to drought stress. Consistently, overexpression of ZmVIL2 enhances drought resistance in maize. Y2H, BiFC, and Co-IP experiments revealed that ZmVIL2 directly interacts with ZmFIP37 (FKBP12-interacting protein of 37). zmfip37 knockout mutants also exhibit decreased drought tolerance. Interestingly, we demonstrated that ZmABF4 directly binds to the ZmVIL2 promoter to enhance its activity in yeast one hybrid (Y1H), electrophoretic mobility shift assay (EMSA) and dual luciferase reporter assays. Therefore, we uncovered a novel model ZmABF4-ZmVIL2/ZmFIP37 that promotes drought tolerance in maize. Overall, these findings have enriched the knowledge of the functions of PHD genes in maize and provides genetic resources for breeding stress-tolerant maize varieties.

Analysis of the molecular mechanisms regulating how ZmEREB24 improves drought tolerance in maize (Zea mays) seedlings.

Drought stress is one of the most limiting factors of maize productivity and can lead to a sharp reduction in the total biomass when it occurs at the seedling stage. Improving drought tolerance at the seedling stage is of great importance for maize breeding. The AP2/ERF transcription factor family plays a critical role in plant response to abiotic stresses. Here, we used a preliminary previously-generated ranscriptomic dataset to identify a highly drought-stress-responsive AP2 gene, i.e., ZmEREB24. Compared to the wild type, the overexpression of ZmEREB24 in maize significantly promotes drought tolerance of transgenic plants at the seedling stage. CRISPR/Cas9-based ZmEREB24-knockout mutants showed a drought-sensitive phenotype. RNA-seq analysis and EMSA assay revealed AATGG.CT and GTG.T.GCC motifs as the main binding sites of ZmEREB24 to the promoters of downstream target genes. DAP-seq identified four novel target genes involved in proline and sugar metabolism and hormone signal transduction of ZmEREB24. Our data indicate that ZmEREB24 plays important biological functions in regulating drought tolerance by binding to the promoters of drought stress genes and modulating their expression. The results further suggest a role of ZmEREB24 in regulating drought adaptation in maize, indicating its potential importance for employing molecular breeding in the development of high-yield drought-tolerant maize cultivars.

ZmC2H2-149 negatively regulates drought tolerance by repressing ZmHSD1 in maize.

Drought is a major abiotic stress that limits maize production worldwide. Therefore, it is of great importance to improve drought tolerance in crop plants for sustainable agriculture. In this study, we examined the roles of Cys2 /His2 zinc-finger-proteins (C2H2-ZFPs) in maize's drought tolerance as C2H2-ZFPs have been implicated for plant stress tolerance. By subjecting 150 Ac/Ds mutant lines to drought stress, we successfully identified a Ds-insertion mutant, zmc2h2-149, which shows increased tolerance to drought stress. Overexpression of ZmC2H2-149 in maize led to a decrease in both drought tolerance and crop yield. DAP-Seq, RNA-Seq, Y1H and LUC assays additionally showed that ZmC2H2-149 directly suppresses the expression of a positive drought tolerance regulator, ZmHSD1 (hydroxysteroid dehydrogenase 1). Consistently, the zmhsd1 mutants exhibited decreased drought tolerance and grain yield under water deficit conditions compared to their respective wild-type plants. Our findings thus demonstrated that ZmC2H2-149 can regulate ZmHSD1 for drought stress tolerance in maize, offering valuable theoretical and genetic resources for maize breeding programmes that aim for improving drought tolerance.

ZmELF6-ZmPRR37 module regulates maize flowering and salt response.

The control of flowering time in maize is crucial for reproductive success and yield, and it can be influenced by environmental stresses. Using the approaches of Ac/Ds transposon and transposable element amplicon sequencing techniques, we identified a Ds insertion mutant in the ZmPRR37 gene. The Ds insertion showed a significant correlation with days to anthesis. Further research indicated that ZmPRR37-CR knockout mutants exhibited early flowering, whereas ZmPRR37-overexpression lines displayed delayed flowering compared to WT under long-day (LD) conditions. We demonstrated that ZmPRR37 repressed the expression of ZmNF-YC2 and ZmNF-YA3 to delay flowering. Association analysis revealed a significant correlation between flowering time and a SNP2071-C/T located upstream of ZmPRR37. The SNP2071-C/T impacted the binding capacity of ZmELF6 to the promoter of ZmPRR37. ZmELF6 also acted as a flowering suppressor in maize under LD conditions. Notably, our study unveiled that ZmPRR37 can enhance salt stress tolerance in maize by directly regulating the expression of ABA-responsive gene ZmDhn1. ZmDhn1 negatively regulated maize salt stress resistance. In summary, our findings proposed a novel pathway for regulating photoperiodic flowering and responding to salt stress based on ZmPRR37 in maize, providing novel insights into the integration of abiotic stress signals into floral pathways.

Molecular mechanism analysis of ZmRL6 positively regulating drought stress tolerance in maize.

MYB-related genes, a subclass of MYB transcription factor family, have been documented to play important roles in biological processes such as secondary metabolism and stress responses that affect plant growth and development. However, the regulatory roles of MYB-related genes in drought stress response remain unclear in maize. In this study, we discovered that a 1R-MYB gene, ZmRL6, encodes a 96-amino acid protein and is highly drought-inducible. We also found that it is conserved in both barley (Hordeum vulgare L.) and Aegilops tauschii. Furthermore, we observed that overexpression of ZmRL6 can enhance drought tolerance while knock-out of ZmRL6 by CRISPR-Cas9 results in drought hypersensitivity. DAP-seq analyses additionally revealed the ZmRL6 target genes mainly contain ACCGTT, TTACCAAAC and AGCCCGAG motifs in their promoters. By combining RNA-seq and DAP-seq results together, we subsequently identified eight novel target genes of ZmRL6 that are involved in maize's hormone signal transduction, sugar metabolism, lignin synthesis, and redox signaling/oxidative stress. Collectively, our data provided insights into the roles of ZmRL6 in maize's drought response.

Single-cell RNA sequencing profiles reveal cell type-specific transcriptional regulation networks conditioning fungal invasion in maize roots.

Stalk rot caused by Fusarium verticillioides (Fv) is one of the most destructive diseases in maize production. The defence response of root system to Fv invasion is important for plant growth and development. Dissection of root cell type-specific response to Fv infection and its underlying transcription regulatory networks will aid in understanding the defence mechanism of maize roots to Fv invasion. Here, we reported the transcriptomes of 29 217 single cells derived from root tips of two maize inbred lines inoculated with Fv and mock condition, and identified seven major cell types with 21 transcriptionally distinct cell clusters. Through the weighted gene co-expression network analysis, we identified 12 Fv-responsive regulatory modules from 4049 differentially expressed genes (DEGs) that were activated or repressed by Fv infection in these seven cell types. Using a machining-learning approach, we constructed six cell type-specific immune regulatory networks by integrating Fv-induced DEGs from the cell type-specific transcriptomes, 16 known maize disease-resistant genes, five experimentally validated genes (ZmWOX5b, ZmPIN1a, ZmPAL6, ZmCCoAOMT2, and ZmCOMT), and 42 QTL or QTN predicted genes that are associated with Fv resistance. Taken together, this study provides not only a global view of maize cell fate determination during root development but also insights into the immune regulatory networks in major cell types of maize root tips at single-cell resolution, thus laying the foundation for dissecting molecular mechanisms underlying disease resistance in maize.

ZmGI2 regulates flowering time through multiple flower development pathways in maize.

GIGANTEA (GI) encodes a component of the circadian clock core oscillator and has been identified as a regulatory pathway of the circadian rhythm and photoperiodic flowering in model plants. However, the regulatory pathway of GI affecting flowering time is unknown in maize. Here, we identified that the zmgi2 mutant flowered earlier than the wild type under long day (LD) conditions, whereas the difference in flowering time was not apparent under short day (SD) conditions. The 24 h optimal expression of the gene in the stem apex meristems (SAM) appeared at 9 h after dawn under LD conditions and at 11 h after dawn under SD conditions. DAP-Seq and RNA-Seq further revealed that ZmGI2 delays flowering by directly binding to the upstream regions of ZmVOZs, ZmZCN8 and ZmFPF1 to repress the expression of these genes and by directly binding to the upstream regions of ZmARR11, ZmDOF and ZmUBC11 to promote the expression of these genes. The genetic and biochemical evidence suggests a model for the potential role of ZmGI2 in regulating the flowering time-dependent photoperiodic pathway. This study provides novel insights into the function of ZmGIs in maize and further demonstrates their potential importance for floral transition. These results contribute to a comprehensive understanding of the molecular mechanisms and regulatory networks of GI transcription factors in regulating flowering time in maize.

Integrative transcriptome and proteome analysis reveals maize responses to Fusarium verticillioides infection inside the stalks.

Fusarium stalk rot caused by Fusarium verticillioides is one of the most devastating diseases of maize that causes significant yield losses and poses potential security concerns for foods worldwide. The underlying mechanisms of maize plants regulating defence against the disease remain poorly understood. Here, integrative proteomic and transcriptomic analyses were employed to identify pathogenesis-related protein genes by comparing differentially expressed proteins (DEPs) and differentially expressed genes (DEGs) in maize stalks after inoculation with F. verticillioides. Functional enrichment analysis showed that DEGs and DEPs were mainly enriched in glutathione metabolism, starch and sucrose metabolism, amino sugar and nucleotide sugar metabolism, linoleic acid metabolism, and phenylpropanoid biosynthesis. Fourteen DEGs and DEGs that were highly elevated after inoculation with F. verticillioides were confirmed with parallel reaction monitoring and reverse transcription-quantitative PCR, demonstrating the accountability and reliability of proteomic and transcriptomic data. We also assessed the potential roles of defence-related genes ZmCTA1, ZmWIP1, and ZmLOX2, identified from the multi-omics analysis, during the process of F. verticillioides infection through virus-induced gene silencing. The elevation of stalk rot symptomatic characteristics in the silenced plants revealed their contribution to resistance. We further functionally characterized the roles of ZmLOX2 expression in the defence response of maize plants conditioning fungal invasion via the salicylic acid-dependent pathway. Collectively, this study provides a comprehensive analysis of transcriptome and proteome of maize stalks following F. verticillioides inoculation, and defence-related genes that could inform selection of new genes as targets in breeding strategies.

De novo genome assembly and analyses of 12 founder inbred lines provide insights into maize heterosis.

Hybrid maize displays superior heterosis and contributes over 30% of total worldwide cereal production. However, the molecular mechanisms of heterosis remain obscure. Here we show that structural variants (SVs) between the parental lines have a predominant role underpinning maize heterosis. De novo assembly and analyses of 12 maize founder inbred lines (FILs) reveal abundant genetic variations among these FILs and, through expression quantitative trait loci and association analyses, we identify several SVs contributing to genomic and phenotypic differentiations of various heterotic groups. Using a set of 91 diallel-cross F1 hybrids, we found strong positive correlations between better-parent heterosis of the F1 hybrids and the numbers of SVs between the parental lines, providing concrete genomic support for a prevalent role of genetic complementation underlying heterosis. Further, we document evidence that SVs in both ZAR1 and ZmACO2 contribute to yield heterosis in an overdominance fashion. Our results should promote genomics-based breeding of hybrid maize.

Functional analysis of a late embryogenesis abundant protein ZmNHL1 in maize under drought stress.

Maize is an important feed and industrial cereal crop and is crucial for global food security. The development of drought-tolerant genotypes is a major aim of breeding programs to fight water scarcity and maintain sustainable maize production. Late embryogenesis abundant (LEA) proteins are a family of proteins related to osmotic regulation that widely exist in organisms. Here, we implemented a previously generated maize transcriptomic dataset to identify a drought-responsive gene designated ZmNHL1. Bioinformatics analysis of ZmNHL1 showed that the protein encoded by ZmNHL1 belongs to the LEA-2 protein family. Tissue specific expression analysis showed that ZmNHL1 is relatively abundant in stems and leaves, highly expressed in tassels and only slightly expressed in roots, pollens and ears. Moreover, the activity of SOD and POD of plants from three 35S::ZmNHL1 transgenic lines under either the induced drought stress conditions (by 20% PEG6000) or the natural water deficit treatment (by water withholding) were higher than that of the WT plants, while the electrolyte leakage of the 35S::ZmNHL1 transgenic plants was lower than that of the WT plants under both drought treatments. Our data further revealed that ZmNHL1 promotes maize tolerance to drought stress in 35S::ZmNHL1 transgenic plants by improving ROS scavenging and maintaining the cell membrane permeability. Overall, our data revealed that ZmNHL1 promotes maize tolerance to drought stress and contributes to provide elite germplasm resources for maize drought tolerance breeding programs.

ZmDWF1 regulates leaf angle in maize.

Leaf angle (LA) is a critical agronomic trait enhancing grain yield under high-density planting in maize. A number of researches have been conducted in recent years to investigate the quantitative trait loci/genes responsible for LA variation, while only a few genes were identified through map-based cloning. Here we cloned the ZmDWF1 gene, which was previously reported to encode Δ24-sterol reductase in the brassinosteroids (BRs) biosynthesis pathway. Overexpression of ZmDWF1 resulted in enlarged LA, indicating that ZmDWF1 is a positive regulator of LA in maize. To reveal the regulatory framework of ZmDWF1, we conducted RNA-Sequencing and yeast-two hybrid (Y2H) screening analysis. RNA-Sequencing analyzing results indicate ZmDWF1 mainly affected expression level of genes involved in cell wall associated metabolism and hormone metabolism including BR, gibberellin, and auxin. Y2H screening with Bi-FC assay confirmed three proteins (ZmPP2C-1, ZmROF1, and ZmTWD1) interacting with ZmDWF1. We revealed a new regulatory network of ZmDWF1 gene in controlling plant architecture in maize.

A Nucleoporin NUP58 modulates responses to drought and salt stress in maize (Zea mays L.).

Nuclear pore complex (NUP) is the main transport channel between cytoplasm and nucleoplasm, which plays an important role in stress response. The function of NUPs was widely reported in yeast and vertebrate but rarely in plants. Here, we identified a nuclear pore complex (ZmNUP58), that is tightly associated with drought and salt tolerance phenotype accompanied with phenotypic and physiological changes under drought and salt stress. The overexpression of ZmNUP58 in maize (Zea mays L.) significantly promotes both chlorophyll content and activities of antioxidant enzymes under drought- and salt-stressed conditions. RNA-Seq analysis showed that ZmNUP58 could regulate the expression of genes related to phytohormone synthesis and signaling, osmotic adjustment substances, antioxidant enzyme system, cell wall biosynthesis, glucose metabolism and aquaporin. The results provide novel insights into the regulatory role of ZmNUP58 in improving drought and salt tolerance through regulating phytohormone and other stress response genes in maize.

Genome-wide identification of HD-ZIP transcription factors in maize and their regulatory roles in promoting drought tolerance.

Drought is the main limiting factor of maize productivity, therefore improving drought tolerance in maize has potential practical importance. Cloning and functional verification of drought-tolerant genes is of great importance to understand molecular mechanisms under drought stress. Here, we employed a bioinformatic pipeline to identify 42 ZmHDZ drought responsive genes using previously reported maize transcriptomic datasets. The coding sequences, exon-intron structure and domain organization of all the 42 genes were identified. Phylogenetic analysis revealed evolutionary conservation of members of the ZmHDZ genes in maize. Several regulatory elements associated with drought tolerance were identified in the promoter regions of ZmHDZ genes, indicating the implication of these genes in plant response to drought stress. 42 ZmHDZ genes were distributed unevenly on 10 chromosomes, and 24 pairs of gene duplications were the segmental duplication. The expression of several ZmHDZ genes was upregulated under drought stress, and ZmHDZ9 overexpressing transgenic plants exhibited higher SOD and POD activities and higher accumulation of soluble proteins under drought stress which resulted in enhanced developed phenotype and improved resistance. The present study provides evidence for the evolutionary conservation of HD-ZIP transcription factors homologs in maize. The results further provide a comprehensive insight into the roles of ZmHDZ genes in regulating drought stress tolerance in maize.

The tomato OST1-VOZ1 module regulates drought-mediated flowering.

Flowering is a critical agricultural trait that substantially affects tomato fruit yield. Although drought stress influences flowering time, the molecular mechanism underlying drought-regulated flowering in tomato remains elusive. In this study, we demonstrated that loss of function of tomato OPEN STOMATA 1 (SlOST1), a protein kinase essential for abscisic acid (ABA) signaling and abiotic stress responses, lowers the tolerance of tomato plants to drought stress. slost1 mutants also exhibited a late flowering phenotype under both normal and drought stress conditions. We also established that SlOST1 directly interacts with and phosphorylates the NAC (NAM, ATAF and CUC)-type transcription factor VASCULAR PLANT ONE-ZINC FINGER 1 (SlVOZ1), at residue serine 67, thereby enhancing its stability and nuclear translocation in an ABA-dependent manner. Moreover, we uncovered several SlVOZ1 binding motifs from DNA affinity purification sequencing analyses and revealed that SlVOZ1 can directly bind to the promoter of the major flowering-integrator gene SINGLE FLOWER TRUSS to promote tomato flowering transition in response to drought. Collectively, our data uncover the essential role of the SlOST1-SlVOZ1 module in regulating flowering in response to drought stress in tomato and offer insights into a novel strategy to balance drought stress response and flowering.

ZmERF21 directly regulates hormone signaling and stress-responsive gene expression to influence drought tolerance in maize seedlings.

Drought stress adversely impacts crop development and yield. Maize frequently encounters drought stress during its life cycle. Improvement of drought tolerance is a priority of maize breeding programs. Here, we identified a novel transcription factor encoding gene, APETALA2 (AP2)/Ethylene response factor (ERF), which is tightly associated with drought tolerance in maize seedlings. ZmERF21 is mainly expressed in the root and leaf and it can be highly induced by polyethylene glycol treatment. Genetic analysis showed that the zmerf21 mutant plants displayed a reduced drought tolerance phenotype, accompanied by phenotypical and physiological changes that are commonly observed in drought conditions. Overexpression of ZmERF21 in maize significantly increased the chlorophyll content and activities of antioxidant enzymes under drought conditions. RNA-Seq and DNA affinity purification sequencing analysis further revealed that ZmERF21 may directly regulate the expression of genes related to hormone (ethylene, abscisic acid) and Ca signaling as well as other stress-response genes through binding to the promoters of potential target genes. Our results thereby provided molecular evidence of ZmERF21 is involved in the drought stress response of maize.

Beyond stress response: OST1 opening doors for plants to grow.

The sucrose non-fermenting 1 (SNF1)-related protein kinase 2 (SnRK2) family members have been discovered to regulate abiotic stress response via the abscisic acid (ABA)-independent and dependent signaling pathways. SnRK2.6, also known as Open Stomata 1 (OST1), is a serine/threonine protein kinase that plays critical roles in linking ABA receptor complexes and downstream components such as transcription factors and anion channels to regulate stress response. Asides from its well-known regulatory roles in stomatal movement and cold stress response, OST1 has also been demonstrated recently to modulate major developmental roles of flowering and growth in plants. In this review, we will discuss about the various roles of OST1 as well as the 'doors' that OST1 can 'open' to help plants perform stress adaptation. Therefore, we will address how OST1 can regulate stomata apertures, cold stress tolerance as well as other aspects of its emerging roles such as balancing flowering and root growth in response to drought.

TEAseq-based identification of 35,696 Dissociation insertional mutations facilitates functional genomic studies in maize.

In plants, transposable element (TE)-triggered mutants are important resources for functional genomic studies. However, conventional approaches for genome-wide identification of TE insertion sites are costly and laborious. This study developed a novel, rapid, and high-throughput TE insertion site identification workflow based on next-generation sequencing and named it Transposable Element Amplicon Sequencing (TEAseq). Using TEAseq, we systemically profiled the Dissociation (Ds) insertion sites in 1606 independent Ds insertional mutants in advanced backcross generation using K17 as background. The Ac-containing individuals were excluded for getting rid of the potential somatic insertions. We characterized 35,696 germinal Ds insertions tagging 10,323 genes, representing approximately 23.3% of the total genes in the maize genome. The insertion sites were presented in chromosomal hotspots around the ancestral Ds loci, and insertions occurred preferentially in gene body regions. Furthermore, we mapped a loss-of-function AGL2 gene using bulked segregant RNA-sequencing assay and proved that AGL2 is essential for seed development. We additionally established an open-access database named MEILAM for easy access to Ds insertional mutations. Overall, our results have provided an efficient workflow for TE insertion identification and rich sequence-indexed mutant resources for maize functional genomic studies.

ZmCCT regulates photoperiod-dependent flowering and response to stresses in maize.

BACKGROUND: Appropriate flowering time is very important to the success of modern agriculture. Maize (Zea mays L.) is a major cereal crop, originated in tropical areas, with photoperiod sensitivity. Which is an important obstacle to the utilization of tropical/subtropical germplasm resources in temperate regions. However, the study on the regulation mechanism of photoperiod sensitivity of maize is still in the early stage. Although it has been previously reported that ZmCCT is involved in the photoperiod response and delays maize flowering time under long-day conditions, the underlying mechanism remains unclear. RESULTS: Here, we showed that ZmCCT overexpression delays flowering time and confers maize drought tolerance under LD conditions. Implementing the Gal4-LexA/UAS system identified that ZmCCT has a transcriptional inhibitory activity, while the yeast system showed that ZmCCT has a transcriptional activation activity. DAP-Seq analysis and EMSA indicated that ZmCCT mainly binds to promoters containing the novel motifs CAAAAATC and AAATGGTC. DAP-Seq and RNA-Seq analysis showed that ZmCCT could directly repress the expression of ZmPRR5 and ZmCOL9, and promote the expression of ZmRVE6 to delay flowering under long-day conditions. Moreover, we also demonstrated that ZmCCT directly binds to the promoters of ZmHY5, ZmMPK3, ZmVOZ1 and ZmARR16 and promotes the expression of ZmHY5 and ZmMPK3, but represses ZmVOZ1 and ZmARR16 to enhance stress resistance. Additionally, ZmCCT regulates a set of genes associated with plant development. CONCLUSIONS: ZmCCT has dual functions in regulating maize flowering time and stress response under LD conditions. ZmCCT negatively regulates flowering time and enhances maize drought tolerance under LD conditions. ZmCCT represses most flowering time genes to delay flowering while promotes most stress response genes to enhance stress tolerance. Our data contribute to a comprehensive understanding of the regulatory mechanism of ZmCCT in controlling maize flowering time and stress response.