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.

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.

PTSA-catalyzed selective synthesis and antibacterial evaluation of 1,2-disubstituted benzimidazoles.

Herein, we developed a convenient and efficient method via protonation of p-toluenesulfonic acid promoted cyclocondensation of o-phenylenediamine and aldehydes for selectively synthesizing 1,2-disubstituted benzimidazoles. This method displayed broad substrate adaptability and afforded the desired products in moderate to excellent yield in short reaction time. The effect of different substituents on the yield was investigated by extending optimum reaction conditions, which was further confirmed by theoretical calculations. It suggested that the surface electrostatic potential of oxygen atom and nitrogen atom on the substrates played important role in the synthesis of 1,2-disubstituted benzimidazoles. Besides, the crystal structure of compound 2t in the orthorhombic space group P2(1)/c was presented. Also, the anti-mycolicibacterium smegmatis (MC2155) activity was evaluated using rifampicin as a positive control. The products (2a, 2b, 2c, 2i, 2j, 2k, 2m) showed good antibacterial activities which were comparable to rifampicin.

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.

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.

Overexpression of ZmPP2C55 positively enhances tolerance to drought stress in transgenic maize plants.

Serine/threonine protein phosphatases play essential roles in plants. PP2C has diverse functions related to development and stress response, while little is known about the functions of PP2C genes with respect to a variety of stresses in maize. In the present study, three ZmPP2C genes, ZmPP2C55, ZmPP2C28, and ZmPP2C71, were identified. Subcellular localization demonstrated that ZmPP2C28 and ZmPP2C71 were nuclear proteins, and ZmPP2C55 was located in both the nucleus and cytoplasm. qRT-PCR analysis showed that ZmPP2C55, ZmPP2C28, and ZmPP2C71 were expressed in roots, leaves and stems, and the three genes were responsive to drought, salt, high-temperature stress and exogenous ABA treatment. To explore the function of the ZmPP2C gene, ZmPP2C55-overexpressing transgenic lines were generated. The transgenic plants exhibited higher RWC, proline content, POD and SOD activities, GSH content and GSH/GSSG ratio and lower MDA content, electrolyte leakage and GSSG content compared with WT plants under natural stress treatment when seedlings were at the three-leaf. Our results illustrated that the overexpression of ZmPP2C55 positively enhanced tolerance to drought stress.

Transcriptional regulatory networks in response to drought stress and rewatering in maize (Zea mays L.).

Drought severely affects the growth and development of maize, but there is a certain degree of compensation effect after rewatering. This study intends to elaborate the response mechanism of maize at the physiological and molecular level as well as excavating potential genes with strong drought resistance and recovery ability. Physiological indexes analysis demonstrated that stomata conductance, transpiration rate, photosynthesis rate, antioxidant enzymes, and proline levels in maize were significantly altered in response to drought for 60 and 96 h and rewatering for 3 days. At 60 h, 96 h, and R3d, we detected 3095, 1941, and 5966 differentially expressed genes (DEGs) and 221, 226, and 215 differentially expressed miRNAs. Weighted correlation network analysis (WGCNA) showed that DEGs responded to maize drought and rewatering through participating in photosynthesis, proline metabolism, ABA signaling, and oxidative stress. Joint analysis of DEGs, miRNA, and target genes showed that zma-miR529, miR5072, zma-miR167e, zma-miR167f, zma-miR167j, miR397, and miR6214 were involved to regulate SBPs, MYBs, ARFs, laccases, and antioxidant enzymes, respectively. Hundreds of differentially expressed DNA methylation-related 24-nt siRNA clusters overlap with DEGs, indicating that DNA methylation is involved in responses under drought stress. These results provide new insights into the molecular mechanisms of drought tolerance, and may identify new targets for breeding drought-tolerant maize lines.

Systematic analysis of differentially expressed ZmMYB genes related to drought stress in maize.

MYB transcription factors play pivotal roles in hormone conduction signaling and abiotic stress response. In this study, 54 differentially expressed ZmMYB genes were identified and comprehensive analyses were conducted including gene's structure, chromosomal localization, phylogenetic tree, motif prediction, cis-elements and expression patterns. The results showed that 54 genes were unevenly distributed on 10 chromosomes and classified into eleven main subgroups by phylogenetic analysis, supported by motif and exon/intron analyses. The mainly stress-related cis-elements were ABRE, ARE, MBS and DRE-core. In addition, 8 core ZmMYB genes were identified by co-expression network. qRT-PCR results showed that the 8 ZmMYB genes exhibited different expression levels under different abiotic stresses, indicating that they were responsive to various abiotic stress. These results will provide insight for further functional investigation of ZmMYB genes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12298-021-01013-2.

Microwave-assisted efficient synthesis of 3-substituted bis-isoxazole ether bearing 2-chloro-3-pyridyl via 1,3-dipolar cycloaddition.

An efficient strategy for synthesizing of 3-substituted bis-isoxazole ether bearing 2-chloro-3-pyridyl under microwave radiation was reported. The reactive regioselectivity was improved by changing mainly the solvent and acid-binding agent. 3-(2-Chloropyridin-3-yl)-5-(((3-substituted phenyl isoxazol-5-yl)methoxy)methyl)isoxazoles were synthesized in 31-92% yields and were characterized by FT-IR, HRMS, 1H and 13C NMR spectroscopy. The single crystal of 3-(2-chloropyridin-3-yl)-5-(((3-(p-tolyl)isoxazol-5-yl)methoxy)methyl)isoxazole was obtained, and the structure of compound has also been determined by X-ray diffraction technique. Weak intra- and intermolecular C-H∙∙∙O interactions and a C-H∙∙∙π interaction link molecules into a three-dimensional network. The results showed that the synthesized compounds belonged to triclinic system, and their regioselectivity depended on the solvent and acid-binding agent. The merits of this method include the environmentally friendly, efficient, simple operation, and higher regional selectivity. An efficient synthesis of 3-substituted bis-isoxazole ethers was developed via 1,3-dipolar cycloaddition reaction starting from 3-substituted phenyl-5-((prop-2-yn-1-yloxy))methyl)isoxazoles and (Z)-2-chloro-N-hydroxynicotinimidoyl chloride using NaHCO3 as an acid-binding agent in THF solvent-dissolved trace water under catalyst-free microwave-assisted conditions.

Design, Microwave-Assisted Synthesis and in Vitro Antibacterial and Antifungal Activity of 2,5-Disubstituted Benzimidazole.

Seventeen novel 2,5-disubstituted benzimidazole derivatives were designed, synthesized and evaluated for their antibacterial activities. The tested compounds B1-B4 and C2-C6 exhibited not only good antifungal activity but also favorable broad-spectrum antibacterial activity. Also, the lowest MIC of antibacterial and antifungal activity was 2 µg/mL and 4 µg/mL, respectively. It suggested that the structure of compound including the different substituent and its sites directly affected the efficacy of the synthesized compounds.