Transcript expression profiling in two contrasting cultivars and molecular cloning of a SKP-1 like gene, a component of SCF-ubiquitin proteasome system from mungbean Vigna radiate L.
ABSTRACT: Protein degradation and turnover under various environmental stresses is basically regulated by ubiquitin-proteasome system (UPS), of which SKP1 is a very essential component. Isolation and cloning of an identified potential stress responsive candidate gene SKP1, was successfully done for the first time to fathom the role of SKP1 in drought tolerance at genetic level in drought tolerant mungbean cultivar Pratap, which was screened after a detailed physio-biochemical screening amongst seven popular mungbean cultivars. The cloned gene SKP1 (accession number KX881912) is 550?bp in length, encodes 114 amino acids. It shows high sequence homology with SKP1 from Zea mays (NP_001148633). The protein expression of isolated SKP1 was confirmed by GUS fused expression using a Histochemical assay under control as well as under drought stress. Further, up-regulation in relative expression level of SKP1 in different plant parts under drought stress confirmed its utility as a potential drought responsive candidate gene certainly demanding extensive genetic research for further incorporation in breeding programs. Moreover, the structure of VrSKP1 (Vigna radiata SKP1) has been modelled, validated and an Essential Dynamics (ED) was done on the Molecular Dynamics (MD) simulation trajectories for filtering large-scale concerted motions. Free-energy calculations on the ED revealed a complex free-energy landscape (FEL) implying the conformational diversity of the modelled VrSPK1 protein.
Project description:Mungbean (Vigna radiate L. Wilczek) is an important legume crop for its valuable nutritional and health benefits. Desiccation tolerance (DT) is a capacity of seeds to survive and maintain physiological activities during storage and under stress conditions. Many studies of DT have been reported in other legume crop, such as soybean and Medicago truncatula with little studies in the mungbean. In this study, the transcript profiles of mungbean seeds under different imbibition times were investigated for DT using RNA-sequencing (RNA-seq). A total of 3210 differentially expressed genes (DEGs) were found at the key period of DT (3-18 h of imbibition). Gene ontology (GO) and KEGG analysis showed that the terms of "response to stimulus," "transcription regulator," "methylation," and "starch and sucrose metabolism" were enriched for DT. Clustering analysis also showed that many transcription factors (MYB, AP2, and NAC), HSPs, embryogenesis abundant (LEA) proteins, and genes encoding methyltransferase and histone were differentially expressed. Nine of these DEGs were further validated by quantitative RT-PCR (qRT-PCR). Our study extends our knowledge of mungbean transcriptomes and further provides insight into the molecular mechanism of DT as well as new strategies for developing drought-tolerant crops.
Project description:Mungbean [Vigna radiata (L.) R. Wilczek var. radiata] is an important food and cash legume crop in Asia. Development of short duration varieties has paved the way for the expansion of mungbean into other regions such as Sub-Saharan Africa and South America. Mungbean productivity is constrained by biotic and abiotic factors. Bruchids, whitefly, thrips, stem fly, aphids, and pod borers are the major insect-pests. The major diseases of mungbean are yellow mosaic, anthracnose, powdery mildew, Cercospora leaf spot, halo blight, bacterial leaf spot, and tan spot. Key abiotic stresses affecting mungbean production are drought, waterlogging, salinity, and heat stress. Mungbean breeding has been critical in developing varieties with resistance to biotic and abiotic factors, but there are many constraints still to address that include the precise and accurate identification of resistance source(s) for some of the traits and the traits conferred by multi genes. Latest technologies in phenotyping, genomics, proteomics, and metabolomics could be of great help to understand insect/pathogen-plant, plant-environment interactions and the key components responsible for resistance to biotic and abiotic stresses. This review discusses current biotic and abiotic constraints in mungbean production and the challenges in genetic improvement.
Project description:Mungbean is an important pulse crop extensively cultivated in Southeast Asia for supply of easily digestible protein. Salinity severely limits the growth and productivity of mungbean, and weeding poses nutritional and disease constraints to mungbean cultivation. To pyramid both salt tolerance and protection against herbicide in mungbean, the AtNHX1 encoding tonoplast Na+/H+ antiporter from Arabidopsis, and bar gene associated with herbicide resistance were co-expressed through Agrobacterium-mediated transformation. Stress inducible expression of AtNHX1 significantly improved tolerance under salt stress to ionic, osmotic, and oxidative stresses in transgenic mungbean plants compared to the wild type (WT) plants, whereas constitutive expression of bar provided resistance to herbicide. Compared to WT, transgenic mungbean plants grew better with higher plant height, foliage, dry mass and seed yield under high salt stress (200 mM NaCl) in the greenhouse. The improved performance of transgenic plants under salt stress was associated with enhanced sequestration of Na+ in roots by vacuolar Na+/H+ antiporter and limited transport of toxic Na+ to shoots, possibly by restricting Na+ influx into shoots. Transgenic plants showed better intracellular ion homeostasis, osmoregulation, reduced cell membrane damage, improved photosynthetic capacity, and transpiration rate as compared to WT when subjected to salt stress. Reduction in hydrogen peroxide and oxygen radical production indicated enhanced protection of transgenic plants to both salt- and methyl vialogen (MV)-induced oxidative stress. This study laid a firm foundation for improving mungbean yield in saline lands in Southeast Asia.
Project description:Mungbean (Vigna radiata (L.) R. Wilzeck var. radiata) is a protein-rich short-duration legume that fits well as a rotation crop into major cereal production systems of East and South-East Asia. Salinity stress in arid areas affects mungbean, being more of a glycophyte than cereals. A significant portion of the global arable land is either salt or sodium affected. Thus, studies to understand and improve salt-stress tolerance are imminent. Here, we conducted a genome-wide association study (GWAS) to mine genomic loci underlying salt-stress tolerance during seed germination of mungbean. The World Vegetable Center (WorldVeg) mungbean minicore collection representing the diversity of mungbean germplasm was utilized as the study panel and variation for salt stress tolerance was found in this germplasm collection. The germplasm panel was classed into two agro-climatic groups and showed significant differences in their germination abilities under salt stress. A total of 5288 SNP markers obtained through genotyping-by-sequencing (GBS) were used to mine alleles associated with salt stress tolerance. Associated SNPs were identified on chromosomes 7 and 9. The associated region at chromosome 7 (position 2,696,072 to 2,809,200 bp) contains the gene Vradi07g01630, which was annotated as the ammonium transport protein (AMT). The associated region in chromosome 9 (position 19,390,227 bp to 20,321,817 bp) contained the genes Vradi09g09510 and Vradi09g09600, annotated as OsGrx_S16-glutaredoxin subgroup II and dnaJ domain proteins respectively. These proteins were reported to have functions related to salt-stress tolerance.
Project description:Heat shock transcription factors (Hsfs) are essential elements in plant signal transduction pathways that mediate gene expression in response to various abiotic stresses. Mungbean (Vigna radiata) is an important crop worldwide. The emergence of a genome database now allows for functional analysis of mungbean genes. In this study, we dissect the mungbean Hsfs using genome-wide identification and expression profiles. We characterized a total of 24 VrHsf genes and classified them into three groups (A, B, and C) based on their phylogeny and conserved domain structures. All VrHsf genes exhibit highly conserved exon-intron organization, with two exons and one intron. In addition, all VrHsf proteins contain 16 distinct motifs. Chromosome location analysis revealed that VrHsf genes are located on 8 of the 11 mungbean chromosomes, and that seven duplicated gene pairs had formed among them. Moreover, transcription patterns of VrHsf genes varied in different tissues, indicating their different roles in plant growth and development. We identified multiple stress related cis-elements in VrHsf promoter regions 2 kb upstream of the translation initiation codons, and the expression of most VrHsf genes was altered under different stress conditions, suggesting their potential functions in stress resistance pathways. These molecular characterization and expression profile analyses of VrHsf genes provide essential information for further function investigation.
Project description:Plant vacuolar NHX exchangers play a significant role in adaption to salt stress by compartmentalizing excess cytosolic Na+ into vacuoles and maintaining cellular homeostasis and ionic equilibrium. We cloned an orthologue of the vacuolar Na+/H+ antiporter gene, VrNHX1 from mungbean (Vigna radiata), an important Asiatic grain legume. The VrNHX1 (Genbank Accession number JN656211.1) contains 2095 nucleotides with an open reading frame of 1629 nucleotides encoding a predicted protein of 542 amino acids with a deduced molecular mass of 59.6 kDa. The consensus amiloride binding motif (84LFFIYLLPPI93) was observed in the third putative transmembrane domain of VrNHX1. Bioinformatic and phylogenetic analysis clearly suggested that VrNHX1 had high similarity to those of orthologs belonging to Class-I clade of plant NHX exchangers in leguminous crops. VrNHX1 could be strongly induced by salt stress in mungbean as the expression in roots significantly increased in presence of 200 mM NaCl with concomitant accumulation of total [Na+]. Induction of VrNHX1 was also observed under cold and dehydration stress, indicating a possible cross talk between various abiotic stresses. Heterologous expression in salt sensitive yeast mutant AXT3 complemented for the loss of yeast vacuolar NHX1 under NaCl, KCl and LiCl stress indicating that VrNHX1 was the orthologue of ScNHX1. Further, AXT3 cells expressing VrNHX1 survived under low pH environment and displayed vacuolar alkalinization analyzed using pH sensitive fluorescent dye BCECF-AM. The constitutive and stress inducible expression of VrNHX1 resulted in enhanced salt tolerance in transgenic Arabidopsis thaliana lines. Our work suggested that VrNHX1 was a salt tolerance determinant in mungbean.
Project description:<h4>Background</h4>Mungbean (Vigna radiata (L.) R. Wilczek, or green gram) is important tropical and sub-tropical legume and a rich source of dietary protein and micronutrients. In this study we employ GWAS to examine the genetic basis of variation in several important traits in mungbean, using the mini-core collection established by the World Vegetable Center, which includes 296 accessions that represent the major market classes. This collection has been grown in a common field plot in southern European part of Russia in 2018.<h4>Results</h4>We used 5041 SNPs in 293 accessions that passed strict filtering for genetic diversity, linkage disequilibrium, population structure and GWAS analysis. Polymorphisms were distributed among all chromosomes, but with variable density. Linkage disequilibrium decayed in approximately 105?kb. Four distinct subgroups were identified within 293 accessions with 70% of accessions attributed to one of the four populations. By performing GWAS on the mini-core collection we have found several loci significantly associated with two important agronomical traits. Four SNPs associated with possibility of maturation in Kuban territory of Southern Russia in 2018 were identified within a region of strong linkage which contains genes encoding zinc finger A20 and an AN1 domain stress-associated protein.<h4>Conclusions</h4>The core collection of mungbean established by the World Vegetable Center is a valuable resource for mungbean breeding. The collection has been grown in southern European part of Russia in 2018 under incidental stresses caused by abnormally hot weather and different photoperiod. We have found several loci significantly associated with color of hypocotyl and possibility of maturation under these stressful conditions. SNPs associated with possibility of maturation localize to a region on chromosome 2 with strong linkage, in which genes encoding zinc finger A20 and AN1 domain stress associated protein (SAP) are located. Phenotyping of WorldVeg collection for maturation traits in temperate climatic locations is important as phenology remains a critical breeding target for mungbean. As demand rises for mungbean, production in temperate regions with shorter growing seasons becomes crucial to keep up with needs. Uncovering SNPs for phenology traits will speed breeding efforts.
Project description:Yellow Mosaic Disease (YMD) in mungbean [Vigna radiata (L.) R. Wilczek] is one of the most damaging diseases in Asia. In the northern part of India, the YMD is caused by Mungbean Yellow Mosaic India Virus (MYMIV), while in southern India this is caused by Mungbean Yellow Mosaic Virus (MYMV). The molecular mechanism of YMD resistance in mungbean remains largely unknown. In this study, RNA-seq analysis was conducted between a resistant (PMR-1) and a susceptible (Pusa Vishal) mungbean genotype under infected and control conditions to understand the regulatory network operating between mungbean-YMV. Overall, 76.8 million raw reads could be generated in different treatment combinations, while mapping rate per library to the reference genome varied from 86.78% to 93.35%. The resistance to MYMIV showed a very complicated gene network, which begins with the production of general PAMPs (pathogen-associated molecular patterns), then activation of various signaling cascades like kinases, jasmonic acid (JA) and brassinosteroid (BR), and finally the expression of specific genes (like PR-proteins, virus resistance and R-gene proteins) leading to resistance response. The function of WRKY, NAC and MYB transcription factors in imparting the resistance against MYMIV could be established. The string analysis also revealed the role of proteins involved in kinase, viral movement and phytoene synthase activity in imparting YMD resistance. A set of novel stress-related EST-SSRs are also identified from the RNA-Seq data which may be used to find the linked genes/QTLs with the YMD resistance. Also, 11 defence-related transcripts could be validated through quantitative real-time PCR analysis. The identified gene networks have led to an insight about the defence mechanism operating against MYMIV infection in mungbean which will be of immense use to manage the YMD resistance in mungbean.
Project description:The beneficial microbial-plant interaction plays important role in the soil health, crop growth and productivity. Plant growth promoting rhizobacteria (PGPR) are such beneficial microorganisms, which in association with plant roots not only promote their growth but also help in counteracting the detrimental effects of soil stresses. Salt stress is one such stress, frequently confronted by the plants. The present study aimed at isolation and identification of PGPR inhabiting the mungbean rhizosphere, testing them for salt (NaCl) tolerance and subsequently in salt-supplemented mungbean crop. For this purpose, two salt-tolerant bacterial strains belonging to genus Pantoea and Enterococcus, characterized for their P-solubilization ability, indole acetic acid and siderophore production were selected. These two PGPR were further evaluated for their effect on the salt-stressed mungbean plants, grown at two salt concentrations (5 and 10 dS/m). The plants treated with the combination of PGPR showed better performance in growth (16-37 %) and yield (22-32 %), under salt stress, as compared with control. The increasing salt concentration was found to increase the membrane damage, Na+ concentration in the plants. PGPR treatments effectively reduced the Na+ concentration (17-41 %), membrane damage (1.1-1.5 folds) and enhanced the antioxidants i.e. ascorbic acid (8-26 %) and glutathione (10-30 %) in salt-stressed plants, in comparison to uninoculated stressed plants. Overall, the results indicated that both PGPR were effective as stress mitigators however, in combination they showed relatively better improvement in growth, yield as well as oxidative parameters of the salt-affected plants. These findings about the effects of native salt-tolerant PGPR Pantoea and Enterococcus sp. in mungbean crop are novel.
Project description:In the majority of wheat growing areas worldwide, the incidence of drought stress has increased significantly resulting in a negative impact on plant development and grain yield. Arbuscular mycorrhizal symbiosis is known to improve drought stress tolerance of wheat. However, quantitative trait loci (QTL) involved in the response to drought stress conditions in the presence of mycorrhizae are largely unknown. Therefore, a diverse set consisting of 94 bread wheat genotypes was phenotyped under drought stress and well watered conditions in the presence and absence of mycorrhizae. Grain yield and yield components, drought stress related traits as well as response to mycorrhizae were assessed. In parallel, wheat accessions were genotyped by using the 90k iSelect chip, resulting in a set of 15511 polymorphic and mapped SNP markers, which were used for genome-wide association studies (GWAS). In general, drought stress tolerance of wheat was significantly increased in the presence of mycorrhizae compared to drought stress tolerance in the absence of mycorrhizae. However, genotypes differed in their response to mycorrhizae under drought stress conditions. Several QTL regions on different chromosomes were detected associated with grain yield and yield components under drought stress conditions. Furthermore, two genome regions on chromosomes 3D and 7D were found to be significantly associated with the response to mycorrhizae under drought stress conditions. Overall, the results reveal that inoculation of wheat with mycorrhizal fungi significantly improves drought stress tolerance and that QTL regions associated with the response to mycorrhizae under drought stress conditions exist in wheat. Further research is necessary to validate detected QTL regions. However, this study may be the starting point for the identification of candidate genes associated with drought stress tolerance and response to mycorrhizae under drought stress conditions. Maybe in future, these initial results will help to contribute to use mycorrhizal fungi effectively in agriculture and combine new approaches i.e., use of genotypic variation in response to mycorrhizae under drought stress conditions with existing drought tolerance breeding programs to develop new drought stress tolerant genotypes.