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Isolation and characterization of a promoter responsive to salt, osmotic and dehydration stresses in soybean.

ABSTRACT: Drought stress is the main limiting factor of soybean yield. Currently, genetic engineering has been one important tool in the development of drought-tolerant cultivars. A widely used strategy is the fusion of genes that confer tolerance under the control of the CaMV35S constitutive promoter; however, stress-responsive promoters would constitute the best alternative to the generation of drought-tolerant crops. We characterized the promoter of ?-galactosidase soybean (GlymaGAL) gene that was previously identified as highly up-regulated by drought stress. The ?-glucuronidase (GUS) activity of Arabidopsis transgenic plants bearing 1000- and 2000-bp fragments of the GlymaGAL promoter fused to the uidA gene was evaluated under air-dried, polyethylene glycol (PEG) and salt stress treatments. After 24 h of air-dried and PEG treatments, the pGAL-2kb led to an increase in GUS expression in leaf and root samples when compared to the control samples. These results were corroborated by qPCR expression analysis of the uidA gene. The pGAL-1kb showed no difference in GUS activity between control and treated samples. The pGAL-2kb promoter was evaluated in transgenic soybean roots, leading to an increase in EGFP expression under air-dried treatment. Our data indicates that pGAL-2kb could be a useful tool in developing drought-tolerant cultivars by driving gene expression.

SUBMITTER: Conforte AJ

PROVIDER: S-EPMC5452143 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Isolation and characterization of a promoter responsive to salt, osmotic and dehydration stresses in soybean.

Conforte Alessandra Jordano AJ Guimarães-Dias Fábia F Neves-Borges Anna Cristina AC Bencke-Malato Marta M Felix-Whipps Durvalina D Alves-Ferreira Márcio M

Genetics and molecular biology 20170327 1 suppl 1

Drought stress is the main limiting factor of soybean yield. Currently, genetic engineering has been one important tool in the development of drought-tolerant cultivars. A widely used strategy is the fusion of genes that confer tolerance under the control of the CaMV35S constitutive promoter; however, stress-responsive promoters would constitute the best alternative to the generation of drought-tolerant crops. We characterized the promoter of α-galactosidase soybean (GlymaGAL) gene that was prev ...[more]

PMID: 28350037

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Project description:BackgroundWater and salt stresses are two important environmental factors that limit the germination of seeds in most ecological environments. Most studies conducted so far to address the genetic basis of the above phenomenon have used stress conditions that are much more extreme than those found in natural environments. Furthermore, although an excess of ions and water restrictions have similar osmotic effects on germination, the common and divergent signalling components mediating the effects of both factors remain unknown.MethodsThe germination of seeds was compared under solutions of NaCl (50 mm) and polyethylene glycol (PEG, -0·6 MPa), that establish mild stress conditions, in 28 Arabidopsis thaliana accessions. Because Bayreuth (Bay) and Shadara (Sha) accessions showed contrasting sensitivity responses to both stresses, a quantitative trait locus (QTL) analysis was carried out using Bay × Sha recombinant inbred lines (RILs) to identify loci involved in the control of germination under mild salt and osmotic stresses.Key resultsTwo loci associated with the salt sensitivity response, named SSR1 and SSR2 QTLs, and four loci for the osmotic sensitivity response, named OSR1-OSR4 QTLs, were mapped. The effects of the SSR1 QTL on toxic salt sensitivity, and the osmotic contribution of OSR1, were confirmed by heterogeneous inbred families (HIFs). Whilst the SSR1 QTL had a significant effect under a wide range of NaCl concentrations, the OSR1 QTL was confirmed only under moderate drought stress. Interestingly the OSR1 QTL also showed pleiotropic effects on biomass accumulation in response to water deficit.ConclusionsThe regulation of germination under moderate salt and osmotic stresses involves the action of independent major loci, revealing the existence of loci specifically associated with the toxic component of salt and not just its osmotic effect. Furthermore, this work demonstrates that novel loci control germination under osmotic stress conditions simulating more realistic ecological environments as found by populations of seeds in nature.

Project description:Biotic and abiotic stresses negatively affect fitness, biomass production, and crop yield in plants. The dehydration-responsive element-binding proteins (DREB) are important transcription factors (TFs), and are induced by abiotic and biotic stresses. In this study, genome-wide identification, in silico sequence, and phylogenetic analyses and expression analyses of DREB2 genes under cadmium (Cd) and salt (NaCl) stresses in sorghum (Sorghum bicolor, Sb) were performed. Six putative SbDREB2 genes were identified in sorghum genome and all contained AP2 domain (PF00847). Nucleotide diversities in SbDREB2 genes were calculated as π: 0.53 and θ: 0.39, respectively. While exon numbers of them were either one or two, length of SbDREB2 proteins ranged from 238 to 388 amino acid residues. Fifty-six cis-acting regulatory elements, which are tissue specific, light, hormone, and stress responsive, were identified in the promotor regions of SbDREB2 genes. Analyses on digital expression data indicated that SbDREB2A and SbDREB2B are more expressed genes than other SbDREB genes in sorghum. Under Cd and NaCl stresses, expressions of SbDREB2 genes were induced at different levels. All SbDREB2 genes in root were up-regulated under salt stress. In case of Cd stress, SbDREB2D gene was particularly up-regulated in leaves and roots. Co-expression analyses revealed four of TFs in co-expression network, indicating that they have roles in transcriptional cascade. Furthermore, five miRNA target regions were identified for four SbDREB2 genes, indicating their roles in post-transcriptional regulation. The predicted 3D structure of SbDREB2 proteins showed some structural divergences and structure overlap between rice and sorghum varied at between 26.58 and 50%. Finally, obtained data could be used in breeding of stress-tolerant plants, particularly genetically engineered DREB2 expressing plants. Findings in this study would also contribute to the understanding of DREB2 genes in plants, especially in sorghum.

Project description:BACKGROUND:Drought is one of the most harmful abiotic stresses for plants, leading to reduced productivity of several economically important crops and, consequently, considerable losses in the agricultural sector. When plants are exposed to stressful conditions, such as drought and high salinity, they modulate the expression of genes that lead to developmental, biochemical, and physiological changes, which help to overcome the deleterious effects of adverse circumstances. Thus, the search for new specific gene promoter sequences has proved to be a powerful biotechnological strategy to control the expression of key genes involved in water deprivation or multiple stress responses. RESULTS:This study aimed to identify and characterize the GmRD26 promoter (pGmRD26), which is involved in the regulation of plant responses to drought stress. The expression profile of the GmRD26 gene was investigated by qRT-PCR under normal and stress conditions in Williams 82, BR16 and Embrapa48 soybean-cultivars. Our data confirm that GmRD26 is induced under water deficit with different induction folds between analyzed cultivars, which display different genetic background and physiological behaviour under drought. The characterization of the GmRD26 promoter was performed under simulated stress conditions with abscisic acid (ABA), polyethylene glycol (PEG) and drought (air dry) on A. thaliana plants containing the complete construct of pGmRD26::GUS (2.054?bp) and two promoter modules, pGmRD26A::GUS (909 pb) and pGmRD26B::GUS (435?bp), controlling the expression of the ?-glucuronidase (uidA) gene. Analysis of GUS activity has demonstrated that pGmRD26 and pGmRD26A induce strong reporter gene expression, as the pAtRD29 positive control promoter under ABA and PEG treatment. CONCLUSIONS:The full-length promoter pGmRD26 and the pGmRD26A module provides an improved uidA transcription capacity when compared with the other promoter module, especially in response to polyethylene glycol and drought treatments. These data indicate that pGmRD26A may become a promising biotechnological asset with potential use in the development of modified drought-tolerant plants or other plants designed for stress responses.

Project description:BACKGROUND: The homeodomain leucine zipper (HD-Zip) transcription factor family is one of the largest plant specific superfamilies, and includes genes with roles in modulation of plant growth and response to environmental stresses. Many HD-Zip genes are characterized in Arabidopsis (Arabidopsis thaliana), and members of the family are being investigated for abiotic stress responses in rice (Oryza sativa), maize (Zea mays), poplar (Populus trichocarpa) and cucumber (Cucmis sativus). Findings in these species suggest HD-Zip genes as high priority candidates for crop improvement. RESULTS: In this study we have identified members of the HD-Zip gene family in soybean cv. 'Williams 82', and characterized their expression under dehydration and salt stress. Homology searches with BLASTP and Hidden Markov Model guided sequence alignments identified 101 HD-Zip genes in the soybean genome. Phylogeny reconstruction coupled with domain and gene structure analyses using soybean, Arabidopsis, rice, grape (Vitis vinifera), and Medicago truncatula homologues enabled placement of these sequences into four previously described subfamilies. Of the 101 HD-Zip genes identified in soybean, 88 exist as whole-genome duplication-derived gene pairs, indicating high retention of these genes following polyploidy in Glycine ~13 Mya. The HD-Zip genes exhibit ubiquitous expression patterns across 24 conditions that include 17 tissues of soybean. An RNA-Seq experiment performed to study differential gene expression at 0, 1, 6 and 12 hr soybean roots under dehydration and salt stress identified 20 differentially expressed (DE) genes. Several of these DE genes are orthologs of genes previously reported to play a role under abiotic stress, implying conservation of HD-Zip gene functions across species. Screening of HD-Zip promoters identified transcription factor binding sites that are overrepresented in the DE genes under both dehydration and salt stress, providing further support for the role of HD-Zip genes in abiotic stress responses. CONCLUSIONS: We provide a thorough description of soybean HD-Zip genes, and identify potential candidates with probable roles in dehydration and salt stress. Expression profiles generated for all soybean genes, under dehydration and salt stress, at four time points, will serve as an important resource for the soybean research community, and will aid in understanding plant responses to abiotic stress.

Dataset Information

Isolation and characterization of a promoter responsive to salt, osmotic and dehydration stresses in soybean.

Publications

Isolation and characterization of a promoter responsive to salt, osmotic and dehydration stresses in soybean.

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