TaSnRK2.4, an SNF1-type serine/threonine protein kinase of wheat (Triticum aestivum L.), confers enhanced multistress tolerance in Arabidopsis.
ABSTRACT: Osmotic stresses such as drought, salinity, and cold are major environmental factors that limit agricultural productivity worldwide. Protein phosphorylation/dephosphorylation are major signalling events induced by osmotic stress in higher plants. Sucrose non-fermenting 1-related protein kinase2 family members play essential roles in response to hyperosmotic stresses in Arabidopsis, rice, and maize. In this study, the function of TaSnRK2.4 in drought, salt, and freezing stresses in Arabidopsis was characterized. A translational fusion protein of TaSnRK2.4 with green fluorescent protein showed subcellular localization in the cell membrane, cytoplasm, and nucleus. To examine the role of TaSnRK2.4 under various environmental stresses, transgenic Arabidopsis plants overexpressing wheat TaSnRK2.4 under control of the cauliflower mosaic virus 35S promoter were generated. Overexpression of TaSnRK2.4 resulted in delayed seedling establishment, longer primary roots, and higher yield under normal growing conditions. Transgenic Arabidopsis overexpressing TaSnRK2.4 had enhanced tolerance to drought, salt, and freezing stresses, which were simultaneously supported by physiological results, including decreased rate of water loss, enhanced higher relative water content, strengthened cell membrane stability, improved photosynthesis potential, and significantly increased osmotic potential. The results show that TaSnRK2.4 is involved in the regulation of enhanced osmotic potential, growth, and development under both normal and stress conditions, and imply that TaSnRK2.4 is a multifunctional regulatory factor in Arabidopsis. Since the overexpression of TaSnRK2.4 can significantly strengthen tolerance to drought, salt, and freezing stresses and does not retard the growth of transgenic Arabidopsis plants under well-watered conditions, TaSnRK2.4 could be utilized in transgenic breeding to improve abiotic stresses in crops.
Project description:Late embryogenesis abundant (LEA) proteins belong to a large family that exists widely in plants and is mainly involved in desiccation processes during plant development or in the response to abiotic stresses. Here, we reported on an atypical LEA gene (IpLEA) related to salt tolerance from Ipomoea pes-caprae L. (Convolvulaceae). Sequence analysis revealed that IpLEA belongs to the LEA_2 (PF03168) group. IpLEA was shown to have a cytoplasmic localization pattern. Quantitative reverse transcription PCR analysis showed that IpLEA was widely expressed in different organs of the I. pes-caprae plants, and the expression levels increased following salt, osmotic, oxidative, freezing, and abscisic acid treatments. Analysis of the 1,495?bp promoter of IpLEA identified distinct cis-acting regulatory elements involved in abiotic stress. Induction of IpLEA improved Escherichia coli growth performance compared with the control under abiotic stresses. To further assess the function of IpLEA in plants, transgenic Arabidopsis plants overexpressing IpLEA were generated. The IpLEA-overexpressing Arabidopsis seedlings and adult plants showed higher tolerance to salt and drought stress than the wild-type. The transgenic plants also showed higher oxidative stress tolerance than the wild-type Arabidopsis. Furthermore, the expression patterns of a series of stress-responsive genes were affected. The results indicate that IpLEA is involved in the plant response to salt and drought, probably by mediating water homeostasis or by acting as a reactive oxygen species scavenger, thereby influencing physiological processes under various abiotic stresses in microorganisms and plants.
Project description:We report here the isolation and functional analysis of AlTMP1 gene encoding a member of the PMP3 protein family. In Aeluropus littoralis, AlTMP1 is highly induced by abscisic acid (ABA), cold, salt, and osmotic stresses. Transgenic tobacco expressing AlTMP1 exhibited enhanced tolerance to salt, osmotic, H?O?, heat and freezing stresses at the seedling stage. Under greenhouse conditions, the transgenic plants showed a higher level of tolerance to drought than to salinity. Noteworthy, AlTMP1 plants yielded two- and five-fold more seeds than non-transgenic plants (NT) under salt and drought stresses, respectively. The leaves of AlTMP1 plants accumulated lower Na? but higher K? and Ca2+ than those of NT plants. Tolerance to osmotic and salt stresses was associated with higher membrane stability, low electrolyte leakage, and improved water status. Finally, accumulation of AlTMP1 in tobacco altered the regulation of some stress-related genes in either a positive (NHX1, CAT1, APX1, and DREB1A) or negative (HKT1 and KT1) manner that could be related to the observed tolerance. These results suggest that AlTMP1 confers stress tolerance in tobacco through maintenance of ion homeostasis, increased membrane integrity, and water status. The observed tolerance may be due to a direct or indirect effect of AlTMP1 on the expression of stress-related genes which could stimulate an adaptive potential not present in NT plants.
Project description:As major environment factors, drought or high salinity affect crop growth, development and yield. Transgenic approach is an effective way to improve abiotic stress tolerance of crops. In this study, we comparatively analyzed gene structures, genome location, and the evolution of syntaxin proteins containing late embryogenesis abundant (LEA2) domain. GmSYP24 was identified as a dehydration-responsive gene. Our study showed that the GmSYP24 protein was located on the cell membrane. The overexpression of GmSYP24 (GmSYP24ox) in soybean and heteroexpression of GmSYP24 (GmSYP24hx) in Arabidopsis exhibited insensitivity to osmotic/drought and high salinity. However, wild type soybean, Arabidopsis, and the mutant of GmSYP24 homologous gene of Arabidopsis were sensitive to the stresses. Under the abiotic stresses, transgenic soybean plants had greater water content and higher activities of POD, SOD compared with non-transgenic controls. And the leaf stomatal density and opening were reduced in transgenic Arabidopsis. The sensitivity to ABA was decreased during seed germination of GmSYP24ox and GmSYP24hx. GmSYP24hx induced up-regulation of ABA-responsive genes. GmSYP24ox alters the expression of some aquaporins under osmotic/drought, salt, or ABA treatment. These results demonstrated that GmSYP24 played an important role in osmotic/drought or salt tolerance in ABA signal pathway.
Project description:Dehydrin (DHN) genes can be rapidly induced to offset water deficit stresses in plants. Here, we reported on a dehydrin gene (IpDHN) related to salt tolerance isolated from Ipomoea pes-caprae L. (Convolvulaceae). The IpDHN protein shares a relatively high homology with Arabidopsis dehydrin ERD14 (At1g76180). IpDHN was shown to have a cytoplasmic localization pattern. Quantitative RT-PCR analyses indicated that IpDHN was differentially expressed in most organs of I. pes-caprae plants, and its expression level increased after salt, osmotic stress, oxidative stress, cold stress and ABA treatments. Analysis of the 974-bp promoter of IpDHN identified distinct cis-acting regulatory elements, including an MYB binding site (MBS), ABRE (ABA responding)-elements, Skn-1 motif, and TC-rich repeats. The induced expression of IpDHN in Escherichia coli indicated that IpDHN might be involved in salt, drought, osmotic, and oxidative stresses. We also generated transgenic Arabidopsis lines that over-expressed IpDHN. The transgenic Arabidopsis plants showed a significant enhancement in tolerance to salt/drought stresses, as well as less accumulation of hydrogen peroxide (H2O2) and the superoxide radical (O2 -), accompanied by increasing activity of the antioxidant enzyme system in vivo. Under osmotic stresses, the overexpression of IpDHN in Arabidopsis can elevate the expression of ROS-related and stress-responsive genes and can improve the ROS-scavenging ability. Our results indicated that IpDHN is involved in cellular responses to salt and drought through a series of pleiotropic effects that are likely involved in ROS scavenging and therefore influence the physiological processes of microorganisms and plants exposed to many abiotic stresses.
Project description:Environmental stresses such as drought, salinity, and cold are major adverse factors that significantly affect agricultural productivity. Protein phosphorylation/dephosphorylation is a major signalling event induced by osmotic stress in higher plants. Sucrose non-fermenting 1-related protein kinase 2 (SnRK2) family members play essential roles in the response to hyperosmotic stresses in plants. In this study, the TaSnRK2.3 gene, a novel SnRK2 member was cloned, and three copies located on chromosomes 1A, 1B, and 1D were identified in common wheat. TaSnRK2.3 was strongly expressed in leaves, and responded to polyethylene glycol, NaCl, abscisic acid, and cold stresses. To characterize its function, transgenic Arabidopsis overexpressing TaSnRK2.3-GFP controlled by the cauliflower mosaic virus 35S promoter was generated and subjected to severe abiotic stresses. Overexpression of TaSnRK2.3 resulted in an improved root system and significantly enhanced tolerance to drought, salt, and freezing stresses, simultaneously demonstrated by enhanced expression of abiotic stress-responsive genes and ameliorative physiological indices, including a decreased rate of water loss, enhanced cell membrane stability, improved photosynthetic potential, and significantly increased osmotic potential and free proline content under normal and/or stressed conditions. These results demonstrate that TaSnRK2.3 is a multifunctional regulator, with potential for utilization in transgenic breeding for improved abiotic stress tolerance in crop plants.
Project description:Late embryogenesis abundant (LEA) proteins are involved in tolerance to drought, cold and high salinity in many different organisms. In this report, a LEA protein producing full-length gene OsLEA3-2 was identified in rice (Oryza sativa) using the Rapid Amplification of cDNA Ends (RACE) method. OsLEA3-2 was found to be only expressed in the embryo and can be induced by abiotic stresses. The coding protein localizes to the nucleus and overexpression of OsLEA3-2 in yeast improved growth performance compared with control under salt- and osmotic-stress conditions. OsLEA3-2 was also inserted into pHB vector and overexpressed in Arabidopsis and rice. The transgenic Arabidopsis seedlings showed better growth on MS media supplemented with 150 mM mannitol or 100 mM NaCl as compared with wild type plants. The transgenic rice also showed significantly stronger growth performance than control under salinity or osmotic stress conditions and were able to recover after 20 days of drought stress. In vitro analysis showed that OsLEA3-2 was able to protect LDH from aggregation on freezing and inactivation on desiccation. These results indicated that OsLEA3-2 plays an important role in tolerance to abiotic stresses.
Project description:Dehydrins (DHNs) play critical roles in plant adaptation to abiotic stresses. The objective of this study was to characterize DHNs in bermudagrass (Cynodon spp.). CdDHN4 gene was cloned from bermudagrass 'Tifway'. Two CdDHN4 transcripts were detected due to alternative splicing (the nonspliced CdDHN4-L and the spliced CdDHN4-S) and both the CdDHN4-S and CdDHN4-L proteins are YSK2-type DHNs, the ?-segment is present in CdDHN4-L and absent in CdDHN4-S. Transgenic Arabidopsis thaliana expressing CdDHN4-L or CdDHN4-S exhibited improved tolerance to salt, osmotic, low temperature and drought stress compared to the wild type (WT). The two transgenic lines did not differ in salt or drought tolerance, while plants expressing CdDHN4-S grew better under osmotic stress than those expressing CdDHN4-L. Both transgenic lines exhibited reduced content of malondialdehyde (MDA) and reactive oxygen species (ROS); and higher antioxidant enzymatic activities than the wild type plants under salt or drought stress. CdDHN4-S exhibited a higher ROS-scavenging capacity than CdDHN4-L.
Project description:BACKGROUND: Aquaporin (AQP) proteins function in transporting water and other small molecules through the biological membranes, which is crucial for plants to survive in drought or salt stress conditions. However, the precise role of AQPs in drought and salt stresses is not completely understood in plants. RESULTS: In this study, we have identified a PIP1 subfamily AQP (MaPIP1;1) gene from banana and characterized it by overexpression in transgenic Arabidopsis plants. Transient expression of MaPIP1;1-GFP fusion protein indicated its localization at plasma membrane. The expression of MaPIP1;1 was induced by NaCl and water deficient treatment. Overexpression of MaPIP1;1 in Arabidopsis resulted in an increased primary root elongation, root hair numbers and survival rates compared to WT under salt or drought conditions. Physiological indices demonstrated that the increased salt tolerance conferred by MaPIP1;1 is related to reduced membrane injury and high cytosolic K+/Na+ ratio. Additionally, the improved drought tolerance conferred by MaPIP1;1 is associated with decreased membrane injury and improved osmotic adjustment. Finally, reduced expression of ABA-responsive genes in MaPIP1;1-overexpressing plants reflects their improved physiological status. CONCLUSIONS: Our results demonstrated that heterologous expression of banana MaPIP1;1 in Arabidopsis confers salt and drought stress tolerances by reducing membrane injury, improving ion distribution and maintaining osmotic balance.
Project description:Aquaporins are channel proteins that facilitate the transmembrane transport of water and other small neutral molecules, thereby playing vital roles in maintaining water and nutrition homeostasis in the life activities of all organisms. <i>Canavalia rosea</i>, a seashore and mangrove-accompanied halophyte with strong adaptability to adversity in tropical and subtropical regions, is a good model for studying the molecular mechanisms underlying extreme saline-alkaline and drought stress tolerance in leguminous plants. In this study, a PIP2 gene (<i>CrPIP2;3</i>) was cloned from <i>C. rosea</i>, and its expression patterns and physiological roles in yeast and <i>Arabidopsis thaliana</i> heterologous expression systems under high salt-alkali and high osmotic stress conditions were examined. The expression of <i>CrPIP2;3</i> at the transcriptional level in <i>C. rosea</i> was affected by high salinity and alkali, high osmotic stress, and abscisic acid treatment. In yeast, the expression of <i>CrPIP2;3</i> enhanced salt/osmotic and oxidative sensitivity under high salt/osmotic and H<sub>2</sub>O<sub>2</sub> stress. The overexpression of <i>CrPIP2;3</i> in <i>A. thaliana</i> could enhance the survival and recovery of transgenic plants under drought stress, and the seed germination and seedling growth of the <i>CrPIP2;3 OX</i> (over-expression) lines showed slightly stronger tolerance to high salt/alkali than the wild-type. The transgenic plants also showed a higher response level to high-salinity and dehydration than the wild-type, mostly based on the up-regulated expression of salt/dehydration marker genes in <i>A. thaliana</i> plants. The reactive oxygen species (ROS) staining results indicated that the transgenic lines did not possess stronger ROS scavenging ability and stress tolerance than the wild-type under multiple stresses. The results confirmed that <i>CrPIP2;3</i> is involved in the response of <i>C. rosea</i> to salt and drought, and primarily acts by mediating water homeostasis rather than by acting as an ROS transporter, thereby influencing physiological processes under various abiotic stresses in plants.
Project description:We characterized an aquaporin gene HvPIP2;5 from Hordeum vulgare and investigated its physiological roles in heterologous expression systems, yeast and Arabidopsis, under high salt and high osmotic stress conditions. In yeast, the expression of HvPIP2;5 enhanced abiotic stress tolerance under high salt and high osmotic conditions. Arabidopsis plants overexpressing HvPIP2;5 also showed better stress tolerance in germination and root growth under high salt and high osmotic stresses than the wild type (WT). HvPIP2;5 overexpressing plants were able to survive and recover after a 3-week drought period unlike the control plants which wilted and died during stress treatment. Indeed, overexpression of HvPIP2;5 caused higher retention of chlorophylls and water under salt and osmotic stresses than did control. We also observed lower accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA), an end-product of lipid peroxidation in HvPIP2;5 overexpressing plants than in WT. These results suggest that HvPIP2;5 overexpression brought about stress tolerance, at least in part, by reducing the secondary oxidative stress caused by salt and osmotic stresses. Consistent with these stress tolerant phenotypes, HvPIP2;5 overexpressing Arabidopsis lines showed higher expression and activities of ROS scavenging enzymes such as catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GR), and ascorbate peroxidase (APX) under salt and osmotic stresses than did WT. In addition, the proline biosynthesis genes, ? 1-Pyrroline-5-Carboxylate Synthase 1 and 2 (P5CS1 and P5CS2) were up-regulated in HvPIP2;5 overexpressing plants under salt and osmotic stresses, which coincided with increased levels of the osmoprotectant proline. Together, these results suggested that HvPIP2;5 overexpression enhanced stress tolerance to high salt and high osmotic stresses by increasing activities and/or expression of ROS scavenging enzymes and osmoprotectant biosynthetic genes.