Overexpression of Arabidopsis acyl‐CoA‐binding protein ACBP2 enhances drought tolerance

Arabidopsis thaliana acyl‐CoA‐binding protein 2 (ACBP2) is a stress‐responsive protein that is also important in embryogenesis. Here, we assign a role for ACBP2 in abscisic acid (ABA) signalling during seed germination, seedling development and the drought response. ACBP2 was induced by ABA and drought, and transgenic Arabidopsis overexpressing ACBP2 (ACBP2‐OXs) showed increased sensitivity to ABA treatment during germination and seedling development. ACBP2‐OXs also displayed improved drought tolerance and ABA‐mediated reactive oxygen species (ROS) production in guard cells, thereby promoting stomatal closure, reducing water loss and enhancing drought tolerance. In contrast, acbp2 mutant plants showed decreased sensitivity to ABA in root development and were more sensitive to drought stress. RNA analyses revealed that ACBP2 overexpression up‐regulated the expression of Respiratory Burst Oxidase Homolog D (AtrbohD) and AtrbohF, two NAD(P)H oxidases essential for ABA‐mediated ROS production, whereas the expression of Hypersensitive to ABA1 (HAB1), an important negative regulator in ABA signalling, was down‐regulated. In addition, transgenic plants expressing ACBP2pro:GUS showed beta‐glucuronidase (GUS) staining in guard cells, confirming a role for ACBP2 at the stomata. These observations support a positive role for ACBP2 in promoting ABA signalling in germination, seedling development and the drought response.     

Overexpression of MtCAS31 enhances drought tolerance in transgenic Arabidopsis by reducing stomatal density

? Dehydrins are a type of late embryogenesis abundant protein. Some dehydrins are involved in the response to various abiotic stresses. Accumulation of dehydrins enhances the drought, cold and salt tolerances of transgenic plants, although the underlying mechanism is unclear. MtCAS31 (Medicago Truncatula cold-acclimation specific protein 31) is a Y(2)K(4)-type dehydrin that was isolated from Medicago truncatula. ? We analyzed the subcellular and histochemical localization of MtCAS31, and the expression patterns of MtCAS31 under different stresses. Transgenic Arabidopsis that overexpressed MtCAS31 was used to determine the function of MtCAS31. A yeast two-hybrid assay was used to screen potential proteins that could interact with MtCAS31. The interaction was confirmed by bimolecular fluorescence complementation (BiFC) assay. ? After a 3-h drought treatment, the expression of MtCAS31 significantly increased 600-fold. MtCAS31 overexpression dramatically reduced stomatal density and markedly enhanced the drought tolerance of transgenic Arabidopsis. MtCAS31 could interact with AtICE1 (inducer of CBF expression 1) and the AtICE1 homologous protein Mt7g083900.1, which was identified from Medicago truncatula both in vitro and in vivo. ? Our findings demonstrate that a dehydrin induces decreased stomatal density. Most importantly, the interaction of MtCAS31 with AtICE1 plays a role in stomatal development. We hypothesize that the interaction of MtCAS31 and AtICE1 caused the decrease in stomatal density to enhance the drought resistance of transgenic Arabidopsis.     

Overexpression of Arabidopsis translationally controlled tumor protein gene AtTCTP enhances drought tolerance with rapid ABA-induced stomatal closure

Translationally controlled tumor protein (TCTP), also termed P23 in human, belongs to a family of calcium- and tubulin-binding proteins, and it is generally regarded as a growth-regulating protein. Recently, Arabidopsis TCTP (AtTCTP) has been reported to function as an important growth regulator in plants. On the other hand, plant TCTP has been suggested to be involved in abiotic stress signaling such as aluminum, salt, and water deficit by a number of microarray or proteomic analyses. In this study, the biological functions of AtTCTP were investigated by using transgenic Arabidopsis plants overexpressing AtTCTP. Interestingly, AtTCTP overexpression enhanced drought tolerance in plants. The expression analysis showed that AtTCTP was expressed in guard cells as well as in actively growing tissues. Physiological studies of the overexpression lines showed increased ABA- and calcium-induced stomatal closure ratios and faster stomatal closing responses to ABA. Furthermore, in vitro protein-protein interaction analysis confirmed the interaction between AtTCTP and microtubules, and microtubule cosedimentation assays revealed that the microtubule binding of AtTCTP increased after calcium treatment. These results demonstrate that the overexpression of AtTCTP confers drought tolerance to plants by rapid ABA-mediated stomatal closure via the interaction with microtubules in which calcium binding enhances the interaction. Collectively, the present results suggest that the plant TCTP has molecular properties similar to animal TCTPs, such as tubulin- and calcium-binding, and that it functions in ABA-mediated stomatal movement, in addition to regulating the growth of plants.     

Overexpression of membrane-associated acyl-CoA-binding protein ACBP1 enhances lead tolerance in Arabidopsis

In Arabidopsis thaliana , a family of six genes encodes acyl-CoA-binding proteins (ACBPs) that show conservation at the acyl-CoA-binding domain. They are the membrane-associated ACBP1 and ACBP2, extracellularly targeted ACBP3, kelch-motif-containing ACBP4 and ACBP5, and 10-kDa ACBP6. The acyl-CoA domain in each of ACBP1 to ACBP6 binds long-chain acyl-CoA esters in vitro , suggestive of possible roles in plant lipid metabolism. We addressed here the use of Arabidopsis ACBPs in conferring lead [Pb(II)] tolerance in transgenic plants because the 10-kDa human ACBP has been identified as a molecular target for Pb(II) in vivo . We investigated the effect of Pb(II) stress on the expression of genes encoding Arabidopsis ACBP1, ACBP2 and ACBP6. We showed that the expression of ACBP1 and ACBP2 , but not ACBP6 , in root is induced by Pb(II) nitrate treatment. In vitro Pb(II)-binding assays indicated that ACBP1 binds Pb(II) comparatively better, and ACBP1 was therefore selected for further investigations. When grown on Pb(II)-containing medium, transgenic Arabidopsis lines overexpressing ACBP1 were more tolerant to Pb(II)-induced stress than the wild type. Accumulation of Pb(II) in shoots of the ACBP1 -overepxressing plants was significantly higher than wild type. The acbp1 mutant showed enhanced sensitivity to Pb(II) when germinated and grown in the presence of Pb(II) nitrate and tolerance was restored upon complementation using an ACBP1 cDNA. Our results suggest that ACBP1 is involved in mediating Pb(II) tolerance in Arabidopsis with accumulation of Pb(II) in shoots. Such observations of Pb(II) accumulation, rather than Pb(II) extrusion, in the ACBP1 -overexpressing plants implicate possible use of ACBP1 in Pb(II) phytoremediation.     

Overexpression of the Arabidopsis 10-kilodalton acyl-coenzyme A-binding protein ACBP6 enhances freezing tolerance

Small 10-kD acyl-coenzyme A-binding proteins (ACBPs) are highly conserved proteins that are prevalent in eukaryotes. In Arabidopsis (Arabidopsis thaliana), other than the 10-kD ACBP homolog (designated Arabidopsis ACBP6), there are five larger forms of ACBPs ranging from 37.5 to 73.1 kD. In this study, the cytosolic subcellular localization of Arabidopsis ACBP6 was confirmed by analyses of transgenic Arabidopsis expressing autofluorescence-tagged ACBP6 and western-blot analysis of subcellular fractions using ACBP6-specific antibodies. The expression of Arabidopsis ACBP6 was noticeably induced at 48 h after 4 degrees C treatment by northern-blot analysis and western-blot analysis. Furthermore, an acbp6 T-DNA insertional mutant that lacked ACBP6 mRNA and protein displayed increased sensitivity to freezing temperature (-8 degrees C), while ACBP6-overexpressing transgenic Arabidopsis plants were conferred enhanced freezing tolerance. Northern-blot analysis indicated that ACBP6-associated freezing tolerance was not dependent on the induction of cold-regulated COLD-RESPONSIVE gene expression. Instead, ACBP6 overexpressors showed increased expression of mRNA encoding phospholipase Ddelta. Lipid profiling analyses of rosettes from cold-acclimated, freezing-treated (-8 degrees C) transgenic Arabidopsis plants overexpressing ACBP6 showed a decline in phosphatidylcholine (-36% and -46%) and an elevation of phosphatidic acid (73% and 67%) in comparison with wild-type plants. From our comparison, the gain in freezing tolerance in ACBP6 overexpressors that was accompanied by decreases in phosphatidylcholine and an accumulation of phosphatidic acid is consistent with previous findings on phospholipase Ddelta-overexpressing transgenic Arabidopsis. In vitro filter-binding assays indicating that histidine-tagged ACBP6 binds phosphatidylcholine, but not phosphatidic acid or lysophosphatidylcholine, further imply a role for ACBP6 in phospholipid metabolism in Arabidopsis, including the possibility of ACBP6 in the cytosolic trafficking of phosphatidylcholine.     

Arabidopsis SDIR1 enhances drought tolerance in crop plants

Arabidopsis E3 ligase salt- and drought-induced RING-finger 1 (SDIR1) has been found to be involved in abscisic acid (ABA)-related stress signaling. SDIR1-overexpressing Arabidopsis plants exhibit improved tolerance to drought. Tobacco (Nicotiana tabacum) and rice (Oryza sativa) are two important agronomic crop plants. To determine whether SDIR1 enhances drought resistance in crop plants, SDIR1 transgenic tobacco and rice plants were generated. Ectopic expression of SDIR1 in both plants conferred improved drought tolerance ability. These results suggest that SDIR1 can function as a drought-tolerance gene in both dicotyledons and monocotyledons, and that it can serve as a drought-tolerance engineering candidate gene in crop plants.     

Overexpression of heat shock protein gene PfHSP21.4 in Arabidopsis thaliana enhances heat tolerance

Nuclear-encoded chloroplast small heat shock proteins (Cp-sHSPs) play important roles in plant stress tolerance due to their abundance and diversity. Their functions in Primula under heat treatment are poorly characterized. Here, expression analysis showed that the Primula Cp-sHSP gene, PfHSP21.4, was highly induced by heat stress in all vegetative and generative tissues in addition to constitutive expression in certain development stages. PfHSP21.4 was introduced into Arabidopsis, and its function was analysed in transgenic plants. Under heat stress, the PfHSP21.4 transgenic plants showed increased heat tolerance as shown by preservation of hypocotyl elongation, membrane integrity, chlorophyll content and photosystem II activity (Fv/Fm), increased seedling survival and increase in proline content. Alleviation of oxidative damage was associated with increased activity of superoxide dismutase and peroxidase. In addition, the induced expression of HSP101, HSP70, ascorbate peroxidase and Δ1-pyrroline-5-carboxylate synthase under heat stress was more pronounced in transgenic plants than in wild-type plants. These results support the positive role of PfHSP21.4 in response to heat stress in plants.     

Overexpression of a western white pine PR10 protein enhances cold tolerance in transgenic Arabidopsis

The PmPR10-1.10 protein from western white pine is known to be associated with frost hardiness, and up-regulated by seasonal cold acclimation and biotic and abiotic stresses. To gain insight into the molecular basis of cold hardiness, we investigated the potential physiological role of PmPR10-1.10 by gene overexpression in transgenic Arabidopsis plants. A binary vector was constructed for PmPR10-1.10 synthesis in higher plants and transgenic Arabidopsis lines were generated by Agrobacterium-mediated transformation. Following Western protein blot analysis confirming target protein production, transgenic Arabidopsis lines were tested for cold tolerance by electrolyte leakage analysis post treatment of different freezing temperatures. Our results demonstrate that accumulation of PmPR10-1.10 protein resulted in significantly greater freezing tolerance in transgenic plants than in wild type plants. This indicates that the transfer and selection of cold acclimation proteins like PmPR10-1.10 may be a breeding strategy for the development of freezing tolerance in conifers.     

Overexpression of a Harpin-encoding gene hrf1 in rice enhances drought tolerance

Harpin proteins are well known as eliciters that induce multiple responses in plants, such as systemic acquired resistance, hypersensitive response, enhancement of growth, resistance to the green peach aphid, and tolerance to drought. Overexpression of Harpin-encoding genes enhances plant resistance to diseases in tobacco, rice, rape, and cotton; however, it is not yet known whether the expression of Harpin-encoding genes in vivo improves plant tolerance to abiotic stresses. The results of this study showed that overexpression of a Harpin-encoding gene hrf1 in rice increased drought tolerance through abscisic acid (ABA) signalling. hrf1- overexpression induces an increase in ABA content and promotes stomatal closure in rice. The hrf1 transgenic rice lines exhibited a significant increase in water retention ability, levels of free proline and soluble sugars, tolerance to oxidative stress, reactive oxygen species-scavenging ability, and expression levels of four stress-related genes, OsLEA3-1, OsP5CS, Mn-SOD, and NM_001074345, under drought stress. The study confirmed that hrf1 conferred enhanced tolerance to drought stress on transgenic crops. These results suggest that Harpins may offer new opportunities for generating drought resistance in other crops.     

Overexpression of SaRBP1 enhances tolerance of Arabidopsis to salt stress

Abiotic stresses are the major concern in recent years as their effect on world food production is constantly increasing. We have obtained salt tolerant Arabidopsis lines overexpressing SaRBP1 (Suaeda asparagoides RNA binding protein 1) of a Korean halophyte, S. asparagoides. Homozygous T3 Arabidopsis transgenic lines were developed and used for salt stress tolerance studies. The transgenic seedlings displayed tolerance to salt and mannitol compared to the wild type (WT) seedlings. Transgenic lines produced longer primary roots, more fresh weight, and higher number of lateral roots than WT. In planta stress tolerance assay results showed that the survival rates of transgenic plants were significantly higher than WT plants. Transgenic lines showed delayed germination under 200 mM NaCl stress. In addition, the transgenics showed higher water retention ability than WT. Subcellular localization results revealed that SaRBP1 was targeted to the cytoplasm. Northwestern blot analysis results confirmed the RNA binding property of SaRBP1. Quantitative Real-Time Polymerase Chain Reaction results revealed that many stress marker genes were upregulated by SaRBP1 overexpression. Thus, our data demonstrate that SaRBP1 overexpression lines are tolerant to salt stress. Hence, this is the first report for the functional characterization of SaRBP1, a novel RBP gene isolated from S. asparagoides cDNA library.     

Overexpression of the wheat salt tolerance-related gene TaSC enhances salt tolerance in Arabidopsis

A novel gene named TaSC was cloned from salt-tolerant wheat. Northern blot showed that the expression of TaSC in salt-tolerant wheat was up-regulated after salt stress. Real-time quantitative PCR analyses showed that TaSC expression was induced by salt and ABA in wheat. Localization analysis showed that TaSC proteins were localized to the plasma membrane in transgenic Arabidopsis thaliana. The overexpression of TaSC in Col-0 and atsc (SALK_072220) Arabidopsis strains resulted in increased salt tolerance of the transgenic plants. TaSC overexpression in Col-0 and atsc signi?cantly up-regulated the expression of AtFRY1, AtSAD1, and AtCDPK2. AtCDPK2 overexpression in atsc rescued the salt-sensitive phenotype of atsc. The TaSC gene may improve plant salt tolerance by acting via the CDPK pathway.     

Overexpression of SmLEA enhances salt and drought tolerance in Escherichia coli and Salvia miltiorrhiza

Salinity and drought are important abiotic stresses limiting plant growth and development. Late embryogenesis abundant (LEA) proteins are a group of proteins associated with tolerance to water-related stress. We previously cloned an LEA gene, SmLEA, from Salvia miltiorrhiza Bunge. Phylogenetic analysis indicated that SmLEA belongs to Group LEA14, which is involved in the dehydration response. To determine its function in detail, we have now overexpressed SmLEA in Escherichia coli and S. miltiorrhiza. The logarithmic increase in accumulations of SmLEA proteins in E. coli occurred earlier under salinity than under standard conditions. SmLEA-transformed S. miltiorrhiza plants also showed faster root elongation and a lower malondialdehyde concentration than the empty vector control plants did when cultured on MS media supplemented with 60 mM NaCl or 150 mM mannitol. Moreover, SmLEA-overexpressing transgenics experienced a less rapid rate of water loss. Under either salinity or drought, overexpressing plants had greater superoxide dismutase activity and a higher glutathione concentration. These results suggest that SmLEA may be useful in efforts to improve drought and salinity tolerance in S. miltiorrhiza. Our data also provide a good foundation for further studies into the stress resistance mechanism and molecular breeding of this valuable medicinal plant.     

Overexpression of OsHsp17.0 and OsHsp23.7 enhances drought and salt tolerance in rice

Heat shock proteins (Hsps) play an important role in plant stress tolerance. We previously reported that expression of OsHsp17.0 and OsHsp23.7 could be enhanced by heat shock treatment and/or other abiotic stresses. In this paper, stress tolerance assays of transgenic rice plants overexpressing OsHsp17.0 and OsHsp23.7 have been carried out. Both OsHsp17.0-OE and OsHsp23.7-OE transgenic lines demonstrated higher germination ability compared to wild-type (WT) plants when subjected to mannitol and NaCl. Phenotypic analysis showed that transgenic rice lines displayed a higher tolerance to drought and salt stress compared to WT plants. In addition, transgenic rice lines showed significantly lower REC, lower MDA content and higher free proline content than WT under drought and salt stresses. These results suggest that OsHsp17.0 and OsHsp23.7 play an important role in rice acclimation to salt and drought stresses and are useful for engineering drought and salt tolerance rice.