Enhanced tolerance to ozone and drought stresses in transgenic tobacco overexpressing dehydroascorbate reductase in cytosol

Ascorbate (vitamin C) is a potent antioxidant protecting plants against oxidative damage imposed by environmental stresses such as ozone and drought. Dehydroascorbate reductase (DHAR; EC 1.8.5.1) is one of the two important enzymes functioning in the regeneration of ascorbate (AsA). To examine the protective role of DHAR against oxidative stress, we developed transgenic tobacco plants overexpressing cytosolic DHAR gene from Arabidopsis thaliana . Incorporation of the transgene in the genome of tobacco plants was confirmed by polymerase chain reaction and Southern blot analysis, and its expression was confirmed by Northern and Western blot analyses. These transgenic plants exhibited 2.3–3.1 folds higher DHAR activity and 1.9–2.1 folds higher level of reduced AsA compared with non-transformed control plants. The transgenic plants showed maintained redox status of AsA and exhibited an enhanced tolerance to ozone, drought, salt, and polyethylene glycol stresses in terms of higher net photosynthesis. In this study, we report for the first time that the elevation of AsA level by targeting DHAR overexpression in cytosol properly provides a significantly enhanced oxidative stress tolerance imposed by drought and salt.     

Functional characterization of an Arabidopsis prolyl aminopeptidase AtPAP1 in response to salt and drought stresses

It has been over 50 years since the first prolyl aminopeptidase gene was identified in Escherichia coli (EC 3.4.11.5). However, up to now, few prolyl aminopeptidases have been reported to regulate osmotic stress tolerance, especially in plant. In this study, we focused on characterization of the biological functions of the Arabidopsis prolyl aminopeptidase AtPAP1 (At2g14260), which positively regulated plant tolerance to salt and drought stresses. Protein sequence alignment revealed that AtPAP1 was evolutionarily conserved among different plant species, and the smaller molecular weight and phylogenetic tree indicated that AtPAP1 belonged to the S33.001 subfamily. By using quantitative real-time PCR assays, we demonstrated that expression of the AtPAP1 gene was rapidly induced by salt and drought stresses. We also found that knockout of the AtPAP1 gene decreased, while AtPAP1 overexpression enhanced plant tolerance to salt and drought stresses. Measurements of the proline contents and the prolyl aminopeptidase activity suggested that the transgenic plants accumulated more free proline and exhibited higher prolyl aminopeptidase activity than the wild type or knockout plants under control conditions, as well as salt and drought stresses. Furthermore, through the GUS activity analysis, we also demonstrated that the AtPAP1 promoter is stress inducible and tissue specific. The AtPAP1-GFP fusion protein was found to localize in the cytoplasm of the onion epidermal cells. In conclusion, we showed that the Arabidopsis AtPAP1 gene could positively regulate plant tolerance to salt and drought stress, maybe by acting as a prolyl aminopeptidase and thereby increasing the concentration of free proline in plant cells.     

水稻NHX1基因启动子和C末端的调控功能研究

为了解水稻Na+/H+逆向转运蛋白(OsNHX1)在植物应答非生物胁迫中的分子调控机制,采用RT-PCR方法克隆OsNHX1基因上游2 000bp的启动子序列,并通过基因枪轰击瞬时转化洋葱表皮细胞,检测不同非生物胁迫下启动子的活性和表达模式;同时,分别克隆全长和C末端缺失的OsNHX1基因,通过花序浸染法转化拟南芥,研究OsNHX1基因及其C末端的功能。结果显示:OsNHX1启动子受逆境胁迫诱导,在盐、干旱、脱落酸胁迫处理下GUS表达活性明显升高;过表达OsNHX1的转基因拟南芥中,种子萌发率、根长、丙二醛含量和相对含水量的测定结果均显示其胁迫耐受性得到改善,但过表达OsNHX1C末端缺失基因对转基因植株的胁迫耐受性无明显影响。研究表明,Na+/H+逆向转运蛋白有助于提高植物耐盐性,且其C末端区域对该转运蛋白活性的发挥具有关键作用。     

Plant Growth-Promoting Rhizobacteria Enhance Abiotic Stress Tolerance in Solanum tuberosum Through Inducing Changes in the Expression of ROS-Scavenging Enzymes and Improved Photosynthetic Performance

In this report we address the changes in the expression of the genes involved in ROS scavenging and ethylene biosynthesis induced by the inoculation of plant growth-promoting rhizobacteria (PGPR) isolated from potato rhizosphere. The two Bacillus isolates used in this investigation had earlier demonstrated a striking influence on potato tuberization. These isolates showed enhanced 1-aminocyclopropane-1-carboxylic acid deaminase activity, phosphate solubilization, and siderophore production. Potato plants inoculated with these PGPR isolates were subjected to salt, drought, and heavy-metal stresses. The enhanced mRNA expression levels of the various ROS-scavenging enzymes and higher proline content in tubers induced by PGPR-treated plants contributed to increased plant tolerance to these abiotic stresses. Furthermore, the photosynthetic performance indices of PGPR-inoculated plants clearly exhibited a positive influence of these bacterial strains on the PSII photochemistry of the plants. Overall, these results suggest that the PGPR isolates used in this study are able to confer abiotic stress tolerance in potato plants.     

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.     

Overexpression of a TFIIIA-type zinc finger protein gene ZFP252 enhances drought and salt tolerance in rice (Oryza sativa L.)

We previously identified a salt and drought stress-responsive TFIIIA-type zinc finger protein gene ZFP252 from rice. Here we report the functional analysis of ZFP252 using gain- and loss-of-function strategies. We found that overexpression of ZFP252 in rice increased the amount of free proline and soluble sugars, elevated the expression of stress defense genes and enhanced rice tolerance to salt and drought stresses, as compared with ZFP252 antisense and non-transgenic plants. Our findings suggest that ZFP252 plays an important role in rice response to salt and drought stresses and is useful in engineering crop plants with enhanced tolerance to salt and drought stresses.     

Plant dihydroorotate dehydrogenase differs significantly in substrate specificity and inhibition from the animal enzymes

The mitochondrial membrane bound dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) catalyzes the fourth step of pyrimidine biosynthesis. By the present correction of a known cDNA sequence for Arabidopsis thaliana DHODH we revealed the importance of the very C-terminal part for its catalytic activity and the reason why--in contrast to mammalian and insect species--the recombinant plant flavoenzyme was unaccessible to date for in vitro characterization. Structure-activity relationship studies explained that potent inhibitors of animal DHODH do not significantly affect the plant enzyme. These difference could be exploited for a novel approach to herb or pest growth control by limitation of pyrimidine nucleotide pools.     

Overexpression of osmotin gene confers tolerance to salt and drought stresses in transgenic tomato (Solanum lycopersicum L.)

Abiotic stresses, especially salinity and drought, are major limiting factors for plant growth and crop productivity. In an attempt to develop salt and drought tolerant tomato, a DNA cassette containing tobacco osmotin gene driven by a cauliflower mosaic virus 35S promoter was transferred to tomato (Solanum lycopersicum) via Agrobacterium-mediated transformation. Putative T0 transgenic plants were screened by PCR analysis. The selected transformants were evaluated for salt and drought stress tolerance by physiological analysis at T1 and T2 generations. Integration of the osmotin gene in transgenic T1 plants was verified by Southern blot hybridization. Transgenic expression of the osmotin gene was verified by RT-PCR and northern blotting in T1 plants. T1 progenies from both transformed and untransformed plants were tested for salt and drought tolerance by subjecting them to different levels of NaCl stress and by withholding water supply, respectively. Results from different physiological tests demonstrated enhanced tolerance to salt and drought stresses in transgenic plants harboring the osmotin gene as compared to the wild-type plants. The transgenic lines showed significantly higher relative water content, chlorophyll content, proline content, and leaf expansion than the wild-type plants under stress conditions. The present investigation clearly shows that overexpression of osmotin gene enhances salt and drought stress tolerance in transgenic tomato plants.     

Functional expression of human dihydroorotate dehydrogenase (DHODH) in pyr4 mutants of ustilago maydis allows target validation of DHODH inhibitors in vivo

Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q6 and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.     

Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants

A rice gene encoding a calcium-dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over-expression of OsCDPK7 enhanced induction of some stress-responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post-translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement.     

Enhancing Arabidopsis salt and drought stress tolerance by chemical priming for its abscisic acid responses

Drought and salt stress tolerance of Arabidopsis (Arabidopsis thaliana) plants increased following treatment with the nonprotein amino acid beta-aminobutyric acid (BABA), known as an inducer of resistance against infection of plants by numerous pathogens. BABA-pretreated plants showed earlier and higher expression of the salicylic acid-dependent PR-1 and PR-5 and the abscisic acid (ABA)-dependent RAB-18 and RD-29A genes following salt and drought stress. However, non-expressor of pathogenesis-related genes 1 and constitutive expressor of pathogenesis-related genes 1 mutants as well as transgenic NahG plants, all affected in the salicylic acid signal transduction pathway, still showed increased salt and drought tolerance after BABA treatment. On the contrary, the ABA deficient 1 and ABA insensitive 4 mutants, both impaired in the ABA-signaling pathway, could not be protected by BABA application. Our data demonstrate that BABA-induced water stress tolerance is based on enhanced ABA accumulation resulting in accelerated stress gene expression and stomatal closure. Here, we show a possibility to increase plant tolerance for these abiotic stresses through effective priming of the preexisting defense pathways without resorting to genetic alterations.     

Stress-inducible expression of barley Hva1 gene in transgenic mulberry displays enhanced tolerance against drought, salinity and cold stress

Coping with different kinds of biotic and abiotic stresses is the foundation of sustainable agriculture. Although conventional breeding and marker-assisted selection are being employed in mulberry (Morus indica L.) to develop better varieties, nonetheless the longer time periods required for these approaches necessitates the use of precise biotechnological approaches for sustainable agriculture. In an attempt to improve stress tolerance of mulberry, an important plant of the sericulture industry, an encoding late embryogenesis abundant gene from barley (HVA1) was introduced into mulberry plants by Agrobacterium-mediated transformation. Transgenic mulberry with barley Hva1 under a constitutive promoter actin1 was shown to enhance drought and salinity tolerance. Here, we report that overexpression of barley Hva1 also confers cold tolerance in transgenic mulberry. Further, barley Hva1 gene under control of a stress-inducible promoter rd29A can effectively negate growth retardation under non-stress conditions and confer stress tolerance in transgenic mulberry. Transgenic lines display normal morphology to enhanced growth and an increased tolerance against drought, salt and cold conditions as measured by free proline, membrane stability index and PSII activity. Protein accumulation was detected under stress conditions confirming inductive expression of HVA1 in transgenics. Investigations to assess stress tolerance of these plants under field conditions revealed an overall better performance than the non-transgenic plants. Enhanced expression of stress responsive genes such as Mi dnaJ and Mi 2-cysperoxidin suggests that Hva1 can regulate downstream genes associated with providing abiotic stress tolerance. The investigation of transgenic lines presented here demonstrates the acquisition of tolerance against drought, salt and cold stress in plants overexpressing barley Hva1, indicating that Arabidopsis rd29A promoter can function in mulberry.     

Over-expression of OsUGE-1 altered raffinose level and tolerance to abiotic stress but not morphology in Arabidopsis

OsUGE-1 is known to be induced by various abiotic stresses, but its exact function in plants is unclear. In the present study, OsUGE-1 was over-expressed in Arabidopsis, transgenic plants conferred tolerance to salt, drought and freezing stress without altering plant morphology. In addition, transgenic plants showed a higher level of the soluble sugar raffinose than did wild-type plants. Our results suggest that elevated level of raffinose with over-expressed OsUGE-1 resulted in enhanced tolerance to abiotic stress. Thus, the gene may be applied to improve tolerance to abiotic stress in crops.     

Modulated fatty acid desaturation via overexpression of two distinct omega-3 desaturases differentially alters tolerance to various abiotic stresses in transgenic tobacco cells and plants

Changes in the degree of fatty acid (FA) desaturation are implicated in plant responses to various abiotic stresses, including heat, salt and drought. However, it is still not known whether decreased levels of linolenic acid, found in many plants subjected to salt and drought stress, reflect a mechanism of defence or damage. We addressed this question by generating tobacco cells and plants ectopically overexpressing two FA desaturases: the cytosolic FAD3 or the plastidic FAD8. A remarkable increase in the ratio of total linolenic to linoleic acids resulted from overexpression of FAD3, whereas ectopic overexpression of FAD8 induced an increased ratio mainly in the plastidic lipids. Here we present evidence that overexpressing FAD8 imposes much greater heat sensitivity than does FAD3 overexpression, in both cultured cells and whole plants. Overexpression of either FAD3 or FAD8 increases tolerance to drought in tobacco plants and to osmotic stress in cultured cells. These findings suggest that a drought-induced decreased level of linolenic acid reflects damage. Our results point to the potential of exploiting FAD overexpression as a tool to ameliorate drought tolerance.     

一个具有S位点的水稻类受体激酶OsSRL参与干旱胁迫反应

植物在进化过程中针对干旱、高盐和高低温等逆境胁迫形成了多种适应机制, 植物类受体激酶作为重要的细胞信号传递分子在植物生长和抗逆境胁迫中发挥着重要功能。该文发现一个具有S位点的类受体激酶基因OsSRL可能参与水稻(Oryza sativa)的干旱胁迫反应。利用RNAi技术降低OsSRL的表达水平后, 转基因植株抗旱性增强, 并表现出幼苗存活率、叶绿素含量及鲜重增加等表型。进一步的研究表明30%PEG和100 μmol·L^–1ABA可诱导OsSRL基因表达, 利用RNAi降低其表达导致干旱诱导基因RAB16A及LEA3表达水平明显增加。表达模式分析发现OsSRL在胚芽、胚根、根、茎节以及花中表达。以上结果表明, OsSRL表达水平的降低增强植物的干旱耐受性, 其作为一个S-位点样类受体激酶可能参与了水稻对干旱胁迫的反应。     

Cloning, expression, purification, and characterization of Leishmania major dihydroorotate dehydrogenase

Leishmania major Friedlin (LmjF) is a protozoan parasite whose genomic sequence has been recently elucidated. Here we have cloned, overexpressed, purified, and characterized the product of the gene from LmjF chromosome 16: LmjF16.0530, which encodes a protein with putative dihydroorotate dehydrogenase activity. Dihydroorotate dehydrogenase (DHODH) is a flavoprotein that catalyses the oxidation of L-dihydroorotate to orotate, the fourth sequential step in the de novo pyrimidine nucleotide synthesis pathway. The predicted enzyme from L. major was cloned and expressed in Escherichia coli strain BL21(DE3) as a histidine-tag fusion protein and purified to homogeneity using affinity chromatography. The final product was homogeneous in SDS-PAGE gel electrophoresis. The dihydroorotate oxidase activity has been assayed and the steady-state kinetic mechanism has been determined using fumarate as the oxidizing substrate. The catalysis by LmDHODH enzyme proceeds by a Ping-Pong Bi-Bi mechanism and the kinetic parameters Km were calculated to be 90 and 418 microM for dihydroorotate and fumarate, respectively, and Vmax was calculated to be 11 micromol min-1 mg-1. Our results confirmed that the product of the gene LmjF16.0530, whose function has previously been predicted based on homology to known proteins, can therefore be positively assigned as L. major DHODH.