JcLEA,a Novel LEA-Like Protein from Jatropha curcas,Confers a High Level of Tolerance to Dehydration and Salinity in Arabidopsis thaliana

Jatropha curcas L. is a highly drought and salt tolerant plant species that is typically used as a traditional folk medicine and biofuel crop in many countries. Understanding the molecular mechanisms that underlie the response to various abiotic environmental stimuli, especially to drought and salt stresses, in J. curcas could be important to crop improvement efforts. In this study, we cloned and characterized the gene for a late embryogenesis abundant (LEA) protein from J. curcas that we designated JcLEA. Sequence analyses showed that the JcLEA protein belongs to group 5, a subgroup of the LEA protein family. In young seedlings, expression of JcLEA is significantly induced by abscisic acid (ABA), dehydration, and salt stress. Subcellular localization analysis shows that that JcLEA protein is distributed in both the nucleus and cytoplasm. Moreover, based on growth status and physiological indices, the overexpression of JcLEA in transgenic Arabidopsis plants conferred increased resistance to both drought and salt stresses compared to the WT. Our data suggests that the group 5 JcLEA protein contributes to drought and salt stress tolerance in plants. Thus, JcLEA is a potential candidate gene for plant genetic modification.     

过表达TaLEA1和TaLEA2基因提高转基因拟南芥的耐盐性

我国土壤盐碱化日益严重,对我国的粮食安全造成了严重威胁。耐盐基因挖掘对作物耐盐育种非常重要。LEA蛋白家族是一个多基因家族,在植物应对非生物胁迫中发挥重要作用。本课题组前期研究阐明小麦TaLEA1基因在拟南芥中过表达可以提高转基因植物的耐盐性和抗旱性。本研究系统分析了小麦TaLEA2基因表达蛋白的理化性质、基因表达模式及启动子功能区域,并在拟南芥中过表达TaLEA2基因及共表达TaLEA1和TaLEA2基因,分析TaLEA2基因的抗逆功能及2个LEA基因的抗逆效果。结果表明,TaLEA2基因的表达产物属于第3组LEA蛋白,是稳定的亲水蛋白,富含α-螺旋、β-转角等结构。TaLEA2基因在小麦根、茎、叶、花、种子等不同组织中均有表达,盐胁迫条件诱导其高表达。在拟南芥中过表达TaLEA2基因,或过表达TaLEA1和TaLEA2基因都能够提高转基因拟南芥的耐盐性和抗旱性,转基因株系的种子萌发率、根长及叶绿素含量显著高于野生型,且双基因过表达的转基因植物的抗逆能力高于单个基因过表达株系。本研究结果为LEA基因抗逆机理的研究和多基因共转提高植物抗逆性提供了重要信息。     

小麦耐逆基因-TaLEA2转化拟南芥的研究

研究小麦第3组LEA基因中T aLEA2对耐旱和耐盐性能的影响.将小麦第3组LEA基因T aLEA2连接在双元表达载体pB I121 C aM V 35S启动子下游,构建了能在植物中高效表达的载体pB I121-T aLEA2.通过农杆菌介导的真空渗透法,将其转入野生拟南芥中,经抗性筛选及PCR验证,获得T0代转基因植株,并用不同浓度的PEG 4000和N aC l对转基因拟南芥的耐逆性进行检测.结果表明,这些转基因植株可明显改进拟南芥在10%PEG及0.8%N aC l培养基上的生长状态.在实验条件下,转基因拟南芥的耐旱性及耐盐性均有所提高,提示T aLEA2基因在植物水分调节方面有重要作用.     

Expression of a calcineurin gene improves salt stress tolerance in transgenic rice

Calcineurin is a Ca²⁺- and calmodulin-dependent serine/threonine phosphatase and has multiple functions in animal cells including regulating ionic homeostasis. We generated transgenic rice plants that not only expressed a truncated form of the catalytic subunit of mouse calcineurin, but also were able to grow and fertilize normally in the field. Notably, the expression of the mouse calcineurin gene in rice resulted in its higher salt stress tolerance than the non-transgenic rice. Physiological studies have indicated that the root growth of transgenic plants was less inhibited than the shoot growth, and that less Na⁺ was accumulated in the roots of transgenic plants after a prolonged period of salt stress. These findings imply that the heterologous calcineurin plays a significant role in maintaining ionic homeostasis and the integrity of plant roots when exposed to salt. In addition, the calcineurin gene expression in the stems of transgenic plants correlated with the increased expression of the Rab16A gene that encodes a group 2-type late-embryogenesis-abundant (LEA) protein. Altogether our findings provide the first genetic and physiological evidence that expression of the mouse calcineurin protein functionally improves the salt stress tolerance of rice partly by limiting Na⁺ accumulation in the roots.     

Two New Group 3 LEA Genes of Wheat and Their Functional Analysis in Yeast

The group 3 late embryogenesis abundant （LEA） proteins are thought to protect cells from stresses associated with dehydration during periods of water deficit. To investigate the functions of different members of the group 3 LEA genes, we isolated and characterized two new group 3 LEA genes, namely TaLEA2 and TaLEA3, from wheat （Triticum aestivum L.） and introduced TaLEA2 and TaLEA3 into Saccharmyces cerevisiae to examine the effect of these genes on yeast cell tolerance to osmotic, salt, and cold stresses. The TaLEA2 gene encoded a protein of 211 amino acids and possessed five repeats of 11-mer amino acid motifs. The TaLEA3 gene encoded a polypeptide of 211 amino acids with nine repeated units. Overexpression of TaLEA2 and TaLEA3 improved stress tolerance in transgenic yeast cells when cultured in medium containing sorbitol, salt and-20℃ freezing treatments respectively. However, the yeast transformants with TaLEA2 seemed to be more tolerant to hyperosmotic and freezing stress than transformants with TaLEA3. This implies that a close relationship exists between function and the number of repeats of the 11- mer amino acid motif in the group 3 LEA protein.     

A comparative study on the protective role of trehalose and LEA proteins against abiotic stresses in transgenic Chinese cabbage (<Emphasis Type="Italic">Brassica campestris</Emphasis>) overexpressing<Emphasis Type="Italic">CaLEA</Emphasis> or<Emphasis Type="Italic">otsA</Emphasis>

Trehalose and LEA proteins, representative low MW chemicals that are synthesized under dehydration, are known to protect plants from drought stress. To compare their effectiveness on enhancing tolerance against various abiotic stresses, we generated transgenic Chinese cabbage plants overexpressingE. ctdi trehalose-6-phosphate synthase gene (otsA) or hot pepper (Capsicum annuum) LEA protein gene(CaLEA). Both transgenic plants exhibited altered phenotype including stunted growth and aberrant root development When subjected to drought, salt or heat stress, these plants showed remarkably improved tolerance against those stresses compared with nontransformants. After dehydration treatment, leaf turgidity and fresh weight was better maintained in both transgenic plants. GaUEA-plants performed somewhat better under dehydrated condition. When treated with 250 mM NaCI, both otsA-plants and CaLEA-plants remained equally healthier than nontransformants in maintaining leaf turgidity and delaying necrosis. Furthermore, leaf Chi content and Fv/Fm was maintained considerably higher in both transgenic plants than nontransformants. After heat-treatment at 45°C, both transgenic plants appeared much less damaged in external shape and PSII function, but LEA proteins were more protective. Our results indicate that although both trehalose and LEA proteins are effective in protecting plants against various abiotic stresses, LEA proteins seem to be more promising in generating stress-tolerant transgenic plants.     

转柽柳晚期胚胎富集蛋白基因烟草T1代的耐盐性评价

目的:验证转柽柳晚期胚胎富集(LEA)蛋白基因烟草T1代的耐盐性。方法:采用盐胁迫方式,对转柽柳LEA蛋白基因烟草T1代的6个株系及非转基因对照烟草T1代进行不同浓度NaCl胁迫处理,分析了NaCl胁迫下转基因烟草的生长量、根系的发育及盐害程度。结果:各转基因烟草T1代组培苗在150mmol/L的NaCl培养基上根系生长良好,平均增重(鲜重)是非转基因对照的7.72倍,平均高生长是非转基因对照的3.51倍,盐害指数低于或等于50%;而非转基因对照烟草T1代组培苗生长缓慢,根系几乎不能生长发育,盐害指数达65%。结论:柽柳LEA蛋白基因的导入提高了T1代烟草的耐盐性。     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

Expression of a Late Embryogenesis Abundant Protein Gene,HVA1, from Barley Confers Tolerance to Water Deficit and Salt Stress in Transgenic Rice

A late embryogenesis abundant (LEA) protein gene, HVA1, from barley (Hordeum vulgare L.) was introduced into rice suspension cells using the Biolistic-mediated transformation method, and a large number of independent transgenic rice (Oryza sativa L.) plants were generated. Expression of the barley HVA1 gene regulated by the rice actin 1 gene promoter led to high-level, constitutive accumulation of the HVA1 protein in both leaves and roots of transgenic rice plants. Second-generation transgenic rice plants showed significantly increased tolerance to water deficit and salinity. Transgenic rice plants maintained higher growth rates than nontransformed control plants under stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by stress and by improved recovery upon the removal of stress conditions. We also found that the extent of increased stress tolerance correlated with the level of the HVA1 protein accumulated in the transgenic rice plants. Using a transgenic approach, this study provides direct evidence supporting the hypothesis that LEA proteins play an important role in the protection of plants under water-or salt-stress conditions. Thus, LEA genes hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance.     

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.     

Ectopic Expression of Arabidopsis Glycosyltransferase UGT85A5 Enhances Salt Stress Tolerance in Tobacco

Abiotic stresses greatly influence plant growth and productivity. While glycosyltransferases are widely distributed in plant kingdom, their biological roles in response to abiotic stresses are largely unknown. In this study, a novel Arabidopsis glycosyltransferase gene UGT85A5 was identified as significantly induced by salt stress. Ectopic expression of UGT85A5 in tobacco enhanced the salt stress tolerance in the transgenic plants. There were higher seed germination rates, better plant growth and less chlorophyll loss in transgenic lines compared to wild type plants under salt stress. This enhanced tolerance of salt stress was correlated with increased accumulations of proline and soluble sugars, but with decreases in malondialdehyde accumulation and Na⁺/K⁺ ratio in UGT85A5-expressing tobacco. Furthermore, during salt stress, expression of several carbohydrate metabolism-related genes including those for sucrose synthase, sucrose-phosphate synthase, hexose transporter and a group2 LEA protein were obviously upregulated in UGT85A5-expressing transgenic plants compared with wild type controls. Thus, these findings suggest a specific protective role of this glycosyltransferase against salt stress and provide a genetic engineering strategy to improve salt tolerance of crops.     

转LEA基因烟草的耐盐性分析

目的:验证柽柳LEA基因的功能,为通过基因工程手段培育耐盐植物提供基础资料。方法:对转LEA基因烟草当代(T0)和子一代(T1)分别进行不同浓度的NaCl胁迫处理,研究转基因烟草的耐盐性。结果:转基因烟草T0代组培苗耐受NaCl的临界浓度为230mmol/L,而对照耐受NaCl的临界浓度为130mmol/L以下;T1代幼苗耐受NaCl的临界浓度为150mmol/L,对照耐受NaCl的临界浓度为100mmol/L以下;在临界浓度转基因烟草的T0代、T1代根系发育良好,生长量明显高于非转基因对照烟草。结论:柽柳LEA基因的转化提高了烟草的耐盐性。     

Expression analysis of rice A20/AN1-type zinc finger genes and characterization of ZFP177 that contributes to temperature stress tolerance

The A20/AN1-type zinc finger protein family is conserved in animals and plants. Using human AWP1 protein as a query, we identified twelve A20/AN1-type zinc finger proteins in japonica rice. Most of these genes were constitutively expressed in leaves, roots, culms and spikes. Through microarray analysis, it was found that four genes (ZFP177, ZFP181, ZFP176, ZFP173), two genes (ZFP181 and ZFP176) and one gene (ZFP157) were significantly induced by cold, drought and H(2)O(2) treatments, respectively. Further expression analysis showed that ZFP177 was responsive to both cold and heat stresses, but down-regulated by salt. The subcellular localization assay indicated that ZFP177 was localized in cytoplasm in tobacco leaf and root cells. Yeast-one hybrid assay showed that ZFP177 lacked trans-activation potential in yeast cells. Overexpression of ZFP177 in tobacco conferred tolerance of transgenic plants to both low and high temperature stresses, but increased sensitivity to salt and drought stresses. Further we found expression levels of some stress-related genes were inhibited in ZFP177 transgenic plants. These results suggested that ZFP177 might play crucial but differential roles in plant responses to various abiotic stresses.     

A novel wheat α‐amylase inhibitor gene,TaHPS, significantly improves the salt and drought tolerance of transgenic Arabidopsis

On the basis of microarray analyses of the salt‐tolerant wheat mutant RH8706‐49, a previously unreported salt‐induced gene, designated as TaHPS [Triticum aestivum hypothetical (HPS)‐like protein], was cloned. Real‐time quantitative polymerase chain reaction analyses showed that expression of the gene was induced by abscisic acid, salt and drought. The encoded protein was found to be localized mainly in the plasma membranes. Transgenic Arabidopsis plants overexpressing TaHPS were more tolerant to salt and drought stresses than non‐transgenic wild‐type (WT) plants. Under salt stress, the root cells of the transgenic plants secreted more Na⁺ and guard cells took up more Ca²⁺ ions. Compared with wild‐type plants, TaHPS‐expressing transgenic plants showed significantly lower amylase activity and glucose and malic acid levels. Our results showed that the expression of TaHPS inhibited amylase activity, which subsequently led to a closure of stomatal apertures and thus improved plant tolerance to salt and drought.     

The pepper late embryogenesis abundant protein CaLEA1 acts in regulating abscisic acid signaling,drought and salt stress response

As sessile organisms, plants are constantly challenged by environmental stresses, including drought and high salinity. Among the various abiotic stresses, osmotic stress is one of the most important factors for growth and significantly reduces crop productivity in agriculture. Here, we report a function of the CaLEA1 protein in the defense responses of plants to osmotic stress. Our analyses showed that the CaLEA1 gene was strongly induced in pepper leaves exposed to drought and increased salinity. Furthermore, we determined that the CaLEA1 protein has a late embryogenesis abundant (LEA)_3 homolog domain highly conserved among other known group 5 LEA proteins and is localized in the processing body. We generated CaLEA1‐silenced peppers and CaLEA1‐overexpressing (OX) transgenic Arabidopsis plants to evaluate their responses to dehydration and high salinity. Virus‐induced gene silencing of CaLEA1 in pepper plants conferred enhanced sensitivity to drought and salt stresses, which was accompanied by high levels of lipid peroxidation in dehydrated and NaCl‐treated leaves. CaLEA1‐OX plants exhibited enhanced sensitivity to abscisic acid (ABA) during seed germination and in the seedling stage; furthermore, these plants were more tolerant to drought and salt stress than the wild‐type plants because of enhanced stomatal closure and increased expression of stress‐responsive genes. Collectively, our data suggest that CaLEA1 positively regulates drought and salinity tolerance through ABA‐mediated cell signaling.     

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.