Over-expression of StAPX in tobacco improves seed germination and increases early seedling tolerance to salinity and osmotic stresses

Ascorbate peroxidase plays a key role in scavenging reactive oxygen species under environmental stresses and in protecting plant cells against toxic effects. The Solanum lycopersicum thylakoid-bound ascorbate peroxidase gene (StAPX) was introduced into tobacco under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that StAPX was transferred into the tobacco genome and StAPX was induced by salt and osmotic stresses in tomato leaves. Over-expression of StAPX in tobacco improved seed germination rate and elevated stress tolerance during post-germination development. Two transgenic lines showed higher APX activity and accumulated less hydrogen peroxide than wild-type plants after stress treatments. The photosynthetic rates, the root lengths, the fresh and dry weights of the transgenic lines were distinctly higher than those of wild-type plants under stress conditions. Results indicated that the over-expression of StAPX had enhanced tolerance to salt stress and osmotic stress in transgenic tobacco plants.     

Transgenic tomato plants overexpressing chloroplastic monodehydroascorbate reductase are resistant to salt- and PEG-induced osmotic stress

RNA gel hybridization showed that the expression of monodehydroascorbate reductase (MDHAR) in the wild type (WT) tomato was decreased firstly and then increased under salt- and polyethylene glycol (PEG)-induced osmotic stress, and the maximum level was observed after treatment for 12 h. WT, sense transgenic and antisense transgenic tomato plants were used to analyze the antioxidative ability to cope with osmotic stresses. After salt stress, the fresh mass (FM) and height of sense transgenic lines were greater than those of antisense lines and WT plants. Under salt and PEG treatments, sense transgenic plants showed a lower level of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), a higher net photosynthetic rate (P _N), and the maximal photochemical efficiency of PSII (F_v/F_m) compared with WT and antisense transgenic plants. Moreover, sense lines maintained higher ascorbate peroxidase (APX) activity than WT and antisense plants under salt- and PEG-induced osmotic stress. These results indicate that chloroplastic MDHAR plays an important role in alleviating photoinhibition of PSII by elevating ascorbate (AsA) level under salt- and PEG-induced osmotic stress.     

过表达LeNLP4转录因子提高番茄抗低温胁迫能力

NAC（NAM-ATAF1,2-CUC2）转录因子在植物胁迫响应中起重要作用。为了探讨三舭丹基因在番茄抗低温胁迫中的功能,分离了番茄LeNLP4转录因子基因,并获得转正义LeNLP4基因番茄植株。荧光定量PCR分析表明,LeNLP4的表达受低温诱导。与野生型植株相比,在4℃胁迫下转基因植株具有较高的生长量和光系统II（PSH）最大光化学效率（Fv／Fm）、过氧化氢（H2O2）和超氧阴离子（O2-）清除速率、抗坏血酸过氧化物酶（APX）和超氧化物歧化酶（SOD）活性,以及较低的丙二醛（MDA）含量和相对电导率（REC）。过表达株系中SICBF1的表达高于野生型。上述结果表明,LeNLP4的过表达提高了转基因番茄抗低温胁迫能力。     

Genetic Engineering of Glycine Betaine Biosynthesis Reduces Heat-Enhanced Photoinhibition by Enhancing Antioxidative Defense and Alleviating Lipid Peroxidation in Tomato

Glycine betaine (GB) is a compatible solute that accumulates rapidly to enhance heat tolerance in many plants grown under heat stress. In this study, a BADH gene (betaine aldehyde dehydrogenase) from spinach was introduced into tomato (Lycopersicon esculentum cv. ‘Moneymaker’) via Agrobacterium-mediated transformation. Transgenic tomato lines expressing BADH exhibited higher capabilities for GB accumulation. Chlorophyll fluorescence analysis of wild type (WT) and transgenic plants exposed to heat treatment (42 °C) showed that transgenic plants exhibited higher photosynthetic capacities than WT plants. This finding suggests that GB accumulation increases tolerance to heat-enhanced photoinhibition. This increased tolerance was associated with an improvement in D1 protein content, which accelerated the repair of photosystem II (PSII) following heat-enhanced photoinhibition. Significant accumulations of hydrogen peroxide (H₂O₂) and superoxide radical (O₂ ^?) were observed in WT plants under heat stress. However, these accumulations were much less for the transgenic plants. An important finding reported herein is that exogenous GB cannot directly reduce the content of reactive oxygen species (ROS). In accordance with a lower relative electrolyte conductivity and malondialdehyde content, the activities of antioxidant enzymes were higher in transgenic lines than in WT plants, indicating that the degree of membrane injury in the transgenic plants was lower compared to the WT plants. These results suggest that GB accumulation in vivo cannot directly eliminate ROS. Rather, higher antioxidant enzyme activities must be maintained to lessen the accumulation of ROS in transgenic plants and to decrease the degree of membrane injury.     

Overexpression of <Emphasis Type="Italic">Populus tomentosa</Emphasis> cytosolic ascorbate peroxidase enhances abiotic stress tolerance in tobacco plants

Reactive oxygen species (ROS) play key roles in plants and are regulated by several ROS-scavenging enzymes. Ascorbate peroxidase (APX), which catalyzes the reduction of hydrogen peroxide to water, a vital part of ROS formation, plays a significant role in higher plants. In this study, a cytosolic APX gene from Populus tomentosa, named PcAPX, was identified and characterized. Recombinant PcAPX had a calculated mass of 33.24 kD and showed high activity towards ascorbic acid (ASA) and hydrogen peroxide (H₂O₂). Real-time PCR analysis showed that APX mRNA expression levels were higher in leaves than roots or stems of P. tomentosa. Compared with wild-type, transgenic tobacco plants overexpressing PcAPX showed no significant difference in morphology under normal conditions. However, the transgenic plants were more resistant to drought, salt and oxidative stress conditions, as shown by decreased levels of malondialdehyde and increased levels of chlorophyll. Moreover, decreased H₂O₂ levels, increased ASA consumption, an increase in the NADP to NADPH ratio, and higher APX activity in the transgenic plants suggested an increased ability to eliminate ROS. These data suggest that PcAPX overexpression in transgenic tobacco plants can enhance tolerance to drought, salt and oxidative stress. Therefore, APX has a crucial role in abiotic stress tolerance in plants.     

过表达番茄GDP-L-半乳糖磷酸酶基因提高烟草抗MV诱导的氧化胁迫能力

为了探讨番茄GDP—L-半乳糖磷酸酶对烟草抗坏血酸（AsA）含量及抗氧化能力的影响,从番茄叶片中分离了GDP-L-半乳糖磷酸酶基因（LeGGP）,并转入到烟草中。以野生型（WT）和转正义LeGGP烟草株系T1-3和T1-15为试材,测定了甲基紫精（MV）处理下AsA、脱氢抗坏血酸（DHA）、H2O2、O2-和叶绿素含量、抗坏血酸过氧化物酶（APX）活性、光合速率和叶绿素荧光参数等。Northem杂交分析表明LeGGP的表达受MV的诱导,在MV处理下,野生型烟草的离体叶圆片发生比转基因烟草更严重的光漂白,转基因烟草的AsA含量及清除H2O2和O2-的能力明显强于野生型,过表达LePGG胀高了烟草的生长量。并且转基因烟草比野生型具有更高的净光合效率（Pn）和光系统Ⅱ（PSII）最大光化学效率（眠）。结果表明,LeGGP的过表达有助于提高烟草AsA含量及抗氧化胁迫能力。     

Constitutive accumulation of zeaxanthin in tomato alleviates salt stress-induced photoinhibition and photooxidation

Zeaxanthin (Z) has a role in the dissipation of excess excitation energy by participating in non‐photochemical quenching (NPQ) and is essential in protecting the chloroplast from photooxidative damage. To investigate the physiological effects and functional mechanism of constitutive accumulation of Z in the tomato at salt stress‐induced photoinhibition and photooxidation, antisense‐mediated suppression of zeaxanthin epoxidase transgenic plants and the wild‐type (WT) tomato were used. The ratio of Z/(V + A + Z) and (Z + 0.5A)/(V + A + Z) in antisense transgenic plants were maintained at a higher level than in WT plants under salt stress, but the value of NPQ in WT and transgenic plants was not significantly different under salt stress. However, the maximal photochemical efficiency of PSII (Fv/Fm) and the net photosynthetic rate (Pn) in transgenic plants decreased more slowly under salt stress. Furthermore, transgenic plants showed lower level of hydrogen peroxide (H₂O₂), superoxide anion radical (O₂^??) and ion leakage, lower malondialdehyde content. Compared with WT, the content of D1 protein decreased slightly in transgenic plants under salt stress. Our results suggested that the constitutive accumulation of Z in transgenic tomatoes can alleviate salt stress‐induced photoinhibition because of the antioxidant role of Z in the scavenging quenching of singlet oxygen and/or free radicals in the lipid phase of the membrane.     

Improved yield,fruit quality,and salt resistance in tomato co-overexpressing LeNHX2 and SlSOS2 genes

The K⁺, Na⁺/H⁺ antiporter LeNHX2 and the regulatory kinase SlSOS2 are important determinants of salt tolerance in tomato plants and their fruit production ability. In this work, we have analyzed the effects of LeNHX2 and SlSOS2 co-overexpression on fruit production, quality in tomato plants (Solanum lycopersicum L. cv. MicroTom), and analyzed physiological parameters related to salt tolerance. Plants overexpressing LeNHX2, SlSOS2 or both were grown in greenhouse. They were treated with 125 mM NaCl or left untreated and their salt tolerance was analyzed in terms of plant biomass and fruit yield. Under NaCl cultivation conditions, transgenic tomato plants overexpressing either SlSOS2 or LeNHX2 or both grew better and showed a higher biomass compared to their wild-type plants. Proline, glucose and protein content in leaves as well as pH and total soluble solid (TSS) in fruits were analyzed. Our results indicate that salinity tolerance of transgenic lines is associated with an increased proline, glucose and protein content in leaves of plants grown either with or without NaCl. Salt treatment significantly reduced yield, pH and TSS in fruits of WT plants but increased yield, pH and TSS in fruits of transgenic plants, especially those overexpressing both LeNHX2 and SlSOS2. All these results indicate that the co-overexpression of LeNHX2 and SlSOS2 improve yield and fruit quality of tomato grown under saline conditions.     

Overexpression of chloroplastic monodehydroascorbate reductase enhanced tolerance to temperature and methyl viologen‐mediated oxidative stresses

A tomato (Lycopersicon esculentum Mill.) monodehydroascorbate reductase gene (LeMDAR) was isolated. The LeMDAR–green fluorescence protein (GFP) fusion protein was targeted to chloroplast in Arabidopsis mesophyll protoplast. RNA and protein gel blot analyses confirmed that the sense‐ and antisense‐ LeMDAR were integrated into the tomato genome. The MDAR activities and the levels of reduced ascorbate (AsA) were markedly increased in sense transgenic lines and decreased in antisense transgenic lines compared with wild‐type (WT) plants. Under low and high temperature stresses, the sense transgenic plants showed lower level of hydrogen peroxide (H₂O₂), lower thiobarbituric acid reactive substance (TBARS) content, higher net photosynthetic rate (P_n), higher maximal photochemical efficiency of PSII (F_v/F_m) and fresh weight compared with WT plants. The oxidizable P700 decreased more obviously in WT and antisense plants than that in sense plants at chilling temperature under low irradiance. Furthermore, the sense transgenic plants exhibited significantly lower H₂O₂ level, higher ascorbate peroxidase (APX) activity, greater P_n and F_v/F_m under methyl viologen (MV)‐mediated oxidative stresses. These results indicated that overexpression of chloroplastic MDAR played an important role in alleviating photoinhibition of PSI and PSII and enhancing the tolerance to various abiotic stresses by elevating AsA level.     

Overexpression of endoplasmic reticulum omega-3 fatty acid desaturase gene improves chilling tolerance in tomato

An endoplasmic reticulum-localized tomato omega-3 fatty acid desaturase gene (LeFAD3) was isolated and characterized with regard to its sequence, response to various temperatures and function in transgenic tomato plants. Northern blot analysis showed that LeFAD3 was expressed in all organs tested and was markedly abundant in roots. Meanwhile, the expression of LeFAD3 was induced by chilling stress (4 °C), but inhibited by high temperature (40 °C). The transgenic plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern and western blot analyses confirmed that sense LeFAD3 was transferred into tomato genome and overexpressed. Level of linolenic acids (18:3) increased and correspondingly level of linoleic acid (18:2) decreased in leaves and roots. After chilling stress, the fresh weight of the aerial parts of transgenic plants was higher than that of the wild type (WT) plants, and the membrane system ultrastructure of chloroplast in leaf cell and all the subcellular organelles in root tips of transgenic plants kept more intact than those of WT. Relative electric conductivity increased less in transgenic plants than that in WT, and the respiration rate of the transgenic plants was notably higher than that of WT. The maximal photochemical efficiency of PSII (F_v/F_m) and the O₂ evolution rate in WT decreased more than those in transgenic plants under chilling stress. Together with other data, results showed that the overexpression of LeFAD3 led to increased level of 18:3 and alleviated the injuries under chilling stress.     

Suppression of tomato <Emphasis Type="Italic">SlGGP</Emphasis> aggravates methyl viologen-mediated oxidative stress

Ascorbate (AsA) is an important antioxidant that can scavenge reactive oxygen species to protect plant cells against oxidative stress. Guanosine 5'-diphosphate (GDP)-L-galactose phosphorylase (GGP) is a key enzyme in the AsA biosynthetic pathway. To investigate the functions of GGP in AsA synthesis and oxidative stress tolerance in tomato, antisense lines with a reduced expression of SlGGP were obtained. Photobleaching after treatment of leaf disks with methyl viologen was more severe in transgenic lines compared to wild type (WT) plants. Moreover, compared with the WT plants, the transgenic plants showed a higher content of hydrogen peroxide, superoxide anion, malondialdehyde, as well as ion leakage, but a lower content of AsA and chlorophylls, ascorbate peroxidase activity, net photosynthetic rate, and maximal photochemical efficiency of photosystem II. Results of real-time quantitative polymerase chain reaction show that suppression of the SlGGP gene in the transgenic plants reduced their oxidative stress tolerance.     

The <Emphasis Type="Italic">SlCMO</Emphasis> Gene Driven by Its Own Promoter Enhances Salt Tolerance of Transgenic Tomato Without Affecting Growth and Yield

Choline monooxygenase (CMO) is a key enzyme involved in betaine synthesis and our preliminary work has shown that the SlCMO gene promoter (pC5: ??267 to +?128 base pair), cloned from Suaeda liaotungensis, is salt-inducible. In the present study, pC5-SlCMO was transferred into tomato (Solanum lycopersicon L. ‘Micro-Tom’) plants via Agrobacterium mediation. Homozygous transgenic plants were selected using quantitative real-time polymerase chain reaction. The expression of SlCMO in pC5-SlCMO transgenic plants was induced by salinity. Under salt tolerance, betaine content, chlorophyll content, and net photosynthetic rate were higher in transgenic plants than in wild-type (WT) plants. Proline content was lower in transgenic plants than in WT plants. Under normal conditions, seed germination, length of the whole plant, dry weight, and fruit products of transgenic plants were the same as in WT plants. These results demonstrated that the pC5 promoter can drive increased expression of SlCMO in transgenic tomato plants under salt stress and increase salt tolerance without affecting plant growth and yield.     

Overexpression of tomato <Emphasis Type="Italic">tAPX</Emphasis> gene in tobacco improves tolerance to high or low temperature stress

In order to investigate the function of chloroplast ascorbate peroxidase under temperature stress, the thylakoid-bound ascorbate peroxidase gene from tomato leaf (TtAPX) was introduced into tobacco. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that TtAPX in tomato was induced by chilling or heat stress. Over-expression of TtAPX in tobacco improved seed germination under temperature stress. Two transgenic tobacco lines showed higher ascorbate peroxidase activity, accumulated less hydrogen peroxide and malondialdehyde than wild type plants under stress condition. The photochemical efficiency of photosystem 2 in the transgenic lines was distinctly higher than that of wild type plants under chilling and heat stresses. Results indicated that the over-expression of TtAPX enhanced tolerance to temperature stress in transgenic tobacco plants.     

Enhancement of low-temperature tolerance in transgenic tomato plants overexpressing Lefad7 through regulation of trienoic fatty acids

We studied how tomato (Lycopersicon esculentum Mill.) chloroplast omega-3 fatty acid desaturase gene (Lefad7) overexpression enhanced low-temperature (LT) tolerance in transgenic tomato plants. In these plants, the content of linolenic acid (18:3) markedly increased and, correspondingly, the content of linoleic acid (18:2) decreased. Similar changes were found after 6 h under LT (4°C) treatment. Under LT stress, wild type (WT) tomato plants showed a much greater increase in relative electrolyte leakage and malondialdehyde (MDA) contents compared with transgenic plants. Transgenic plants exhibited higher activities of antioxidative enzymes and a lower content of reactive oxygen species (ROS). Transgenic plants maintained a relatively higher level of the net photosynthetic rate (P _N) and chlorophyll (Chl) content than WT plants under LT stress. Taken together, we suggested that overexpression of Lefad7 enhanced LT tolerance by changing the composition of membrane lipids in tomato plants, with the increased content of trienoic fatty acids and reduced content of dienoic fatty acids that led to series of physiological alterations.     

Overexpression of Rice CBS Domain Containing Protein Improves Salinity, Oxidative, and Heavy Metal Tolerance in Transgenic Tobacco

We have recently identified and classified a cystathionine ??-synthase domain containing protein family in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa L.). Based on the microarray and MPSS data, we have suggested their involvement in stress tolerance. In this study, we have characterized a rice protein of unknown function, OsCBSX4. This gene was found to be upregulated under high salinity, heavy metal, and oxidative stresses at seedling stage. Transgenic tobacco plants overexpressing OsCBSX4 exhibited improved tolerance toward salinity, heavy metal, and oxidative stress. This enhanced stress tolerance in transgenic plants could directly be correlated with higher accumulation of OsCBSX4 protein. Transgenic plants could grow and set seeds under continuous presence of 150?mM NaCl. The total seed yield in WT plants was reduced by 80%, while in transgenic plants, it was reduced only by 15?C17%. The transgenic plants accumulated less Na⁺, especially in seeds and maintained higher net photosynthesis rate and Fv/Fm than WT plants under NaCl stress. Transgenic seedlings also accumulated significantly less H₂O₂ as compared to WT under salinity, heavy metal, and oxidative stress. OsCBSX4 overexpressing transgenic plants exhibit higher abiotic stress tolerance than WT plants suggesting its role in abiotic stress tolerance in plants.