Increased Chilling Tolerance Following Transfer of a betA Gene Enhancing Glycinebetaine Synthesis in Cotton (Gossypium hirsutum L.)

A betA gene encoding choline dehydrogenase from Escherichia coli was transformed into cotton (Gossypium hirsutum L.) via Agrobacterium-mediated transformation. Transgenic cotton plants exhibited improved tolerance to chilling due to accumulation of glycinebetaine (GB). The results of our experiment showed that GB contents of leaves of transgenic lines 1, 3, 4, and 5, both before and after chilling stress, were significantly higher than those of wild-type (WT) plants. At 15°C, transgenic lines 1, 3, 4, and 5 exhibited higher germination capacity as determined by the germination speed and final germination percentage and, displayed less inhibition in seedling shoot growth rate than WT plants. Under chilling stress, transgenic lines 4 and 5 maintained higher relative water content, upper carbon dioxide (CO₂) fixation capacity and PSII electron transfer rate, better osmotic adjustment (OA), a lower percentage of ion leakage, and less lipid membrane peroxidation when compared with WT plants. Chilling resistance of the transgenic lines was demonstrated to be positively correlated with GB content under chilling stress. The high levels of GB in transgenic cotton plants might not only protect the integrity of cell membrane from chilling damage, but also be involved in OA which alleviated chilling induced water stress. Moreover, under chilling-stressed conditions, transgenic cotton plants enhanced stomatal conductance, PSII electron transport rate, and further leaf photosynthesis through accumulating high levels of GB.     

Heterologous expression of Arabidopsis C-repeat binding factor 3 (AtCBF3) and cold-regulated 15A (AtCOR15A) enhanced chilling tolerance in transgenic eggplant (Solanum melongena L.)

Key message

Our study shows that the expression of AtCBF3 and AtCOR15A improved the chilling tolerance in transgenic eggplant.

Abstract

In an attempt to improve chilling tolerance of eggplant (Solanum melongena L) plants, Arabidopsis C-repeat binding factor 3 (AtCBF3) and cold-regulated 15A (AtCOR15A) genes both driven by an Arabidopsis RESPONSIVE TO DESSICATION 29A promoter (AtRD29A) were transferred into the plants of eggplant cultivar Sanyueqie. Two independent homozygous transgenic lines were tested for their cold tolerance. The leaves of the transgenic plants in both lines withered much slower and slighter than the wild-type plants after exposure to cold stress treatment at 2 ± 1 °C. The gene expression of AtCBF3 and AtCOR15A was significantly increased as well as the proline content and the levels of catalase and peroxidase activities, while the relative electrical conductivity and the malondialdehyde content were remarkably decreased in the transgenic plants compared with the wild type at 4 ± 0.5 °C. The results showed that the expression of the exogenous AtCBF3 and AtCOR15A could promote the cold adaptation process to protect eggplant plants from chilling stress.     

Overexpression of Loquat Dehydrin Gene <Emphasis Type="Italic">EjDHN1</Emphasis> Promotes Cold Tolerance in Transgenic Tobacco

Dehydrins (DHNs) play vital roles in response to dehydration stress in plants. To examine the contribution of EjDHN to low-temperature stress in loquat (Eriobotrya japonica Lindl.), EjDHN1 was overexpressed in tobacco (Nicotiana tabacum L.). The plant growth of transgenic lines was significantly better than wild type (WT) after 4 d of recovery from cold stress. Cold stress led to membrane lipid peroxidation and reduced photosystem II (PSII) activity in leaves, and these were less severe in transgenic lines. To examine oxidative stress tolerance, the plants were treated with different concentrations of methyl viologen (MV), which inhibited plant growth both in WT and transgenic lines. After exposure to 2.0 μM MV for 10 d, the WT plants had a dramatically lower survival rate. MV treatment in leaf disks confirmed that transgenic lines accumulated less reactive oxygen species (ROS) and suffered less lipid peroxidation. The results suggested that the tolerance of the transgenic plants to cold was increased, and EjDHN1 could protect cells against oxidative damage caused by ROS production under cold stress. It also provided evidences that the enhanced cold tolerance resulted from EjDHN1 overexpression could be partly due to their protective effect on membranes by alleviating oxidative stresses.     

Constitutive expression of a fungal glucose oxidase gene in transgenic tobacco confers chilling tolerance through the activation of antioxidative defence system

Scientific evidences in the literature have shown that plants treated exogenously with micromole concentration of hydrogen peroxide (H₂O₂) acquire abiotic stress tolerance potential, without substantial disturbances in the endogenous H₂O₂ pool. In this study, we enhanced the endogenous H₂O₂ content of tobacco (Nicotiana tabaccum L. cv. SR1) plants by the constitutive expression of a glucose oxidase (GO; EC 1.1.3.4) gene of Aspergillus niger and studied their cold tolerance level. Stable integration and expression of GO gene in the transgenic (T₀–T₂) tobacco lines were ascertained by molecular and biochemical tests. Production of functionally competent GO in transgenic plants was confirmed by the elevated levels of H₂O₂ in the transformed tissues. When three homozygous transgenic lines were exposed to different chilling temperatures for 12 h, the electrolyte conductivity was significantly lower in GO-expressing tobacco plants than the control plants; in particular, chilling protection was more prominent at −1°C. In addition, most transgenic lines recovered within a week when returned to normal culture conditions after −1°C–12 h cold stress. However, control plants displayed symptoms of chilling injuries such as necrosis of shoot tip, shoots and leaves, consequently plant death. The protective effect realized in the transgenic plants was comparable to cold-acclimatized wild tobacco. The chilling tolerance of transgenic lines was found associated, at least in part, with elevated levels of total antioxidant content, CAT and APX activities. Based on our findings, we predict that the transgenic expression of GO may be deployed to improve cold tolerance potential of higher plants.     

<Emphasis Type="Italic">CbLEA</Emphasis>, a Novel<Emphasis Type="Italic">LEA</Emphasis> Gene from<Emphasis Type="Italic">Chorispora bungeana</Emphasis>, Confers Cold Tolerance in Transgenic Tobacco

A novel late embryogenesis abundant (LEA) gene (AY804193), namedCbLEA, has now been isolated fromChorispora bungeana. This rare alpine subnival plant can survive sudden snowstorms and low temperatures. The full-lengthCbLEA is 842 bp, with an open reading frame encoding 169 ami no acids. The putative molecular weight ofCbLEA protein is 17.9 kDa, with an estimatedpl of 6.45. To investigate the functioning of thisCbLEA protein in cold-stress tolerance,CbLEA was introduced into tobacco under the control of the CaMV35S promoter. Second-generation (R₁) transgenic tobacco plants exhibited significantly increased tolerance to cold. These transgenics maintained lower malondialdehyde (MDA) contents and electrolyte leakage (EL) but their relative water content (RWC) was significantly higher compared with non-transgenic plants under chilling stress. Further experimental results showed that non-transgenic plants had severe freezing damage after exposure to -2°C for 1 h, whereas the transgenics suffered only slight injury under the same conditions. Moreover, survival was longer in the latter genotype at that temperature. The extent of increased cold tolerance was positive correlated with the level ofCbLEA protein accumulation, and was also reflected by the delayed development of damage symptoms. This indicates thatCbLEA is an excellent stress tolerance gene, and holds considerable potential as a new molecular tool for engineering improved plant genetics.     

Down-Regulation of OsSPX1 Causes High Sensitivity to Cold and Oxidative Stresses in Rice Seedlings

Rice SPX domain gene, OsSPX1, plays an important role in the phosphate (Pi) signaling network. Our previous work showed that constitutive overexpression of OsSPX1 in tobacco and Arabidopsis plants improved cold tolerance while also decreasing total leaf Pi. In the present study, we generated rice antisense and sense transgenic lines of OsSPX1 and found that down-regulation of OsSPX1 caused high sensitivity to cold and oxidative stresses in rice seedlings. Compared to wild-type and OsSPX1-sense transgenic lines, more hydrogen peroxide accumulated in seedling leaves of OsSPX1-antisense transgenic lines for controls, cold and methyl viologen (MV) treatments. Glutathione as a ROS scavenger could protect the antisense transgenic lines from cold and MV stress. Rice whole genome GeneChip analysis showed that some oxidative-stress marker genes (e.g. glutathione S-transferase and P450s) and Pi-signaling pathway related genes (e.g. OsPHO2) were significantly down-regulated by the antisense of OsSPX1. The microarray results were validated by real-time RT-PCR. Our study indicated that OsSPX1 may be involved in cross-talks between oxidative stress, cold stress and phosphate homeostasis in rice seedling leaves.     

Expression of Arabidopsis DREB1C improves survival, growth, and yield of upland New Rice for Africa (NERICA) under drought

Dehydration-responsive-element-binding protein 1 genes have important roles in response to stress. To improve the drought tolerance of an upland rice cultivar NERICA1, we introduced Arabidopsis AtDREB1C or rice OsDREB1B driven by a stress-inducible rice lip9 promoter. Plants of some transgenic lines survived better than non-transgenic plants under severe drought. AtDREB1C transgenic plants had higher dry weights than non-transgenic plants when grown under moderate drought until the late vegetative growth stage. On the other hand, OsDREB1B transgenic plants had lower dry weights than non-transgenic plants under the same condition. Similar results were obtained under osmotic stress. The AtDREB1C transgenic plants headed earlier, had a larger sink capacity, and had more filled grains than non-transgenic plants. These results suggest that AtDREB1C expressed in NERICA1 improves not only survival under severe drought, but also growth and yield under moderate drought.     

Overexpression of HVA1 gene from barley generates tolerance to salinity and water stress in transgenic mulberry (Morus indica)

Late embryogenesis abundant (LEA) proteins are members of a large group of hydrophilic proteins found primarily in plants. The barley hva1 gene encodes a group 3 LEA protein and is induced by ABA and water deficit conditions. We report here the over expression of hva1 in mulberry under a constitutive promoter via Agrobacterium-mediated transformation. Molecular analysis of the transgenic plants revealed the stable integration and expression of the transgene in the transformants. Transgenic plants were subjected to simulated salinity and drought stress conditions to study the role of hva1 in conferring tolerance. The transgenic plants showed better cellular membrane stability (CMS), photosynthetic yield, less photo-oxidative damage and better water use efficiency as compared to the non-transgenic plants under both salinity and drought stress. Under salinity stress, transgenic plants show many fold increase in proline concentration than the non-transgenic plants and under water deficit conditions proline is accumulated only in the non-transgenic plants. Results also indicate that the production of HVA1 proteins helps in better performance of transgenic mulberry by protecting membrane stability of plasma membrane as well as chloroplastic membranes from injury under abiotic stress. Interestingly, it was observed that hva1 conferred different degrees of tolerance to the transgenic plants towards various stress conditions. Amongst the lines analysed for stress tolerance transgenic line ST8 was relatively more salt tolerant, ST30, ST31 more drought tolerant, and lines ST11 and ST6 responded well under both salinity and drought stress conditions as compared to the non-transgenic plants. Thus hva1 appears to confer a broad spectrum of tolerance under abiotic stress in mulberry.     

Increased glycine betaine synthesis and salinity tolerance in AhCMO transgenic cotton lines

Glycine betaine is an osmoprotectant that plays an important role and accumulates rapidly in many plants during salinity or drought stress. Choline monooxygenase (CMO) is a major catalyst in the synthesis of glycine betaine. In our previous study, a CMO gene (AhCMO) cloned from Atriplex hortensis was introduced into cotton (Gossypium hirsutum L.) via Agrobacterium mediation to enhance resistance to salinity stress. However, there is little or no knowledge of the salinity tolerance of the transgenic plants, particularly under saline-field conditions. In the present study, two transgenic AhCMO cotton lines of the T₃ generation were used to study the AhCMO gene expression, and to determine their salinity tolerance in both greenhouse and field under salinity stress. Molecular analysis confirmed that the transgenic plants expressed the AhCMO gene. Greenhouse study showed that on average, seedlings of the transgenic lines accumulated 26 and 131% more glycine betaine than those of non-transgenic plants (SM3) under normal and salt-stress (150 mmol l⁻¹ NaCl) conditions, respectively. The osmotic potential, electrolyte leakage and malondialdehyde (MDA) accumulation were significantly lower in leaves of the transgenic lines than in those of SM3 after salt stress. The net photosynthesis rate and Fv/Fm in transgenic cotton leaves were less affected by salinity than in non-transgenic cotton leaves. Therefore, transgenic cotton over-expressing AhCMO was more tolerant to salt stress due to elevated accumulation of glycine betaine, which provided greater protection of the cell membrane and photosynthetic capacity than in non-transgenic cotton. The seed cotton yield of the transgenic plants was lower under normal conditions, but was significantly higher than that of non-transgenic plants under salt-stressed field conditions. The results indicate that over-expression of AhCMO in cotton enhanced salt stress tolerance, which is of great value in cotton production in the saline fields.     

过表达IbOr基因甘薯增强抗旱性的生理机制

以超表达甘薯橙色基因(IbOr)的转基因甘薯(TS)以及非转基因甘薯(NT)为实验材料,通过15%聚乙二醇6000(PEG-6000)模拟干旱条件,研究转基因与非转基因甘薯幼苗在水分胁迫不同时间的光合系统,膜脂过氧化及抗氧化防御系统中主要指标的变化情况,探讨转基因甘薯耐旱性的生理机制。结果显示:(1)随PEG-6000胁迫时间延长,甘薯叶片的叶绿素、类胡萝卜素含量及其叶片净光合速率、气孔导度、胞间CO2浓度、蒸腾速率都显著降低,但转基因株系降低幅度小于非转基因植株。(2)在正常供水和水分胁迫下,超表达IbOr基因甘薯叶片中O-·2、MDA含量均低于非转基因甘薯,即转基因甘薯具有较低的活性氧水平且脂膜受损伤较小。(3)PEG-6000胁迫24h后,甘薯叶片中SOD、POD酶活性均增加,48h达到最大值,且转基因甘薯中2种酶活性显著高于非转基因甘薯。研究表明,过表达IbOr基因可以有效减轻甘薯在水分胁迫条件下受损害的程度,且可能主要通过提高甘薯的抗氧化胁迫能力来完成。     

Wheat catalase expressed in transgenic rice can improve tolerance against low temperature stress

We examined whether the expression of wheat catalase (EC 1.11.1.6) cDNA in transgenic rice ( Oryza sativa L.) could enhance tolerance against low temperature injury. Transgenic rice plants expressing wheat CAT protein showed an increase of activities in leaves at 25°C, 2- to 5-fold that in non-transgenic rice. At 5°C, catalase activities were about 4–15 times higher than those in non-transgenic rice were. A comparison of damage observed in leaves as they withered due to chilling at 5°C showed that transgenic rice displayed an increased capability to resist low temperature stress. The exposure of these plants to low temperature at 5°C for 8 days resulted in decreased catalase activities in leaves at 25°C, but the transgenic plants indicated 4 times higher residual catalase activities than those of non-transgenic ones. The concentration of H₂O₂ in leaves was kept lower in transgenic rice than that of the control plants during the 8 days chilling. These results suggest that the improved tolerance against low temperature stress in genetically engineered rice plants be attributed to the effective detoxification of H₂O₂ by the enhanced catalase activities.     

Protein Kinase LTRPK1 Influences Cold Adaptation and Microtubule Stability in Rice

Rice LTRPK1, which encodes a member of the casein kinase I family, has been reported to be involved in root development, hormone response, and metabolic processes. Here we further show that LTRPK1 participates in stress resistance by regulating cytoskeleton rearrangement and formation of cold tolerance and adaptation. Semiquantitative RT-PCR analysis revealed enhanced expression of LTRPK1 in plants subject to low-temperature stress at 4 °C, suggesting a role in low-temperature-related cell responses and signal transduction pathways. Further analysis of LTRPK1-deficient transgenic plants showed that under low-temperature treatment, the growth rate of transgenic plant primary roots, which is commonly used as an indicator for cold stress response abilities, was less inhibited than that of control plants. Moreover, damage to the plasma membrane of root cells in LTRPK1-deficient plants was greater than that of controls as measured by relative electrical conductivity (REC). The malondialdehyde (MDA) content of LTRPK1-deficient plants also increased over that of the control, indicating increased plasma membrane permeability. Further immunofluorescence localization observations indicated that microtubules of transgenic plants subject to low temperature disassembled more rapidly, whereas the control plant microtubules in most cells of the root elongation zone kept their normal habitus, which suggested that LTRPK1-deficient plants had reduced capacity to resist low-temperature stress through regulation of microtubule assembly. These results demonstrate involvement of LTRPK1 in low-temperature stress and provide new insight for rice breeding and germplasm innovation to improve crop cold tolerance.