Overexpression of the Wheat Expansin Gene TaEXPA2 Improved Seed Production and Drought Tolerance in Transgenic Tobacco Plants

Expansins are cell wall proteins that are grouped into two main families, α-expansins and β-expansins, and they are implicated in the control of cell extension via the disruption of hydrogen bonds between cellulose and matrix glucans. TaEXPA2 is an α-expansin gene identified in wheat. Based on putative cis-regulatory elements in the TaEXPA2 promoter sequence and the expression pattern induced when polyethylene glycol (PEG) is used to mimic water stress, we hypothesized that TaEXPA2 is involved in plant drought tolerance and plant development. Through transient expression of 35S::TaEXPA2-GFP in onion epidermal cells, TaEXPA2 was localized to the cell wall. Constitutive expression of TaEXPA2 in tobacco improved seed production by increasing capsule number, not seed size, without having any effect on plant growth patterns. The transgenic tobacco exhibited a significantly greater tolerance to water-deficiency stress than did wild-type (WT) plants. We found that under drought stress, the transgenic plants maintained a better water status. The accumulated content of osmotic adjustment substances, such as proline, in TaEXPA2 transgenic plants was greater than that in WT plants. Transgenic plants also displayed greater antioxidative competence as indicated by their lower malondialdehyde (MDA) content, relative electrical conductivity, and reactive oxygen species (ROS) accumulation than did WT plants. This result suggests that the transgenic plants suffer less damage from ROS under drought conditions. The activities of some antioxidant enzymes as well as expression levels of several genes encoding key antioxidant enzymes were higher in the transgenic plants than in the WT plants under drought stress. Collectively, our results suggest that ectopic expression of the wheat expansin gene TaEXPA2 improves seed production and drought tolerance in transgenic tobacco plants.     

Effects of water-deficit stress and paclobutrazol on growth,relative water content,electrolyte leakage,proline content and some antioxidant changes in Curcuma alismatifolia Gagnep. cv. Chiang Mai Pink

Effects of water-deficit stress and paclobutrazol (PBZ) on the physiological and biochemical changes in Curcuma alismatifolia Gagnep. cv. Chiang Mai Pink (Zingiberaceae) were investigated. One hundred rhizomes were grown for 30–35 days and then divided into the following 4 treatments: (1) well-watered, (2) not watered, (3) well-watered and treated with 1500 ppm PBZ being applied once to the soil, and (4) not watered but treated with 1500 ppm PBZ. After 50 days of growth, watering was withheld for 30 days. After water stress was initiated, plant height, plant fresh weight, soil water content, relative water content (RWC), electrolyte leakage (EL), proline content, vitamin C and E content, as well as the activities of catalase (CAT) and superoxide dismutase (SOD) in the leaves were determined every 10 days. The results showed that water-deficit stress decreased plant height and plant fresh weight, whereas this stress and PBZ did not result in a decrease in these parameters. Water stress reduced RWC, but induced EL and proline content in the leaves. However, the leaves showed opposite results when PBZ was added to the treatments. Some antioxidants such as vitamin C, vitamin E, and the activities of CAT and SOD were induced in the leaves by PBZ. Moreover, the content of vitamin C, vitamin E and CAT activity were higher in relation to water-deficit stress and PBZ treatments. This indicates that PBZ induced a number of some physiological and biochemical adaptations (maintaining growth and RWC, decreasing EL and proline content, increasing the vitamin C and vitamin E levels, and CAT and SOD activities) that enable the Curcuma plant to tolerate drought.     

Engineering stress tolerance in transgenic plants

Research in our laboratory has focused on the analysis of the functions of a variety of enzymes that are involved in the scavenging of reactive oxygen intermediates (ROI) such as superoxide radicals (·O ₂ ⁻ ) and hydrogen peroxide (H₂O₂). Recent work has been on transgenic plants that over-express glutathione S-transferases (GST) that also have glutathione peroxidase activity. Transgenic tobacco plants that contain gene constructs that encode two different tobacco GST’s had elevated levels of both GST and GPX activity. Analysis of mature vegetative transgenic tobacco plants that over-express GST/GPX failed to show any increase in paraquat tolerance or protection from photooxidative stress. However, seeds of these GST/GPX-expressing tobacco lines are capable of more rapid germination and seedling growth at low temperatures and at elevated salt concentrations. Reduced levels of lipid peroxidation were noted in GST/GPX-expressing seedling compared to control seedlings under both stressful and non-stressful conditions. In addition, GST/GPX-expressing seedlings significantly accumulated more oxidized glutathione (GSSG) than control seedlings during stress. These characteristics clearly indicate that over-expression of GST/GPX in transgenic seedlings can have substantial effects on their stress tolerance. Furthermore, it appears that this effect is due primarily to the elevated levels of GPX activity.     

Evaluation of the stress-inducible production of proline in transgenic sugarcane (Saccharum spp.): osmotic adjustment, chlorophyll fluorescence and oxidative stress

Proline accumulates in a variety of plant species in response to stresses such as drought, salinity and extreme temperatures. Although its role in plant osmotolerance remains controversial, proline is thought to contribute to osmotic adjustment, detoxification of reactive oxygen species and protection of membrane integrity. In the present study, we evaluated the effects of stress-inducible proline production on osmotic adjustment, chlorophyll fluorescence and oxidative stress protection in transgenic sugarcane transformed with a heterologous P5CS gene. In well-watered conditions, free proline, malondialdehyde (MDA) levels, Fv/Fm ratios and chlorophyll contents (Chls) in transgenic sugarcane were not statistically different from non-transformed control plants. After 9 days without irrigation, proline content in transgenic events was on the average 2.5-fold higher than in controls. However, no osmotic adjustment was observed in plants overproducing proline during the water-deficit period. The photochemical efficiency of PSII observed was higher (65%) in the transgenic events at the end of the water-deficit experiment. The effects of proline on lipid peroxidation as MDA levels and on the decline of Chl in paraquat-treated leaf discs along the drought period suggest that proline protected the plants against the oxidative stress caused by the water deficit. The overall capacity of transgenic plants to tolerate water-deficit stress could be assessed by the significantly higher biomass yields 12 days after withholding water. These results suggest that stress-inducible proline accumulation in transgenic sugarcane plants under water-deficit stress acts as a component of antioxidative defense system rather than as an osmotic adjustment mediator.     

The garlic NF-YC gene, <Emphasis Type="Italic">AsNF-YC8</Emphasis>, positively regulates non-ionic hyperosmotic stress tolerance in tobacco

To investigate the relationship between nuclear factor Y (NF-Y) and stress tolerance in garlic, we cloned a NF-Y family gene AsNF-YC8 from garlic, which was largely upregulated at dehydrate stage. Expression pattern analyses in garlic revealed that AsNF-YC8 is induced through abscisic acid (ABA) and abiotic stresses, such as NaCl and PEG. Compared with wild-type plants, the overexpressing-AsNF-YC8 transgenic tobacco plants showed higher seed germination rates, longer root length and better plant growth under salt and drought stresses. Under drought stress, the transgenic plants maintained higher relative water content (RWC), net photosynthesis, lower levels of malondialdehyde (MDA), and less ion leakage (IL) than wild-type control plants. These results indicate the high tolerance of the transgenic plants to drought stress compared to the WT. The transgenic tobacco lines accumulated less reactive oxygen species (ROS) and exhibited higher antioxidative enzyme activities compared with wild-type (WT) plants under drought stress, which suggested that the overexpression of AsNF-YC8 improves the antioxidant defense system by regulating the activities of these antioxidant enzymes, which in turn protect transgenic lines against drought stress. These results suggest that AsNF-YC8 plays an important role in tolerance to drought and salt stresses.     

Enhanced sensitivity to oxidative stress in transgenic tobacco plants with decreased glutathione reductase activity leads to a decrease in ascorbate pool and ascorbate redox state

To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses of CO₂ assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of H₂O₂ in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate redox state.     

Exogenous Selenium Mitigates Salt Stress in Soybean by Improving Growth,Physiology, Glutathione Homeostasis and Antioxidant Defense

The mechanism of selenium (Se)-induced salt tolerance was studied in moderately sensitive soybean (Glycine max L.) plants. To execute this view, soybean plants were imposed with salt stress (EC 6 dS m⁻¹ ) applying NaCl. In other treatments, Se (0, 25, 50 and 75 µM Na₂SeO₄) was sprayed as co-application with that level of salt stress. Plant height, stem diameter, leaf area, SPAD value decreased noticeably under salt stress. Altered proline (Pro) level, together with decreased leaf relative water content (RWC) was observed in salt-affected plants. Salt stress resulted in brutal oxidative damage and increased the content of H₂O₂, MDA level and electrolyte leakage. Exogenous Se spray alleviated oxidative damage through boosting up the antioxidant defense system by increasing the activity of antioxidant enzymes such as catalase (CAT), peroxidase (POD) and glutathione reductase (GR), as well as by improving non-enzymatic antioxidants like glutathione (GSH) and GSH/glutathione disulfide (GSSG). The upregulated antioxidant defense system, restored Pro and leaf RWC, higher SPAD value conferred better growth and development in Se-sprayed salt-affected soybean plants which altogether put forth for the progressive yield contributing parameters and finally, seed yield. Among different doses of Se, soybean plants sprayed with 50 µM Na₂SeO₄ showed better salt tolerance.     

Increased tolerance of rice to cold, drought and oxidative stresses mediated by the overexpression of a gene that encodes the zinc finger protein ZFP245

ZFP245 is a cold- and drought-responsive gene that encodes a zinc finger protein in rice. The ZFP245 protein localizes in the nucleus and exhibits trans-activation activity. Transgenic rice plants overexpressing ZFP245 were generated and found to display high tolerance to cold and drought stresses. The transgenic plants did not exhibit growth retardation, but showed growth sensitivity against exogenous abscisic acid, increased free proline levels and elevated expression of rice pyrroline-5-carboxylatesynthetase and proline transporter genes under stress conditions. Overproduction of ZFP245 enhanced the activities of reactive oxygen species-scavenging enzymes under stress conditions and increased the tolerance of rice seedlings to oxidative stress. Our data suggest that ZFP245 may contribute to the tolerance of rice plants to cold and drought stresses by regulating proline levels and reactive oxygen species-scavenging activities, and therefore may be useful for developing transgenic crops with enhanced tolerance to abiotic stress.