| Within their natural habitat, plants are often subjected to a combination of various abiotic stress conditions such as drought and high temperature. The effects of individual drought or high temperature stress on plant have been extensively studied. However, only a little is known about how their combination impacts on plant. Glycine betaine (GB) plays an important role in protecting plant cell from damage by various single stresses, but what does it under the combination of drought and high temperature stresses? In the present study, to investigate the different responses of wheat to drought, high temperature stress and their combination, and analyze the physiological mechanisms of GB improvement involved in the wheat tolerance to stress conditions, a transgenic wheat line T6 and the wild-type (WT) line Shi4185 were used. The transgenic line was generated by introducing a BADH gene into wheat by microprojectile bombardment, the BADH gene encoded a betaine aldehyde dehydrogenase, which was cloned from Atriplex hortensis L. in the plasmid pABH9 containing maize ubiquitin promoter and bar gene, the transgenic wheat plants T6 exhibits higher BADH activity and GB accumulation than WT. The preparation of experimental treatments in this study was conducted by three ways: laboratory cultivation, pot cultivation outroom and field cultivation. There were three treatments in laboratory test and pot experiment, including single drought (DS), heat stress (HS) and their combination (DS+HS), Field investigation was conducted on 20 and 28 may 2009 under natural condition. In this time, air dry, high temperature and high light stresses occurred simultaneously. Photosynthetic gas exchange, water status, and lipid peroxidation of the wheat leaves were examined in all experiments.1. The effects of over-accumulated GB on photosynthesis of wheat seedlings leaveWhen the 3 rd leaf was expanded fully, all treatments were performed with at least three parallels. Drought was imposed by 30% (w/v) PEG-6000 (osmotic potential -1.88 MPa) until the relative water content (RWC) of leaves reached to 83% to 86% (continual 3 days typically). A combination of drought and high temperature stress was performed by subjecting the drought-stressed plants to a high temperature of 40°C for 3 h. High temperature stress was applied by raising the temperature in artificial chamber to 40°C for 3 h at the same time when stress combination was executed. The results are as follows:(1) The effects of different stresses on the photosynthesis of wheat seedlings leaves were different. Chlorophyll content, net photosynthesis rates (Pn), the apparent quantum yield (AQY) and the carboxylation efficiency of photosynthesis (CE) were decreased significantly in both wheat lines (P<0.05) under both drought and heat stress, and they were greatly aggravated by a combination of drought and heat stress. Compared the results under drought stress to high temperature stress; the decrease of these parameters was greater under high temperature than that under drought stress. But the effect of drought stress on transpiration rate (Tr), stomatal conductance (Gs) and intercellular CO2 concentration (Ci) were more serious than high temperature stress. Individual drought stress resulted in opening of stomata, but under individual high temperature stress, that was opened as non-stressed control. However, after drought stress treatment, the succedent high temperature stress can not lead to opening stomata. Introducing foreign BADH gene into wheat induced visibly the overaccumulation of GB, and GB accumulation level was also induced by drought,high temperature stress and their combination. Overexpression of GB in T6 increased the tolerance of photosynthesis not only to individual drought or high temperature stress but also to their combination.(2) Over accumulated GB can help the transgenic wheat leaves to maintaining well water status compared to WT. Both drought and a combination of drought and heat stresses resulted in a significant (P<0.05) decrease in RWC of leaves, but heat stress can not gave significant effect on it. Besides as an osmolyte, overaccumulated GB could increase the level of osmotic adjustment (OA) via accumulating some other compatible solutes such as proline and soluble sugar, all them can decrease osmotic potential of the cell and enhance water-absorbing, and consequently maintain a better water status in wheat leaves. Otherwise, the improvement of GB on water status may be related to protecting the aquaporin (AQP) in cell membrane.(3) Overaccumulated GB enhanced the antioxidant ability of the transgenic wheat. Stresses caused the increases of reactive oxygen species (O2 and H2O2) content, malondialdehyde (MDA) content, and ultimately resulted in electrolyte leakage of plant cell in wheat leaves. Both drought and high temperature stress increased ion leakage of the cell significantly (p<0.05), but the increase was slightly greater under heat than drought stress, and the most increase was observed under stress combination. The activity observations of several main antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) suggested that overaccumulated GB can maintain higher enzyme activity in transgenic wheat leaves under stresses, and the increased accumulations of two non-enzyme antioxidants, namelyascirbic acid (AsA) and glutathione (GSH) were also higher in T6 than that in WT. The increased antioxidant enzymes activity and non-enzyme antioxidant accumulation may be the major cause that GB can help to scavenge O2 and H2O2 indirectly, because GB is ineffective in scavenging O2 and H2O2 directly.(4) Overaccumulated GB alleviates the damage of functional protein and the thylakoid membrane integrity under stresses through maintaining lipid components and polypeptides. The index of unsaturated fatty acids (IUFA) of thylakoid membrane lipids decreased under almost all stresses. Transgenic line T6 with overaccumulated GB had a relatively stable status under stresses in components and levels of thylakoid membrane lipids. Similarly, the abundance of 20~60 kDa polypeptides was affected by stresses and some new polypeptide appeared under stresses. However, the influences were smaller in T6 than that in WT.(5) Overaccumulated GB protected chloroplast ultrastructure in wheat seedling leaves. The shape of the chloroplast was spherical, the thylakoid lamellae was swollen under drought stress, and thylakoid lamellae was scattered under high temperature; the most severe damage was found under combined stress. However, over accumulated GB can protect chloroplast ultrastructure from damage by stresses. The chloroplast and thylakoid lamella ultrastructure were damaged by all kinds of stresses, but the demolition in WT was more serious than that in T6 under stresses, suggesting the protection of overexpressed GB to photosynthetic apparatus of wheat leaves.2. The effects of overaccumulated GB on photosynthesis of wheat flag leaves at flowering stage.Seeds of both WT and T6 were sown in earthenware pots (high, 18cm; diameter, 24 cm) filled with 3 kg soil composed of loam and organic fertilizer by the ratio in 7:3 and 250 g complete fertilizer (NH4NO3:K3PO4:KNO3 = 20:10:20). Seven seeds were sown in each pot initially, and five plants per pot were left after germination. Then, all plants were grown in a greenhouse with conventionality cultivation until flowering stage. All treatments were performed in parallel. Drought stress was imposed by withdrawing water from plants until the flag leaves reached relative water content (RWC) of 76% to 84% (typically 6–7 d). High temperature was applied by raising the temperature in the growth chamber to 40°C for 4 h. A combination of drought and high temperature was performed by subjecting drought-stressed plants (RWC of 76%–84%) to a high temperature treatment (40°C for 4 h). After treatments, the relative indexes were examined. The results are as follows:(1) Over accumulated GB increased photosynthesis of the wheat flag leaves. Introducing of foreign BADH gene visibly enhanced the accumulation of GB in wheat flag leaves, and the contents were higher than that in wheat seedling leaves, this may be related to the developmental stages and environment of wheat growth. Similarly, overaccumulated GB in T6 increased the tolerance of wheat flag leaves photosynthesis not only to individual drought or high temperature stress but also to their combination.(2) Under stresses, the T6 plants maitained the higher nitrogen assimilation than WT. Drought stress induced the increase of the activities of nitrate reductase (NR) and glutamine synthetase (GS), but high temperature and combined stress significantly decreased them (P < 0.05), indicating the sensitivity of two enzymes to high temperature. The activities of NR and GS were significantly higher in T6 than that in WT under different stresses. The higher proline and free amino acid level in T6 may be involved in the higher nitrogen (N) metabolism under stresses. (3) Under stresses, overaccumulated GB increased the wheat flag leaves PSII photochemistry activity. Drought had no significant effect on the maximal efficiency (Fv/Fm), however, the high temperature and the combined stress significantly decreased the Fv/Fm and actual efficiency (ФPSII). Overaccumulated GB in T6 alleviated the decrease of both Fv/Fm andФPSII under stress conditions, suggesting the higher activity of PSII reaction center in T6 than WT. The results showed that the PSII complex in T6 is better in with standing photoinduced inactivation than WT under stress conditions.(4) Drought stress increases thermostability of PSII. An increased resistance of PSII to high temperature in drought-stressed leaves was observed, which was associated with an improvement of the resistance of PSII reaction centre to high temperature, as indicated by a smaller increase in the proportion of QB-non-reducing PSII reaction centre during high temperature in drought-stressed leaves than in well-watered plants. In addition, these results demonstrate that increased thermostability of PSII in drought-stressed plants was also associated with an improvement in thermostability of the O2-evolving complex (OEC), as shown by a less pronounced phase K in the polyphasic fluorescence transients in combination-stressed plants than in the high temperature -stressed plants.(5) Overaccumulated GB improved xanthophyll cycle-dependent energy dissipation. Under all three stress conditions, the NPQs of two wheat plants were significantly (P<0.05) increased, and the NPQ was relatively higher in T6 than WT. Meanwhile, the changes of the (A + Z) / (V + A + Z) in wheat leaves were consistent with the NPQ under different stresses. Because (A + Z) / (V + A + Z) is positive correlation to the energy dissipation ability, the results suggested that overaccumulated GB can enhance the thermal dissipation in the T6 leaves by increasing xanthophyll cycle.3. The effects of overaccumulated GB on diurnal variation of photosynthetic rate in wheat flag leaves during grain-filling period in the field condition.Under natural field condition, diurnal variation of photosynthesis in wheat flag leaves during grain-filling period was measured on a sunny day. The results are as follows:(1) Under the field condition, diurnal variation of stomatal conductance and net photosynthetic rate revealed two peaks at about 10:00 and 16:00 separately because of the strong intensity of sunlight, high temperature and relatively low humidity. The maximum at 10:00 was higher than that at 16:00 with a minimum at 12:00. The changes of other gas parameters were almost consistent with the net photosynthetic rate. Overaccumulated GB significantly decreased the gap between the maximum and minimum of net photosynthetic rate.(2) Overaccumulated GB can alleviate the photoinhibition of photosynthesis in wheat flag leaves in the filed. We compared the photoinhibition of photosynthesis of T6 leave to WT subjected to midday strong light stress under field conditions. This was done by measuring diurnal variation of Fv/Fm andФPSII of PSII. The diurnal changes of the Fv/Fm andФPSII were similar to that of net photosynthetic rate. The Fv/Fm andФPSII in WT were much lower than those of T6 leaves in both maxima and minimum level, showing a more severe photoinhibition in WT. Based on these results, we concluded that the overaccumulated GB may be benefit to increase the quality and yield of wheat under stress or in the years which occurre Dry-heat Wind.(3) The effects of overaccumulated GB on the yield parameter. Over accumulated GB had no significant effects on wheat yield (P>0.05); however, there were more seeds per spike in T6 than in WT, significantly (P<0.05), indicating that overaccumulated GB maybe affect on the spike development of the wheat.(4) At wheat filling stage, the non-photochemical quenching (NPQ) was higher in T6 than that in WT. During the wheat filling stage,"Midday Depression of wheat Photosynthesis"was serious in further, the depression of the PSII reaction center in the wheat flag leaves aggravated, and the energy dissipation in wheat flag leaves of two wheats decreased, too, with a minimum at 12:00. But the NPQ was higher in T6 than that in WT during a day. These results show that overaccumulated GB can alleviate"Midday Depression of wheat Photosynthesis"in the later growing period of wheat by decreasing the photoinhibition. |
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