| Salt stress is one of the major stresses that depress plant growth and limit crop production, and has been a great threat to agricultural development all over the word. To cope with salt stress, some plants have the ability of either extruding Na+out of cells or sequestering Na+into the vacuole in order to decrease excessive Na+in the cytoplasm. The compartmentation of Na+into vacuoles is implemented by Na+/H+antiporter located in tonoplast. The importance of vacuolar sequestration to plant salinity resistance has been underlined by experiments in which constitutive overexpression of different vacuolar Na+/H+antiporter genes can greatly increase salinity resistance in a wide range of plant species.Poplar is one of the most important timber tree species planted in China. Therefore, understanding of the mechanism of its response to salt stress is of paramount importance to agriculture application and plant improvement. Overexpression of Na+/H+antiporters have been reported to increase the salt resistance of various herbaceous or shrub species, but very little is known about the role of these antiporters in the salt resistance of trees. Successful Agrobacterium-mediated transformation of AtNHXl to the shoot stem explants of Populus×euramericana ’Neva’ and regeneration of transgenic plants has been previously performed. In order to elucidate mechanism of overexpressing the AtNHXl gene response to salt-tolerance in woody plants, the wild-type plants (WT)(Populus×euramericana’Neva’) and its transgenic varieties (TR) that overexpress the AtNHX1gene were investigated to identify their growth and physiological character responses to salt stress. Moreover, the WT and TR were also exposed to different concentrations of seawater to clarify the correlation between overexpressing the AtNHXl gene and seawater-tolerance in poplars. All these would help to develop strategies for improving the understanding of transgenic plants response to salt stress. The main results obtained were shown as follows: 1. All tests using PCR and RT-PCR indicated that the AtNHX1gene was integrated into the genome of Populus×euramericana ’Neva’ and transcribed in transgenic poplar plants.2. Seedlings of WT and TR were subjected to different concentrations of NaCl (from0to150mmol·L-1) stress for30days. Plant growth was inhibited by salinity in both WT and TR but in different degrees, and salt-resistance of TR was much higher than that of WT. The relative growth rate of stem diameter (RGRH) values of TR were46%and44%and the relative growth rate of shoot height (RGRD) values were36%and61%more than that of WT at75and150mM NaCl treatments, respectively. Moreover, TR produced much more total leaf area and dry weight than WT.Under the same NaCl level treatments, TR maintained higher RGR, total leaf area, and dry weight than WT because TR had a higher Pn. The Pn values in TR were22%and9%higher than that of WT when grown at75and150mmol·L-1NaCl for5days, and13%and48%higher for30days. Stomatal limitation was the primary factor limiting Pn in Populus×euramericana ’Neva’ under salt stress. At the same time, maximum photochemical efficiency of PS Ⅱ (Fv/Fm), actual quantum yield of PS Ⅱ (Φps Ⅱ), photochemical quenching coefficient (qP) and relative electron transport rate (ETR) in both WT and TR decreased while non-photochemical quenching coefficient (NPQ) increased in various degrees. However, under the same level of NaCl treatment, TR maintained higher Fv/Fm, ΦpsⅡ qP, ETR and lower NPQ than those of WT. These suggest that TR could better protect PSⅡ from injury induced by the salt stress. So it had higher PSⅡ activity and light energy transform efficiency, which could turn the absorbed light energy into chemical energy effectively, and then increased the transmitting rate of photosythetic electrons, thereby enhancing biomass. To some extent, the propertites of TR decribed above improved salt resistance.The activity of antioxidant enzymes such as SOD, POD and CAT were induced to increase greatly in the leaf of TR. To WT. salt stress for30days induced obvious decline on the activity of these enzymes. These indicate that TR was likely to be more effective in removing H2O2than WT under salt stress.Differences in the pattern of K+accumulation between WT and TR were apparent under NaCl stress. In general, TR accumulated more Na+in the root, stem, and leaf than WT at both75and150mmol·L-1NaCl concentrations. The K+uptake in TR was much more than that in WT under the same NaCl level. For example, the K+contents of TR in the root, stem, and leaf were32%,38%, and35%more than those of WT under the150mmol·L-1NaCl treatment. Despite a higher accumulation of Na+in TR, it maintained relatively higher K+and K+/Na+ratio compared to WT. Results show that plants that TR was markedly more resistant to NaCl, possibly because of intracellular K+content regulation.3. Seedlings of WT and TR were exposed to different seawater concentrations (from0to30%) for30days to determine the effects of seawater on seedling growth, photosynthetic productivity, and ion content. Plant growth was inhibited by seawater in both WT and TR but in different degrees, and seawater-resistance of TR was much higher than that of WT. TR could maintain higher RGR, total leaf area, Pn, and Fv/Fm, so that it is able to accumulate more dry mass than WT at the same seawater level. TR accumulated more Na" and K+contents in plant tissue than WT. At10%seawater treatment, the dry weight of TR was unaffected, but decreased by8%and22%at20%and30%seawater treatments, respectively. These findings indicate that10%-20%seawater could be used to irrigate TR plants, thus implying the viable utilization of seawater. However, further investigations of the effects of stresses relative to transgenic poplar ontogeny are necessary to assess realistic stress pressures that occur naturally in the field.4. Seedlings of WT and TR which cultivated in pot with soil were subjected to different concentrations of NaCl (from0to150mmol·L-1) stress for30days. We investigated the salt-resistance of the WT and TR plants by measuring growth parameters, chlorophyll (Chl) and carotenoid (Car) content, Pn, ion content, leaf malondialdehyde (MDA) and electrolytic leakage. Compared with the control, the growth of WT was restrained significantly in the presence of both low salt and high salt. The dry weight of WT was significantly lower under salt stress than that of control, the dry weight decreased gradually with increasing NaCl concentration and their dry weight under high salt was just50%of the control. However, the dry weight of TR in low salt was similar to the control, up to the high salt treatment, where their dry weight was74%of the control. Moreover, the dry weight of TR was significant higher than that of WT in both NaCl treatments, and the discrepancy of dry weight was increased with increasing NaCl concentration. In the presence of NaCl, Chl and Car content of TR were significantly higher than that of WT, and the TR maintained a remarkably high Pn and Fv/Fm. Although TR accumulated more Na+in their roots and leaf tissues under salinity conditions compared with the WT, they absorbed more K+and maintained a higher K+/Na+ratio. Moreover, these TR plants kept a lower MDA and electrolytic leakage level than that of wild-type. Under salt stress, the chloroplasts of TR under low salt stress were not different from the control, and the structure of chloroplasts under high salt was better than that of WT. Under the same salinity level, the TR can maintain a relative integrated chloroplast, high chlorophyll contents and preferable net photo synthetic rate, so its salt tolerance were improved.5. These findings indicate that transformation of AtNHXl gene into Populus×euramericana ’Neva’ can confer plants more tolerance to salinity than its wild-type plants. These transgenic poplars provided a feasible way for improving important trees to adapt to salt stress conditions that are common in saline and arid soils. Our result also proved that transformation of single AtNHXl gene into plants can confer plants more resistance to salinity. |
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