【作者】 王茜；

【导师】 王锁民；

【作者基本信息】 兰州大学 ， 草业科学， 2014， 博士

【摘要】 土壤盐渍化已成为世界范围内限制作物质量和产量的重要环境因子。植物体内积累过量的Na+会扰乱细胞离子稳态平衡、损伤细胞膜并抑制代谢酶活性,从而导致植物生长受到抑制甚至引起死亡。其中由低亲和性吸收系统介导的毒性Na+吸收是造成植物盐害的诱因,因此减少有害Na+进入植物体内是解决这一问题的关键。然而,截至目前为止,关于植物根系低亲和性Na+吸收的途径却仍不清楚。与此同时,毒性Na+和营养性K+在植物体内的分配更是植物耐盐性的关键所在,因此,在了解Na+吸收途径的同时,探讨Na+、K+在植物体内的稳态平衡过程,可为提高植物耐盐性提供重要理论依据。本论文以拟南芥(Arabidopsis thaliana)野生型(WT)、hktl突变体(athkt1;1)及sosl突变体(atsos1)为研究材料,首先运用各类K+通道或转运蛋白抑制剂,对不同Na+、K+处理下,WT和athkt1;1根系22Na+内流、Na+、K+净吸收速率、Na+、K+积累及相关基因的表达模式进行分析,初步探讨了拟南芥根系低亲和性Na+吸收途径；随后,再对不同Na+、K+处理下,WT、athkt;1和atsos1体内Na+、K+积累及分配进行分析,进而对AtHKT1;1和AtSOS1如何协同调节拟南芥体内Na+、K+稳态平衡进行了探讨。取得如下主要结果：1.在正常K+(2.5mM)加盐(25mM NaCl)条件下,10mM Ca2+口5mMBa2+能显著抑制拟南芥野生型(WT)根系22Na+内流及其Na+净吸收速率,但不影响野生型(WT)中K+的净吸收速率；同时,AtHKT1;1受25mM NaCl的短期诱导,其表达量在盐处理3h后急剧上调。由此可见,在正常K+(2.5mM)加盐(25mM NaCl)条件下,NSCC是拟南芥野生型(WT)根系主要的低亲AtHKT1;1和性Na+吸收途径。2.在正常K+(2.5mM)加盐(25mM NaCl)条件下,Ca2+、Ba2+、TEA+和NH4+均能显著抑制拟南芥hktl；1突变体(athkt1;1)根系22Na+内流及其Na+净吸收速率；同时,AtHAK5受25mM NaCl的短期诱导,其表达量在盐处理3h后急剧上调。由此可见,在正常K+(2.5mM)加盐(25mM NaCl)条件下,NSCC和AtHAK5是拟南芥hkt1；1突变体(athkt1;1)根系主要的低亲和性Na+吸收途径。3.低K+(0.01mM)胁迫7d后,拟南芥野生型(WT)和hkt1；1突变体(athkt1;1)的Na+净吸收速率、根及地上部Na+浓度均急剧增加,但其组织含水量却无显著差异。由此可见,K+亏缺条件下,植物会吸收部分Na+代替K+行使保持体内渗透势平衡及细胞膨压等生理过程的功能。4.在低K+(0.0l mM)加盐(25mM NaCl)条件下,尽管各抑制剂对拟南芥野生型(WT) Na+、K+净吸收速率无显著影响,但与正常K+(2.5mM)加盐(25mM NaCl)相比,其Na+净吸收速率由50nmol/g.RFW/min增至近130nmol/g. RFW/min;同时,与正常K+(2.5mM)加盐(25mM NaCl)相比,拟南芥野生型(WT)中,AtHKT1;1的表达水平在低K+(0.01mM)加盐(25mM NaCl)处理下显著下调,而AtHAK5则显著上调。以上结果表明,在低K+(0.01mM)加盐(25mM NaCl)条件下,主要由AtHAK5调节拟南芥野生型(WT)根系低亲和性Na+的吸收。5.在低K+(0.0l mM)加盐(25mM NaCl)条件下,与拟南芥野生型(WT)相似,尽管各抑制剂对拟南芥hktl；1突变体(athkt1;1)Na+、K+净吸收速率无显著影响,但与正常K+(2.5mM)加盐(25mM NaCl)相比,其Na+净吸收速率由50nmol/g. RFW/min曾至100nmol/g. RFW/min;同时,与正常K+(2.5mM)加盐(25mM NaCl)相比,拟南芥hkt1；1突变体(athkt1;1)中,AtHAK5的表达水平显著上调,并随着盐处理时间的延长,其表达量持续增加。以上结果表明,在低K+(0.01mM)加盐(25mM NaCl)条件下,主要时由AtHAK5介导拟南芥hktl,1突变体(athkt1;1)根系低亲和性Na+的吸收。6.正常K+(2.5mM)加轻度盐胁迫(25mM NaCl)下,拟南芥hktl；1突变体(athkt1;1)拥有更高的地上部Na+浓度和更低的根Na+浓度,及更低的地上部K+浓度和更高的根K+浓度,但整体长势与拟南芥野生型(WT)相似；而拟南芥sosl突变体(atsosl)则拥有更高的根Na+浓度和更低的K+净吸收速率,且整体长势显著受抑。由此可见,AtHKT1;1与AtSOS1协同调节正常K+加轻度盐胁迫下拟南芥体内Na+、K+平衡：其中AtHKT1;1主导Na+从木质部中的卸载过程,而AtSOS1主导Na+外排过程,进而共同维持植物体内低Na+浓度。7.正常K+(2.5mM)加重度盐胁迫(100mM NaCl)下,拟南芥hkt1；1突变体(athkt1;1)拥有更高的地上部Na+浓度和更低的根Na+浓度,及更低的K+净吸收速率；而拟南芥SOS1突变体(atsos1)则拥有更高的根及地上部Na+浓度。由此可见,AtHKT1;1与AtSOS1协同调节正常K+加重度盐胁迫下拟南芥体内Na+、K+平衡：其中AtHKT1;1主导Na+从木质部中的卸载过程,而AtSOS1主导Na+外排过程,进而共同维持植物体内低Na+浓度。8.低K+(0.01mM)加轻度盐胁迫(25mMNaCl)下,拟南芥hkt1；1突变体(athkt1;1)与拟南芥野生型(WT)在长势及离子吸收积累方面均无显著差异；而拟南芥sos1突变体(atsos1)则拥有更高的根Na+浓度和更低的地上部Na+浓度,且整体长势显著受抑。由此可见,AtHKT1;1与AtSOS1协同调节低K+加轻度盐胁迫下拟南芥体内Na+、K+平衡：其中AtHKT1;1功能受低K+条件抑制,AtSOS1主导Na+在木质部的装载,进而在K+亏缺环境下,利用Na+代替K+维持植物体的渗透平衡。9.低K+(0.01mM)加重度盐胁迫(100mM NaCl)下,拟南芥hktl；1突变体(athktl;1)拥有更低的Na+净吸收速率；拟南芥sos1突变体(atsos1)则拥有更低的地上部Na+浓度和更低的K+净吸收速率,整体长势同样显著受抑。由此可见,AtHKT1;1与AtSOS1协同调节低K+加重度盐胁迫下拟南芥体内Na+、K+平衡：其中AtHKT1;1主导根系Na+吸收,AtSOS1主导Na+在木质部的装载,进而协同利用Na+代替K+维持植物体的渗透平衡,从而改善低K+高盐对植物造成的双重伤害。综上所述,NSCC、AtHKT1;1和AtHAK5协同调节不同Na+、K+环境下根系对Na+的吸收；同时,AtHKT1;1与AtSOS1构成的调控网络对植物体在各种Na+、K+胁迫下,维持其体内Na+、K+稳态平衡具有重要作用。更多还原

【Abstract】 Salinity is one of adverse environments that limits production of crop plants worldwide. High [NaCl]ext disturbs intracellular ion homeostasis, leads to cell membrane dysfunction and attenuation of metabolic activity, which cause growth inhibition and lead ultimately to plant death. Low-affinity Na+uptake is toxic to the cytoplasm of most of crop plant, so reducing Na+influx must be the key step for controlling Na+accumulation in plants and improving salt tolerance of crop plants. However, the pathways by which plants take up Na+are still unclear. At the same time, the distribution of toxic Na+and nutrient K+is still the key of salt tolerance of crop plants. Therefore, we not only research the pathway of low-affinity Na+uptake in plants, but also study on the mechanism of Na+, K+homeostasis in plants, which is also important to improve salt resistance of crop plants. Nevertheless, there is little research on this field. The aims of this Ph.D work, therefore, are to discuss the pathway of low-affinity Na+uptake in Arabidopsis thaliana by analyzing22Na+influx, net Na+and K+uptake rate, Na+and K+accumulations, and the expression patterns of relevant genes in both WT and athkt1;1under different treatments. Simultaneously, we investigated the roles of AtHKT1;1and AtSOS1in mediating homeostasis of Na+and K+in plants. The main foundings and conclusions are as follows:1. Under2.5mM K+plus25mM NaCl,10mM Ca2+and5mM Ba2+could inhibit22Na+influx and net Na+uptake rate in WT; Meanwile, AtHKT1;1was induced by short-term of25mM NaCl, and its expression level up-regulated sharply after3h treatment. The results indicated that NSCC and AtHKT1;1might are main pathway of low-affinity Na+uptake in WT under2.5mM K+plus25mM NaCl. 2. Under2.5mM K+plus25mM NaCl,10mM Ca2+,5mM Ba2+,10mM TEA+and5mM NH4+could inhibit22Na+influx and net Na+uptake rate in athkt1;1significantly; Meanwile, AIHAK5was induced by short-term of25mM NaCl, and its expression level up-regulated sharply after3h treatment. The results indicated that NSCC and AtHAK5might are main pathway of low-affinity Na+uptake in athkt1;1under2.5mM K+plus25mM NaCl.3. After low K+(0.01mM) for7d, net Na+uptake rate, root and shoot Na+concentrations were increased greatly in both WT and athkt1;1, while the tissue water had no difference. These results implied that Na+uptake by roots was induced by K+deficiency, because Na+could instead of K+to keep the osmotic equilibrium and cell turgor under low K+condition.4. Under0.01mM K+plus25mM NaCl, the inhibitors had on effect on net Na+, K+uptake rate in WT. However, compared with2.5mM K+plus25mM NaCl, Na+uptake rate was increased from50nmol/g. RFW/min to130nmol/g. RFW/min in WT under0.01mM K+plus25mM NaCl; Meanwile, compared with2.5mM K+plus25mM NaCl, AtHKT1;1down-regulated as well as AtHAK5up-regulated under0.01mM K+plus25mM NaCl. The results indicated that AtHAK5might are main pathway of low-affinity Na+uptake in WT under0.01mM K+plus25mM NaCl.5. Under0.01mM K+plus25mM NaCl, the inhibitors had on effect on net Na+, K+uptake rate in athkt1;1, which was similar to WT. However, compared with2.5mM K+plus25mM NaCl, Na+uptake rate was increased from50nmol/g. RFW/min to100nmol/g. RFW/min in athkt1;1under0.01mM K+plus25mM NaCl; Meanwile, AtHAK5up-regulated significantly under0.01mM K+plus25mM NaCl, and its expression level increased continuously with prolonging of salt stress. The results indicated that AtHAK5might are main pathway of low-affinity Na+uptake in athkt1;1under0.01mM K+plus25mM NaCl.6. Under2.5mM K+plus25mM NaCl, there were the less root Na+concentration and the more shoot Na+concentration in athkt1;1, and there were the more root K+ concentration and the less shoot K+concentration in athktl;1, however, there was no difference on growth conditon between WT and athktl;1. While atsosl possessed the more root Na+concentration and the lower net K+uptake rate, meanwhile, compared with WT, the growth conditon of atsosl was inhibited significantly. These results implied that coordination between AtHKT1;1and AtSOS1in mediating Na+、K+homeostasis in Arabidopsis thaliana under the normal K+with mild salt stress: AtHKT1;1mainly involved in Na+unloading and AtSOS1mainly involved in Na+exclusion, which kept the low Na+level in plants.7. Under2.5mM K+plus100mM NaCl, there were the less root Na+concentration and the more shoot Na+concentration in athktl;1, and there were the lower net K+uptake rate in athkt1;1. While atsosl possessed the more root and shoot Na+concentrations. These results implied that coordination between AtHKT1;1and AtSOS1in mediating Na+、K+homeostasis in Arabidopsis thaliana under the normal K+with severe salt stress:AtHKT1;1mainly involved in Na+unloading and AtSOS1mainly involved in Na+exclusion, which kept the low Na+level in plants.8. Under0.01mM K+plus25mM NaCl, there were no difference between WT and athkt1;1on both growth conditon and ions accumulations. While atsosl possessed the more root Na+concentration and the less shoot Na+concentration, meanwhile, compared with WT, the growth conditon of atsosl was inhibited significantly. These results implied that coordination between AtHKT1;1and AtSOS1in mediating Na+、K+homeostasis in Arabidopsis thaliana under the low K+with mild salt stress:the activity of AtHKT1;1was inhibted by low K+, AtSOS1mainly involved in Na+loading into the xylem, which could insure Na+insteaded of K+to keep the osmotic equilibrium and cell turgor under low K+condition.9. Under0.01mM K+plus100mM NaCl, there were the less root Na+concentration and the more shoot Na+concentration in athkt1;1, and there were the more root K+concentration and the less shoot K+concentration in athkt1;1, at the same time, it growth conditon was inhibited significantly. While atsosl possessed the less root Na+concentration and net K+uptake rate, meanwhile, compared with WT, the growth conditon of atsosl was inhibited significantly. These results implied that coordination between AtHKTl;1and AtSOS1in mediating Na+、K+homeostasis in Arabidopsis thaliana under the low K+with severe salt stress:AtHKT1;1mainly involved in Na+uptake and AtSOS1mainly involved in Na+loading into the xylem, which could insure Na+insteaded of K+to keep the osmotic equilibrium and cell turgor under low K+condition.In summary, our study suggest that the NSCC, AtHKT1;1and AtHAK5mediate low-affinity Na+uptake together in Arabidopsis thaliana under different Na+, K+conditons; In addition, AtHKT1;1and AtSOSl play the key roles in keeping Na+, K+homeostasis in Arabidopsis thaliana under salt stress and K+deficiency conditions.更多还原