【作者】 李红；

【导师】 武维华； 王毅；

【作者基本信息】 中国农业大学 ， 植物学， 2016， 博士

【摘要】 钾和氮是植物生长发育所必需的矿质营养元素。在农作物生产中,钾和氮的供给水平直接影响农作物的产量和品质。然而,我国耕地土壤普遍缺钾和氮,农作物生产中需大量施用钾肥和氮肥。已有的研究报道显示,植物可以协同吸收利用钾和氮。长期的农业生产实践早已证明,按比例施用钾肥和氮肥可以显著提高肥料的吸收利用效率。然而,长久以来植物协同吸收利用钾和氮的分子机制仍不清楚。本实验室前期筛选拟南芥EMS诱变群体获得低钾敏感突变体lks2。图位克隆结果显示,lks2中NPF家族成员基因NRT1.5/NPF7.3发生单碱基突变,导致氨基酸序列的第209位甘氨酸突变为谷氨酸。本论文工作以此为基础,深入研究了NRT1.5参与钾离子(K+)在木质部装载的分子机制,探讨了K+和NO3-在木质部协同运输的分子调控机制。低钾移苗实验结果显示,与野生型相比,lks2和nrtl.5突变体表现出冠部提前发黄、根部持续生长的低钾敏感表型。回补材料lks2/NRT1.5, nrtl.5/NRT1.5能将突变体的低钾敏感表型恢复至野生型水平,表明Iks2、nrtl.5的低钾敏感表型是由NRT1.5的功能缺失导致。钾离子含量测定结果显示,无论在正常条件还是低钾条件下,nrtl.5、lks2冠部的K+含量都较野生型显著降低,而根部K+含量则较野生型显著升高,说明K+在突变体根部积累而不能被转运至冠部。硝酸根含量测定结果显示,在突变体中N03-的分布也呈现出根部高冠部低的特点。进一步检测发现,nrt1.5和lks2突变体木质部伤流液中K+和N03-含量都显著低于野生型,表明NRT1.5可能参与调控K+和NO3-由根部向冠部协同运输的生理过程。离子依赖性实验结果显示,nrt1.5和lks2突变体冠部发黄的敏感表型只依赖于外界的低钾条件,而并不依赖于外界的N03-浓度。植株体内的阳离子含量测定结果显示,nrtl.5和lks2突变体的Ca2+和Mg2+的分布并未受到显著影响,说明NRT1.5特异地调控K+由根部向冠部的转运,而不影响其它阳离子的转运。利用非洲爪蟾卵母细胞异源表达系统分析NRT1.5的离子转运功能,结果显示NRT1.5可以直接介导细胞内的K+向细胞外转运,并且NRT1.5向细胞外转运K+的活性依赖于胞外的酸性条件,而不依赖于胞内外的NO3-浓度,说明NRT1.5作为一个H+/K+反向转运体向细胞外转运K+。进一步研究显示,来自拟南芥、玉米和水稻中的NRT1.5同源蛋白都具有外向转运K+的活性。本论文工作的研究结果表明,拟南芥NRT1.5作为K+外向转运体介导K+在木质部的装载,并调控K+从根部到冠部的长距离运输,同时也影响N03-在根冠的分配。本论文研究结果还提示,NRT1.5及其同源蛋白可能是植物中一类新的K+转运蛋白,并可能参与调控植物体内K+长距离运输的生理过程。本论文的研究结果对于认知植物长距离转运钾的分子调控机制具有重要意义,也为将来利用分子遗传手段改良作物钾和氮的养分吸收利用效率提供了重要的理论依据。更多还原

【Abstract】 As essential macronutrients, potassium and nitrogen play crucial roles in plant growth and development. Sufficient supply of potassium and nitrogen also determine crop yield and quality. In China, the large areas of arable land are deficient in potassium and nitrogen, so that a large amount of potassium and nitrogen fertilizers is required for crop production. Previous studies have indicated that the absorption and translocation of potassium (K+) and nitrogen (NO3) are correlated each other. However, the molecular mechanism for the coordination between K+and NO3- transport remains unclear. Our previous work has isolated an Arabidopsis low-K+-sensitive mutant lks2 from EMS-mutagenized seedlings. Map-based cloning showed that LKS2 encodes NPF family member NRT1.5/NPF7.3. A single-nucleotide mutation (G3242A) in NRT1.5 gene was found in lks2 mutant, which resulted in a change of Gly209 to Glu (G209E) in NRT1.5 amino acid sequence. The focus of this dissertation work is to analyze the function and molecular mechanism of NRT1.5 in K+ loading in xylem in Arabidopsis.Phenotype test indicated that lks2 and nrtl.5 mutants showed similar K+-sensitive phenotype on low K+(LK) medium, whose shoots became yellow compared with wild-type plants after the plants were transferred to LK medium for 7 days. However, the primary roots of lks2 and nrtl.5 still kept growing on LK medium, while the root growth of wild-type plants stopped. The complementation lines were able to resuce the low-K+-sensitive phenotype of both nrtl.5 and Iks2, which confirmed that the low-K+-sensitive phenotype of mutants was due to the loss-of-function of NRT1.5. The ion content measurements showed that the K+ contents in mutant shoots were lower than that in wild-type plants, while the K+ contents in mutant roots were greater. Similar to K+, the NO3- contents were increased in mutant roots and reduced in mutant shoots on LK medium. Morevere, both K+ and NO3- concentrations in xylem exudate from mutant plants were significantly reduced under LK condition. These results indicated that the translocation of both K+ and NO3- from root to shoot was impaired in mutant plants, suggesting that NRT1.5 not only participates in K+ translocation, but also regulating NO3- transport from root to shoot.The ion dependence analyses indicated that the leaf chlorosis phenotype of nrtl.5 and lks2 specifically depends on external low K+, but not on NO3- or NH4+. In addition, the results of ion content measurement showed that only the root/shoot distribution of K+, but not Ca2+ or Mg2+, was significantly changed in lks2 and nrtl.5 mutants, suggesting that NRT1.5 specifically regulates K+ transport rather than any other cation transport.Using Xenopus laevis oocytes, the ion transport activity of NRT1.5 was analyzed. The results showed that NRT1.5 directly mediated K+ efflux out of the oocytes, and the K+ efflux activity of NRT1.5 relied on the external acidic pH but not on the NO3- concentrations. It is suggested that NRT1.5 functions as an H+/K+ antiporter that mediates K+ efflux from cells. In addition, the NRT1.5 orthologues from Arabidopsis, rice and maize can also function as K+ efflux transportersIn summary, the results presented in this dessertation reveals that NRT1.5 functions as a K+ efflux transporter not only involving in K+ loading into the xylem, but also regulating NO3- translocation. The results also suggests that NRT1.5 as well as its orthologues may be a new type of K+ transporters in plants, which play important roles in K+ distribution in plants. This study not only reveals the mechanism for K+ loading in the xylem, but aslo provides the theoretical basis for improving corp potassium and nitrogen utilization efficiency.更多还原