| 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. |
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