Roles Of Nitrate Transporter 1.1 In Plant Tolerace To Stresses Of H~+ And Low K~+ And Their Underlying Mechanisms

Abiotic stress is the primary factor of crop loss worldwide,causing average yield losses of more than 50%for major crops.Among various abiotic stresses,proton?H⁺?toxicity and potassium?K⁺?deficiency are two most widespread stressing factors in acid soil.Plants have developed a series of adaptive mechanisms in response to H⁺stress and low-K⁺stress during the long period of evolution.As an essential macronutrient for plant growth and development,nitrogen was found to be closely related to the ability of plants to adapt to H⁺stress and low-K⁺stress in many physiological studies,however,the molecular mechanisms are not fully understood.Given the basic theory that plants absorb NO₃^-together with an equal amount of H⁺but excreting H⁺when they take up K⁺,we proposed that NRTs?nitrate transporters?may play an important role in plant tolerating H⁺stress and low-K⁺stress.Therefore,we used Arabidopsis thaliana wild-type,and NRTs?nitrate transporters?-related mutants and transgenic plants to investigate the roles and the underlying mechanisms of NRTs,especially the NRT1.1,in improving the plant tolerance to H⁺stress and low-K⁺stress by using several physiological and molecular biological approaches.The main results are as follows:1.We firstly studied the effect of loss of NRT1.1 function on tolerance to H⁺stress in Arabidopsis plants grown on agar medium.Under H⁺stress,the root elongation and biomass production of NRT1.1-null mutants nrt1.1-1 and chl1-5 were significantly reduced compared with those of Col-0 plants.Similar results were also observed in another NRT1.1-null mutants chl1-6.We then examined H⁺resistance of another NRT knockout mutants including nrt1.2,nrt2.1,nrt2.2,nrt2.4,and nrt2.5.However,these nitrate uptake-related mutants showed similar biomass production and root elongation to their corresponding wild-type plants.These results showed that only NRT1.1 is involved in Arabidopsis H⁺resistance.By comparing the biomass production of Col-0 plants and nrt1.1-1,chl1-5 mutants in a low-NO₃^-or NO₃^--removed media,we found that NRT1.1-conferred H⁺resistance requires a sufficient NO₃^-in the growth medium.To clarify the function of NO₃^-in NRT1.1-conferred plant H⁺resistance,we examined the H⁺resistance of nlp7-2mutants,which shows common characteristics with NRT1.1-null mutants in loss function of numerous NO₃^-sensing processes,but has a normal NO₃^-uptake activity and chl1.9 mutants,whose NO₃^-uptake is reduced,but the NO₃^-sensing function is normal.The results indicated that the NO₃^--uptake activity,rather than the NO₃^--sensing process,may be a part of the NRT1.1-conferred H⁺resistance.2.We further investigated the mechanism underling how NRT1.1 regulates plants to adapt to H⁺stress.The results showed that the expression of NRT1.1 was significantly increased after low-pH treatments,GUS staining was also analyzed in the roots of pNRT1.1::NRT1.1-GUS transgenic plants,and showed that low-pH treatments caused a clear increase in NRT1.1-GUS protein levels.Furthermore,H⁺stress obviously increased the uptake rate of NO₃^-in Col-0,but had little effect on the nrt1.1 mutants.These results suggested that an induction of NRT1.1 activity resulted from H⁺stress was responsible for the increase of root NO₃^-uptake.We then measured the pH in the growth medium and found that the pH of the Col-0 rooting medium but not the nrt1.1 rooting medium,markedly increased under H⁺stress.However,there was no clearly difference in biomass production between Col-0 and nrt1.1 mutant when the pH in the growth media was buffered at low-pH.Thus,H⁺stress stimulates the NRT1.1-mediated NO₃^-uptake by roots,which consumes H⁺and elevates rhizospheric pH.3.We evaluated the effect of NO₃^-supply on K⁺uptake in the roots of Col-0plants using NMT.The results showed that an appropriate increase of NO₃^-in the culture media may favor the uptake and accumulation of K⁺by roots.We then investigated the effect of loss of NRTs function on tolerance of plants to low-K⁺stress.After low-K⁺treatment,the fresh weight,root elongation,and K⁺levels of nrt1.1-1,chl1-5,and chl1-6 were greatly lower than those of their corresponding wild-type plants,while the nrt1.2,nrt2.1,nrt2.2,nrt2.4,and nrt2.5 mutants showed similar phenotypic features and K⁺levels to their corresponding wild-type plants.Furthermore,in pNRT1.1::NRT1.1-GFP transgenic plants,the reduction of fresh weight,root elongation,and K⁺levels resulted from lack of NRT1.1 could be rectified by complementation with NRT1.1.Therefore,among the 6 NRTs responsible for NO₃^-uptake in roots,the NRT1.1-confered resistance to K⁺deficiency may be a relatively specific manner in Arabidopsis.We than analyzed the fresh weight,root elongation,and K⁺levels of Col-0 and nrt1.1-1,chl1-5 in a low-NO₃^-or NO₃^--removed media,and found that NRT1.1-confered resistance to K⁺deficiency in plants also needs a sufficient NO₃^-supply.Real-time quantitative PCR and histochemical staining results indicated that low-K⁺treatment clearly enhanced the gene expression of NRT1.1 in Col-0 roots and the protein levels of NRT1.1-GFP and NRT1.1-GUS protein levels in the roots of pNRT1.1::NRT1.1-GFP and pNRT1.1::NRT1.1-GUS transgenic plants,respectively.Meanwhile,the rates of net NO₃^-influx of in Col-0 treated with low-K⁺were significantly higher than that of the plants treated with normal K⁺level,which proves that an increase of NO₃^-uptake activity should be linked with the up-regulation of NRT1.1 due to low-K⁺stress.The above results indicate that an induction of NRT1.1 activity in NO₃^-transmembrane transport may be critical for plants to adapt to low-K⁺stress.4.We next analyzed the uptake rates of K⁺in plants using NMT.Under both sufficient and low K⁺testing media,the rates of net K⁺influx in nrt1.1-1 and chl1-5mutants were greatly decreased than those in Col-0 plants.This indicated that NRT1.1contribute to K⁺uptake in the roots.In addition,the proportion of K⁺distribution in roots and shoots were also calculated,and showed that a K⁺-insufficient status of plants facilitates NRT1.1 to involve in root-to-shoot K⁺allocation,which also depends on a sufficient NO₃^-supply.Further analysis of K⁺levels in grafted plants generated by Col-0 and nrt1.1-1,and found that root part is the action point for NRT1.1 to improve K⁺nutrition.The calculation of K⁺levels in pPHO1::NRT1.1 and pSultr1;2::NRT1.1 transgenic plants revealed that the complementation of NRT1.1 in epidermis and cortex of roots may contribute to the K⁺uptake,and the complementation of NRT1.1 in central vasculature of roots is responsible for transferring K⁺to the shoots.This indicated that the contributions of NRT1.1 in improving K⁺uptake and translocation to the shoots depends its spatial expression in epidermis-cortex tissue and central vasculature,respectively.The expression of NRT1.1 in the yeast mutant R5421,lacking the main K⁺uptake systems,indicated that NRT1.1 could not transport K⁺directly.Thus,a series of dual mutants of nrt1.1-1with K⁺transport channels/proteins mutants?nrt1.1-1/akt1,nrt1.1-1/hak5-3,nrt1.1-1/kup7,and nrt1.1-1/skor-2?were constructed,and two-way analysis of variance showed that NRT1.1 should respectively cooperate with the K⁺transport channels/proteins in root epidermis-cortex and central vasculature to mediate K⁺uptake and transfer to the shoots.In conclusion,both H⁺stress and low-K⁺stress stimulate NRT1.1-mediated NO₃^-uptake by roots,which,in turn,improves the plant tolerance to these two abiotic stress.Thus,our findings indicate that using biotechnology or genetic breeding methods for enhancing the activity of NRT1.1 homologous protein in crops might be a promising strategy for improving the utilization efficiencies of N and K fertilizers and the adaptability of plants in acid soil.