The Molecular Mechanism Of Abscisic Acid Signaling Regulating Nitrate Deficiency In Arabidopsis

N(Nitrogen)is an essential macronutrient for plant growth and productivity.Since the N supply is an important factor to ensure high yield of crops,farmers have to fertilize with large amounts of N to enable maximal crop yield,and the use of synthetic N has increased remarkably in recent years.However,crops use only 30%-50%of the N applied.The overuse of nitrogen fertilizer not only increases the cost of crop production but also easily causes serious environmental problems.Therefore,reducing the use of nitrogen fertilizer,while continuously improving crop yield,has become a key issue in sustainable development of agriculture.It is of great significance to deeply understand the mechanism of plant N uptake and to analyze the regulatory network of N uptake for improving crop yield,improving agronomic traits of crops,reducing excessive application of nitrogen fertilizer and environmental pollution,and promoting sustainable development of agriculture.The ABA(phytohormone abscisic acid)have been suggested to play a vital role in development responses and a variety of biotic and abiotic stresses response in plants.ABA receptor protein PYR/PYL/RCAR family,phosphatase PP2Cs and protein kinase SnRK2s constitute the core pathways of ABA signal pathway in plants,regulating many downstream factors that are widely involved in plant growth,development,and stress adaptation.However,the molecular mechanisms underlying the involvement of ABA in N deficiency responses are largely unknown,which poses barriers for application of molecular technology in agricultural breeding.In this study,we found new players in response to nitrate deficiency stress by a reverse genetic stragegy using various mutants of Arabidopsis thaliana.Our results showed that the ABA-deficient mutant aba2-1 and the pyr1 pyl1 pyl2 pyl4 mutant grew longer primary roots and had a higher lateral root density compared with the WT(wild type)under N deficiency.By contrast,the abi1-2 abi2-2 hab1-1 pp2ca-1 mutant grew shorter primary roots and had a lower root density compared with the WT under N deficiency.The snrk2.2 snrk2.3 snrk2.6triple mutant grew a longer primary root.The snrk2.2 snrk2.3 snrk2.6 had a higher rate of nitrate influx and accumulation compared with WT plants under nitrate deficiency.Strikingly,SnRK2.2/2.3/2.6 proteins interacted with the nitrate transceptor NRT1.1(nitrate transporter 1.1)in vitro and in vivo as evidenced by Y2H,Bi FC,LCA and Co-IP methods.SnRK2s phosphorylated both the NT and CT cytoplasmic segments of NRT1.1,and Ser8 and Ser585 are key phosphorylation sites.The phosphomimetic NRT1.1^S585D was impaired in both low-and high-affinity transport activities in oocytes,while NRT1.1^S8D had no difference.The NRT1.1^S585D/chl1-5 lines grew an obviously shorter primary root both under control and N-deficiency conditions compared with the WT,which was a similar phenotype to that of the chl1-5 mutant.By contrast,the NRT1.1^S585A/chl1-5,NRT1.1^S8D/chl1-5 and NRT1.1^S8A/chl1-5 transgenic lines displayed no significant differences compared with the WT.These results showed that phosphorylation of NRT1.1 by SnRK2s resulted in a significant decrease of nitrate uptake and impairment of root growth and identified NRT1.1^Ser585 as a previously unknown functional site.SnRK2-mediated phosphorylation might influence transport activity,rather than localization or protein stability of NRT1.1,and NRT1.1^S585D/chl1-5 lines are slightly insensitive to ABA.In addition,sequence alignment of NRT1.1 protein in different species showed that Ser585 was functionally conserved to a certain extent as a serine/threonine site.Taken together,we demonstrated that ABA signaling components,particularly the three-subclass?SnRK2s,functioned in root foraging and nitrate uptake under nitrogen deficiency in Arabidopsis.We propose that SnRK2.2/2.3/2.6 negatively regulate the activity of NRT1.1via phosphorylation,which alters the nitrate transport activity of NRT1.1 and root foraging under N deficiency.Moreover,we identified NRT1.1^Ser585 as a previously unknown functional site:the phosphomimetic NRT1.1^S585D was impaired in both low-and high-affinity transport activities.Our findings in this study provide new insight into how plants fine-tune growth via ABA signaling under N deficiency.These results expanded the regulation network of plant response to N deficiency,and laid a theoretical foundation for crop improvement with both stress resistance and high nitrogen use efficiency.