Function Analyses Of A Dual-affinity Nitrate Transporter Gene OsNRT2.4 In Rice

Two major forms of inorganic nitrogen(N)available for plant roots are nitrate(NO3-)in aerobic soil and ammonium(NH4+)in flooding paddy soil.In plants,NO3-can serve as a signaling molecule to break seed dormancy,regulate lateral root development and leaf growth,alter flowering time,and integrate the expression of NO3--induced genes for plant growth and development.In many plants the larger part of NO3-acquired by roots is transported to the shoots before being assimilated.To cope with varied concentrations of NO3-in soils,plant roots have developed two NO3-uptake systems,high-affinity N03-transport systems(HATS)and low-affinity NO3-transport system(LATS)operating at less and larger than 1 mM,respectively,for NO3-acquisition.Plant nitrate transporters were first identified and functionally characterized more than 20 years ago.They are encoded at least by four gene families,NPF(NO3-/peptide transporter)(originally named as NRT1/PTR),NRT2,CLC(chloride channel),and SLAC1/SLAH(slow anion channel associated homologues).The processes of NO3-uptake,translocation,and storage in plants are very sophisticated and are delicately controlled by the regulation and cross-talk between four main types of transporters.The substrate specificity and affinity values of these transporter family members are not strict as originally predicted.For example,the NPF members are suggested to function as the main components of the LATS for NO3-at high concentrations,while NRT1.1(NPF6.3,or CHL1)in Arabidopsis and MtNRT1.3 in Medicago truncatula,actually serve as a dual-affinity nitrate transporter involved in both HATS and LATS,NRT1.1 can also transport auxin.In contrast a Medicago truncatula protein belonging to NPF family encoded by NIP/LATD(for Numerous Infections and Polyphenolics/Lateral root-organ Defective)gene functions as a high affinity nitrate transporter.NRT 1.1 has been proposed to be a ’transceptor’(a protein with dual transport/sensing functions).,.The NRT2 transporter family has at least seven members in Arabidopsis,five in rice and three in Medicago truncatula genome.Notably,many NRT2 family members are unable to transport NO3-alone;they require a partner protein,NAR2,which is also named NRT3.In Arabidopsis,six of the seven NRT2 family members except AtNRT2.7 require NAR2.1 for transporting NO3-.In rice,three of the OsNRT2 members need OsNAR2.1 for root acquisition of NO3-.The key amino acids of OsNAR2.1(arginine 100 and aspartic acid 109)interacting with OsNRT2.3a have been identified,and such interaction was critical for OsNRT2.3a plasma membrane localization and NO3-transport activity.Interestingly,unlike NPF members which transport a diverse range of substrates,all the characterized plant NRT2 or NAR2 members function as components of HATS,specifically in N03-influx.Previously,we have shown that in rice OsNRT2.1,OsNRT2.2 and OsNRT2.3a are transcriptionally up-regulated in roots by NO3-treatment,while OsART2.3b and OsNRT2.4 are expressed mainly in shoot.OsNRT2.1 and OsNRT2.2 have relatively higher affinity with nitrate than OsNRT2.3a and OsNRT2.3b.OsNRT2.3a is involved in long-distance NO3-transport from root to shoot at low NO3-concentration,while OsNRT2.3b plays a critical role in sensing cytosolic pH of phloem cells for balancing NO3-and NH4+uptake.In this study,we focus on the characterization of OsNRT2.4 gene in rice.The expression patterns of the gene were analyzed by qRT-PCR,GUS reporter gene and eGFP reporter gene;the Xenopus oocytes heterologous expression system was used to analyze the absorption characteristics of NO3-and IAA;The function of OsNRT2.4 in NO3-regulated lateral root(LR)initiation and growth was characterized using independent CRSPR/CAS9 mutated osnrt2.4 lines and its wild type(WT)in solid phytagel medium.The uptake and redistribution of NO3-in the CRSPR/CAS9 mutated osnrt2.4 lines and WT were analyzed by 15N labeled NO3-.The main results were listed as follows:1.We performed the qRT-PCR analysis of OsART2.4 expression pattern in rice(cv.Nipponbare).OsNRT2.4 was expressed abundantly in shoots and faintly in entire roots.Its expression was up-regulated by increasing NO3-supply levels and down-regulated by presence of NH4+.Interestingly,OsNRT2.4 expression was strongly enhanced by exogenous treatment of seedlings with 10 μM indole-3-acetic acid(IAA)and 10 μM especially jasmonic acid(JA),while 0.1 abscisic acids(ABA),1μN-I-naphthylphthalamic acid(NPA)and 50μM salicylic acid(SA)did not have effect on its expression.2.To determine the subcellular location of OsNRT2.4,eGFP fused at either the N or C terminus of OsNRT2.4 was transiently expressed in rice protoplasts by cauliflower mosaic virus 35S promoter.The green fluorescence of OsNRT2.4-eGFP or eGFP-OsNPF2.4 was observed at the protoplast plasma membrane compared with the diffuse cytoplasmic localization of the eGFP control,confirming that OsNRT2.4 was a plasma membrane-localized transporter.3.To analyze the expression pattern in different rice tissues,the 1351 bp upstream region of OsNRT2.4-coding region was fused to the GUS reporter gene and introduced into rice(cv.Nipponbare).The GUS staining showed that OsNRT2.4 in root was expressed in the base of the lateral root primordia,not in other parts including root hair zone and tip.It was also detected in root-shoot junction,sheath and blade,but not in embryo.To examine further the OsNRT2.4 expression in different types of cells,GUS protein was detected by its antibody.Within a root,the immunostaining was only observed in the cells at the base of lateral root primordia,but not in any other cells.In shoots,the staining was shown in vascular cells of basal node,sheath and blade.4.The functional analysis of the transporter in Xenopus laevis Oocytes heterologous expression system showed that OsNRT2.4 is a pH-independent dual-affinity NO3-transporter.It can work alone without OsNAR2.1 and it has no NO3-efflux activity and IAA uptake activity.5.In view of the specific localization of OsNRT2.4 in the base of lateral root primordia in rice,we characterized its function in NO3-regulated lateral root(LR)initiation and growth using independent CRSPR/CAS9 mutated osnrt2.4 lines and WT in solid phytagel medium.The number and length of the lateral roots of osnrt2.4 lines was significantly lower than those of WT when grown in both low(0.25 mM)and high(2.5 mM)NO3-levels,while no significant difference of the LR number and length was observed between the mutants and WT when grown in the NH4+(2.5 mM)culture medium.6.In comparison to WT,the disruption of OsNRT2.4 significantly inhibited root and shoot growth at both low(0.25 mM)and high(2.5 mM NO3-supplies.The mutants showed lower dry weight and less total N accumulation than WT irrespective of NO3-levels.In contrast,there was no significant difference of the root and shoot dry weight and the total N concentrations between the osnrt2.4 mutants and WT when grown in 2.5 mM NH4+solution.7.Since OsNRT2.4 was detected in vascular cells at root-shoot junction and leaves,we analyzed the effect of OsNRT2.4 knockout on the distribution of the 155NO3-from root or source leaf to the sink organs of N-starved rice.We found that the relative distribution ratios of root acquired 15N-NO3-in root,culm and sheath,and leaf blade were nearly the same for WT and osnrt2.4 mutant at 0.25mM NO3-supply,while the 15N-NO3-distribution was increased in culm and leaf sheath but decreased in leaf blade of the mutants in comparison with WT at 5 mM NO3-supply.The results indicate that OsNRT2.4 is involved in the re-localization of nitrate in rice.8.The effect of OsNRT2.4 overexpression on rice growth and N acquisition was also examined at the maturity stage in paddy field.In comparion with WT,the OsNRT2.4 overexpressing lines showed lower plant height,grain yield,the 1000-grain weight,and seed setting rate.In comparision to WT,OsNRT2.4 overexpression decreased N concentration in the 1st.2nd and 3rd leaf of but increased N concentration in the culm and grains.The OsNRT2.4 overexpression in rice down-regulated the expression of OsNAR2.1,OsnAR2.2,OsNRT2.1 and OsNRT2.2.Taken together,we conclude that OsNRT2.4 encodes a dual-affinity nitrate transporter.In rice OsNRT2.4 is expressed in lateral root primordia in addition to abundant expression at multiple aerial organs.OsNRT2.4 plays important roles in maintaining the NO3--mediated growth of lateral roots and shoots of rice.