Characterization Of OsNAR2.1 Interaction With OsNIT1 And OsNIT2 In Rice Uncovers Their Different Roles In Mediating Root Growth Response To Nitrate And Ammonium

The main forms of nitrogen(N)available for plant root acquisition are nitrate(NO3-)in upland soil and ammonium(NH4+)in flooding paddy soil.For efficient acquisition of varied N forms and concentrations,plant roots have developed sophisticated regulatory systems including local(available N on root surface)and systematic(total N nutrition status)signaling pathways for enhancing N acquisition.Rice is commonly grown in paddy soil with frequent alteration of drying and flooding during its grown season.The abundant aerenchyma allows oxygen delivered from shoot to roots and released to root surface,resulting in strong nitrification in the rhizosphere even under flooding condition.Therefore,rice is actually supplied with both ammonium and nitrate.Auxin(indole-3-acetic acid,IAA)is one of key players involved in plant root growth adaptation to altered supply of nitrate.There are several proposed pathways for the tryptophan-dependent IAA biosynthesis in Arabidopsis.Indole-3-acetonitrile(IAN)has been identified as one of two key intermediates in Indole-3-acetaldoxime(IAOx)-mediated IAA biosynthesis.The conversion of IAN to the basal production of IAA requires nitrilases for the nitriles hydrolysis in kernels of maize(Zea mays)and Arabidopsis.However,it is not known if nitrilases are involved in N-induced change of root growth and N acquisition.Previous studies in our laboratory have shown that the OsNAR2/OsNRT2 two components of nitrate transporters play the major role in nitrate acquisition at both low and high supply range in rice.The elongation of lateral roots in osnar2.1 mutant was less sensitive to local NO3-supply than wild-type rice,suggesting that OsNAR2.1 may be involved in the NO3-signaling pathway that controlled lateral root formation.For further elucidating the regulatory mechanism of OsNAR2.1 dependent root growth response to varied N supply,we first performed Pull Down experiment using OsNAR2.1 protein as a bait,and obtained several candidate proteins which were predicted to be interacted with OsNAR2.1.In this study,we focused on characterization of two nitrilase proteins OsNIT1 and OsNIT2 functions in root growth response to nitrate and ammonium.The main results are summarized as follows:1.OsNAR2.1 interacts with OsNIT1 and OsNIT2 at nitrate supplied roots.The protein interaction between OsNAR2.1 and OsNIT1,OsNIT2 was further confirmed by yeast twohybridization and co-immunoprecipitation(Co-IP).In addition,as quantified by qPCR,nitrate supply enhanced transcription expression of OsNAR2.1,OsNITl and OsNIT1 in rice roots.In situ hybridization showed the nearly same expression pattern of three genes,they all located in the root tip and lateral roots under nitrate conditions.2.Knockout of OsNIT1 or OsNIT2 resulted in the same root phenotype with osnar2.1 mutant at nitrate supply.The physiological functions of OsNIT1 and OsNIT2 and their interaction with OsNAR2.1 were characterized by comparison of their single knockout mutant,double mutation of OsNAR2.1 and OsNIT2.1 and wild type.The osnitl,osnit2 mutants and the osnit2-2×osnar2.1-1 double mutant were obtained by T-DNA insertion,CRISPR/Cas9 system and hybridization technique.Knockout of OsNAR2.1 resulted in downregulation of OsNIT1 and OsNIT2 gene expression in nitrate supplied roots.Knockout of OsNIT1,OsNIT2 and OsNAR2.1 all lead to the phenotype of shorten primary root and lower root density.The root phenotype of the double mutant osnit2-2×osnar2.l-1 was similar to that of osnit2-2,while the lateral root density of the double mutation was significantly less than that of osnit2-2 under nitrate supply condition.3.Knockout of OsNIT1 and OsNIT2 did not alter nitrate uptake rate of unit weight root.Knockout of OsNIT1 and OsNIT2 did not affect the expression of OsNAR2.1 and nitrate transporter protein gene OsNRT2.1,OsNRT2.3 in roots.Using 15N tracer,we detected that unlike OsNAR2.1 mutation,knockout of OsNITl and OsNIT2 did not significantly affect nitrate uptake rate of unit root weight in short term(5 min),indicating that OsNIT1 and OsNIT2 did not generate feedback to OsNAR2.1 function in N uptake.There was no significant difference of total N concentration,but not the N content,of osnitl and osnit2 mutants and wild type grown in N-rich soil at the flowering stage,further indicating that OsNIT1 and OsNIT2 did not directly contribute N acquisition.Both osnitl and osnit2 showed lower N concentration in their flag leave than that in the wild type at the maturity stage mainly due to the knock-out effect of OsNITl or OsNIT2 on the ability of transport nitrogen from the 1St leaf to the grain.Unlike osnar2.1,both osnitl and osnit2 showed the same root nitrate acquisition rate with wild type.4.Knockout of OsNIT1 or OsNIT2 decreased the root growth sensitivity to IAN,but not to NAA.Knockout of OsNITl enhanced the resistance of primary root length and lateral root density to external IAN in both Nipponbare and Hwayoung backgrounds.The length of primary root(PR)and the lateral root(LR)density of osnitl was about 140%longer and 40%less than its Nipponbare wild-type(WT).osnit2 showed the similar phonotype in the alteration of primary root length with osnitl,but the effect was much less significant.Knockout of OsNIT1 or OsNIT2 did not affect the root responses to external NAA.The data indicate that IAN which can be hydrolyzed by nitrilase could play the regulatory role in rice root growth.5.Knockout of OsNIT1 or OsNIT1 resulted in low acropetal IAA transport,but not total concentration of IAA in roots.In view of the significant responses of rice root growth to external IAN and both OsNIT1 and OsNIT2 were involved in the responses,we made the effort to analyze the IAN and IAA concentration in osnitl or osnit2 mutants.We did not get IAN in the seedling of Nipponbare,indicating that IAN was neither present or little to be detectable or IAN can be hydrolyzed very rapidly in rice.In addition,the total concentration of IAA in the osnitl,osnit1 mutants and WT was similar,indicating that OsNITI and OsNIT2 are not the major factors for total IAA synthesis or accumulation in rice seedlings.We also compared the[3H]-IAA transport in the nitrate supplied roots of WT and mutants.Notably,as quantified by amount of[3H]-IAA in the 0-3 cm root tips,knockout OsNAR2.1 very significantly confined both root-ward and shoot-ward movement[3H]-IAA,while knockout OsNITI or knockout OsNIT2 only inhibited the[3H]-IAA root-ward movement,but not shoot-ward movement.In comparison to WT,both and lines showed the suppressed expression of OsPIN1c and OsPIN1d while up-regulation of OsPIN2,OsGH3-2,OsGH3-8 and OsGH3-13 in their roots.The data demonstrate the OsNITI or OsNIT2 regulation of root growth via alteration of auxin root-ward transport and local distribution.6.OsNITI showed the enzyme activity for IAN hydrolysis which could be significantly improved by co-expression of OsNIT2 and OsNAR2.1 in vitro.In short-term(1 hour)vitro assay of IAN conversion rate quantified by liquid chromatography indicated that OsNITI,but not OsNIT2 and OsNAR2.1,showed the activity in hydrolyzing IAN.Remarkably,coexpression of OsNIT1 and OsNIT2,in particular,together with OsNAR2.1 enhanced OsNITI activity in hydrolyzing IAN by 3.2-and 5.3-fold,respectively.The result indicated that biological significance of the interaction between OsNAR2.1 and two NIT proteins in activating the rice nitrilase.7.Unlike OsNAR2.1,OsNIT1 and OsNIT2 were upregulated by ammonium and functioned in ammonium sensitive root growth.Both OsNIT1 and OsNIT2 were upregulated in the root tip and lateral roots by ammonium.Both osnitl and osnit2 mutants supplied with 0.25 mM NH4+had the phenotype of short primary root and low lateral root density,about 3 5-45%less than that in WT.In contrast,knockout of OsNAR2.1 did not alter the expression of OsNITI and OsNIT2 and root growth at ammonium supply.Moreover,double mutation of OsNAR2.1 and OsNIT2 showed the similar root phenotype with osnit2,confirming that OsNAR2.1 did not involve in the function of NIT proteins in the root responses to ammonium supply.The altered acropetal transport of[3H]-IAA was observed in the roots of osnitl and osnit2 lines,but not in osnar2.1 mutant grown in ammonium solution.The hyper-sensitivity of root growth of osnit1 and osnit2 to external ammonium demonstrated that OsNIT1 and OsNIT2 functioned in regulating root growth independent of OsNAR2.1 under ammonium supply.In summary,OsNAR2.1 regulates root growth not only through the nitrate uptake but also directly by activating the interaction with OsNIT1 and OsNIT2 at nitrate supply.OsNAR2.1 and OsNIT2 enhanced the OsNIT1 activity in hydrolyzing IAN to IAA.Inactivation of OsNIT1 and OsNIT2 did not affect total IAA content,but inhibited acropetal transport of IAA.OsNIT1 and OsNIT2 functioned in root growth independent of OsNAR2.1 under ammonium supply.