The Responses Of Nitrate Transport Accessory Protein Gene OsNAR2.1 To Nitrate And Its Interaction With OsNRT2 Members

As one of the most important staple food crops,rice(Oryza sativa L.)provides the food for more than half of the world's population.In addition,rice is also an important model plant of monocot species.Nitrogen(N)is the major factor limiting crop growth and yield among all the essentaial nutrient elements for plant.Both ammonium(NH4+)and nitrate(NO3-)are the major sources of soil N for plants,however,ammonium is the predominant form of N in a paddy soils.Some physiological experiments have shown that lowland rice was exceptionally efficient at acquiring NO3-formed by nitrification in the rhizosphere,so both NH4+ and NO3-are the major sources of soil N for rice.It has been predicted that rice roots may take up 20-40%of total N in the form of NO3-in waterlogged paddy soil.There are two different uptake systems in plants to cope with low or high NO3-concentrations in soil,the high affinity systems(HATS)and low affinity NO3-uptake systems(LATS).It is reasonable to predict that NO3-is taken up mainly by the HATS rather than the LATS of rice roots since NO3-concentration found in rhizosphere of flooded paddy soil is low.Our previous studies have shown that the HATS for nitrate uptake in rice is mediated by a two-component NRT2/NAR2 transport system.In this study,we focused on the regulation of the partner protein named OsNAR2.1 and its interaction with OsNRT2 members.Firstly,we screened and identified the NO3-responsive cis-elements by detecting the GUS reporter signals driven by different mutated promoter fragments of OsNAR2.1 located in the position of-311/-1bp in rice.Secondly,we identified the key motifs of OsNAR2.1 interacting with OsNRT2.3a by using the membrane yeast two-hybrid system,BiFC,and 15N-NO3-uptake in Xenopus oocytes.Thirdly,we characterized the preliminary functions of OsNAR2.1 and its interacting OsNRT2 members in improving rice growth and N uptake by generating the stably inherited transgenic lines of their single and double over-expressions.The main results were as follows:1.Different lengths and site mutations of the-311 bp/-1 bp region in OsNAR2.1 promoter which has been previously proved to be responsive to nitrate signaling were fused to the GUS reporter gene and introduced in transgenic rice.To identify the cis-acting DNA elements necessary for nitrate induced gene expression,we detected the expression of beta-glucuronidase(GUS)reporter in the transgenic rice.We found that-129 to-1bp region is necessary for the nitrate-induced full activation of OsNAR2.1 Besides,the site mutations showed that the 20bp fragment between-191 and-172bp contains an enhancer binding site necessary to fully drive the OsNAR2.1 expression.However its four copies of the 20bp fragment fused to 35S minimum promoter did not have the function in mediating the nitrate induced gene expression.These findings thus reveal that the presence of not only one cis-acting element for mediating nitrate responses in plants.2.Analyses by using the membrane yeast two-hybrid system and Xenopus oocytes for N15 uptake demonstrated that either R100 or D109 point mutations impaired the OsNAR2.1 interaction with OsNRT2.3a.Western Blot and visualization using green fluorescent protein fused to either the N-or C-terminus of OsNAR2.1 indicated that OsNAR2.1 is expressed in both the PM and cytoplasm.The split-yellow fluorescent protein(YFP)/BiFC analyses indicated that OsNRT2.3a was targeted to PM in the presence of OsNAR2.1,while either R100 or D109 mutation resulted in the loss of OsNRT2.3a-YFP signal in PM.Based on these results,arginine 100 and aspartic acid 109 of the OsNAR2.1 protein are key amino acids in the interaction with OsNRT2.3a,and their interaction occurs in PM but not cytoplasm.3.Over expression of OsNAR2.1 alone,OsNRT2.1/2.1 alone,OsNRT2.3a alone and joint over-expression of OsNRT2.1/2.2 and OsNAR2.1,joint over-expression of OsNRT2.3a and OsNAR2.1 transgenic plants were successfully obtained through transgenic manipulation.RT-PCR,TAIL-PCR,Southern blot and Western Blot methods were employed to identify the stably inherited transgenic lines.The over-expression of OsNAR2.1 alone in rice(cv.Nipponbare)increased the expression of OsNRT2.1,OsNRT2.2 and OsNRT2.3a.Over expression of OsNAR2.1 could enhance the nitrate uptake rate and improve the growth of rice plants.Over-expression of OsNRT2.1/2.2 or OsNRT2.3a alone in rice did not generate the significant difference in nitrate concentration and the growth phenotype.However,joint over-expression of OsNRT2.1/2.2 or OsNRT2.3a with OsNAR2.1 in rice increased nitrogen concentration and yield higher than those obtained by over-expression of OsNAR2.1 alone.Taken together,the cis-acting DNA elements are localized in the promoter of rice nitrate transport accessory protein gene OsNAR2.1 rather than OsNRT2 members,indicating that either OsNAR2.1 or OsNRT2s is regulated by different transcription factors or OsNAR2.1 transfers the nitrate signal for upregulating the expression of OsNRT2 members.Besides,the R100 or D109 in the OsNAR2.1 protein are important not only for establishing HATS activity,but also for co-localization of OsNAR2.1 with OsNRT2.3a at the plasma membrane.The physiological function analysis showed that OsNAR2.1 is not only required for OsNRT2.1/OsNRT2.2 and OsNRT2.3a for function in nitrate uptake,but also may directly or indirectly regulate the expression of OsNRT2.1,OsNRT2.2 and OsNRT2.3a.