Molecular Mechanisms Of Higher Nitrogen Utilization Efficiency Mediated By Protein Phosphatase PP2C9 In Rice(Oryza Sativa L.)

Nitrogen(N)is an essential element usually limiting in plant growth and a basic factor for increasing the input cost in agriculture.In the last 40 years,the application of synthetic nitrogen(N)to crops has been increased drastically,resulting in significant increase in yield but with considerable impacts on the environment.To ensure the food security and environmental sustainability it is urgently required to manage the N fertilizer.The identification or development of genotypes with high nitrogen utilization efficiency(NUE)which can grow efficiently and sustain yield in low N conditions is a possible solution.In China,rice is a staple food and by 2030,rice demand will have increased up to 14%,to fulfill the increasing demand farmers are extensively using N fertilizers which results in fairly low NUE.This study was conducted to understand the underlying mechanisms involved in the regulation of NUE in rice(Oryza sativa L.).We used two isogenic rice genotypes,i.e.,wild-type rice Kitaake and its transgenic line PP2C9TL overexpressed protein phosphatase gene(PP2C9)to evaluate the NUE under different N levels(zero N,control(mid),low and high levels).The reversible proteins phosphorylation by protein phosphatases is an essential mechanism to regulate the different biological processes.In plants,PP2Cs is a major phosphatase-encoding gene family that has emerged as a key regulator of stress signaling.Protein phosphatases(PP2Cs)negatively regulate abscisic acid(ABA)signaling.In Arabidopsis,PP2C proteins such as ABA-insensitive 1(ABI1),ABI2,and Hypersensitive to ABA 1(HAB1)have been identified to regulate ABA-induced signaling under biotic and abiotic stresses by interacting with SnRK2s and PYR/PYL/RCARs.The activity of nitrate transporters(NPF6.3)is regulated by CBL9(Calcineurin B like protein 9)and CIPK23 complex(CBL interacting protein kinase 23),CBL9 phosphorylates the CIPK23 activating the protein complex.The activated CIPK23 inhibits the activity of NPF6.3.However,protein phosphatase(PP2C9s)enhances the NPF6.3-dependent nitrate sensing by dephosphorylating the CIPK23 and CBL9 complex,nitrate signaling,and nitrate transport.Based on PP2C dephosphorylation ability of CIPK23 and CBL9 complex to enhance nitrate sensing and transport we hypnotized that the overexpression of PP2C9 gene in rice could increase the NUE.In our preliminary study,we have found that the protein phosphatase(PP2C9)was closely related with the improvement of the NUE in rice by enhancing N uptake and assimilation,however,the appropriate information still lacks,especially regarding the PP2C9 regulatory mechanism for high NUE in rice plants.We designed this study keep in mind the following objectives,to evaluate the physiological performance of rice(Oryza sativa L.)mediated by protein phosphatase(PP2C9)gene,to identify the genes and their network involved in NUE through differential proteomics between WT and PP2C9TL using 2 dimensional gel electrophoresis technique,western blot,and COIP,to observe the N related ions influx and efflux in roots of PP2C9TL and WT through non-invasive micro test technology(NMT),and to evaluate the interaction of rice plants with rhizosphere microbial community mediated by PP2C9 gene through the high throughput sequencing technology of bacterial 16S rRNA gene,which in turn affects the mechanism of nitrogen uptake and utilization in rice.Firstly,we evaluated the physiological response of two genotypes under different level of N treatments.The result shows that the overexpression of protein phosphatase(PP2C9)significantly improves rice plant performance under N deficient conditions.As compared to WT,in PP2C9TL the dry matter and nonstructural carbohydrates(sugars and starch)significantly increased up to 10 DAF,after that it slowly started to decrease from 15 DAF to 20 DAF and finally,a significant decline was observed from 20 DAF up to 30 DAF under control and N deficient conditions.Similarly,higher chlorophyll content was observed in PP2C9TL than WT.The enzymatic activities of NR,GOGAT and GS were higher in PP2C9TL than WT at heading stage and decreased at maturity,however the GDH activity was higher at the maturity in PP2C9TL under low N condition.In order to estimate the nitrogen uptake and NUE,we have measured the N content of PP2C9TL and WT plants under control and low N conditions.The significant differences in N content were found at heading stage between the two genotypes under low N conditions.The PP2C9TL showed efficient N uptake compared to WT.The PP2C9TL exhibits higher NUE and produces higher yield than WT.The difference between yields of PP2C9TL and WT is higher without N than that with a low level of N,i.e.,the yield differences decreased with the increase in N level.PP2C9TL produced a higher yield(58 g/pot)than WT(43 g/pot)at low N condition.The yield of PP2C9TL in low N level is almost equal to WT yield at the control level of N,these results indicate that PP2C9TL produced a high yield by consuming less N.There was no significant increase in yield under high level of N due to production of nonproductive tillers and inefficient transport of photo assimilates to grains at maturity.The differences in yield at given levels of N confirmed the genetic variation for NUE between the two genotypes.Physiological attributes showed significant differences at 10 DAF between the two genotypes,clearly the PP2C9TL performed better than the WT so,to investigate the molecular pathway involved for higher NUE in PP2C9TL at low N,we designed differential proteomic experiments at 10 DAF.Secondly,the two isogenic genotypes i.e.WT and PP2C9TL were used for comparative proteomics analysis at control and low level of N to identify specific proteins and encoding genes related to high NUE.2D gel electrophoresis was used to perform the differential proteomic analysis.In the leaf proteome,30 protein spots were differentially expressed between the two isogenic lines under low N level,which were involved in the process of energy,photosynthesis,N metabolism,signaling,and defense mechanisms.These identified proteins were enriched in several pathways,including glycolysis,Calvin cycle,TCA cycle,glutamate synthase and nitrate reductase,carbohydrate metabolism and defense,of which the three interested regulatory proteins were identified as the PP2C9,14-3-3 family,and SnRK.PP2C9 deals with phosphorylation/dephosphorylation of several proteins,so it is quite possible to alter the signaling pathways.In addition,we have found that protein phosphatase enhances nitrate reductase activation by downregulation of SnRK1 and 14-3-3 proteins.Our results suggest that the 14-3-3s regulate the C and N metabolism through interaction with SnRK1 and PP2C9.The 14-3-3 protein is a highly conserved protein in crop plants and is directly involved in the NR inactivation mechanism.NR activity is considered as the most important factor for nitrogen uptake and assimilation.The mechanism involved in the process is the most probable reason for higher NUE of PP2C9TL in low nitrogen.We confirmed the downregulation of 14-3-3 protein by using western blot analysis.Furthermore,in order to find the PP2C9-associated protein interactions and to pull out all protein complexes among PP2C9TL and WT,we carried out the immunoprecipitation assay.The Co-IP validates the results obtained by comparative proteomics as most of the identified proteins were similar with 2DE proteins and involved in photosynthesis,energy,N metabolism,signaling cascades,and defense mechanisms.The findings suggests that these identified differential proteins from Co-IP and 2DE were responsible for higher NUE of PP2C9TL than WT through the highly expressed PP2C9 interaction with the related proteins especially under low N treatment.Further,to investigate the mutual effect between plants and rhizosphere microbial activities mediated by PP2C9 we carried out the high throughput sequencing technology of bacterial 16S rRNA gene to analysis the bacterial communities of rhizosphere,rhizoplane,endosphere of rice under low nitrogen and normal nitrogen treatment.The result showed that the PP2C9TL can increase diversity of bacteria communities in rice rhizosphere and rhizoplane under low nitrogen treatment.The relative abundance of Proteobacteria,Firmicutes,Actinobacteria,Bacteriodetes,Chloroflexi and Acidobactria were increased,which can promote the circulation and transformation of nitrogen in rhizosphere soil.PP2C9TL can increase diversity of bacteria communities in rice rhizosphere and rhizoplane compared with WT under low nitrogen treatment.The ion fluxes at root apical were detected by Non-invasive Micro-test Technique;it was found that root PP2C9 transgenic rice could absorb more Ca2+ and NO2-under low nitrogen condition.Finally,through investigating the growth of rice combined with the results of COIP and 2D performance,it was concluded that the PP2C9 transgenic rice has more flourishing roots,more actively growing shoot,yield and nitrogen uptake were significantly increased through the higher expression of PP2C9 and its interaction with the related proteins,while there was no very much significant difference at high and normal level of nitrogen treatment due to the revise case in the protein expression pattern.This study provides new insights on the rice proteome,which would be useful in the development of new strategies to increase NUE in cereal crops.