Mechanisms Of Nitrate And Brassinosteroid Regulating Plant Growth And Development

Nitrogen(N)is an essential macronutrient for plants,and the main component of protein,chlorophyll,amino acid,nucleic acid and secondary metabolites.N nutrients can be provided as ammonium or nitrate forms,and nitrate is the main source of nitrogen.Besides its role as a nutrient,nitrate treatments trigger local and systemic signaling pathways that modulate gene expression,metabolism,physiology as well as growth and developmental processes in plants.Brassinosteroid(BR),a class of plantspecific steroid hormones,play important role in cell elongation,root development and seed germination.However,how nitrate and BR co-regulate plant growth and development is still poorly understood.In our research we determined how nitrate and brassinosteroid regulate plant growth and development through cell biology,molecular biology,biochemistry and genetics methods.The main results are as follows:1.Brassinosteroid is required for nitrate-mediated plant growth.Nitrate supply significantly promoted plant growth in wild type plants,which was significantly inhibited in BR-deficient mutant det2-1;BR gain-of-function mutant bzr1-1D was more sensitive to nitrate,and bzr1-1D could partially restore the phenotype of det2-1.After PPZ treatment,the effect of nitrate on plant growth was significantly reduced,while BL treatment restored response of plant to nitrate.Results of diverse nitrogen form treatment indicated that nitrate specifically regulate bzr1-1D growth.These suggest that nitrate-mediated plant growth and development need BZR1.2.Nitrate induce expression of BR synthesis genes and promote BZR1 accumulation.The transcript levels of CPD and ROT3 significantly increased after nitrate treatment,while nitrate promoted protein accumulation of BZR1 as well.In addition,analysis of growth-related genes indicated that nitrate and brassinosteroid induce expression of EXP1,EXP8 and PRE1 and regulate plant growth.3.Brassinosteroid induce expression of NRT2.1 and influence nuclear localization of NLP7.Analysis of RT-qPCR showed that expression of nitrate transporter NRT2.1 is significantly increased by brassinosteroid.Under normal operation,nitrate treatment the nuclear localization of NLP7.After PPZ treatment,protein was transported from nucleus to cytoplasm.However,co-treatment with BL and PPZ attenuated PPZ-induced transport of NLP7,indicating that brassinosteroid promoted the nitrate-triggered nuclear-localization of NLP7.Furthermore,BR acceptor BRI1 interact with nitrate acceptor NRT1.1,while effect of brassinosteroid on nitrogen absorption and nitrate content remains to be studied.4.RGA play important roles in systemic nitrate signaling.RGA,a negative regulator of GA signaling,interacts with TCP20 in vivo and in vitro.proRGA:GFP(rga-Δ17),a RGA gain-of-function allele was grown on the medium of split-root system,which has been extensively used to study root foraging as it mimics the heterogeneous distribution of nutrients in the natural soil.The result indicated that RGA accumulation regulated systemic nitrate signaling.Similar results were obtained after PAC treatment,indicating that RGA play important roles in systemic nitrate signaling.Protoplast transient gene-expression assays showed that TCP20 induced expression of luciferase reporter driven by the CEP5,while RGA inhibited TCP20-mediated CEP5 expression.In summary,nitrate treatment induces expression of BR synthesis genes and promote BZR1 accumulation;brassinosteroid induces expression of NRT2.1 and influence nuclear localization of NLP7,these results suggest that brassinosteroid is required for nitrate-mediated plant growth.On the other hand,nitrate promotes RGA accumulation by means of inhibiting expression of GA synthesis genes;RGA inhibited TCP20-mediated CEP5 expression via interacting with TCP20.Nitrate enhances the activity of brassinosteroid and gibberellin,and finally promotes plant growth and development,but detailed mechanisms of their crosstalk need further exploration.