The Molecular Mechanism Of Rice MADs-box Transcription Factors OsMADS25,OsMADS27 And OsMADS57 Regulating Root Growth And Development By Responding To Nitrate Signals

Plant root system is of vital importance for terrestrial plants to anchor and effectively absorb nutrients and water from the soil,thus play a crucial role in the adaptability of plants to the external environment.Nitrogen is an essential nutrient for plant growth,it mainly has two forms:nitrate（NO₃^-）and ammonium（NH₄⁺）.Nitrate is not only the main source of nitrogen for plants,but also an important signaling factor that regulate plant growth and development.In agricultural production,the application of nitrogen fertilizer has become the main way to increase the yield of crops.However,excessive use of nitrogen leads to eutrophication that threatens the lives of animals and plants.Therefore,it is very important to study the molecular mechanism of nitrate utilization in plants and to improve the regulation network of nitrogen efficient utilization,which also provide important theoretical and genetic basis for improving the nitrogen use efficiency（NUE）of crops,thus promoting the sustainable development of agriculture and protecting the ecological environment.In this study,we investigated the biological functions of three homologous genes OsMADS25,OsMADS27 and OsMADS57 in rice root,and their important roles in promoting root growth and development in response to nitrogen signal.In addition,we also revealed the molecular mechanism of OsMADS25,OsMADS27 and OsMADS57involved in nitrate signal pathway to regulate the growth and development of primary and lateral roots in rice.Moreover,we demonstrated the positive roles of OsMADS25and OsMADS57 genes in response to salt stress.The main results of the study are as follows:1)OsMADS25 interacts with OsNAR2.1 and regulates root growth and development by responding to nitrate signal.OsMADS25 was specifically expressed in roots and was strongly induced by nitrate signals.By detecting the expression level of OsMADS25 in nar2.1 mutant after nitrate induction,it was found that the response of OsMADS25 to nitrate signal was strongly inhibited,indicating that the response of OsMADS25 to nitrate signal was dependent on OsNAR2.1.In addition,yeast two-hybrid（Y2H）,bimolecular fluorescence complementary（Bi FC）and co-immunoprecipitation（Co-IP）assays showed that OsMADS25 interacts with OsNAR2.1.Moreover,by observing the localization of OsMADS25 and OsNAR2.1 in rice protoplasts,it was found that these two proteins were co-localized in cytoplasm and nucleus.In addition,the protoplasts of wild type were extracted to observe the effect of NO₃^-on OsMADS25 protein,it was found that the stimulation of NO₃^-signal could promote the transfer of OsMADS25 protein from cytoplasm to nucleus,but this phenomenon was not observed in the nar2.1 mutant.Thus,the results indicated that OsMADS25 was localized to the nucleus only in the presence of OsNAR2.1.Auxin response factor OsARF7 was screened by ChIP-Seq analysis.It was further confirmed by ChIP-q PCR and dual-luciferase assay which illustrated OsMADS25 could directly bind to the promoter region of OsARF7 to activate its expression,thus responding to auxin signal to regulate root development.In conclusion,OsMADS25 is transferred from cytoplasm to nucleus with the assistance of OsNAR2.1 under nitrate treatment,and regulate the development of rice root by directly regulating the expression of auxin-responsive factor ARF7.2)OsMADS27 regulates root growth in response to nitrate signal and directly regulates the expressions of OsNRT1 and OsNLP2 to promote nitrate uptake and transport in rice.To evaluate the biological function of OsMADS27 in root development,the expression level of OsMADS27 in different tissues was detected.It was found that OsMADS27 was mainly expressed in roots and was strongly induced by nitrate signal.Overexpression of OsMADS27 in Arabidopsis could promote the growth of lateral root in a nitrate-dependent manner.Overexpression of OsMADS27 in rice could significantly promote the growth of primary root and increase the number of lateral root.In addition,overexpression of OsMADS27 in rice promoted the accumulation of NO₃^-and the expression of nitrate transporter genes.Moreover,Y2H,Bi FC and Co-IP assays showed that OsMADS27 interacted with OsNAR2.1 protein.It was further found that OsMADS27 and OsNAR2.1 were co-localized in rice protoplasts.Additionally,it was found that the stimulation of NO₃^-signal could promote the transfer of OsMADS27 protein from cytoplasm to nucleus,while it was not found in nar2.1mutant.By detecting the expression level of OsMADS27 in nar2.1 mutant under nitrate treatment,it was found that the response of OsMADS27 to nitrate was inhibited,indicating that the response of OsMADS27 to nitrate signal was dependent on OsNAR2.1.Two genes related to nitrate signaling pathway,OsNRT1 and OsNLP2,were screened by ChIP-Seq analysis,and it was further confirmed by ChIP-q PCR and dual-luciferase assay which demonstrated that OsMADS27 could directly bind to the promoter of OsNRT1and OsNLP2 to activate their expressions.In conclusion,after nitrate induction,OsMADS27 requires the participation of OsNAR2.1 to transfer from cytoplasm to nucleus,which promotes nitrate uptake by affecting the expression of downstream genes OsNRT1 and OsNLP2,thus regulating roots growth and development.3)OsMADS57 is an important regulator of root development in response to nitrate signal.In order to explore the role of OsMADS57 in root growth and development,the tissue expression profile of OsMADS57 in rice was analyzed.It was found that OsMADS57 was mainly expressed in leaves and roots.The treatment of exogenous nitrate induced the expression of OsMADS57.Overexpression of OsMADS57 in Arabidopsis significantly promoted lateral root growth induced by nitrate.The transgenic lines of OsMADS57 in rice showed that the overexpressed lines exhibited increased lateral root primordium,primary root length and lateral root length under nitrate treatment,while the interference lines illustrated opposite phenotypes.In addition,overexpression of OsMADS57 promoted the accumulation of nitrate and the expression of nitrate transport genes in rice.In conclusion,OsMADS57 is an important transcription factor that regulates root development in response to nitrate signal.4)OsMADS25 and OsMADS57 improve the tolerance of plants to salt stress.In order to explore the role of OsMADS25,OsMADS27 and OsMADS57 in response to salt stress,the expression levels of these three genes in salt treatment were analyzed,and it was found that all three genes were strongly induced by Na Cl treatment.The transgenic lines of OsMADS25 and OsMADS57 in Arabidopsis and rice were used to investigate the function of OsMADS25 and OsMADS57 in salt stress.It was found that overexpression of OsMADS25 and OsMADS57 in both Arabidopsis and rice could improve plant salt tolerance.Compared to WT,the overexpression lines had higher seed germination rate,better root growth condition and higher survival rate under salt stress.On the contrary,the RNAi lines showed higher sensitivity to salt stress.In addition,the histochemical staining analysis of 3,3’-diaminobenzidine（DAB）and nitroblue tetrazole（NBT）showed that the accumulation of reactive oxygen species in the overexpression lines of OsMADS57 under salt stress was less than control.Further experiments revealed that overexpression of OsMADS57 in rice significantly improved the ability of plants to resist oxidative damage under salt stress,mainly by increasing the activities of antioxidant enzymes such as superoxide dismutase（SOD）and peroxidase（POD）,reducing the content of malondialdehyde（MDA）,and increasing the expression of stress-related genes.Collectively,OsMADS25 and OsMADS57 play positive roles in enhancing plant salt tolerance by activating the antioxidant defense system against oxidative damage induced by salt stress.5)The OsMADS25-OsMADS27-OsMADS57 regulatory module plays a key role in plant root growth and development.In order to further explore the upstream and downstream relationship among the three homologous genes OsMADS25,OsMADS27 and OsMADS57,the data of ChIP-Seq was analyzed.It showed that OsMADS27 was downstream of OsMADS25 and OsMADS57 was downstream of OsMADS27.ChIP-q PCR and dual-luciferase assays further confirmed that OsMADS25 could directly bind to the promoter region of OsMADS27 to activate its expression,and OsMADS27 could directly bind to the promoter region of OsMADS57to regulate its expression.In conclusion,these three homologous genes have regulatory relationships,and play a key role in regulating root growth by responding to nitrogen signal.In summary,we investigated the role of OsMADS25,OsMADS27 and OsMADS57genes regulating root growth in response to nitrogen signal.Furthermore,a MADS-box regulation network was established to regulate root growth and improve nitrate uptake in response to nitrogen signal.The molecular mechanism of OsMADS25 and OsMADS27 responding to nitrate signal in an OsNAR2.1-dependent manner was proposed,which further supplemented the key components in the regulation network for efficient nitrogen utilization,thus provided important theoretical basis and potential genetic resources for improving the nitrogen use efficiency in crops.