Rice Transcription Factor OsMADS61 Mediates The Low-Nitrate Response By Promoting Nitrate Transport,Assimilation And Root Elongation

Rice is one of the most important crops in the world,feeding about half of the world’s population.A large number of research results show that soil fertility and nitrogen fertilizer application are the most important guarantees for achieving high yields of rice.However,excessive application of nitrogen fertilizer has caused serious environmental problems.In addition to continuously improving soil fertility and implementing scientific comprehensive management techniques for nutrient resources,it is a major task for plant biologists to fully tap the genetic potential of rice to efficiently absorb and utilize N.In recent years,increasing the expression of NO₃^-transporter genes and the expression of genes which involved in regulating nitrogen absorption can improve rice yields at low nitrogen levels,and achieve reduction of nitrogen fertilizer and efficient use of nitrogen fertilizer at the same time.Although the signaling pathways that regulate NO₃^-uptake and transport in Arabidopsis have been deeply studied,the regulatory network of NO₃^-transport and assimilation in rice is still unclear.In Arabidopsis,AtANR1 has been reported to involve in the regulatory network of nitrate absorption.In rice,among five homologous genes of AtANR1（OsMADS23,OsMADS25,OsMADS27,OsMADS57 and OsMADS61）,the relative expression of OsMADS61 is only induced in roots by low nitrate.It’s unclear what the biological functions are involved.In this study,OsMADS61 was taken as the research object and it’s important role was clarified in rice growth and development through genetics,physiology and molecular biology.The main findings are as follows:1.On the RAP-DB website,OsMADS61 has two gene accession numbers.After RT-qPCR verification,it is confirmed that the gene sequences under the two accession numbers together constitute the complete gene sequence of OsMADS61.After bioinformatics analysis,it is inferred that OsMADS61 is a MIKC^c type MADS-Box transcription factor,which is located in the nucleus through subcellular localization.And the relative expression of OsMADS61 was analyzed in rice plants under two nitrogen forms（NH₄⁺,NO₃^-）and three N concentrations（0.2,2.5 and 5 mM）.The results showed that,compared with N treatments with 2.5 and 5 mM,OsMADS61expression was the highest under 0.2 mM nitrate condition,and was mainly expressed in rice roots;the results from GUS staining are consistent in that from RT-qPCR.2.Multiple lines of homozygous OSMADS61 mutants were obtained through CRISPR/Cas9 gene editing technology and from the Korean Rice Mutant Library（RiceGE）;the pTCK303 vector was used to construct overexpression lines（T2-T3generations）.Compared with the wild type,the N content in the OsMADS61 mutant was significantly reduced under the condition of 0.2 mM NO₃^-,but the difference was not significant under the condition of 2.5 mM NO₃^-;while the overexpression lines had higher nitrogen content than that of the wild type under the two nitrogen concentrations,which indicates that OsMADS61 may be involved in NO₃^-absorption or transport in rice under low nitrate conditions.The results of the ¹⁵N-NO₃^-isotopic tracing method showed that,compared with the wild type,the ¹⁵N content in the OsMADS61 mutant was significantly reduced when treated with 0.2 mM ¹⁵N-NO₃^-for 5 min,while the ¹⁵N content in the overexpression Lines was significantly reduced.These indicates that OsMADS61 is involved in regulating the absorption of NO₃^-by rice.The results from the high-resolution scanning ion-selective electrode technique（SIET）further confirmed that OsMADS61 participates in NO₃^-absorption in rice under low NO₃^-conditions.3.In order to clarify which NO₃^-transport protein gene that OsMADS61 directly interacts with,we analyzed relative expression of high-affinity nitrate transporters and their partners.The results of RT-qPCR showed that compared with wild-type rice,lower relative expression of OsNRT2.1,OsNRT2.2,OsNRT2.4 and OsNAR2.1 was recorded in roots of the mutants and higher in overexpression lines.However,only one CArG-Box motif was found in the promoters of OsNRT2.1/OsNRT2.2,so it is speculated that OsNRT2.1/OsNRT2.2 may be the downstream target genes of OsMADS61.Through the yeast one-hybrid system（Y1H）,tobacco transient expression system and chromatin immunoprecipitation-qPCR system（ChIP-qPCR）,it was confirmed that OsMADS61 can directly bind to the promoters of OsNRT2.1/OsNRT2.2,thereby modulating the transcription level of OsNRT2.1/OsNRT2.2. And the OsMADS61 mutant supplementing OsNRT2.1 can significantly increase the nitrogen content in OsMADS61 mutant,which further confirms that OsMADS61 directly regulates the transcription level of OsNRT2.1/OsNRT2.2.4.In addition to positively regulating the transcription level of nitrate transporter genes OsNRT2.1/OsNRT2.2,OsMADS61 has also been found that it is also involved in modulating the activity of nitrate reductase.Compared with the wild type,the nitrate reductase activity was significantly reduced in roots of the mutant roots,while was increased in the overexpression lines.Then we detect the expression of two nitrate reductase genes OsNIA1 and OsNIA2 in rice.The results from RT-qPCR showed that,compared with wild-type rice,the expression level of OsNIA2 in roots of the mutants and overexpression lines was significantly reduced and increased,respectively;however,the expression level of OsNIA1 was not affected in transgenic plants.After analyzing the promoter of rice nitrate reductase gene OsNIA2,we found six CArG-Box motifs that can be recognized by OsMADS genes.Studies using Y1H,ChIP-qPCR and tobacco transient expression system showed that OsMADS61 recognizes and binds to the CArG-Box motif at the site of-1051bp～-1042bp of OsNIA2 promoter.These confirm that OsMADS61 can directly bind to the promoter of the OsNIA2 gene and directly regulates the expression of OsNIA2.5.Interestingly,compared with wild-type rice,the length of seminal and adventitious roots was significantly reduced in mutants under the condition of 0.2 mM NO₃^-,and the difference was not recorded under the condition of 2.5 mM NO₃^-;while the overexpression lines under the two nitrogen concentrations had longer roots.These indicate that OsMADS61 may be involved in the elongation process of rice roots under low nitrate conditions.Compared with wild-type plants,the number of cells in the root meristem was significantly reduced in mutants under 0.2 mM NO₃^-treatment,while was increased in the overexpression,indicating that the change of root elongation in transgenic plants is due to the size of root tip meristem.6.Studies have shown that rice nitrate reductase is a key synthetase of nitric oxide（NO）,and the level of NO concentration in rice root tips directly affects the elongation of rice roots.The results of the pharmacological experiment show that the addition of NO donor can recover the phenotype of mutant roots to the similar level as that of the wild-type plants,and the addition of the NO scavenger reduces the elongation of the wild-type root to the similar level as that of the mutant plants,so it is speculated that the change of NO content may result in the changes in the root length of the osmads61mutants.The results from time-course of NO content in the root tip of rice（0～1 hour）showed that compared with wild-type rice,the NO content in the root tip of the mutant decreased significantly when treated by 0.2 mM NO₃^-for 5 minutes while the overexpression lines increased significantly the NO content.Furthermore,the change of NO content in rice root tip parallels with the expression of nitrate reductase gene OsNIA2 and nitrate reductase activity.Moreover,the addition of exogenous nitrate reductase inhibitor can significantly decrease NO content in root tips and root elongation,which further proves that NO synthesized by the nitrate reductase pathway regulated by OsMADS61 participates in root elongation.7.Previous studies have found that the change of NO₃^-content can affect the content of NO in plant roots,so whether the NO synthesized by OsNIA2 pathway regulated by OSMADS61 can regulate the absorption of NO₃^-in rice needs further verification.The results showed that the addition of exogenous NO donor under 0.2m M NO₃^-treatment significantly promoted the expression of OsNRT2 family genes in wild-type and mutant plants,and no significant difference was recorded between them;correspondingly,exogenous addition of NO scavenger significantly inhibited the expression of OsNRT2 family genes in wild-type and mutant plants.These indicates that the NO synthesized by the nitrate reductase pathway regulated by OsMADS61 can regulate the NO₃^-uptake of rice.In summary,OsMADS61 enhances nitrogen acquisition capacity by regulating root elongation and high-affinity nitrate transport genes OsNRT2.1 and OsNRT2.2.In addition,OsMADS61 promotes N assimilation by promoting the expression of the nitrate reductase gene OsNIA2.The NO produced by OsNIA2 promotes the expression of OsNRT2.1 and OsNRT2.2.Therefore,the nitrogen absorption assimilation network regulated by OsMADS61 may be a comprehensive strategy for rice to adapt to changes in NO₃^-supply.