The Mechanism Of OsMADS57,a Transcription Factor,regulating Rice Root Morphology And Architecture Respones To Nitrogen Nutrition

Nitrogen(N)is an essential macronutrient for plant growth and development.It plays key roles in the entire life cycle of the plant.N deficiency dramatically affects plant growth and crop productivity.Ideotype root architecture is important for efficient N acquisition in plant.The development of plant root systems is sensitive to the availability and distribution of nutrients within the soil.Rice plants have aerenchymatous tissue in their roots that allow partial oxygen to release into the soil.Thus nitrification occurs immediately in the aerobic niche of the rhizosphere or on the surface of the roots.Therefore,even in flooded paddy soil,rice roots are actually exposed to the mixture of NH4+and low NO3-concentration.Numerous study showed molecular mechanism for NO3-regulating root growth especially for lateral root proliferation in Arabiopsis.However,mechanism for NO3-regulating rice root growth is poorly understood,mainly due to ignoring nitrate nutrition in rice plant.A NO3--inducible Arabidopsis gene(AtANR1),belong to MADS-box family,was identified as a key component of the signal transduction pathway by which NO3-stimulates lateral root proliferation.Our previous results showed that complementary nitrate high-affinity transport protein OsNAR2.1 participates in NO3--regulating root growth under low NO3-concentration.Interestingly,the expression of(MADS-box transcription factor 57)OsMADS57,one of AtANR1’s analoge genes,is down-regulated in osnar2.1 mutants under 0.2 mM NO3-supply.These suggested that OsMADS57 might be involved in NO3--regulating rice root growth.Thus,on the base of illustrating the mechanism of OsNAR2.1 in regulating rice root growth,we emphasize on clarifying whether OsMADS57 play an important role in regulating rice root growth.Results were listed as follows.1.Bioinformatic analysis showed that OsMADS57 localizes in chromosome 2;its open reading frame is composed of seven introns and eight exons,encoding a protein composed of 241 amino acids.Two key domains are predicted in OsMADS57 protein:amino acids from 1 to 60 as M domain and those from 79 to 113 as K domain.Results of qRT-PCR showed that OsMADS57 is expressed in leaves,stems,shoot base and roots.In addition,OsMADS57 is induced by nitrate and responsive to NAA treatment.2.Spatial expression pattern of OsMADS57 was detected in transgenic rice with promoter of OsMADS57 fusing GUS.GUS staining experiment showed that OsMADS57 is expressed in callus,germinated cotyledons,roots,shoot base and leaves.Interstingly,OsMADS57 is expressed in the root’s central cylinder,which is induced by nitrate supply.3.Three osmads57 mutant lines were obtained from RiceGE,the Rice Functional Genomics Express Database of Korea.Gene-silencing effect and the T-DNA insertion position of the osmads57 mutants were identified through RT-PCR and two-round PCR methods.The expression of OsMADS57 was down-regulated by 80-90%as compared with wild-type plants.At the same time,ten lines of OsMADS5 7 transgenic overexpression were obtained through transgenic manipulation technology.The most importantly,two one-copy positive over-expression lines were obtained at T2 generation through southern-blot method.4.Compared with wild-type plants,higher N concentration in rice root was observed in two osmads57 mutants under 0.2 mM NO3-.However,no difference of N concentration was observed in shoot under 0.2 mM NO3-and in shoot and root among mutants and wild-type plants under 5 mM NO3-and 0.2 mM NH4+.Results from 5-minutes 0.2 mM 15N-NO3--uptake assay showed no significant difference in nitrate influx rate among two osmads57 mutants and wild-type plants,which indicated that OsMADS57 mutation didn’t affect nitrate uptake.Results from 60-minutes 0.2 mM 15N-NO3--uptake treatment showed that higher 15N-NO3-concentration in rice root and lower 15N-NO3-concentration in rice shoot were recorded in two osmads57 mutants as comparison with wild-type plants.Furthermore,lower NO3-content in xylem sap was observed in two osmads57 mutants than wild-type plants.And we analyzed 15N-NO3-concentration in two over-expression lines and wild-type plants for an hour 15N-NO3--uptake experiments.Higher 15N-NO3-concentration was observed in shoots and slightly lower in roots of two overexpression lines than wild-type plants.These results suggested that OsMADS57 maybe invovled in regulating nitrate transport from root to shoot.5.Results from qRT-PCR showed that,compared with wild-type plants,the expression of OsNRT2.1/2.2/2.3a/2.4 is down-regulated in two mutants under 0.2 mM NO3-;contrarily,the expression of OsNRT2.1/2.2/2.3a/2.4 is up-regulated in two overexpression lines under 0.2 mM N03-.OsNRT2.3a has been reported to participate in nitrate transport from root to shoot.One-hibrid yeast assay showed that OsMADS57 can bind to the cis-elements in the promotor of OsNRT2.3a in yeast.These results suggested that OsMADS57 affect nitrate transport from the root to shoot through modulating OsNRT2.3a expression.6.Compared with wild-type plants,elongation of seminal and adventitious roots was inhibited in osmads57 mutants under 0.2 mM NO3-.However,no difference ws observed in formation of adventitious and lateral roots and in elongation of lateral roots under 0.2 mM NO3-and in rice root morphology under 0.2 mM NH4+among mutants and wild-type plants.Further results showed that the mutation of OsMADS57 enhanced auxin acropetal transport from the shoot to root rather than auxin basipetal transport,which resulted in higher auxin concentration in root tip.These might be the reason why the mutation of OsMADS57 only affected elongation of seminal and adventitious roots rather than lateral root.7.Results from field experiments showed that,compared with wild-type,the tillering number of osmads57 mutants increased significantly when high level N fertilizer applied.However,no difference was observed in tillering number between mutants and WT under under low nitrogen condition.Compared with wild-type,the expression of OsFCland D14/17/2 7 in osmads57 mutants was down-regulated when high level N fertilizer applied.and up-regulated under low nitrogen condition.These results suggested that OsMADS57 affect rice tillers through modulating expression of OsFCland D14/17/27.8.knockdown of OsNAR2.1 inhibited lateral root(LR)formation under low NO3-concentrations,but not under low NH4+concentrations.15N-labelling NO3-supplies(provided at concentrations of 0-10 mM)demonstrated that(i)defects in LR formation in mutants subjected to low external NO3-concentrations resulted from impaired NO3-uptake,and(ii)the mutants had significantly fewer LRs than the WT plants when root N contents were similar between genotypes.LR formation in osnar2.1 mutants was less sensitive to localised NO3-supply than LR formation in WT plants,suggesting that OsNAR2.1 may be involved in a NO3--signalling pathway that controls LR formation.Knockdown of OsNAR2.1 inhibited LR formation by decreasing auxin transport from shoots to roots.Taken together,transcription factor OsMADS57 is invovled in nitrate transport from root to shoot through modulating nitrate high-affinity transporter OsNRT2.3a under low NO3-condition.And OsMADS57 participate in elongation of seminal and adventitious roots through regulating auxin acropetal transport.OsMADS57 regulate rice tillers depende on N supply level.