Characterization Of Symbiotic Function Of The Tyrosine Sulfonyltransferase Mt Tpst In Medicago Truncatula

Legumes can interact with rhizobia and form symbiotic organ nodule in plant roots.Rhizobia immobilize nitrogen in the atmosphere into reductive nitrogen which can be used by plants.This biological process is of great significance to the sustainable development of agriculture and ecological balance.It has been found that some plant peptide hormones(such as PSK,RGF,etc.)can regulate the formation of root nodules in legumes,thus affecting the growth and development of plants.However,it is difficult to study the biological functions of these small peptides from the perspective of genetics because of their large number of members and functional redundancy.It has been reported that the sulfonation modification mediated by tyrosine sulfotransferase TPST is crucial to the maturation and activity of these peptides.Therefore,we can analyze the biological function of sulfonated peptides in symbiotic nodulation and nitrogen fixation by studying the function of TPST gene in legumes.First,we analyzed the expression pattern of MtTPST gene by quantitative PCR,and the results showed that the expression level of MtTPST was the highest in the young nodules of Medicago truncatula.Promoter-GUS activity analysis showed that MtTPST was mainly expressed in the nodule primordium,meristematic region of developing nodules and meristematic region of root tip.It indicates that MtTPST regulates the initiation and development of nodules.In addition,auxin treatment could significantly induce MtTPST gene expression.In order to study the biological function of MtTPST gene,we used the Tnt1 insertion mutant of MtTPST gene to carry out the reverse genetics experiment.We analyzed the number of infection thread(IT)one week after inoculation with rhizobia and the growth phenotype of Medicago truncatula two weeks and three weeks after inoculation.It was found that the formation of nodule primordia of MtTPST gene deletion mutant was affected,resulting in a significant reduction in the number of nodules.In addition,we found that the nodule of mttpst mutant became smaller and shorter.The paraffin section of nodule showed that this phenotype was caused by the decrease of the number of cell layers in the apical meristem of the mutant nodule.Combined with the Promoter-GUS staining and quantitative PCR results,we concluded that MtTPST gene affected the initiation and development of nodule of Medicago truncatula.At the same time,the root growth,lateral root formation,fresh weight,pod and seed growth of mttpst mutant were also severely inhibited,which was consistent with the results of At TPST homologous gene in Arabidopsis.After applying nitrogen fertilizer to wild type and mutant Medicago truncatula,we found that the growth of the mutant,especially the upper part of ground,basically returned to wild type.It was found that the main reason for the difference between mutant and wild type was the effect of MtTPST on the formation and development of nodule.We used p MtTPST:MtTPST stable transgenic successfully complements the mttpst mutant phenotype,genotypic and phenotypic co segregation of offspring derived from backcross between mttpst mutant and wild type.It is fully indicated that the nodulation symbiosis defect phenotype of mttpst mutant is caused by MtTPST mutation.In addition,p At TPST:MtTPST stable transgenic successfully restored the growth and development phenotype of Arabidopsis tpst-1 mutant,which proved that MtTPST has the function of conservative tyrosine sulfotransferase.Finally,we studied the molecular mechanism of MtTPST regulating nodulation and symbiosis of Medicago truncatula.Quantitative PCR was used to analyze the expression level of MtTPST in the mutants of genes related to nodule factor signaling pathway.The results showed that MtTPST was involved in the regulation of nodule formation downstream of nodule factor signaling pathway.At the same time,we used synthetic sulfated PSK? and RGF3 for peptide treatment experiments.PSK? significantly inhibited the defective phenotype of mttpst mutant,indicating that MtTPST mainly promoted the nodulation and symbiosis of legumes by sulfonating PSK? peptide.