Mechanism Of LIN Regulating Delayed Nitrogen Fixation Of Medicago Truncatula

Legumes can form a reciprocal symbiotic relationship with the rhizobia in the soil,forming a special tissue-nodules in the roots.The formation of nodular organs is subjected to a series of complex regulatory processes.There are two main aspects involved,namely the infection of rhizobia and the development of rhizobia primordia.After inoculation with rhizobia,wild-type plants can form pink cylindrical nodules at 2 wpi.In our laboratory,we discovered a new mutant that exhibited a delayed nitrogen fixation phenotype.Only small white protrusions can be formed at 2 wpi,and pink cylindrical functional nodules cannot be formed until 5 wpi.Through map-based cloning,we founded that the gene is Mt LIN,but it lost 2.3 Kb of the promoter of ubiquitin ligase at E3.Since this mutant was a homologous mutant of lin,we named it lin-5.In order to clarify the causes of delayed nitrogen fixation of lin-5 mutants and the role of Mt LIN in nitrogen fixation,this study makes a series of observations on WT and lin-5 mutants,including the infection events,the development of nodule and the release process of rhizobia,and further analyzes the expression and tissue location of Mt LIN.The specific research results are as follows:1.The phenotype of lin-5 mutant is complemented by Mt LIN gene.When the construct LIN-p KGWRR is introduced into the lin-5 mutant,the transformed plants returned to the wild-type phenotype,forming pink cylindrical nodules.2.The lin-5 mutant infection event is completely normal.The infection threads of lin-5mutant plants can extend to the cortical cells normally,and the number of infection threads is equivalent to the wild type;while the infection process of lin-2 is damaged,only a small number of infection chambers can be formed.3.The formation of lin-5 nodule primordium is normal.The formation process of lin-5nodule primordium is normal.The lin-5 mutant mesothelial cells can normally differentiate to form nodule primordia,and the nodule bacteria can infect the nodule primordia.The differentiation process of lin-5 mutants in the late stage of nodule is normal,and can develop into mature nodules with different divisions.The differentiation process of lin-5 mutants in the late stage of nodule is normal,and could be differentiated into different zones mature nodules.4.Rhizobia enters plant nodule cells more slowly in lin-5 mutant than wild type..Therefore,tthe slow release rate of rhizobium in lin-5 mutants leads to delayed nitrogen fixation.5.Mt LIN delayed expression in lin-5.There is no significant difference in the expression of Mt LIN in WT and lin-5 uninoculated roots;however,the expression of Mt LIN in lin-5 is significantly lower than that in WT at early stage.As time goes by,the expression level of Mt LIN is gradually accumulated in lin-5,the expression level in nodules at 5 wpi has no significant difference from WT.7.The tissue localization of Mt LIN is specific.The construct p LIN:GUS and plin-5:GUS fusion expression vectors are introduced into WT,lin-5 and vapyrin by A.rhizogenes-mediated hairy root transformation.After inoculation,the transformed roots were subjected to GUS staining for tissue localization.The results show that,(1)when introduced into the WT,p LIN::GUS and plin-5::GUS are highly expressed in the occurrence site of the lateral roots without inoculation of rhizobium;after inoculation,p LIN::GUS is found in the root zone of the nodule primordia,the entire new root nodule and the mature root nodule.In mature nodules,it expresses in meristem,infection zone and part of the nitrogen-fixing zone;However,plin-5::GUS is not expressed in the early stage,it begin to express in the root nodule meristem and infection zone until 5wpi.(2)When introduced into the lin-5,the expression of p LIN::GUS and plin-5::GUS are similar,they are expressed in the occurrence site of the lateral roots,nodule primordia and small nodules,indicating that p LIN::GUS has a certain inhibitory effect on its own expression.(3)When introduced into the vapyrin,p LIN::GUS and plin-5::GUS are highly expressed in the occurrence site of the lateral roots without inoculation of rhizobium.After inoculation,p LIN::GUSis expressed in the nodule primordia and entire small nodule,while the expression of plin-5::GUS in the nodule primordia and nodule is not detected.This study found that the delayed nitrogen fixation of lin-5 is caused by the slow release rate of rhizobia to the nodule primordia.Mt LIN has a specific spatiotemporal expression pattern and tissue localization pattern,which plays important roles in the formation of infection threads and the developmen of nodules.This helps us to analyze the coordinated mechanism between epidermal rhizobia infection and cortical nodule development,and provides new ideas for the study of nitrogen-fixing symbiotic signaling pathways.