Identification Of Phytocyanin Gene Family In Legumes And Their Functional Analyses In Nodulation Of Medicago Truncatula

During the long-term evolution,legumes have established a symbiotic relationship with rhizobia that leads to the formation of nodules.In nodules,rhizobia reduce the atmospheric nitrogen to ammonia,which is provided to the host plants so that the plants are able to survive in nitrogen-deficient environment.Nodule formation is a complicated biological process involving the infection of rhizobia,the differentiation of plant root cortex cells and the development of nodules.Although the research on the molecular mechanisms of symbiotic interaction between legumes and rhizobia has made great progress,the exact functions of some genes related to nodulation are still unknown.Phytocyanins(PCs)are a class of plant-specific blue copper proteins,playing critical roles in plant development including nodule formation.Although a few PC genes have been isolated from legume nodules,their exact functions in the symbiotic nodulation are largely unexplored.In this study,genome-wide identification and evolution analysis of PC gene family were performed in seven sequenced legumes(Medicago truncatula,Glycine max,Cicer arietinum,Cajanus cajan,Lotus japonicus,Vigna angularis and Phaseolus vulgaris).In the model plant Medicago truncatula,three nodule-specific genes(MtPLC1,MtENODL27 and MtENODL28)were identified from the PC gene family and the function of MtPLC1 in nodulation was analyzed in detail.The main results are as follows:(1)Genome-wide identification and gene expression analyses of PC genes:(i)The evolution analysis of PC gene family in seven sequenced legumes showed that PCs were significantly expanded in M.truncatula and G.max.Specifically,the expansion of PCs in M.truncatula was mainly derived from tandem duplication events.In M.truncatula,82 MtPCs could be divided into four subfamilies and clustered into seven branches.Expression analyses showed MtPCs had different cross-tissues expression patterns.(ii)Spatial-temporal expression analyses showed that MtPLC1,MtENODL27 and MtENODL28 were nodule-specific genes.The co-expression analysis indicated that MtPLC1,MtENODL27 and MtENODL28 were co-expressed with the genes related to symbiotic nitrogen fixation and grouped into two different functional modules,suggesting that the three genes play different roles in nodulation.(2)Localization analysis of MtPLC1:(i)Tissue localization analysis showed that in the inoculated roots,MtPLC1 was firstly expressed in the inner cortex,then in the cortex.It was also expressed in the infection chamber.In nodules,MtPLC1 was mainly expressed in the transition zone and nitrogen fixation zone.(ii)Subcellular localization: Sequence analysis revealed that MtPLC1 contains an N-terminal signal peptide,suggesting that the MtPLC1 protein may be secreted.The subcellular localization showed that MtPLC1 was retained in the cell of tobacco leaf and alfalfa root rather than secreted out of the cell.The immunofluorescence localization in nodule showed that MtPLC1 was localized in the peribacterial membrane of the symbiosomes in the transition zone and nitrogen fixation zone.Further,the symbiosomes were isolated from the mature nodule and immunoblotted with antibody,and the result showed that the MtPLC1 protein was expressed in the symbiosome.(3)RNAi(RNA interference)analyses of MtPLC1:(i)Statistics on infection events showed that the number of microcolonies,elongating infection threads(EITs),ITs and nodule primordia were significantly reduced in the RNAi plants compared with the control group.The expression analyses of the marker genes associated with infection showed that there was no obvious difference in the transcriptional level of DIM1 and DIM2 between the RNAi plants and the control,while DIM3,NSP1,NSP2,ERN1,ERN2,ERN3,ENOD11 and NIN were significantly downregulated,indicating that MtPLC1 was located downstream of the calcium peak and involved in the infection of rhizobia.The analyses of plant growth showed that plant height,root length and nodule number in the RNAi group were significantly reduced at 28 day post inoculation(dpi)compared with the control group.Chlorophyll content,nitrogenase activity and transcription level of the leghemoglobin were also strongly decreased,indicating that the interference of MtPLC1 had a negative effect on symbiotic nodulation.(ii)At 10 dpi,there were a large number of ITs in the RNAi nodule cells,indicating the release of rhizobia was inhibited.At 14 dpi,it was observed that several symbiosomes were surrounded by the membrane-like structure in the RNAi nodules,suggesting that the bacteroids began to degrade.At 28 dpi,nodule cells of the nitrogen-fixing zone in RNAi nodules became irregular,and the bacteroids had been degraded,indicating that the silence of MtPLC1 resulted in nodule premature senescence.(iii)Activity of cytochrome oxidase was significantly reduced in the RNAi nodules compared with the control,indicating that the down-regulation of MtPLC1 might affect the energy metabolism and nitrogen-fixing environment of the bacteroids.(iv)The contents of hydrogen peroxide and superoxide anion in the RNAi nodules were increased significantly at 15 dpi.Moreover,superoxide anion in the RNAi nodules was accumulated substantially compared with that in the control group at 14 dpi and 28 dpi,indicating MtPLC1 was involved in ROS homeostasis.Although the hydroxyl radical scavenging ability,superoxide anion scavenging ability and superoxide dismutase activity in RNAi plants were increased significantly compared with the control group at 15 dpi,the total antioxidant capacity was significantly lower than that in the control group,indicating that the scavenging ability in the RNAi plants could not impair the accumulation of ROS;(v)The results of RNA-seq analysis showed that differentially expressed genes(DEGs)were dominantly related to morphological development of nodules,redox reaction,regulation of transcription and protein phosphorylation in the RNAi roots at 10 dpi and 28 dpi.Also,the qPCR results of the DEGs related to cell differentiation,ROS production and antioxidants were consistent with the transcriptome data,indicating that the interference of MtPLC1 affected the homeostasis of the oxidoreduction in nodules.(4)Overexpression analyses of MtPLC1:(i)The number of EITs and ITs were increased significantly in the over-expression plants compared with the control group,indicating the over-expression of MtPLC1 promoted the rizobia infection.The chlorophyll content,plant height,root length,nodule number,nitrogenase activity and transcription level of the leghemoglobin were also increased dominantly in the over-expression plants at 45 dpi,indicating that the over-expression of MtPLC1 contributed to plant nodulation and promoted the nitrogen fixation ability of nodule.(iii)At 45 dpi,the senescence zone occurred in the nodules of the control plants,while there was no senescence zone and still a large area of nitrogen fixation zone in the over-expression nodules,indicating that the over-expression of MtPLC1 delayed nodule senescence.In summary,the genome-wide identification and evolution analysis of PC family members were performed in legumes.The phylogenetic characteristics were clarified and three nodule-specific genes were identified in M.truncatula.The cellular localization and functional studies by RNAi and over-expression confirmed that MtPLC1,targeted to the peribacterial membrane of the symbiosomes in transition zone and nitrogen fixation zone of nodules,was essential for rhizobia infection,bacteroid differentiation and nodule development.Further,the mechanism analysis suggested that MtPLC1 might regulate nodule formation by affecting the ROS homeostasis.These results laid a foundation for further elucidating the functions of PC family genes in the symbiotic interaction between legumes and rhizobia,and provided a reference for illuminating the biological functions of PC family genes in plants.