The Evolution Of BHLH Family In Legumes And Functional Validation Of Soybean Candidate BHLH In Nodulation

bHLH transcription factors are the second largest family of transcription factors in plants,which play important roles in various biological processes.To date,understanding of bHLH transcription factors is basically from model plants.Soybean has experienced two genome-wide replication events with unique evolutionary characteristics,however,little is known about the bHLH transcription factors,their functions and evolution in soybean.In this study,332 members of the bHLH family were identified from the soybean genome.Among them,263 members have DNA binding ability,while the remaining 69 members do not have DNA binding ability.Phylogenetic analysis showed that soybean bHLH are divided into 27 subfamilies,24 of which are consistent with Arabidopsis,and the remaining 3 subfamilies are unique to soybean.Further collinearity analysis results showed that there were 22 conserved members in plants,suggesting their conserved functions in plant growth and adaptation in plants.Indeed,loss of function in Gm AIBa resulted in reduced expression of the nodulation genes and decreased nodule number,indicating a positive role of Gm AIBa in soybean nodulation.In addition,there are 197 legume unique and 96 bHLH members unique to legumes and soybean,respectively.Expression analysis showed that half of the legume-specific bHLHs in soybean were expressed in underground organs,among which 31 members were responsive to rhizobia,while only one third of the soybean-specific bHLHs were expressed in the underground organs,among which 7 members responded to the rhizobial infection.Two legume-specific bHLH(Gm NSE1 and Gm NSE2)was specifically expressed in nodule and the preliminary results showed that knockout of Gm NSE1 lead to increased number of nodules in soybean,suggesting a positive role of Gm NSE1 in nodulation.Interestingly,there were 449 bHLH members in the wild soybean,117 more than in the cultivated soybean,suggesting that many bHLH members was lost during soybean domestication.Further analysis showed that the lost members are mainly from tandem replicated genes with high protein similarity to their parent proteins,indicating that functional redundancy may be the main cause of the gene loss during soybean demestication.In summary,we have systemically analyzed soybean bHLHs,evolution of bHLHs in legume and non-legumes and during soybean domestication.These results will provide a deeper understanding of the evolution and function characteristics of bHLH family in soybean and Leguminosae.