Transcription factor bHLH2 represses CYSTEINE PROTEASE 77 to negatively regulate nodule senescence

Legume-rhizobia symbiosis is a time-limited process due to the onset of senescence, which results in the degradation of host plant cells and symbiosomes. A number of transcription factors, proteases, and functional genes have been associated with nodule senescence; however, whether other proteases or transcription factors are involved in nodule senescence remains poorly understood. In this study, we identified an early nodule senescence mutant in Medicago truncatula, denoted bhlh2 (basic helix-loop-helix transcription factor 2), that exhibits decreased nitrogenase activity, acceleration of plant programmed cell death (PCD), and accumulation of reactive oxygen species (ROS). The results suggest that MtbHLH2 plays a negative role in nodule senescence. Nodules of wild-type and bhlh2-TALEN mutant plants at 28 days post inoculation were used for transcriptome sequencing. The transcriptome data analysis identified a papain-like cysteine protease gene, denoted MtCP77, that could serve as a potential target of MtbHLH2. Electrophoretic mobility shift assays and chromatin immunoprecipitation analysis demonstrated that MtbHLH2 directly binds to the promoter of MtCP77 to inhibit its expression. MtCP77 positively regulates nodule senescence by accelerating plant PCD and ROS accumulation. In addition, the expression of MtbHLH2 in the nodules gradually decreased from the meristematic zone to the nitrogen fixation zone, whereas the expression of MtCP77 showed enhancement. These results indicate that MtbHLH2 and MtCP77 have opposite functions in the regulation of nodule senescence. These results reveal significant roles for MtbHLH2 and MtCP77 in plant PCD, ROS accumulation, and nodule senescence, and improve our understanding of the regulation of the nodule senescence process.