Identification of regulatory promoter sequences directing MtCP6 transcription at the onset of nodule senescence in Medicago truncatula

Abstract The symbiotic association of legumes with rhizobia results in the formation of new root organs called nodules. However, the lifespan of nodules is limited by the senescence process. Increased proteolytic activity is one of the hallmarks of nodule senescence. In Medicago truncatula, a papain cysteine protease encoding gene, MtCP6, is a marker for the onset of nodule senescence under both developmental and stress-induced pathways. To identify the promoter regions responsible for the senescence-related expression of MtCP6, progressive MtCP6 promoter deletions were generated and fused with the GUS reporter for promoter::GUS activity analysis in transgenic M. truncatula roots. In planta, a minimal promoter sequence of 67 bp was identified as sufficient for specific spatiotemporal transcriptional activation of MtCP6 in nodules. The functionality of this promoter regulatory module, thereafter named ‘nodule senescence (NS) promoter regulatory module’, was validated by both gain- and loss-of-function approaches in M. truncatula. A yeast-one-hybrid (Y1H) screen identified the AP2/ERF transcription factor ERF091, shown to positively regulate nodulation in Lotus japonicus, as an NS- interacting factor. Further Y1H and Nicotiana transactivation assays demonstrated the specificity of ERF91 to interact with and mediate transcription activation of the NS promoter regulatory motif. This work has uncovered a new senescence-related nodule-specific regulatory region and provides evidence for the likely involvement of a stress-related ERF family member in the regulation of MtCP6, at the onset of nodule senescence.