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

Abstract

O_LIThe 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. C_LIO_LITo identify promoter regions conferring MtCP6 senescence-related expression, progressive MtCP6 promoter deletions were generated and the resulting sequences were fused with a reporter gene for promoter::GUS fusion analysis in transgenic M. truncatula roots. C_LIO_LIIn 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 cis-regulatory sequence, thereafter named Nodule Senescence (NS), was validated by both gain- and loss-of-function approaches. C_LIO_LIERF091, an AP2/ERF family transcription factor, was identified in a yeast one-hybrid (Y1H) screen as an NS-box interacting factor, and shown to mediate transcription activation of a NS-box:GUS reporter in transactivation assays in Nicotiana benthamiana. C_LIO_LIThis work uncovered a new senescence-related nodule specific cis-regulatory region (NS-box) and provided evidence for the likely involvement of a stress-related ERF family member in the regulation of MtCP6, at the onset of nodule senescence. C_LI