Soybean nodule development and senescence : the role of cysteine proteases and their inhibitors

Abstract

Background: Root nodules in soybean play an important role in fixation of atmospheric nitrogen used for plant growth. Premature senescence of nodules can negatively impact on nitrogen availability for plant growth and, as such, a better understanding of nodule development and senescence is required. Cysteine proteases are known to play a role in nodule senescence, but knowledge is still fragmented regarding the function of their inhibitors (cystatins) during the development and senescence of soybean nodules. Results: RNA-Seq expression analysis showed that transcription of cystatins Glyma05g28250, Glyma15g12211, Glyma15g36180 increased during onset of senescence, possibly regulating proteolysis when nodules senesce and undergo programmed cell death. Biochemical inhibitory assays with recombinantly expressed and purified cystatins showed that most cystatins had preferential affinity to cathepsin L-like cysteine proteases. Both actively- and non-actively transcribed nodule cystatins inhibited cathepsin-L- and B-like activities in different age nodules and they also inhibited papain and cathepsin-L activity. The localisation of these proteins could not be determined with the approach that was followed. Conclusions: This PhD study provided the first evidence with regard to cystatin expression during nodule development combined with biochemical characterization of their inhibition strength. Knowledge about the expression, localization and possible function of the cysteine protease-cystatin system during soybean nodule development was generated. Overlap in activities and specificities of actively and non-actively transcribed cystatins, raises the question if non-transcribed cystatins provide a reservoir for response to particular environments. This data might be applicable to the development of strategies to extend the active life span of nodules or prevent environmentally induced senescence.