Abstract:
BACKGROUND : Nodules play an important role in fixing atmospheric nitrogen for soybean growth. Premature
senescence of nodules can negatively impact on nitrogen availability for plant growth and, as such, we need a
better understanding of nodule development and senescence. Cysteine proteases are known to play a role in
nodule senescence, but knowledge is still fragmented regarding the function their inhibitors (cystatins) during the
development and senescence of soybean nodules. This study provides the first data with regard to cystatin
expression during nodule development combined with biochemical characterization of their inhibition strength.
RESULTS : Seventy nine non-redundant cysteine protease gene sequences with homology to papain, belonging to
different subfamilies, and several legumain-like cysteine proteases (vacuole processing enzymes) were identified
from the soybean genome assembly with eighteen of these cysteine proteases actively transcribed during nodule
development and senescence. In addition, nineteen non-redundant cystatins similar to oryzacystatin-I and belonging
to cystatin subgroups A and C were identified from the soybean genome assembly with seven actively transcribed
in nodules. Most cystatins had preferential affinity to cathepsin L-like cysteine proteases. Transcription of cystatins
Glyma05g28250, Glyma15g12211, Glyma15g36180 particularly increased during onset of senescence, possibly
regulating proteolysis when nodules senesce and undergo programmed cell death. Both actively transcribed 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 when expressed and purified from bacterial cells.
CONCLUSIONS : 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.
