Abstract
BackgroundNodules 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.ResultsSeventy 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.ConclusionsOverlap 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0294-3) contains supplementary material, which is available to authorized users.
Highlights
Nodules play an important role in fixing atmospheric nitrogen for soybean growth
Cystatin identification All expressed nodule cystatins were identified from our RNAseq data
When we carried out a phylogenetic genetic analysis of cystatins by comparison with cystatins from different I25 cystatin subfamilies (Figure 1), Glyma13g04250 and Glyma20g08800, transcribed in nodules during nodule development and senescence, had high similarity to group A cystatins (Vigna unguiculata cystatin, OCI, HvCPI-1 and HvCPI-2) [20]
Summary
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. Several cystatin functions have been proposed, but all involve a balanced interplay with a cysteine protease to regulate proteolytic activity [2,3]. Despite strong evidence for cysteine protease involvement in nodule development and senescence, only limited detailed information is currently available on any specific cystatin function and activity in these development and senescence processes [6,8,11,12]. Further evidence of the in vivo regulation of cysteine protease have been provided by Pillay et al [14] showing that co-expression of the rice cystatin OCI in tobacco plants protected recombinant proteins from degradation by lowering overall cysteine protease activity
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