Cysteine protease and cystatin expression and activity during soybean nodule development and senescence

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.

Figure 1

Mapping of transcribed soybean nodule cystatins to different I25 cystatin subfamilies.

Figure 2

Mapping of transcribed cysteine proteases to sub-families and functional groups with similarity to the C1 cysteine protease papain.

Figure 3

Expression changes of cystatins, cysteine proteases and vacuolar processing enzymes. (A) Expression of cystatins (CYS) (B) cysteine proteases (CYP) and (C) vacuolar processing enzymes (VPE) in 4, 8 and 14 week old nodules expressed as FPKM (transcript abundances in fragments per kilobase of exon per million fragments mapped). Colour scale represents transcription for each time point normalized by subtracting the mean/median of three values from each individual value for each gene reduced by SD/RMS. * indicates significant change (p < 0.05) in transcription between individual time points. Multi-experiment viewer (MeV v4.8.1) software package was applied to graphically represent data [52].

Figure 4

Relative expression measured by quantitative real-time PCR of soybean cysteine proteases, cystatins, leghemoglobin and a VPE at each time point (4, 8 and 14 weeks) and corresponding FPKM abundance estimates derived from RNA-Seq mapping.