BACKGROUND
in plants, cystatins are natural and specific inhibitors of cysteine proteases of the papain c1a family that generally block c1a proteases by a tight and reversible interaction  <cit> .
several cystatin functions have been proposed, but all involve a balanced interplay with a cysteine protease to regulate proteolytic activity  <cit> .
research has so far provided strong evidence that plant cystatins regulate endogenous protein turnover during growth and developmental processes, including senescence and programmed cell death, and are further involved in accumulation and mobilization of storage proteins.
a further key function is protection against plant pests where cystatins prevent cysteine protease activity required for protein digestion in pests  <cit> .
cysteine protease expression during nodule senescence has been previously reported  <cit> .
proteolytic activity in infected nodules limits the bacterial symbiosis and nitrogen fixation, with cytosolic leghemoglobin and the bacteriod as targets.
in medicago trunctula anti-sense inhibition of the cysteine protease cyp15a caused a delay in nodule senescence  <cit>  and nodule lifespan was prolonged, when a nodule-specific papain-like cysteine protease  was silenced  <cit> .
however, 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  <cit> .
the most detailed analysis of participation of an endogenous cystatin in interaction with an endogenous cysteine protease during senescence has been the coordinated expression of the mrnas of a cysteine protease and a cystatin in senescent spinach leaves where a senescence-related cysteine protease–cystatin complex was identified  <cit> .
further evidence of the in vivo regulation of cysteine protease have been provided by pillay et al.
<cit>  showing that co-expression of the rice cystatin oci in tobacco plants protected recombinant proteins from degradation by lowering overall cysteine protease activity.
the phytozome database  currently contains over  <dig> cystatin-like sequences from the viridiplantae kingdom,  <dig> c <dig> cysteine protease sequences and  <dig> c <dig> cysteine protease  sequences.
the recent release of the complete soybean genome  <cit>  as well as the release of a rnaseq atlas of genes expressed in fourteen different soybean tissues including nodules  <cit>  has further allowed identification and characterization of all  <dig> soybean cystatins, irrespective of transcriptional activity, and  <dig> active cysteine proteases.
accurate studies are now possible to determine the cystatin and cysteine protease classes expressed in nodules and also to investigate if endogenous cystatins preferentially interact with specific target cysteine proteases in nodules.
our study was therefore aimed to provide a first insight into such interactions by identifying and characterizing all members of the cystatin and cysteine protease gene families in soybean nodules.
we included both actively and non-actively transcribed cystatins and cysteine proteases identified through homology searches in the soybean genomic database.
the nodule transcription profiles were developed with the technique of rnaseq  <cit>  which allowed us to determine the expression of all oryzacystatin i-like cystatins, papain-like cysteine proteases, as well as vacuole vpe-type cysteine proteases in determinate soybean crown nodules during nodule development and senescence.
such vpe cysteine proteases resemble mammalian caspases and they contribute to the senescence process and pcd   <cit> , but might further activate pre-proteases by post-translational modification  <cit> .
in our characterization, we were also interested to determine to which families and functional groups nodule cystatins and cysteine proteases belong as well as the cystatin substrate preference by testing in vitro produced cystatin proteins with various cysteine protease-containing extracts.
cystatins are part of subfamily b of the i <dig> cystatin family and in cereals they can be divided into various functional groups  with most cystatins belonging to groups a and c  <cit> .
group a cystatins, which efficiently inhibit cathepsin l-like cysteine-proteases, are preferentially expressed in dry and germinating seeds whereas group c <dig> cystatins, which are potent inhibitors of c1a peptidases, are mostly expressed in developing seed endosperms.
cysteine proteases cluster into different subfamilies  <cit>  with cysteine proteases closest to papain clustering with subfamily xcp <dig> represented by the arabidopsis thaliana genes at1g <dig> and at4g <dig>  cysteine proteases with cathepsin-l-like activity can closely cluster with subfamily rd <dig> consisting of rd21a , rd21b  and rd21c .
a c-terminal granulin domain is characteristic of the rd <dig> subfamily.
cysteine proteases with cathepsin-l-like activity can further cluster with the sag <dig> subfamily.
cysteine proteases with cathepsin-f-like activity cluster with subfamily rd <dig> with members rd19a , rd19b  and rd19c  and rd <dig> members have a characteristic erfnaq motif in the pro-domain.
cysteine proteases with cathepsin-h-like activity cluster with members of the aalp  and alp <dig>  subfamily.
we were finally also interested to determine the interaction affinities between selected actively and non-actively transcribed cystatins during nodule development and senescence.
this should provide information about the relative activities and specificities of both expressed and non-expressed cystatin genes in soybean.
in our study, we found an overlap in the activities and specificities of the expressed and non-expressed cystatin genes raising the question of whether the non-transcribed cystatins provide a reservoir for responses to particular environments.
RESULTS
cystatin identification
all expressed nodule cystatins were identified from our rnaseq data.
when the oryzacystatin-i  was used for comparison as a model cystatin,  <dig> cystatin sequences were identified in the assembled genome; of these  <dig> were non-redundant sequences .
when we carried out a phylogenetic genetic analysis of cystatins by comparison with cystatins from different i <dig> cystatin subfamilies , glyma13g <dig> and glyma20g <dig>  transcribed in nodules during nodule development and senescence, had high similarity to group a cystatins   <cit> .
glyma13g <dig> was further paralogous to glyma14g <dig> with identical location, but on a different chromosome.
also, the two cystatins glyma13g <dig> and glyma15g <dig> were highly similar to cystatin b  and hvcpi- <dig>  and glyma05g <dig> was further highly similar to group b cystatins , hvcpi- <dig> and hvcpi-9).
they also contained a c-terminal extension with a snsl amino acid motif enabling them to inhibit legumain c <dig> cysteine proteases  <cit> .
finally, glyma15g <dig>  which was the most abundant cystatin in nodules, was similar to group c  cystatins , hvcpi- <dig> and hvcpi-8).figure  <dig>
mapping of transcribed soybean nodule cystatins to different i <dig> cystatin subfamilies.
we also searched all cystatin sequences for signal peptides indicating their possible cellular localisation .
glyma05g <dig>  glyma07g <dig> and glyma13g <dig> might be localised in the secretory pathway, whereas glyma13g <dig>  glyma14g <dig> and glyma20g <dig> are localised to any location, except the chloroplast, mitochondrion or secretory pathway.
localisation of glyma15g <dig> was not reliable and the cystatin could be located in either the mitochondrion or the secretory pathway.
cysteine protease identification
a total of  <dig> cysteine protease sequences with homology  to the model cysteine protease papain  were further identified from the soybean genome assembly .
several sequences were alleles, paralogos and orthologos of other cysteine protease gene sequences.
from these we identified  <dig> non-redundant cysteine protease gene sequences which had similarity to members of eight different cysteine protease sub-families.
seven sub-families were distinguished from our expression data and we identified confidently five functional groups .
however, none of the identified soybean cysteine proteases clustered with papain .
cysteine proteases with cathepsin-l-like activity included glyma04g <dig> , as well as the two proteases glyma14g <dig> and glyma17g <dig> .
we also confirmed the c-terminal granulin domain, characteristic of the rd <dig> subfamily, in these cysteine proteases.
glyma04g <dig>  had highest similarity to rdl <dig>  and closely clustered with the rd <dig> subfamily members.
finally, glyma04g <dig> and glyma06g <dig>  were highly similar to members of the sag <dig> subfamily despite absence of the additional c amino acid in the cgccwafs motif.figure  <dig>
mapping of transcribed cysteine proteases to sub-families and functional groups with similarity to the c <dig> cysteine protease papain.
seven proteases with cathepsin-f-like activity  were highly similar to subfamily rd <dig> members.
however, the erfnaq motif  characteristic of the rd <dig> subfamily, was absent.
glyma08g <dig>  which had no significant similarity to any specific subfamily, was closest to the two subfamilies rd <dig> or ctb <dig>  further cysteine proteases with cathepsin-h-like activity included glyma09g <dig>  glyma15g <dig> and glyma17g <dig>  which had high similarity to aalp and alp <dig>  the three proteases also had an n-terminal npir vacuolar targeting signal and other rd <dig> subfamily motifs .
although glyma03g <dig> and glyma19g <dig> had similarity to this subfamily, they contained an ecgie motif in the c terminus, characteristic of subfamily ctb <dig>
cystatin transcription
we then investigated the nodule cystatin and cysteine protease transcriptome at various time points  of soybean nodule development and senescence .
the time point at 4 weeks represents initial nodule development, 8 weeks mature nodules actively fixing nitrogen, and 14 weeks senescing nodules.
after three biological replicates were produced for each time point and pooled, rna was sequenced producing a total of ~ <dig> million paired reads for each time point.
a cystatin, or cysteine protease, was considered transcriptionally active if a fpkm ≥ <dig>  was obtained in any of the three time points  <cit> .
if a cystatin, or cysteine protease, was not transcriptionally active  at all  <dig> of the time points, the cystatin, or cysteine protease, was considered transcriptionally inactive.figure  <dig>
expression changes of cystatins, cysteine proteases and vacuolar processing enzymes.
expression of cystatins   cysteine proteases  and  vacuolar processing enzymes  in  <dig>   <dig> and  <dig> week old nodules expressed as fpkm .
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  in transcription between individual time points.
multi-experiment viewer  software package was applied to graphically represent data  <cit> .
we first compared our fpkm data with previous published data available online at soyseq database  on the soybase website  <cit>  where various tissue types have been analysed 20– <dig> days after inoculation .
transcript abundance estimates from the two studies were directly comparable .
from a total of  <dig> putative soybean cystatins identified with the model i25b cystatin oc-i, only seven cystatins were transcriptionally active in nodules .
glyma13g <dig> and glyma20g <dig> had highest expression after 4 weeks but their expression decreased when nodules aged .
in contrast, transcription of glyma05g <dig>  glyma15g <dig>  and glyma15g <dig> increased in the later stages of nodule development , although none of these cystatins had statistically significant  transcriptional changes.
the two remaining cystatins, glyma13g <dig> and glyma14g <dig>  did either not change  or expression was below, or close to, the detectable threshold level .
we also validated our rnaseq data by quantitative real-time pcr where tested transcripts were selected on the basis of being representative for each investigated gene family.
determination of relative fold-expression of transcripts during development confirmed our rnaseq data indicating the fidelity of our rnaseq analysis approach .figure  <dig>
relative expression measured by quantitative real-time pcr of soybean cysteine proteases, cystatins, leghemoglobin and a vpe at each time point  and corresponding fpkm abundance estimates derived from rna-seq mapping.
cysteine protease transcription
from the initial  <dig> putative cysteine protease sequences homologous to the model c <dig> cysteine protease papain,  <dig> cysteine proteases were transcriptionally active in nodules during at least one time point .
glyma15g <dig>  was the most abundant cysteine protease in 4 weeks old nodules with glyma17g <dig>  the most abundant at 14 weeks.
transcription of the majority of cysteine proteases increased with the onset of senescence, with five cysteine proteases  highly expressed in  <dig> and 8 weeks old nodules.
none of the cysteine protease transcription changed significantly  except glyma06g <dig> transcription, with a very low relative abundance, which changed  due to senescence .
we also investigated vpe protease  transcription .
these proteases resemble mammalian caspases.
vpe transcription significantly increased during nodule senescence and transcription of four sequences  significantly  increased  for glyma14g <dig> and glyma17g <dig>  with glyma17g <dig> having the largest increase in transcription due to senescence .
from the seven vpe gene sequences identified in the genome, only glyma16g <dig> was not transcribed during nodule development.
cystatin inhibition strength and specificity
in a next step, we carried out cysteine protease activity measurements with nodule extracts to determine potency of transcribed cystatins.
fluorometric interaction assays were used with either commercially available cathepsin-l or cathepsin-b as well as isolated nodule protein extracts representing the total proteolytic complement active in nodules.
to establish a preferential binding for each cystatin, we first tested cystatin potency with commercially available enzyme preparations for cathepsin-l and cathepsin-b.
cystatins transcribed in nodules had generally stronger affinity for cathepsin-l than cathepsin-b, with glyma13g <dig> and glyma14g <dig> equally effective in preventing both cathepsin activities .
further, glyma15g <dig> inhibited cathepsin-l, but was unable to inhibit cathepsin-b, even when an inhibitor concentration of 1 mm was used.
in contrast, cystatins not transcriptionally active in nodules showed higher inhibition rates of cathepsin-l, with glyma18g <dig> inhibiting both cathepsin-l and -b. glyma14g04260’s second domain and both domains of glyma14g <dig> were further unable to inhibit cathepsin-b, even at a concentration of 1 mm .table  <dig>
inhibition  of protease activity by actively and non-actively transcribed cystatins during nodule life-span
cystatin
cathepsin l-like activity
cathepsin b-like activity
4 weeks
8 weeks
14 weeks
p ≤  <dig>
4 weeks
8 weeks
14 weeks
p ≤  <dig>
actively transcribed
non-actively transcribed
*a ; *b ; *c ; ni represents inhibition ≥20%; *significant at p ≤ <dig> .
blank values for cathepsin l-like activity and cathepsin b-like activity was  <dig>  ±  <dig>  fu/sec and  <dig>  ±  <dig>  fu/sec, respectively.
the negative control values for cathepsin l-like activity and cathepsin b-like activity was  <dig>  ±  <dig>  fu/sec and  <dig>  ±  <dig>  fu/sec, respectively.
we then tested cystatin potency against various nodule extracts .
we first used the model rice cystatin oc-i as well as the cysteine protease inhibitor e <dig>  oc-i and e <dig> both prevented cathepsin-l-like activity in 4 weeks old nodules but were less efficient against extracts derived from  <dig> and 14 weeks old nodules .
both inhibitors also prevented cathepsin-b-like activity in an extract of 4 weeks old nodules.
we then compared oc-i and e <dig> potency with the potency of various recombinant soybean cystatins either actively transcribed or non-active in nodules .
cystatins tested were generally more active against extracts from younger nodules .
five of the cystatins actively transcribed in nodules blocked cysteine protease activity in nodule extracts.
however, only glyma05g <dig> inhibited cathepsin-l-like activity in nodule extracts from all three time points  and cathepsin-b-like activity in extracts derived from  <dig> and 8 weeks old nodules.
the most potent cystatin among the expressed cystatins was glyma15g <dig> and potency of this cystatin was comparable to oc-i and e <dig> when either cathepsin–l or b activity was measured in an extract derived from 4 weeks old nodules.table  <dig>
expression and inhibitory potency of cystatins against proteases from different aged nodules
cystatin
expression
cat-l inhibition
cat-b inhibition
active
4 weeks
14 weeks
non-active
4 weeks
14 weeks
++ strong, + medium.
low and  no cystatin expression/or activity .
expression indicated as measured fpkm abundances and activity indicated as% inhibition.
finally, we were also interested in testing cystatins not actively transcribed during nodule development.
these cystatins were generally more active against nodule extracts than cystatins actively transcribed in nodules .
all non-transcribed cystatins had potency comparable to oc-i and e <dig> when tested against an extract derived from 4 weeks old nodules.
among them, glyma14g <dig> domain  <dig> and glyma18g <dig> had highest inhibition of all tested cystatins with  <dig> % and 54% inhibition respectively.
three cystatins  inhibited cathepsin-l as well as cathepsin-b like activity in extracts derived from all three time points.
glyma07g <dig> was further the most potent of all tested cystatins against cathepsin-l and b activity in an extract derived from 14 weeks old senescent nodules.
discussion
we identified  <dig> cystatin genes expressed in soybean nodules using rnaseq.
since we carried out macro-dissection of crown nodule tissue, and not micro-dissection with selected tissues, rnaseq data represented transcription profiles of the entire nodule and also contained transcripts from areas surrounding the senescing nodule cortex.
when we compared their transcription with already published rnaseq data from various other tissue types  <cit> , none of the identified nodule cystatins was uniquely transcribed.
several cystatins were further actively transcribed during nodule development and senescence but not exclusively transcribed at a particular time point like senescence.
only glyma05g <dig> was actively transcribed, and also inhibited cathepsin-l-like activity in nodule extracts, at all three selected time points.
the cystatin very likely plays a maintenance role and regulates cysteine protease activity throughout nodule development and senescence.
the other actively transcribed cystatins were only capable of inhibiting specific types of cysteine proteases activity  at specific time points.
cathepsin b is a member of the peptidase c <dig> family and this cysteine protease is required for pcd involved in the plant disease resistance hypersensitive response  <cit> .
transcription of cystatins glyma05g <dig>  glyma15g <dig>  glyma15g <dig> increased by about two-fold during the onset of senescence with concurrent co-induction of several cysteine proteases.
these cystatins very likely regulate proteolysis when nodules senesce and undergo pcd to maintain nitrogen fixation in determinate soybean nodules for as long as possible.
cystatins, which are part of subfamily b of the i <dig> cystatin family, belong to various groups  from to our phylogenetic analysis  <cit> .
however, any classification solely based on phylogenetic analysis might not accurately provide information about a particular function  <cit> .
several nodule cystatins, almost equally transcribed during nodule development and senescence, had high similarity to group a cystatins.
in cereals, group a cystatins, including rice cystatins, efficiently inhibit cathepsin l-like cysteine-proteases and they are preferentially expressed in dry and germinating cereal seeds.
they possibly regulate endogenous enzymes involved in the mobilization of stored proteins upon germination  <cit> .
the nodule group a cystatin cluster also contained two cystatins, glyma13g <dig> and glyma15g <dig>  with a c-terminal extension.
such an extension was also found in glyma05g <dig>  highly similar to group b cystatins.
plant cystatins with a carboxy-terminal extension contain a snsl amino acid motif and inhibit cysteine proteases of the legumain c <dig> family   <cit> .
their consistent transcription during nodule development and increase of transcription found for glyma15g <dig> and glyma05g <dig> when nodules senesce, indicates that they are very likely produced to tightly control cell disruption and activation of any cysteine proteases that may compromise nitrogen fixation.
vpe proteases resemble mammalian caspases and they contribute to the senescence process and pcd by contributing to the collapse of the vacuole membrane with release of proteases into the cell  <cit> .
there is also evidence that vpes play a regulatory role activating pre-proteases by post-translational modification, leading to maturation and proteolytic activity upon removal on the i <dig> inhibitory domain  <cit> .
cysteine proteases, expressed as pre-proteins, consist of an i <dig> inhibitor domain preventing non-specific activity  <cit> .
in our study, transcription of the entire set of nodule vpe cysteine proteases strongly increased coinciding with the progression of senescence.
vpes are therefore predominantly transcribed in senescent nodules and might play an important role in the activation of cysteine proteases.
these activated cysteine proteases finally degrade both the bacteroids and nodule cells  <cit>  and correlates with nitrogenase activity decrease  <cit>  as well as decrease in both crown nodule biomass and nodule number  <cit> .
glyma15g <dig>  identified in the phytozome database, was the most abundant nodule cystatin with similarity to group c <dig> cystatins.
this cystatin was almost four-times higher transcribed than all other actively transcribed cystatins in nodules.
the glyma15g <dig> was identical to the previously described glyma15g <dig>  <cit>  which was found to be highly transcribed both in nodules and in other tissues including seeds.
in cereals, group c <dig> cystatins are preferentially expressed in seeds, particularly in developing endosperms, and are potent inhibitors of c1a peptidases  <cit> .
future research is needed to identify the specific target cysteine proteases and why glyma15g <dig> is preferentially expressed in nodules.
we also identified cystatins not actively transcribed in nodules.
when expressed in vitro, these cystatins had a much broader range of inhibitory activity against the nodule proteolytic complement than actively transcribed cystatins.
they had nearly double the inhibitory capacity towards cathepsin-l-like cysteine protease activity, and also partially towards cathepsin-b-like cysteine protease activity, compared to actively transcribed cystatins.
this might indicate that proteolytic activity should not be compromised by extensive cystatin expression with the onset of senescence and remobilization of nitrogen.
however, these non-actively-transcribed cystatins might also have other specific functions and are only activated under certain biotic and abiotic stress conditions to prevent premature nodule death.
based on our rnaseq data,  <dig> cysteine proteases were actively transcribed in nodules during development and senescence.
identified cysteine proteases were further functionally diverse belonging to different proteolytic sub-families.
transcript abundance of cysteine proteases at early and mature nodule development was relatively constant, with different cysteine proteases contributing toward the overall proteolytic complement .
most of our tested nodule cystatins had preferential affinity to cathepsin l-like cysteine proteases.
with the onset of senescence, however, cysteine protease transcript abundance was nearly doubled and correlated with senescence progression.
however, only transcription of glyma06g <dig>  which was very lowly transcribed, changed significantly due to the onset of senescence.
this cysteine protease is homologous to senescence-related sag <dig> .
however, in a previous comprehensive transcriptomics study in medicago truncatula to understand medicago nodule senescence, four cysteine protease genes highly homologous to sag <dig> were the most abundant  <cit> .
future research has to determine the reason for such transcription difference of sag <dig> homologous cysteine proteases in soybean determinate nodules and medicago indeterminate nodules.
to analyze any endogenous cystatin function in nodules, it is crucial to identify their possible endogenous target cysteine proteases.
only little is so far known about any possible direct interaction between cystatins and their target proteases in vivo  <cit> .
we therefore searched cystatin and cysteine proteases sequences for signal peptides to obtain some information about their cellular localisation.
cystatins glyma05g <dig>  glyma07g <dig> and glyma13g <dig> are likely localised in the secretory pathway.
the er has a vast protein storage protein capacity and relatively low proteolytic activity and cystatins might contribute to low proteolytic activity in this organelle  <cit> .
nodule cystatins, such as glyma05g <dig>  glyma07g <dig> and glyma13g <dig> with signal peptides, would be able to interact with c1a proteases that also have secretion sequences in their gene sequences  <cit> .
mj chrispeels and nv raikhel  <cit>  further predicted that the open reading frame of two cysteine proteases contain a putative vacuolar targeting signal.
we also found such signal for glyma04g <dig> and glyma06g <dig>  despite that glyma06g <dig> was explicitly expressed during nodule senescence which raises the question of the particular glyma06g <dig> function when targeted to the vacuole.
one possible explanation is that this vacuolar targeting signal directs these cysteine proteases to the bacteroid-containing symbiosome compartment for bacteroid protein degradation affecting peribacteriod membrane stability.
rupture of the peribacteriod membrane then eliminates the microbial partner  <cit> .
CONCLUSIONS
in our study the phylogenetic relationship and transcription of individual components of the cysteine protease–cystatin system in soybean nodules during nodule development and senescence were characterised.
several of these components, including legumains, had coordinated transcription during nodule senescence strongly indicating their direct involvement in nodule senescence.
our study has, overall, created new knowledge about types of cystatin and cysteine protease transcribed, timing of coordinated transcription of the two components of the system and inhibitory activity of actively transcribed and non-transcribed cystatins in nodules for more future detailed interaction studies.
application of this knowledge might ultimately allow development of engineering strategies to circumvent particularly stress-induced premature senescence in nodules  <cit> .
the potential of using cystatins to regulate cysteine protease activity and thereby agronomical important traits  was demonstrated in a transgenic soybean line overexpressing a cystatin  <cit> .
prolonging the period of active nitrogen fixation, due to delayed nodule senescence by regulating cysteine protease activity, might provide the benefit of better soybean growth and improving yield  <cit>  and potentially contributing to future food security  <cit> .
however, since still little is currently known about the in vivo interaction between the two components of the system, we are currently determining, as a next step, their interaction by in vitro assays with both purified recombinant cystatin and purified cysteine proteases and investigating their individual localisation by immuno-histochemistry.

 in plants, cystatins are natural and specific inhibitors of cysteine proteases of the papain c1a family that generally block c1a proteases by a tight and reversible interaction. several cystatin functions have been proposed, but all involve a balanced interplay with a cysteine protease to regulate proteolytic activity. research has so far provided strong evidence that plant cystatins regulate endogenous protein turnover during growth and developmental processes, including senescence and programmed cell death, and are further involved in accumulation and mobilization of storage proteins. a further key function is protection against plant pests where cystatins prevent cysteine protease activity required for protein digestion in pests. cysteine protease expression during nodule senescence has been previously reported. proteolytic activity in infected nodules limits the bacterial symbiosis and nitrogen fixation, with cytosolic leghemoglobin and the bacteriod as targets. in medicago trunctula anti-sense inhibition of the cysteine protease cyp15a caused a delay in nodule senescence and nodule lifespan was prolonged, when a nodule-specific papain-like cysteine protease was silenced. however, 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. the most detailed analysis of participation of an endogenous cystatin in interaction with an endogenous cysteine protease during senescence has been the coordinated expression of the mrnas of a cysteine protease and a cystatin in senescent spinach leaves where a senescence-related cysteine protease–cystatin complex was identified. further evidence of the in vivo regulation of cysteine protease have been provided by pillay et al. showing that co-expression of the rice cystatin oci in tobacco plants protected recombinant proteins from degradation by lowering overall cysteine protease activity. the phytozome database currently contains over cystatin-like sequences from the viridiplantae kingdom, c cysteine protease sequences and c cysteine protease sequences. the recent release of the complete soybean genome as well as the release of a rnaseq atlas of genes expressed in fourteen different soybean tissues including nodules has further allowed identification and characterization of all soybean cystatins, irrespective of transcriptional activity, and active cysteine proteases. accurate studies are now possible to determine the cystatin and cysteine protease classes expressed in nodules and also to investigate if endogenous cystatins preferentially interact with specific target cysteine proteases in nodules. our study was therefore aimed to provide a first insight into such interactions by identifying and characterizing all members of the cystatin and cysteine protease gene families in soybean nodules. we included both actively and non-actively transcribed cystatins and cysteine proteases identified through homology searches in the soybean genomic database. the nodule transcription profiles were developed with the technique of rnaseq which allowed us to determine the expression of all oryzacystatin i-like cystatins, papain-like cysteine proteases, as well as vacuole vpe-type cysteine proteases in determinate soybean crown nodules during nodule development and senescence. such vpe cysteine proteases resemble mammalian caspases and they contribute to the senescence process and pcd, but might further activate pre-proteases by post-translational modification. in our characterization, we were also interested to determine to which families and functional groups nodule cystatins and cysteine proteases belong as well as the cystatin substrate preference by testing in vitro produced cystatin proteins with various cysteine protease-containing extracts. cystatins are part of subfamily b of the i cystatin family and in cereals they can be divided into various functional groups with most cystatins belonging to groups a and c. group a cystatins, which efficiently inhibit cathepsin l-like cysteine-proteases, are preferentially expressed in dry and germinating seeds whereas group c cystatins, which are potent inhibitors of c1a peptidases, are mostly expressed in developing seed endosperms. cysteine proteases cluster into different subfamilies with cysteine proteases closest to papain clustering with subfamily xcp represented by the arabidopsis thaliana genes at1g and at4g cysteine proteases with cathepsin-l-like activity can closely cluster with subfamily rd consisting of rd21a, rd21b and rd21c. a c-terminal granulin domain is characteristic of the rd subfamily. cysteine proteases with cathepsin-l-like activity can further cluster with the sag subfamily. cysteine proteases with cathepsin-f-like activity cluster with subfamily rd with members rd19a, rd19b and rd19c and rd members have a characteristic erfnaq motif in the pro-domain. cysteine proteases with cathepsin-h-like activity cluster with members of the aalp and alp subfamily. we were finally also interested to determine the interaction affinities between selected actively and non-actively transcribed cystatins during nodule development and senescence. this should provide information about the relative activities and specificities of both expressed and non-expressed cystatin genes in soybean. in our study, we found an overlap in the activities and specificities of the expressed and non-expressed cystatin genes raising the question of whether the non-transcribed cystatins provide a reservoir for responses to particular environments.  cystatin identification all expressed nodule cystatins were identified from our rnaseq data. when the oryzacystatin-i was used for comparison as a model cystatin, cystatin sequences were identified in the assembled genome; of these were non-redundant sequences. when we carried out a phylogenetic genetic analysis of cystatins by comparison with cystatins from different i cystatin subfamilies, glyma13g and glyma20g transcribed in nodules during nodule development and senescence, had high similarity to group a cystatins. glyma13g was further paralogous to glyma14g with identical location, but on a different chromosome. also, the two cystatins glyma13g and glyma15g were highly similar to cystatin b and hvcpi- and glyma05g was further highly similar to group b cystatins, hvcpi- and hvcpi-9). they also contained a c-terminal extension with a snsl amino acid motif enabling them to inhibit legumain c cysteine proteases. finally, glyma15g which was the most abundant cystatin in nodules, was similar to group c cystatins, hvcpi- and hvcpi-8).figure mapping of transcribed soybean nodule cystatins to different i cystatin subfamilies. we also searched all cystatin sequences for signal peptides indicating their possible cellular localisation. glyma05g glyma07g and glyma13g might be localised in the secretory pathway, whereas glyma13g glyma14g and glyma20g are localised to any location, except the chloroplast, mitochondrion or secretory pathway. localisation of glyma15g was not reliable and the cystatin could be located in either the mitochondrion or the secretory pathway. cysteine protease identification a total of cysteine protease sequences with homology to the model cysteine protease papain were further identified from the soybean genome assembly. several sequences were alleles, paralogos and orthologos of other cysteine protease gene sequences. from these we identified non-redundant cysteine protease gene sequences which had similarity to members of eight different cysteine protease sub-families. seven sub-families were distinguished from our expression data and we identified confidently five functional groups. however, none of the identified soybean cysteine proteases clustered with papain. cysteine proteases with cathepsin-l-like activity included glyma04g, as well as the two proteases glyma14g and glyma17g. we also confirmed the c-terminal granulin domain, characteristic of the rd subfamily, in these cysteine proteases. glyma04g had highest similarity to rdl and closely clustered with the rd subfamily members. finally, glyma04g and glyma06g were highly similar to members of the sag subfamily despite absence of the additional c amino acid in the cgccwafs motif.figure mapping of transcribed cysteine proteases to sub-families and functional groups with similarity to the c cysteine protease papain. seven proteases with cathepsin-f-like activity were highly similar to subfamily rd members. however, the erfnaq motif characteristic of the rd subfamily, was absent. glyma08g which had no significant similarity to any specific subfamily, was closest to the two subfamilies rd or ctb further cysteine proteases with cathepsin-h-like activity included glyma09g glyma15g and glyma17g which had high similarity to aalp and alp the three proteases also had an n-terminal npir vacuolar targeting signal and other rd subfamily motifs. although glyma03g and glyma19g had similarity to this subfamily, they contained an ecgie motif in the c terminus, characteristic of subfamily ctb cystatin transcription we then investigated the nodule cystatin and cysteine protease transcriptome at various time points of soybean nodule development and senescence. the time point at 4 weeks represents initial nodule development, 8 weeks mature nodules actively fixing nitrogen, and 14 weeks senescing nodules. after three biological replicates were produced for each time point and pooled, rna was sequenced producing a total of ~ million paired reads for each time point. a cystatin, or cysteine protease, was considered transcriptionally active if a fpkm ≥ was obtained in any of the three time points. if a cystatin, or cysteine protease, was not transcriptionally active at all of the time points, the cystatin, or cysteine protease, was considered transcriptionally inactive.figure expression changes of cystatins, cysteine proteases and vacuolar processing enzymes. expression of cystatins cysteine proteases and vacuolar processing enzymes in and week old nodules expressed as fpkm. 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 in transcription between individual time points. multi-experiment viewer software package was applied to graphically represent data. we first compared our fpkm data with previous published data available online at soyseq database on the soybase website where various tissue types have been analysed 20– days after inoculation. transcript abundance estimates from the two studies were directly comparable. from a total of putative soybean cystatins identified with the model i25b cystatin oc-i, only seven cystatins were transcriptionally active in nodules. glyma13g and glyma20g had highest expression after 4 weeks but their expression decreased when nodules aged. in contrast, transcription of glyma05g glyma15g and glyma15g increased in the later stages of nodule development, although none of these cystatins had statistically significant transcriptional changes. the two remaining cystatins, glyma13g and glyma14g did either not change or expression was below, or close to, the detectable threshold level. we also validated our rnaseq data by quantitative real-time pcr where tested transcripts were selected on the basis of being representative for each investigated gene family. determination of relative fold-expression of transcripts during development confirmed our rnaseq data indicating the fidelity of our rnaseq analysis approach.figure relative expression measured by quantitative real-time pcr of soybean cysteine proteases, cystatins, leghemoglobin and a vpe at each time point and corresponding fpkm abundance estimates derived from rna-seq mapping. cysteine protease transcription from the initial putative cysteine protease sequences homologous to the model c cysteine protease papain, cysteine proteases were transcriptionally active in nodules during at least one time point. glyma15g was the most abundant cysteine protease in 4 weeks old nodules with glyma17g the most abundant at 14 weeks. transcription of the majority of cysteine proteases increased with the onset of senescence, with five cysteine proteases highly expressed in and 8 weeks old nodules. none of the cysteine protease transcription changed significantly except glyma06g transcription, with a very low relative abundance, which changed due to senescence. we also investigated vpe protease transcription. these proteases resemble mammalian caspases. vpe transcription significantly increased during nodule senescence and transcription of four sequences significantly increased for glyma14g and glyma17g with glyma17g having the largest increase in transcription due to senescence. from the seven vpe gene sequences identified in the genome, only glyma16g was not transcribed during nodule development. cystatin inhibition strength and specificity in a next step, we carried out cysteine protease activity measurements with nodule extracts to determine potency of transcribed cystatins. fluorometric interaction assays were used with either commercially available cathepsin-l or cathepsin-b as well as isolated nodule protein extracts representing the total proteolytic complement active in nodules. to establish a preferential binding for each cystatin, we first tested cystatin potency with commercially available enzyme preparations for cathepsin-l and cathepsin-b. cystatins transcribed in nodules had generally stronger affinity for cathepsin-l than cathepsin-b, with glyma13g and glyma14g equally effective in preventing both cathepsin activities. further, glyma15g inhibited cathepsin-l, but was unable to inhibit cathepsin-b, even when an inhibitor concentration of 1 mm was used. in contrast, cystatins not transcriptionally active in nodules showed higher inhibition rates of cathepsin-l, with glyma18g inhibiting both cathepsin-l and -b. glyma14g04260’s second domain and both domains of glyma14g were further unable to inhibit cathepsin-b, even at a concentration of 1 mm.table inhibition of protease activity by actively and non-actively transcribed cystatins during nodule life-span cystatin cathepsin l-like activity cathepsin b-like activity 4 weeks 8 weeks 14 weeks p ≤  4 weeks 8 weeks 14 weeks p ≤  actively transcribed non-actively transcribed *a; *b; *c; ni represents inhibition ≥20%; *significant at p ≤. blank values for cathepsin l-like activity and cathepsin b-like activity was  ±  fu/sec and  ±  fu/sec, respectively. the negative control values for cathepsin l-like activity and cathepsin b-like activity was  ±  fu/sec and  ±  fu/sec, respectively. we then tested cystatin potency against various nodule extracts. we first used the model rice cystatin oc-i as well as the cysteine protease inhibitor e oc-i and e both prevented cathepsin-l-like activity in 4 weeks old nodules but were less efficient against extracts derived from and 14 weeks old nodules. both inhibitors also prevented cathepsin-b-like activity in an extract of 4 weeks old nodules. we then compared oc-i and e potency with the potency of various recombinant soybean cystatins either actively transcribed or non-active in nodules. cystatins tested were generally more active against extracts from younger nodules. five of the cystatins actively transcribed in nodules blocked cysteine protease activity in nodule extracts. however, only glyma05g inhibited cathepsin-l-like activity in nodule extracts from all three time points and cathepsin-b-like activity in extracts derived from and 8 weeks old nodules. the most potent cystatin among the expressed cystatins was glyma15g and potency of this cystatin was comparable to oc-i and e when either cathepsin–l or b activity was measured in an extract derived from 4 weeks old nodules.table expression and inhibitory potency of cystatins against proteases from different aged nodules cystatin expression cat-l inhibition cat-b inhibition active 4 weeks 14 weeks non-active 4 weeks 14 weeks ++ strong, + medium. low and no cystatin expression/or activity. expression indicated as measured fpkm abundances and activity indicated as% inhibition. finally, we were also interested in testing cystatins not actively transcribed during nodule development. these cystatins were generally more active against nodule extracts than cystatins actively transcribed in nodules. all non-transcribed cystatins had potency comparable to oc-i and e when tested against an extract derived from 4 weeks old nodules. among them, glyma14g domain and glyma18g had highest inhibition of all tested cystatins with % and 54% inhibition respectively. three cystatins inhibited cathepsin-l as well as cathepsin-b like activity in extracts derived from all three time points. glyma07g was further the most potent of all tested cystatins against cathepsin-l and b activity in an extract derived from 14 weeks old senescent nodules. discussion we identified cystatin genes expressed in soybean nodules using rnaseq. since we carried out macro-dissection of crown nodule tissue, and not micro-dissection with selected tissues, rnaseq data represented transcription profiles of the entire nodule and also contained transcripts from areas surrounding the senescing nodule cortex. when we compared their transcription with already published rnaseq data from various other tissue types, none of the identified nodule cystatins was uniquely transcribed. several cystatins were further actively transcribed during nodule development and senescence but not exclusively transcribed at a particular time point like senescence. only glyma05g was actively transcribed, and also inhibited cathepsin-l-like activity in nodule extracts, at all three selected time points. the cystatin very likely plays a maintenance role and regulates cysteine protease activity throughout nodule development and senescence. the other actively transcribed cystatins were only capable of inhibiting specific types of cysteine proteases activity at specific time points. cathepsin b is a member of the peptidase c family and this cysteine protease is required for pcd involved in the plant disease resistance hypersensitive response. transcription of cystatins glyma05g glyma15g glyma15g increased by about two-fold during the onset of senescence with concurrent co-induction of several cysteine proteases. these cystatins very likely regulate proteolysis when nodules senesce and undergo pcd to maintain nitrogen fixation in determinate soybean nodules for as long as possible. cystatins, which are part of subfamily b of the i cystatin family, belong to various groups from to our phylogenetic analysis. however, any classification solely based on phylogenetic analysis might not accurately provide information about a particular function. several nodule cystatins, almost equally transcribed during nodule development and senescence, had high similarity to group a cystatins. in cereals, group a cystatins, including rice cystatins, efficiently inhibit cathepsin l-like cysteine-proteases and they are preferentially expressed in dry and germinating cereal seeds. they possibly regulate endogenous enzymes involved in the mobilization of stored proteins upon germination. the nodule group a cystatin cluster also contained two cystatins, glyma13g and glyma15g with a c-terminal extension. such an extension was also found in glyma05g highly similar to group b cystatins. plant cystatins with a carboxy-terminal extension contain a snsl amino acid motif and inhibit cysteine proteases of the legumain c family. their consistent transcription during nodule development and increase of transcription found for glyma15g and glyma05g when nodules senesce, indicates that they are very likely produced to tightly control cell disruption and activation of any cysteine proteases that may compromise nitrogen fixation. vpe proteases resemble mammalian caspases and they contribute to the senescence process and pcd by contributing to the collapse of the vacuole membrane with release of proteases into the cell. there is also evidence that vpes play a regulatory role activating pre-proteases by post-translational modification, leading to maturation and proteolytic activity upon removal on the i inhibitory domain. cysteine proteases, expressed as pre-proteins, consist of an i inhibitor domain preventing non-specific activity. in our study, transcription of the entire set of nodule vpe cysteine proteases strongly increased coinciding with the progression of senescence. vpes are therefore predominantly transcribed in senescent nodules and might play an important role in the activation of cysteine proteases. these activated cysteine proteases finally degrade both the bacteroids and nodule cells and correlates with nitrogenase activity decrease as well as decrease in both crown nodule biomass and nodule number. glyma15g identified in the phytozome database, was the most abundant nodule cystatin with similarity to group c cystatins. this cystatin was almost four-times higher transcribed than all other actively transcribed cystatins in nodules. the glyma15g was identical to the previously described glyma15g which was found to be highly transcribed both in nodules and in other tissues including seeds. in cereals, group c cystatins are preferentially expressed in seeds, particularly in developing endosperms, and are potent inhibitors of c1a peptidases. future research is needed to identify the specific target cysteine proteases and why glyma15g is preferentially expressed in nodules. we also identified cystatins not actively transcribed in nodules. when expressed in vitro, these cystatins had a much broader range of inhibitory activity against the nodule proteolytic complement than actively transcribed cystatins. they had nearly double the inhibitory capacity towards cathepsin-l-like cysteine protease activity, and also partially towards cathepsin-b-like cysteine protease activity, compared to actively transcribed cystatins. this might indicate that proteolytic activity should not be compromised by extensive cystatin expression with the onset of senescence and remobilization of nitrogen. however, these non-actively-transcribed cystatins might also have other specific functions and are only activated under certain biotic and abiotic stress conditions to prevent premature nodule death. based on our rnaseq data, cysteine proteases were actively transcribed in nodules during development and senescence. identified cysteine proteases were further functionally diverse belonging to different proteolytic sub-families. transcript abundance of cysteine proteases at early and mature nodule development was relatively constant, with different cysteine proteases contributing toward the overall proteolytic complement. most of our tested nodule cystatins had preferential affinity to cathepsin l-like cysteine proteases. with the onset of senescence, however, cysteine protease transcript abundance was nearly doubled and correlated with senescence progression. however, only transcription of glyma06g which was very lowly transcribed, changed significantly due to the onset of senescence. this cysteine protease is homologous to senescence-related sag. however, in a previous comprehensive transcriptomics study in medicago truncatula to understand medicago nodule senescence, four cysteine protease genes highly homologous to sag were the most abundant. future research has to determine the reason for such transcription difference of sag homologous cysteine proteases in soybean determinate nodules and medicago indeterminate nodules. to analyze any endogenous cystatin function in nodules, it is crucial to identify their possible endogenous target cysteine proteases. only little is so far known about any possible direct interaction between cystatins and their target proteases in vivo. we therefore searched cystatin and cysteine proteases sequences for signal peptides to obtain some information about their cellular localisation. cystatins glyma05g glyma07g and glyma13g are likely localised in the secretory pathway. the er has a vast protein storage protein capacity and relatively low proteolytic activity and cystatins might contribute to low proteolytic activity in this organelle. nodule cystatins, such as glyma05g glyma07g and glyma13g with signal peptides, would be able to interact with c1a proteases that also have secretion sequences in their gene sequences. mj chrispeels and nv raikhel further predicted that the open reading frame of two cysteine proteases contain a putative vacuolar targeting signal. we also found such signal for glyma04g and glyma06g despite that glyma06g was explicitly expressed during nodule senescence which raises the question of the particular glyma06g function when targeted to the vacuole. one possible explanation is that this vacuolar targeting signal directs these cysteine proteases to the bacteroid-containing symbiosome compartment for bacteroid protein degradation affecting peribacteriod membrane stability. rupture of the peribacteriod membrane then eliminates the microbial partner.  in our study the phylogenetic relationship and transcription of individual components of the cysteine protease–cystatin system in soybean nodules during nodule development and senescence were characterised. several of these components, including legumains, had coordinated transcription during nodule senescence strongly indicating their direct involvement in nodule senescence. our study has, overall, created new knowledge about types of cystatin and cysteine protease transcribed, timing of coordinated transcription of the two components of the system and inhibitory activity of actively transcribed and non-transcribed cystatins in nodules for more future detailed interaction studies. application of this knowledge might ultimately allow development of engineering strategies to circumvent particularly stress-induced premature senescence in nodules. the potential of using cystatins to regulate cysteine protease activity and thereby agronomical important traits was demonstrated in a transgenic soybean line overexpressing a cystatin. prolonging the period of active nitrogen fixation, due to delayed nodule senescence by regulating cysteine protease activity, might provide the benefit of better soybean growth and improving yield and potentially contributing to future food security. however, since still little is currently known about the in vivo interaction between the two components of the system, we are currently determining, as a next step, their interaction by in vitro assays with both purified recombinant cystatin and purified cysteine proteases and investigating their individual localisation by immuno-histochemistry. 
nodules play an important role in fixing atmospheric nitrogen for soybean growth. seventy nine non-redundant cysteine protease gene sequences with homology to papain, belonging to different subfamilies, and several legumain-like cysteine proteases 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. transcription of cystatins glyma05g glyma15g glyma15g particularly increased during onset of senescence, possibly regulating proteolysis when nodules senesce and undergo programmed cell death. cystatincysteine proteaseprogrammed cell deathrnaseqsenescencesoybeansymbiotic nitrogen fixationtranscriptomeissue-copyright-statement© the author 2014 cysteine proteases are known to play a role in nodule senescence, but knowledge is still fragmented regarding the function their inhibitors during the development and senescence of soybean nodules. 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. 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. most cystatins had preferential affinity to cathepsin l-like cysteine proteases. this study provides the first data with regard to cystatin expression during nodule development combined with biochemical characterization of their inhibition strength. 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.
 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 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.  seventy nine non-redundant cysteine protease gene sequences with homology to papain, belonging to different subfamilies, and several legumain-like cysteine proteases 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 glyma05g glyma15g glyma15g 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.  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. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords cystatincysteine proteaseprogrammed cell deathrnaseqsenescencesoybeansymbiotic nitrogen fixationtranscriptomeissue-copyright-statement© the author 2014 