Subfamily RD21 members). We also confirmed the C-terminal granulin domain, characteristicSubfamily RD21 members). We also

Subfamily RD21 members). We also confirmed the C-terminal granulin domain, characteristic
Subfamily RD21 members). We also confirmed the C-terminal granulin domain, characteristic on the RD21 subfamily, in these cysteine proteases. Glyma04g04400 (cathepsin-L-like activity) had highest similarity to RDL2 (Arabidopsis gene At3g19400) and closely clustered with the RD21 subfamily members. Ultimately, Glyma04g36470 and Glyma06g18390 (cathepsinL-like activity) had been extremely similar to members in the SAG12 subfamily despite absence of the extra C amino acid within the CGCCWAFS motif. Seven proteases with cathepsin-F-like activity (Glyma04g03020, Glyma06g03050, Glyma10g35100, Glyma11g12130, Glyma12g04340, Glyma14g40670, Glyma17g37400) were highly related to subfamily RD19 members. Having said that, the ERFNAQ motif (as an alternative with the ERFNIN motif inside the pro-domain) characteristic in the RD19 subfamily, was absent. Glyma08g12340, which had no important similarity to any certain subfamily, was closest for the two subfamilies RD19 or CTB3. Further cysteine proteases with cathepsin-H-like activity integrated Glyma09g08100, Glyma15g19580 and Glyma17g05670, which had high similarity to AALP and ALP2. The three proteases also had an N-terminal NPIR vacuolar targeting signal andvan Wyk et al. BMC Plant Biology 2014, 14:294 http:biomedcentral1471-222914Page 4 ofSAG12 XCPRDRD21 XBCP3 AALPFigure 2 Mapping of transcribed cysteine proteases to sub-families and functional groups with similarity towards the C1 cysteine protease papain.other RD21 subfamily motifs (except that the ATC motif was lacking in Glyma09g08100). While Glyma03g38520 and Glyma19g41120 had similarity to this subfamily, they contained an ECGIE motif within the C terminus, characteristic of subfamily CTB3.Cystatin transcriptionWe then investigated the nodule cystatin and cysteine protease transcriptome at several time points (four, eight and 14 weeks) of soybean nodule improvement and senescence (Figure 3). The time point at 4 weeks represents initial nodule improvement, eight weeks mature nodules actively fixing nitrogen, and 14 weeks senescing nodules. Just after three biological ACAT Accession replicates were made for every single time point and pooled, RNA was sequenced generating a total of 40 million paired reads for each and every time point. A cystatin, or cysteine protease, was considered CDK3 manufacturer transcriptionally active if a FPKM 5.0 was obtained in any of your 3 time points [23]. If a cystatin, or cysteine protease, was not transcriptionally active (FPKM 5) at all 3 in the time points, the cystatin, or cysteine protease, was regarded transcriptionally inactive.We initial compared our FPKM data with prior published information obtainable online at SoySeq database (http: soybase.orgsoyseq) on the SoyBase site [16] exactly where different tissue forms happen to be analysed 205 days just after inoculation (comparable to our four weeks information). Transcript abundance estimates from the two research had been directly comparable (information not shown). From a total of 20 putative soybean cystatins identified together with the model I25B cystatin OC-I, only seven cystatins had been transcriptionally active in nodules (Figure 3A). Glyma13g04250 and Glyma20g08800 had highest expression soon after 4 weeks but their expression decreased when nodules aged (Figure 3A). In contrast, transcription of Glyma05g28250, Glyma15g12211 (the most abundant cystatin) and Glyma15g36180 improved inside the later stages of nodule development (Figure 3A), though none of these cystatins had statistically substantial (p 0.05) transcriptional changes. The two remaining cystatins, Glyma13g25870 and Glyma14g04250, did either no.