Subfamily RD21 members). We also confirmed the C-terminal CCR9 Purity & Documentation 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 together with the RD21 subfamily members. Ultimately, Glyma04g36470 and Glyma06g18390 (cathepsinL-like activity) were very equivalent to members with the SAG12 subfamily in spite of absence of your further C amino acid inside the CGCCWAFS motif. Seven proteases with cathepsin-F-like activity (Glyma04g03020, Glyma06g03050, Glyma10g35100, Glyma11g12130, Glyma12g04340, Glyma14g40670, Glyma17g37400) were extremely related to subfamily RD19 members. On the other hand, the ERFNAQ motif (as an alternative of the ERFNIN motif in the pro-domain) characteristic from the RD19 subfamily, was absent. Glyma08g12340, which had no substantial similarity to any particular subfamily, was closest to the two subfamilies RD19 or CTB3. Additional cysteine proteases with cathepsin-H-like activity included Glyma09g08100, Glyma15g19580 and Glyma17g05670, which had higher similarity to AALP and ALP2. The 3 proteases also had an N-terminal NPIR vacuolar targeting signal andvan Wyk et al. BMC Plant Biology 2014, 14:294 http:biomedcentral1471-222914Page four ofSAG12 XCPRDRD21 XBCP3 AALPFigure 2 Mapping of transcribed cysteine proteases to sub-families and functional groups with similarity to the C1 cysteine protease papain.other RD21 subfamily motifs (except that the ATC motif was lacking in Glyma09g08100). Even though Glyma03g38520 and Glyma19g41120 had similarity to this subfamily, they contained an ECGIE motif in 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, 8 weeks mature nodules actively fixing nitrogen, and 14 weeks senescing nodules. Following 3 biological replicates have been produced for every time point and pooled, RNA was sequenced producing a total of 40 million paired reads for every single time point. A cystatin, or cysteine protease, was deemed transcriptionally active if a FPKM five.0 was obtained in any with the three time points [23]. If a cystatin, or cysteine protease, was not transcriptionally active (FPKM five) at all 3 of the time points, the cystatin, or cysteine protease, was considered transcriptionally inactive.We 1st compared our FPKM data with prior published data offered on the web at SoySeq database (http: soybase.orgsoyseq) around the SoyBase site [16] exactly where several tissue sorts have already been analysed 205 days following inoculation (comparable to our 4 weeks information). Transcript abundance estimates in the two research were 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 immediately after four weeks but their expression decreased when nodules aged (Figure 3A). In contrast, transcription of Glyma05g28250, Glyma15g12211 (the most abundant cystatin) and Glyma15g36180 enhanced within the later stages of nodule improvement (Figure 3A), despite the fact that none of those cystatins had statistically considerable (p 0.05) transcriptional changes. The two remaining cystatins, Glyma13g25870 and GlyT2 Species Glyma14g04250, did either no.
