br Discussion This study is the first analysis

Discussion This study is the first analysis of the transcriptome of drought-stressed soybean crown nodules. Previously, only the transcriptome of developing non-stressed crown nodules has been investigated (van Wyk et al., 2014). In our study drought stress decreased soybean plant growth but resulted in larger root weight (Appendix Table 3A) which has previously also been reported (Fenta et al., 2011). As the roots, due to drought stress, become deeper and denser, the what is c2 increases but negatively affected the formation of root nodules and nodule activity. Following drought exposure, nodule formation terminated and nodules were inactivated, resulting in a colour change in nodule tissue from red to greenish (Puppo et al., 2005; Fenta et al., 2011 and Fenta et al., 2014). Further, the nodule water potential decreased due to drought. A low water potential negatively affects nodule respiratory capacity resulting in a decline in nodule permeability (Purcell and Sinclair, 1995). When cell turgor is lost in the nodule cortex, the microbial partner is eliminated due to limited O2 diffusion (Guerin et al., 1990). The induction of changes in expression of a high number of genes under severe drought conditions has recently been reported for soybean roots (Song et al., 2016). In this study, drought also induced higher expression of nodule genes including various cysteine proteases. The majority of cysteine proteases expressed in nodules belonged to the C1 cysteine protease (papain-like) family. Contribution of C1 cysteine proteases to the total cysteine protease transcriptome further changed when a more severe drought treatment was applied (30% mWHC). C1 cysteine proteases represent about 50% of all cysteine proteases in the soybean transcriptome (Severin et al., 2010). Only the expression of one C1 cysteine protease Glyma.14G085800 and of two VPEs, Glyma.17G230700 and Glyma.05G055700, were highly up-regulated under our most severe drought condition (30% mWHC). However, although expressed in non-stressed nodules, expression of the C1 cysteine protease Glyma.14G085800 did not increase in our study when nodules senesced. The C1 cysteine protease Glyma.14G085800 is also highly expressed in all other investigated soybean tissues (Severin et al., 2010; van Wyk et al., 2014) and is also responsive to phosphorus deficiency (Sha et al., 2016). Furthermore we found that expression of two C1 nodule cysteine proteases, Glyma.06G283100 and Glyma.06G174800, changed very little under drought despite the two proteases having similarity to the Arabidopsis senescence-related SAG12 gene with 62% similarity by Glyma.06G283010 and 58% by Glyma.06G174800. The senescence-specific cysteine protease SAG12 is involved in developmental senescence specific cell death and does not accumulate, for example, until a leaf develops chlorosis (Weaver et al., 1998; Gepstein et al., 2003). However, except for some very low expression due to drought at 40% mWHC, both were not prominently expressed in our study under drought conditions. The C1 protease, Glyma.08G116900, was the only C1 cysteine protease which decreased in our study due to drought exposure. However, since Glyma.08G116900 expression also decreases in older nodules, this protease might have more a function in earlier plant development and not in senescence or in response to drought induced senescence.