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  • Furthermore we found that expression of two C

    2020-01-14

    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. C13 cysteine proteases or VPEs, which are similar to mammalian caspases, are involved in senescence as well as programmed cell death. Very little is currently known about the exact function of specific VPEs. Previous research has shown that they are responsible for the collapse of the vacuole membrane which leads to the release of different enzymes, including proteases, into the K03861 (Hara-Nishimura et al., 2005). VPEs are also responsible for activation of cysteine proteases due to their ability to remove the I19 inhibitory domain of pre-proteases (Roberts et al., 2012). Results from a study investigating the processing of pro2S albumins by VPEs in seeds further indicated that vegetative VPEs, α-VPE and γ-VPE, are not directly required for precursor processing in the presence of β-VPE, but partly compensate for any lack in β-VPE activity (Shimada et al., 2003). Interestingly, whereas the α-type VPE Glyma.17G230700 was highly expressed in our study under drought and during senescence, Glyma.14G085800, β-type VPE Glyma.05G055700 was expressed under natural senescence and drought but only highly expressed under drought. The α-type VPE Glyma.17G230700 is quite distinct from other VPEs. This VPE lacks a GmNAC81/GmNAC30 binding site transducing a cell death signal (Mendes et al., 2013) and the VPE Glyma.17G230700 function is possibly in controlling protein formation and cysteine protease activity. We draw this conclusion from our results of characterizing an α-type VPE Arabidopsis mutant. Arabidopsis plants contain an α-type VPE which has similarity with the soybean α-type VPE Glyma.17G230700. Arabidopsis mutant plants had a higher protein content associated with lower cysteine protease activity. Lower cysteine protease activity in the α-VPE Arabidopsis mutant was possibly caused by lower α-VPE-dependant cysteine protease maturation. Reduced cysteine protease activity very likely resulted in less cellular proteolysis and consequently more protein and plant biomass formation. Arabidopsis plants were stressed with PEG 8000 to mimic osmotic stress conditions by inducing low water potential similar to the vermiculite drying system (Sharp et al., 2004). The decrease in water availability caused by PEG-mediated treatment was efficient for the biochemical analysis due to a constant water potential and low transpiration rate over all plates which makes drought avoidance improbable (Verslues and Bray, 2004). While there is much debate with regards to the use of PEG in osmotic or drought stress studies, our main aim was to induce a senescence proses and measure VPE-dependant cysteine protease activity and not drought stress specific responses. Our study also provided a first insight with regards to the up-regulation of nodule genes following drought treatment. These genes might be candidates for future more detailed investigations. The most prominent drought-induced up-regulated gene, based on our RNA-seq analysis, was a gene for a defensin-like protein (Glyma.13G35320), which is normally expressed in soybean flowers and roots, but only lowly expressed in nodules (Severin et al., 2010). This gene transcribes small cysteine-rich compounds (Lay and Anderson, 2007). Most plant genomes consist of up to 300 defensin genes, which are induced by pathogen inoculation or environmental stresses (Graham et al., 2007). Another strongly up-regulated nodule gene was a LEA-D11 protein (Glyma.05G112000). LEA (Late Embryogenesis Abundant proteins) genes are divided into six groups and D11-LEA group 2 proteins, also called dehydrins, which are plant-specific proteins. Soybean varieties differ in their LEA-D11 sequence. Such sequence variation was found when leaves of different drought-sensitive and drought-tolerant soybean varieties were compared (Savitri et al., 2013). These LEA proteins, produced to survive stressful environments like plant growth under low temperature or drought, possibly stabilize membranes, proteins or other cellular structures under stress (Eriksson and Harryson, 2011).