Regulation of the chloroplastic copper chaperone (CCS) and cuprozinc superoxide dismutase (CSD2) by alternative splicing and copper excess in Glycine max

Metal homeostasis is an important aspect of plant physiology, and the copper transport into the chloroplast and its fate after delivery is of special relevance for plants. In this work, the regulation of the chloroplastic copper chaperone for the cuprozinc superoxide dismutase (GmCCS) and its target, the cuprozinc superoxide dismutase (GmCSD2), was investigated in photosynthetic cell suspensions and entire plants from Glycine max (L.) Merr. Both genes were expressed in cell suspensions and in all plant tissues analysed, and their RNAs matured by alternative splicing with intron retention (IntronR). This mechanism generated a spliced and three non-spliced mRNAs in the case of GmCCS but only a spliced and a non-spliced mRNAs in GmCSD2. Copper excess strongly upregulated the expression of both fully spliced mRNAs but mostly unaffected the non-spliced forms. In entire plants, some tissue specificity was also observed depending on copper content status. At the protein level, the GmCCS was mostly unaffected but the GmCSD2 was strongly induced under copper excess in all subcellular fractions analysed, suggesting a post-transcriptional regulation for the former. This different protein regulation of the chaperone and its target may indicate some additional function for the CSD2 protein. In addition to its well-known superoxide dismutase (SOD) activity, it may also function as a metal sink in copper excess availability to avoid metal cell damage. Furthermore, the GmCCS seems to be present in the stroma only but the GmCSD2 was present in both stroma and thylakoids despite the general idea that the SOD enzymes are typically soluble stroma proteins. The presence of the SOD enzyme on the surface of the thylakoid membranes is reasonable considering that the superoxide radical (O2-) is preferentially formed at the acceptor side of the PSI.