Involvement of MicroRNA in Copper Deficiency-Induced Repression of Chloroplastic CuZn-Superoxide Dismutase Genes in the Moss Physcomitrella patens

Abstract

Superoxide dismutases (SODs) are metallo-enzymes that catalyze the dismutation of superoxide radicals. In Arabidopsis thaliana, the expression of CuZn-SOD in both the chloroplast and cytosol was reported to be down-regulated by microRNA398 (miR398) during growth on low copper. The moss Physcomitrella patens contains chloroplastic and cytosolic CuZn-SOD genes, but lacks miR398. From analysis of P. patens microRNA, miR1073 was predicted to target CuZn-SOD mRNAs. We noticed that two chloroplastic CuZn-SOD genes contain the miR1073 target sequence in the 3' untranslated region; however, the cytosolic isozyme genes lack this sequence. In this study, we investigated the involvement of miR1073 in the expression of CuZn-SOD genes in P. patens. When protonemata of P. patens were cultured on a copper-depleted medium, SOD activity and mRNA levels of chloroplastic CuZn-SODs were decreased markedly. In contrast, cytosolic CuZn-SODs showed little or no change in mRNA levels or SOD activity. The precursor transcript and the mature form of miR1073 were induced by copper deficiency. The chloroplastic CuZn-SOD (PpCSD1) mRNA was cleaved at the miR1073 target site under copper deficiency. These results suggest that miR1073 is involved in the down-regulation of PpCSD1 expression. In addition to PpCSD1 mRNA, antisense RNAs of PpCSD1 were also detected under normal conditions; however, under copper deficiency, they were cleaved within the open reading frame (ORF) region. The cleavage of sense PpCSD1 mRNA was also detected within the ORF region. Although only miR1073 exists in the database, it is presumed that RNA cleavage, other than that mediated by miR1073, is involved in the regulation of PpCSD1 expression.

Maintenance of Normal Stress Tolerance in the Moss Physcomitrella patens Lacking Chloroplastic CuZn-Superoxide Dismutase

“…In contrast to these examples, copper deficiency in P. patens did not induce Fe-SOD activity and led to an abrupt reduction of the chloroplast SOD level. Therefore, it was expected that the P. patens chloroplasts are sensitive to various stressors under copper deficient conditions [8]. In our preliminary experiments, P. patens protonemata grown on copper-deficient medium showed the similar tolerance to high-light stress.…”

“…Since chloroplasts are subjected to oxidative stresses under these stress conditions, it was expected that the moss chloroplasts would have a high stress scavenging system. Recently, we analyzed SOD isozymes of P. patens and found several unusual properties in the expression of chloroplastic SOD isozymes [8]. From the genome database, four CuZn-SOD genes (two cytosolic and two chloroplastic isoforms), two Fe-SOD genes (extracellular and chloroplastic isoforms), and two Mn-SOD genes (a previously identified mitochondria isoform and a newly identified but unexpressed isoform) were predicted [8].…”

“…Recently, we analyzed SOD isozymes of P. patens and found several unusual properties in the expression of chloroplastic SOD isozymes [8]. From the genome database, four CuZn-SOD genes (two cytosolic and two chloroplastic isoforms), two Fe-SOD genes (extracellular and chloroplastic isoforms), and two Mn-SOD genes (a previously identified mitochondria isoform and a newly identified but unexpressed isoform) were predicted [8]. Based on SOD activity staining, isolated chloroplasts showed activity of CuZn-SOD but not other SOD isozymes, suggesting that CuZn-SODs are key SOD isozymes for oxidative stress response in chloroplasts [8].…”

“…From the genome database, four CuZn-SOD genes (two cytosolic and two chloroplastic isoforms), two Fe-SOD genes (extracellular and chloroplastic isoforms), and two Mn-SOD genes (a previously identified mitochondria isoform and a newly identified but unexpressed isoform) were predicted [8]. Based on SOD activity staining, isolated chloroplasts showed activity of CuZn-SOD but not other SOD isozymes, suggesting that CuZn-SODs are key SOD isozymes for oxidative stress response in chloroplasts [8]. Under copper deficient conditions, chloroplast CuZn-SOD isozymes were preferentially inhibited, while the activities of other SOD isozymes were not affected.…”

“…In P. patens, there are several possibilities as to why chloroplasts with reduced level of CuZn-SOD are able to maintain stress tolerance. After repeated subcultures of P. patens protonemata under copper-deficient conditions, trace amounts of CuZn-SOD activity were detected [8]. Therefore, it is possible that a small amount of CuZn-SOD is able to maintain normal function in the chloroplast.…”