Alternative Splicing of Rice Chloroplastic CuZn Superoxide Dismutase, OsCSD2: Impact on expression and protein characteristics.

Abstract

Superoxide dismutases (SODs) are involved in cellular reactive oxygen species (ROS) homeostasis under diverse conditions. Among different plant SODs, CuZnSODs (CSDs) are most abundant and localized to multiple compartments, including chloroplast. Rice (Oryza sativa) genome encodes a chloroplastic CuZnSOD (OsCSD2, LOC_Os08g44770), which undergoes alternative splicing (AS), generating two splice variants (SVs), OsCSD2-SV1 (constitutive) and OsCSD2-SV2 (alternative). The AS-event remove 12 nucleotides in OsCSD2-SV2, causing loss of four amino acids (29GPTT32) in OsCSD2-SV2. The two OsCSD2 isoforms were analyzed at RNA and protein levels. The OsCSD2-SV1 and OsCSD2-SV2 transcripts showed comparable stability but differed in expression pattern in certain tissues and stress conditions. For assessment of 'GPTT loss', OsCSD2 isoforms were over-expressed in E. coli, purified, and compared for biochemical-biophysical characteristics. Both isoforms were found to be homodimers (subunit molecular weight: ~19 kDa), and enzymatically active; however, OsCSD2-SV2 (lacking GPTT) showed substantially lower SOD activity than OsCSD2-SV1. Both isoforms showed comparable pH optima, but differed in SOD activity and stability at higher pH and temperature (OsCSD2-SV1 > OsCSD2-SV2), sensitivity to H2O2 (OsCSD2-SV1 > OsCSD2-SV2), and Cu cofactor content. Circular dichroism (CD) showed discernible differences between the isoforms, while differential scanning fluorimetry (DSF) revealed lower thermostability of OsCSD2-SV2, likely due to reduced H-bonds/other interactions. OsCSD2 isoforms showed heteromeric interaction with each other and with the SOD-specific chaperone. Analysis of purified homo/heterodimers indicated that heteromeric interaction reduces the SOD activity of OsCSD2. The study showed several new insights into AS-mediated modulation of rice OsCSD2, which might be important for its chloroplastic functions.