Tracking copper-zinc and manganese superoxide dismutase in Avicennia marina reveals time-dependent expression of SOD isoforms in response to salt and lead stress

Salinity and heavy metals significantly impact plant growth and development. However, mangroves demonstrate remarkable tolerance to these stresses. This study focuses on tracking superoxide dismutase (SOD), a key enzyme responsible for neutralizing superoxide anions. Gene expression analysis revealed that the genes encoding CuZnSOD (AmSOD1) and MnSOD are upregulated in Avicennia marina seedlings exposed to salt and lead stress. To investigate the protein-level expression of these isoforms, CuZnSOD and MnSOD (AmSOD2) from A. marina were heterologously expressed, purified, and used to produce specific antibodies in rabbits. These antibodies successfully distinguished between the two isoforms. Using 2D gel electrophoresis, western blotting combined with mass spectrometry, AmSOD1 was identified as two distinct spots with different molecular weights. Notably, the intensity of the high-molecular-weight spot increased in the leaves of A. marina seedlings during the early stages of salt and lead exposure, while the low-molecular-weight spot became more pronounced at later stages. AmSOD2 appeared as a single spot whose intensity increased at various time points in response to salt and lead, compared to controls. Protein extracts from the leaves displayed enhanced SOD activity under salt and lead stress. Additionally, Escherichia coli strains expressing AmSOD1 and AmSOD2 further confirmed the functional activity of these isoforms.