Full-length cDNA cloning, characterization of catalase from Ulva prolifera and antioxidant response to diphenyliodonium

Abstract

Here, we present a method for the acquisition of the catalase gene, among the most important enzymes involved in the elimination of reactive oxygen species, from Ulva prolifera, and investigate the relationship of NADPH oxidase, salicylic acid, and high-temperature stress on U. prolifera. The full-length complementary DNA (cDNA) of the catalase gene from the green alga U. prolifera (UpCAT) was cloned by polymerase chain reaction using rapid amplification of cDNA ends. The activity of superoxide dismutase, catalase, NADPH oxidase, and phenylalanine ammonia-lyase and the expression level of UpCAT and glutathione reductase (GR) were investigated following treatment with the NADPH oxidase inhibitor diphenyliodonium and salicylic acid at high temperature. The UpCAT cDNA sequence consisted of 1919 bp containing a 5′-untranslated region of 133 bp, a 3′-untranslated region of 307 bp with a polyA tail, and an open reading frame of 1479 bp encoding a polypeptide of 492 amino acids with molecular weight of 56.88 kDa and theoretical pI of 6.658. The deduced amino acid sequence of UpCAT had significant homology to catalases from other plants. The UpCAT protein sequence contained a proximal heme-ligand signature of 344RIFSYPDTQ352 and a proximal active-site signature of 54FDRERIPERVVHARGAAA71. Additionally, three key residues correlating to catalytic activity, His65, Asn139, and Tyr348, were in the UpCAT sequence. Phylogenetic-tree analysis showed that U. prolifera grouped with Ulva fasciata, Chlamydomonas reinhardtii, Haematococcus pluvialis, and Volvox carteri. Furthermore, we observed that under the high-temperature treatment condition, diphenyliodonium treatment (0.01–0.5 mg L−1) reduced NADPH oxidase activity over the entire time, reduced superoxide dismutase, catalase, and phenylalanine ammonia-lyase activity at the early time and induced the expression of UpCAT. Additionally, the expression level of glutathione reductase was suppressed after DPI (0.5 mg L−1) treatment. Analysis of the UpCAT gene substantially advanced our understanding of green-lineage evolution. The results indicated that NADPH oxidase is involved in the process of salicylic acid-induced alleviation of high-temperature stress; however, the specific mechanism requires further study.