Characterization and expression analysis of ascorbate peroxidase from the moss Dicranum scoparium during abiotic stresses

Abstract

Ascorbate peroxidase (APX) is an important antioxidant enzyme responsible for the conversion of H2O2 to H2O and O2. In this study, APX was studied in the widespread boreal cushion moss Dicranum scoparium. Native PAGE of crude extracts of moss thalli revealed the presence of APX isoforms in D. scoparium with a range of molecular masses. An APX complementary DNA (cDNA) gene of 771-bp length was cloned and designated as DsAPX. The cloned coding domain sequence (CDS) encoded a 256 amino acid polypeptide, and the predicted protein product was calculated to have a molecular mass of 28.4 kDa with an isoelectric point of 5.7. Several highly conserved sites important for enzyme activity were predicted using bioinformatic tools. The DsAPX protein has similarities of 91%, 78%, 67% and 66% with APX homologs from Grimmia pilifera, Physcomitrella patens, Zea mays and Nicotiana tomentosiformis, respectively. The high homology with cytosolic APX from G. pilifera strongly suggests that the cloned DsAPX gene encodes a cytosolic APX. We studied the role of APX in the tolerance of this moss to abiotic stresses. Changes in both APX activity and DsAPX gene expression, estimated using reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR), showed that the gene was up-regulated in response to desiccation/rehydration, and expression was maintained during a heat stress of +50°C. By contrast, DsAPX was down-regulated by a freezing (–20°C) treatment. Results obtained extend our knowledge of the diversity of APX isoforms that occur in bryophytes and suggest that APX may be involved in the tolerance of D. scoparium to abiotic stresses.