Liquid chromatography-tandem mass spectrometry-assisted identification of two salinity-inducible ascorbate peroxidases in a salt-sensitive rice cultivar (Oryza sativa L. cv. ‘IR-29’)

Abstract

Ascorbate peroxidases (APXs) play an essential role in H2O2 scavenging by converting it into H2O in living cells, and eight APXs have been annotated in the rice genome (Oryza sativa L. cv. ‘Nipponbare’). We conducted mass spectrometric sequencing of a protein pool, obtained using an APX in-gel assay, to retrieve salt-inducible genes from a salt-sensitive rice cultivar (O. sativa L. cv. ‘IR-29’) using liquid chromatography–tandem mass spectrometry. We selected seven candidates from the peptide sequencing output, and performed reverse transcription-polymerase chain reaction to evaluate the candidates for inducible expression against salt treatment (100 mM NaCl). Two orthologs of OsAPX1 and OsAPX2 (OsAPX1-IR29 and OsAPX2-IR29, respectively) were found, and in vitro recombinant enzyme assays revealed that both OsAPX1-IR29 and OsAPX2-IR29 could convert H2O2 into H2O by using ascorbate as the electron donor, with 10- to 15-fold higher enzymatic activity than that of a crude extract. OsAPX2-IR29 transcripts were significantly more abundant than OsAPX1-IR29 transcripts in the shoot. However, after salt treatment, OsAPX1-IR29 transcripts were highly induced, depending on salt concentration (greater than threefold) and time (greater than sixfold). Furthermore, we observed a similar APX protein accumulation pattern using isoform-specific antibodies. Importantly, transcript levels markedly increased 5 days after salt treatment, and were consistently maintained until 7 days after treatment, although the seedlings sustained serious damage and showed no indication of viability. Our results suggest that OsAPX1-IR29 and OsAPX2-IR29 are the major APX isozymes, and play significant roles in scavenging H2O2 during salt stress in the IR-29 cultivar.