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.