Isolation of a cDNA encoding cytosolic ascorbate peroxidase in tobacco.

Abstract

APX is a hydrogen peroxide-scavenging enzyme whose presumed function is to protect the cell from hydrogen peroxide accumulation, particularly under stress conditions. APX catalyzes the reduction of hydrogen peroxide using ascorbate as an electron donor, to yield water and oxidized ascorbate. In plants, three different isoforms of APX are known: a cytosolic form and two chloroplastic forms. The cytosolic form is quite distinct from the other two (Miyake et al., 1993). The cytosolic APX cDNA nucleotide sequences from six different species are known (Pisum sativum L. [Mittler and Zilinskas, 19911; Arabidopsis thaliana var Columbia [Kubo et al., 19921; Spinacia oleracea [GenBank accession No. L208641; Glycine max IChatfield and Dalton, 19931; Daucus cavota [GenBank accession No. 2173981; and Rapkanus staivus [GenBank accession No. X784521). As part of our examination of the role of APX in tolerance of tobacco to ozone stress, we report here the nucleotide and amino acid sequence from a cDNA encoding cytosolic APX in tobacco (Nicotiana tabacum cv Xanthi). Degenerate primers were designed by comparing the APX cDNA sequences from P. sativum and A. tkaliana. These primers were used to amplify a 338-bp fragment from a single-stranded cDNA made from RNA extracted from ozone-exposed tobacco leaves. A nested primer, based on the sequence of the 338-bp fragment, and a poly(C) primer were then used to amplify a 5’ end G-tailed cDNA made from the same RNA, using a modified method from Gurr and McPherson (1991). Based on the resulting 5’ end sequence, another nested primer, -53 bp to the start codon, and a poly(T) primer were then used to amplify a full-length cDNA. Clones from three individual PCR amplifications were sequenced from both ends of these cDNAs. The full-length clones contain a cDNA that is 1040 bp in length, consisting of an 83-bp leader sequence (sequence -83 to -53 is based on a single clone), an open reading frame of 750 bp coding for 250 amino acids, and an untranslated tail of 207 bp (Table I). The predicted molecular