Ascorbate peroxidase from soybean root nodules.

AP is involved in the destruction of harmful H202. In soybean (Glycine m a x [LI Merr.) root nodules, AP initiates a sequence of coupled redox reactions (ascorbate-GSH pathway) that results in peroxide scavenging (Dalton et al., 1986). The ascorbate-GSH pathway occurs in other plant tissues and has been extensively studied in chloroplasts where photoreducing conditions lead to the production of H202 (Asada, 1992). Two forms of AP are present in plant cells, a chloroplast form and a cytosol form. Both forms of AP are quite distinct from other types of plant peroxidases that occur in the vacuole, cell wall, or cytosol (Asada, 1992). The molecular cloning and sequencing of cytosolic AP cDNA from pea (Mittler and Zilinskas, 1991) and a cDNA from Arabidopsis (Kubo et al., 1992) as well as a characterization of the pea gene (Mittler and Zilinskas, 1992) have been described. We report the isolation of a full-length cDNA clone that encodes an AP subunit (Table I). The isolation strategy was to screen a nodule cDNA expression library with a polyclonal antibody prepared from purified (Dalton et al., 1987) root nodule AP. Putative AP clones were isolated at a frequency of 1 in 800, and 80% of the isolates contained a 1.1-kb insert. Double-stranded dideoxy sequencing of one clone resulted in a 1054-nucleotide sequence with a 3' poly(A) tail and an open reading frame encoding 249 amino acids. Comparison of the N-terminal amino acid sequence obtained by automated Edman sequencing (20 residues) confirmed that the clone was authentic AP. A comparison of deduced protein sequences (249 amino acids) revealed a sequence identity of 90% with that of pea (Mittler and Zilinskas, 1991) and 78% with that of Arabidopsis (Kubo et al., 1992). The high activity of AP (Dalton et al., 1987) in nodules suggests that this pathway may provide an essential protective action in processes related to nitrogen fixation. This analysis was a first step to studies addressing the genetic regulation of the ascorbate-GSH pathway and ultimately the criticalness of this pathway to nitrogen fixation. ~~~