Ascorbate Peroxidase from Soybean Root Nodules - NCBI

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Department of Biology, West Virginia State College, Institute, West Virginia 251 12 (M.C.); and. Biology Department, Reed College, Portland, Oregon 97202 ...
Plant Physiol. (1993) 103: 661-662

Plant Gene Register

Ascorbate Peroxidase from Soybean Root Nodules' Mark Chatfield* and David A. Dalton

Department of Biology, West Virginia State College, Institute, West Virginia 251 12 (M.C.); and Biology Department, Reed College, Portland, Oregon 97202 (D.A.D.)

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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 H 2 0 2(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.

Table 1. Characteristics of soybean AP cDNA (pSOYAP75)

Organ ism : Soybean (Glycine max [LI Merr. cv Hobbit). Function: A P (EC 1.1 1.1.1 1 ) catalyzes t h e reduction of HzOz to H20 with the concomitant oxidation of L-ascorbate to 2-monodehydroascorbate. Source: A-gtl1 cDNA library constructed with soybean root nodule polyadenylated RNA. Sequencing Strategy: Double-stranded pBluescript plasmid-based sequencing of exonuclease III deletions was used to sequence both strands. Subcellular Location: Enzyme activity was detected in the soluble fraction of nodule plant cells and isolated mitochondria but not in peroxisomes or bacteroids. lmmunogold microscopy indicated that soybean AP was located in the cytosol (Dalton et al., 1993). Structural Features of Deduced Protein: 249 amino acids ( M , 26,923); isoelectric point of 5.60. Antibodies: Polyclonal antisera available.

ACKNOWLEDCMENTS

We thank Dr. Lin Ji and Dr. Robert Klucas (University of Nebraska) for providing a cDNA library from soybean nodules. Sequence analysis was performed at the National Center for Biotechnology Information using the BLAST network service.

Received February 8, 1993; accepted February 11, 1993. Copyright Clearance Center: 0032-0889/93/103/0661/02. The GenBank accession number for the sequence reported in this article is L10292.

Supported by National Science Foundation grants Nos. DCB8903254 and IBN-9206453 and a Howard Hughes Medica1 Institute grant to Reed College (1991 Undergraduate Biosciences Initiative). * Corresponding author; fax 1-304-766-4127.

Abbreviation: AI', ascorbate peroxidase. 661

Chatfield a n d Dalton

662 LITERATURE CITED

Asada K (1992) Ascorbate peroxidase-a hydrogen-scavenging enzyme in plants. Physiol Plant 8 5 235-241 Dalton DA, Baird LM, Langeberg L, Taugher CT, Anyan WR, Vance CP, Sarath G (1993) Subcellular localization of oxygen defense enzymes in soybean (Glycine max [LI Merr.) root nodules. Plant Physiol102: 481-489 Dalton DA, Hanus FJ, Russell SA, Evans HJ (1987) Purification, properties and distribution of ascorbate peroxidase in legume root nodules. Plant Physiol83 789-794 Dalton DA, Russell SA, Hanus FJ, Pascoe GA, Evans HJ (1986)

Plant Physiol. Vol. 103, 'I 993

Enzyniatic reactions of ascorbate and glutathione that prevent peroxide damage in soybean root nodules. Proc Natl Acad Sci USA 8 3 3811-3815 Kubo A, Saji H, Tanaka Ki, Tanaka Ku, Kondo N (1992) Cloning and sequencing of a cDNA encoding ascorbate peroxidase from Arabidopsis thaliana. Plant Mo1 Biol 18: 691-701 Mittler R, Zilinskas BA (1991) Molecular cloning and nucleotide sequence analysis of a cDNA encoding pea cytosolic ascorbate peroxidase. FEBS 289 257-259 Mittler R, Zilinskas BA (1992) Molecular cloning and characterization of a gene encoding pea cytosolic ascorbate peroxidase. I Biol Chem 267: 21802-21807