Tissue Specificity of Tobacco Peroxidase Isozymes and Their Induction by Wounding and Tobacco Mosaic Virus Infection

Abstract

ABSTRACrPeroxidases (EC 1.11.1.7) have been implicated in the responses of plants to physical stress and to pathogens, as well as in a variety of cellular processes including cell wall biosynthesis. Tissue samples from leaf, root, pith, and callus of Nicotiama tabacum were assayed for specific peroxidase isozymes by analytical isoelectric focusing. Each Xanthi plants were grown from seed in the greenhouse with 14-h daily light periods. Tobacco callus cultures were derived from sections ofpith from N. tabacum L. cv Xanthi. Callus was grown on agar plates and subcultured every 14 d. The medium contained Murashige-Skoog salts, B5 vitamins, 2% sucrose, 0.4 mg/ L 2,4-D, 0.05 mg/L kinetin, and 0.8% agar, and callus was grown in fluorescent light at 28°C (6).Wounding and TMV' Infection. Tobacco leaves were wounded by crushing with a hemostat at a 45°angle to the midvein. Pith was wounded by cutting out 5-mm wedges from the stem at 5-cm intervals or by aseptically removing the pith with a cork borer and slicing into 3-mm cross-sections. These sections were incubated for various times in a moist dark chamber either in water alone or water containing 5 jug/ml cycloheximide. In all cases tissue samples were combined from three or more mature healthy plants. All experiments were repeated at least three times with similar results.Eight-week-old Xanthi plants were infected with TMV by gently rubbing the leaves with carborundum and then applying an aqueous virus suspension with a cotton applicator. Approximately 50 necrotic lesions were detected 2 d after infection. Excessive wounding of the leaves was avoided.Extraction Procedure, Protein Assay, and Enzyme Assay. Tissue samples were homogenized on ice with a Polytron homogenizer (Brinkman Industries) at setting 8 for 20 s in 10 mm sodium phosphate buffer (pH 6.0). The ratios of buffer volume to tissue mass were as follows: 3 to 1, for leaftissue; 3 to 1 for root tissue; 2 to 1 for pith tissue; and 1 to 1 for callus tissue. The homogenate was centrifuged for 20 min at 15,000g, and the cleared supernate was saved for analysis. Protein content was determined by the Bradford reagent method (BioRad). Peroxidase activity was assayed in 0.28% guaiacol, 0.05 M sodium phosphate buffer (pH 6.0), and 0.3% H202. The increase in absorbance was monitored at 470 nm in a Gilford spectrophotometer. Samples prepared for isoelectric focusing included 1% PVP to decrease binding by phenolic compounds which may affect the number of isozymes (21).Flat Bed Isoelectric Focusing and in Situ Peroxidase Staining. Tissue extracts were subjected to analytical flat bed isoelectric focusing on polyacrylamide gels containing ampholines in the pH range 3.5 to 9.5 (LKB, Bromma, Sweden). The samples were subjected to electrophoresis for 1.5 h at 0.125 W/cm2 at 10°C. After focusing, the gels were soaked for 30 min in 500 ml of PBS (10 mm sodium phosphate buffer [pH 6.0], 150 mM NaCl) to remove the ampholines and equalize the pH throughout the gel. The isozymes at the extremes ofthe pH gradient would otherwise ...

Differential Activity of Peroxidase Isozymes in Response to Wounding, Gypsy Moth, and Plant Hormones in Northern Red Oak (Quercus rubra L.)

“…Changes in the activities of POD isozymes have been observed in response to pathogen infection and wounding in several plant species (Espelie et al, 1986;Bashan et al, 1987;Lagrimini and Rothstein, 1987;Svalheim and Robertsen, 1990;Ye et al, 1990). In addition, generalized soluble or wall-bound POD activity increases in other plant species in response to wounding (Birecka and Miller, 1974;Kawaoka et al, 1994), pathogens (Bashan et al, 1987), salicylic acid (Rao et al, 1997), and jasmonates (Thaler et al, 1996;Moore et al, 2003).…”

“…Each plant species typically displays a unique pattern and number of soluble and wall-bound isozymes that may respond differentially to environmental stimuli. Some of the factors known to influence POD isozyme expression are mechanical wounding (Birecka and Miller, 1974;Svalheim and Robertsen, 1990), mite feeding (Bronner et al, 1991), pathogen infection (Lagrimini and Rothstein, 1987;Ye et al, 1990), plant hormones (Ridge and Osborne, 1970;Birecka and Miller, 1974;Neuman et al, 1992), and plant developmental stage (Pao and Morgan, 1988;Biles and Martyn, 1993).…”

“…Although POD functions have been well studied in tobacco, horseradish, and other herbaceous species (Birecka and Miller, 1974;Lagrimini and Rothstein, 1987;Kawaoka et al, 1994), activity studies in woody plants are uncommon (Goodin et al, 1993;Dowd et al, 1998a). Since insect attack elicits changes in leaf polyphenols in some oak species (e.g., Rossiter et al, 1988), it is likely that the activities of oak phenolic-oxidizing enzymes and isozymes also change in response to wounding.…”

Peroxidase-Induced Wilting in Transgenic Tobacco Plants.

“…The tobacco anionic peroxidase (M, 36,000, pl 3.5) is the best characterized of all tobacco peroxidases (Mader, Nessel, and Bopp, 1977;Nessel and Mader, 1977;Mader, Ungemach, and Schloss, 1980;Lagrimini and Rothstein, 1987).…”

Differential Activation of Expression of a Suberization-Associated Anionic Peroxidase Gene in Near-Isogenic Resistant and Susceptible Tomato Lines by Elicitors of Verticillium albo-atratrum

“…Such enzymes probably catalyze the polymerization of the phenolic components of lignin (13,20) and suberin (2,17). The deposition of suberin monomers in wound-healing potato (Solanum tuberosum) tuber slices was shown to be associated with induction of a highly anionic peroxidase (2,7).…”