Influence of Iron Supply on Peroxidase Activity and Peroxidase Isozymes in Corn (Zea mays)

Plants of Oh43 X Wf9 corn (Zea mays L.), grown in a sand culture system, were watered daily with nutrient solution containing 0.025, 1.25, or 2.5 ppm Fe. Plants also were grown in a soil-sand mixture. Leaf extracts were assayed quantitatively for peroxidase activity, and samples of the extracts were subjected to gel electrophoresis. A positive relationship was observed between Fe supply and proxidase activity. Numerous peroxidase isozymes were detected by gel electrophoresis. As judged by intensity of the bands, all isozymes appeared to be in£Iuenced by Fe supply. ---------A W ALA et al. (1) and Brown and Hendricks (2) reported reduced peroxidase activity in preparations of corn (Zea mays L.) leaves from plants grown under low-iron conditions. Their work did not include an investigation of the isozymes of peroxidase. Other studies have shown peroxidase isozyme patterns to be influenced by such factors as growth stage, genetic constitution, and disease (4, 10). This report deals with the peroxidase isozyme patterns of corn plants differing, because of level of Fe supply, in total peroxidase activity. MATERIALS AND METHODS Corn (Oh43 X 'V(9) was grown in the greenhouse in a freedrainage sand culture system. Clay pots (16 cm in diameter) were lined with plastic and filled with white silica sand that had been washed with 2 N HCl and thoroughly rinsed with distilled water. Kernels were planted at the rate of four per pot, and plants were thinned, about 2 weeks after planting, to one per pot in Experiment I and two per pot in Experiment 2. In both experiments the sand was watered daily with distilled water during the 1st week after plauting. Thereafter, the sand was saturated daily with nut ricnt solution. Except for concentrations of Zn and Fe, the nutrient solution was identical to O.25-strength Hoagland and Arnon Solution I (6). The zinc concentration was 1.0 ppm; Fe levels were varied as indicated in Table I. The source of iron was Geigy's Sequestrene 330 Fe (monosodium ferric dicthylenetriaminepentaacetate or NaFeDPTA). Other chemicals used in the solution were reagent grade. . .., For purposes of companson, plants grmnl in a soil-sand nuxture (approximately 60% greenhouse soil and 40% sand) were included in the studv, These plants, referred to as "Controls," received distilled water onlv, except for several applications of 0.00375 Af KNO", made on successive days when the plants were about 4 weeks old, to guard against possible N deficiency. At the time of sampling, plant height was measured from the surface of the sand or soil to the tip of the longest leaf. Fresh weight determinations included only th~ aboveground portions of the plants. Samples used for protem and enzyme assays consisted of leaf tissue Crable I) with the midrib and tip removed. In a coldroom (2 C) these samples were ground in a mortar with a little sand and 10 ml of buffer (0.1 M tris, 0.5 M sucrose, pH 8.0)/g of fresh tissue. These crude preparations were centrifuged (39,000 g, 30 min, 2 C), and supernatant fractions were used as enzyme preparations. Preliminary experiments indicated that these fractions contained more than 90% of the peroxidase activity of the crude prepara· tions. The procedure of Lowry et al. (7) was used to determine the concentration of trichloroacetic acid-precipitable pro· tein in the enzyme preparations of Experiment I, and Potty', modification (9) of the Lowry procedure was used in Experiment 2. Peroxidase activity was assayed by a modification of the spectrophotometric procedure described by Haskins (5). The reaction mixture consisted of 2.65 ml of 0.01 1\;[ sodium acetate buffer, pH 5.6; 0.70 ml of O.I8 M guaiacol; O.IO ml of 0.145 M hydrogen peroxide; and 0.05 ml of diluted enzyme preparation. On the basis of preliminary trials, dilutions of enzyme preparat ions were adjusted to provide approximately equal rates of absorbance change in the various reaction mixtures. Each pre· paration was assayed in duplicate. Buffer and guaiacol solutions were brought to 30 C before reaction constituents were mixed; hydrogen peroxide and enzyme preparations were held in ice unt.il used. _\ modification of the procedure of Davis (3) was used for gel electrophoresis. A 7"/" acrylamide running gel was employed. The gel buffer was 0.378 A[ tris, pH 8.9. Spacer and sample gels were e not used; rather, the enzyme preparation was applied in the extraction buffer directly to the surface of the running gel. The chamber buffer was 0.0124 }I;[ tris, 0.096 M glycine, adjusted to pH 8.3 with HC!. The power supply provided constant voltage (180 volts), and current was varied from an initial value of about 2.7 millamperes/gel to about 2.0 milliamperes/gel at the end of the nm. Duration of electrophoresis was about 90 min. All electrophoretic runs were done in a coldroorn (2 C). '\fter electrophoresis g'els were incubated with guaiacol or benzidine-HCI to detect bands having peroxidase activity (4). \Vhen gnaiacol was used as the sU.bstrate, gels we~e immersed for ,3{) min at room temperature in 0.018 Af guaiacol, rinscc twice with deionized water, and then immersed in 0.015(;0 hydrogen peroxide-I % acetic acid. Bands developed in aboui 10 min. With benzidine-HCI (8) as the substrate, gels were immersed in a mixture of 0.02% bcnzidine-HtI, 1% acetic acid, and 0.015% hydrogen peroxide. An immersion time of 3 to "' min was used for distinguishing' between slow-migrating bauds, and approximately 15 min was required for de~e~opmellt of the more mobile but less intense bands. After stammg, gels were transferred to a fixing solution consisting of methanol, water, and glacial acetic acid (5:5: I, v/vIv). RESULTS AND DISCUSSION At the time of sampling, plants had 7 to 9 visible leaves. The three oldest leaves on all plants in both Table 1. Description of treatments and samples. See text for further details. 1.,':lf I1U I w.ts conside-re-d to hl' lilt' uppennosl lc:tf on the plu nt , In ">qwl'inll'llt 2 onh ,'veil ntln~l.ll't·('d I[':\\'('S wen' t.rkon n-om one plant .md onlv odd number-ed from til,' nth"1 rhus, eac-h s.rmplc «ouststed of Four l.-avcs 1 Published with the approval of the Director as Paper No. ,\072, Journal Series, Nebraska Agr. Exp. Sta. Research reported was conducted under Projects 12-1 and 12-50 and was supported in part by a grant from the Rockefeller l'-oundation to the Cor~l research program at Nebraska. Data were .take~l from a thesis submitted by the senior author to the University of Nebraska in partial fulfillment of the requirements for the M.S. degree. Received Feb. 26, 1971. 2 Graduate Assistant, Bert Rodgers Professor, and Assistant Professor, respectively, Department of Agronomy, University of Nebraska, Lincolu. Current address 01 Dr. .\keson: Gre.u Western Sugar Co .. Longmont, Colo. 8Hi .17 ~:llllpl"'.,, u-c.unu-nt I1Lll1[;'; samptc 1.(':11",,'.';