Similar hypocotyl samples taken as

Similar hypocotyl samples taken as described above were immediately frozen in liquid nitrogen. They were then processed to obtain cell wall ionically bound peroxidases as described by Lin and Kao [32]. Briefly, 1.2 g apical or basal tissue was homogenized in a cold mortar with pestle along with 1% polyvinyl pyrrolidone in 50 mM phosphate buffer (pH 5.8). The homogenate was centri- fuged at 22 000 g for 15 min and then washed four times with the buffer solution. The first supernatant (FS) was used for the cytosolic protein control. The final pellet was resuspended in 1M NaCl for 2 h at 20 C and finally filtered. This last supernatant (LS) was used to determine cytosolic protein contamination and as protein extract for peroxidase activity.
Cytosolic protein contamination was determined by measuring glucose-6-P-dehydrogenase (EC 1.1.1.49) activity, a cytosolic protein marker as previously described [8]. Two to 15 mg of protein present in FS or LS were used for glucose-6-P-dehydrogenase assay. The reaction mixture contained 100 mM TRIS-HCl buffer (pH 8), 1 mM MgCl2, 0.2 mM NADP and 1 mM Glucose-6-P. Results were expressed as units of Glucose-6-P-dehydrogenase activity (U. g DW 1). One U 1⁄4 nmol of Glucose-6-P metabolized min 1.
Peroxidase activities were determined by measuring the initial rate of the decrease of absorbance at corresponding maxima. Total peroxidase activity was measured using guaiacol as substrate. Reactions containing 20–50 mg of sample protein, 50 mM sodium phosphate buffer (pH 5.8), 11.8 mM H2O2 and 7.2 mM guaiacol were followed spectrophotometrically at 470 nm for 5 min at 30 C (extinction coefficient for tetraguaiacol 1⁄4 26.6 mM 1 cm 1) [51]. Blank was measured without H2O2. Results were expressed as units of total peroxidase activity (U. g DW 1). One U 1⁄4 mmol of tetra- guaiacol min 1. Ferulic acid peroxidase was assayed according to Sa ́nchez et al. [51]. The oxidation of ferulic acid was measured spectrophotometrically following the absorbance decrease at 310 nm in a reaction mixture containing 1.35 mL Na-phosphate buffer (0.2 M, pH 5.8), 0.5 mL ferulic acid (240 mM), 0.5 mL H2O2 (3 mM) and 0.15 mL enzyme extract. One unit of Ferulic acid POD was defined as a decrease of A310 min 1.
NADH oxidation by peroxidases was measured at 340 nm in mixtures containing 50 mM NADH in 30 mM sodium acetate buffer