Changes in the specific activity and multiple forms of peroxidase along the differentiating root of Pisum sativum have been examined. The specific activity of the total homogenate increased steadily along the root, although that of the low speed supernatant fraction showed a fall in the first 20 mm. This finding supports previous cytochemical studies which showed low activity in the meristem and increased cell wall activity in the older regions. Acid phosphatase also increased gradually along the root, although phenylalanine ammonia lyase (PAL) showed a peak in the 20-40 mm region which corresponded with the first positive staining for lignin. When stelar and cortical tissues were compared, peroxidase specific activity was highest in a cortical supernatant fraction while PAL was highest in the stelar tissues. The separation of peroxidase by gel electrophoresis showed the presence of a number of bands which varied both along the root and between stele and cortex. The results are discussed in relation to the possible functions of peroxidase and, in particular, to its proposed role in lignification. INTRODUCTION Changes in biochemical activity can be examined in relation to changes in structure and function by taking serial sections along the root axis. However, these changes may only be an indication of the activity of the most dominant cell type in each region. If possible, the findings should be related to a parallel histochemical and cytochemical survey which follows qualitatively the changes occurring in each cell type. The serial sectioning technique has been used for various enzymes and aspects of metabolism and has provided valuable information relating structure to function (Brown and Broadbent, 1950; Cook, 1959; Hellebust and Forward, 1962; Sexton and Sutcliffe, 1969; Fowler and Ap Rees, 1970). To understand the seemingly diverse functions of peroxidase a biochemical survey of the enzyme has been carried out in the differentiating root of Pisum sativum and the results discussed in relation to previous histochemical and cytochemical studies. In addition, the distribution of phenylalanine ammonia lyase (PAL) and acid phosphatase have been examined. PAL is an enzyme known to be concerned with secondary metabolism and, in particular, is thought to be involved 1 Present address: Department of Botany, Imperial College, London SW7 2BB. This content downloaded from 207.46.13.109 on Sun, 24 Apr 2016 06:27:18 UTC All use subject to http://about.jstor.org/terms 984 Fielding and Hall—Peroxidase in Pisum Roots in lignification (Rubery and Northcote, 1968). In this respect, a comparison with peroxidase is of considerable interest. Acid phosphatase is a hydrolytic enzyme with uncertain function but may act as a marker for cellular autolysis in senescing or differentiating cells (Gahan and Maple, 1966; Sexton and Sutcliffe, 1969). MATERIALS AND METHODS Roots of Pisum sativum var. Alaska were grown as previously described (Fielding and Hall, 1978). Tissue preparation Serial sectioning was carried out in a precooled microtome similar to that described by Sexton and Sutcliffe (1969). Generally 100 roots of similar length were sectioned, homogenized in 50 mM TES/Tris buffer (pH 7-0) using a ground glass homogenizer, and assayed. Stripping the stele from the cortex was facilitated by the excision of the root tip to avoid contamination of either fraction. Figures for cell numbers were obtained from Sexton and Sutcliffe (1969). Enzyme assays Peroxidase was assayed using guaiacol as donor and acid phosphatase using p-nitrophenyl phosphate as substrate as described previously (Fielding and Hall, 1978). PAL was assayed using a modification of the radiochemical technique of Koukol and Conn (1961). The incubation medium contained the following solutions in a final volume of 5-05 ml: 50 mM Tris/HCl (pH 8-8), 1 mM L-phenylalanine, and 0-1 iuCi U-L-[14C]phenylalanine, and enzyme (extracted in the presence of 0-05% mercaptoethanol and 0-05% EDTA). After incubation for 90 min at 35 °C, the reaction was stopped by the addition of 0-5 ml 0-1% cinnamic acid in 0-05 M NaOH, immediately followed by 0-5 ml 50% (w/v) TCA. The acidified mixture was then extracted once with 2-0 ml toluene. After centrifugation, 1 ml aliquots of toluene were placed in scintillation vials containing 2-0 ml scintillation fluid and counted in a Beckman scintillation counter. Protein was assayed using the Folin Ciocalteau reagent after precipitation in 10% TCA as described by Lowry, Rosebrough, Farr, and Randall (1951). Gel electrophoresis Gels were run, prepared, and stained as described previously (Fielding and Hall, 1978). Microscopy Lignin was located by incubation of tissue sections in a saturated solution of phloroglucinol in 20% (w/v) HC1.