HORMONAL CONTROL OF PEROXIDASE ACTIVITY IN CULTURED PELARGONIUM PITH

Freshly excised Pelargonium pith tissue lacks peroxidase activity toward guaiacol or benzidine, but it develops such activity within 24-36 hr in aseptic culture. All the activity is manifested as a single enzyme moving toward the cathode during electrophoresis on starch gel at pH 9.0. This development of peroxidase activity is at first (up to ca. 50 hr in culture) inhibited and later (ca. 100-150 hr in culture) promoted by IAA. This dual effect of IAA resembles that previously reported for specific isoperoxidases in tobacco pith cells. Kinetin alone also inhibits peroxidase formation, but in the presence of IAA those concentrations which enhance growth enhance peroxidase formation as well. RECENTLY it has been shown that the various organs of peas (Ockerse, Siegel, and Galston, 1966; Siegel and Galston, 1967) and tobacco (Galston, Lavee, and Siegel, in press) contain distinctive patterns of electrophoretically separable isoperoxidases, and that auxin can both enhance and inhibit the formation of specific members of the isozyme complex. The results with cultured tobacco pith were particularly instructive, since these initially homogeneous cells can be caused to differentiate into lignified cells, roots and shoots (Skoog and Miller, 1957), and the development of particular isoperoxidases is frequently correlated with the new growth and differentiation patterns. Since similar morphogenetic changes were recently elicited in cultured Pelargonium pith (Chen and Galston, 1967), experiments were undertaken to determine the pattern of peroxidase activity in this tissue, and its changes under various culture conditions. The results are detailed below. M\ETHODs-Plant material-Shoots of greenhouse-grown Pelargonium 'Nittany Lion' were excised at the base and the pith removed aseptically from the middle 10 cm of stem by means of a sterile no. 1 cork borer (diam 2.5 mm). The excised cylinders were sliced transversely every 2-3 mm before implantation on the medium. In vitro cultures-A slightly modified semisolid White's medium (21-MW of Lavee, 1960) was used throughout. The cultures were initiated by explants weighing ca. 25 mg and were maintained for 6 weeks at 25 C in ca. 100 ft-c white fluorescent light in tubes containing 10 ml of medium to which were added 2 mg/liter of an I Received for publication 24 November 1967. 2 Permanent address: The Volcani Institute of Agricultural Research, P.O. Box 15, Rehovot, Israel. appropriate auxin, such as indole-3-acetic acid (IAA) or a-naphthaleneacetic acid (NAA) and, where appropriate, 0.2 mg/liter kinetin. Peroxidase activity-Sap was expressed from ca. 20 mg of tissue by squeezing the tissue directly onto a porcelain plate with a spatula. Three 5-mm2 pieces of 3 MM Whatman filter paper were then used to absorb the expressed sap. Each square, containing 6,u liter sap, was separately analyzed. Total peroxidase was determined by eluting the enzyme from one such filter paper square in 1 ml 0.1 M pH 5.8 phosphate buffer at room temperature for 2 hr, then adding 3 ml of 5 mm guaiacol and H202 (Galston et al., in press) in phosphate buffer. Optical density readings at 470 nm, taken at 15to 60-sec intervals for 3-10 -min in a Bausch and Lomb Spectronic-20 colorimeter, indicated the course of the reaction. Results are expressed as A OD/min. For electrophoretic separation and detection of isoperoxidases, one filter paper square was introduced into the middle of an 18 X 14 X 0.6cm starch-gel plate, prepared as previously described (Siegel and Galston, 1967), and containing NaOHborate buffer at pH 9.0. The electrophoretic separation proceeded for 70 min in a gradient of 1OV/cm. To visualize the separated isoperoxidases, the gels were sliced transversely and each half developed by flooding with guaiacol-H202 reagent. The resulting brown spots were photographed for the permanent records, and the intensity of the reaction estimated visually directly from the stained gel. Three replicate cultures were sampled for each value; all experiments were repeated at least three times and all yielded similar results. Data of representative experiments were chosen for presentation. RESULTS-Pith segments removed at 6-cm