Effect of Cultivation Temperature on Peroxidase Isozymes of Plant Cells Grown in Suspension

Peroxidase has been implicated in a number of diverse phenomena observed in plants including black rot in sweet potato and in lignification. As obtained from plant sources the enzyme will generally consist of a number of isozvmes. So far, the factors governing the distribution of these isozvmes is not well known or understood. Accordingly, the peroxidase isozvmes from a plant tissue culture and the effect of temperature on the peroxidase isozvme distribution w-ere investigated. Stable tobacco suspension cells (WR-132 cultures) were grown at 130, 250, and 350. Previous work had shown that peroxidase activity can be recovered in the medium of non-lignifving WR432 cells grown at 250 (2). 'The present report establishes that the most prevalent peroxidase isozyme secreted into the media was different for each temperature. Additional peroxidase isozymes with similar electrophoretic mobility were secreted at each temperature of growth in different proportions. Preliminarv results indicate that the temperature of growth also influences the peroxidase isozvme pattern of intracellular peroxidase. The specific activity of intracellular, buffersoluble peroxidase was highest in cells grown at 13?. Tobacco cells (WR-132) used in the present investigation were grown in liquid medium under conditions described previously (2, 11) with cultivation maintained at the 3 temperature levels. These cultures have been shown to grow reproduciblv at these temperatures for an indefinite period. The media and cells used in this work were from cultuires grown over at least 10 consecutive passages at a given temperature. In a typical experiment, cells were grown in 500 ml of media for 6 days at the selected temperatures. Cells were separated from the media bv filtration through Miracloth3. The protein was precipitated from the 3 media by 90 % saturation with (NH4)2SOX and the protein collected by centrifugation at 12,000 X g for 30 minutes. The precipitates wvere dissolved in a minimum amount of water and dialyzed against water in the cold in 5 ml Technilab Model F5 flow-cells to remove (NH4)2SO04. The peroxidase isozymes in the media were analvzed by means of (isc electrophoresis, using the standard Canalco system. Gels containing 7 % acrylamide and tris buffer at pH 9.5 were polymerized in 3 stages. The sample gel portion was made last to minimize possible loss of enzyme resulting from prolonged exposure to room temperature. Gels were removed from the electrophoresis ttubes and placed in staining tubes which contained 1 ml 1% H290 and 1 ml 0.1 M benzidine-HCI. After bands were sufficiently well developed (15-30 min) the gels were rinsed several times with distilled water and scanned with a Canalco Model F Microdensitometer to record the absorbance peaks. Densitometer scans of stained gels after disc electrophoresis of peroxidases secreted at the 3 temperatures of growth are shown in figure 1. The relative electrophoretic mobilities for the major peroxidase isozyme at each temperature of growth were 0.05 for 130, 0.30 for 250, and 0.10 for 350. A peroxidase isozvme secreted at 130 with relative mobility of 0.15 was absent at the other 2 temperatures of growth. The major 250 isozyme was not found in medium from either 13?or 350-grown cultures. Two faintly stained isozvme bands in the distal region of the gel (at 0.60 and 0.80 relative mobility) were apparently secreted at all 3 temperatures of growth, although at different levels of relative activity. Growth temperature also affects the peroxidases within the cultured cells. Partially fractionated cell extracts were tused to demonstrate this. After harvesting and Miracloth filtration, cells were suspended in a solution consisting of 75 mm Na2HPO4-KH,PIO4 buffer at pH 6.5, mannitol (0.35 M), EDTA (0.5 mM), cysteine (1 mM), and polyvinylpyrrolidone (360,000 mol xvt at 0.1 %). Cells and homogenizing medium were combined in proportions of 10 g/10 ml. The Virtis homogenizer with a single 1 A preliminary report on this work was presented at the Annual Meeting of the Institute of Food Technologists, Minneapolis, Min.nesota, May 14 to 19, 1967. 2 National Research Council Postdoctoral Res,eaTch Associate. 3 Reference to a company or product name does not imply approval or recommendation o;f the product by the Department of Agriculture to the exclusion of others that may also be suitable.