Contamination of oat mesophyll protoplasts by apoplastic polyamine oxidase

Abstract

W. 1989. Contamination of oat mesophyll protoplasts by ,apoplastic polyamine oxidase. -Physiol. Plant. 77: 347-351.Mesophyll protoplasts isolated from peeled oat (Avena sativa L. cv Victory) leaves with 1% (w/v) Cellulysin in 20 mM KPO<, pH 5.5 and 0.6 M sorbitol retain ahout 6% of the polyamine oxidase (PAO, EC 1.4.3.4) activity of the whole peeled leaf. However, more than 99% of the oat leaf PAO activity is apoplastic and can be extracted by vacuum infiltration with 200 mM NaCl and this procedure extracts no activity for the cytoplasmic marker enzyme glucose-6-phosphate dehydrogenase (G6PD, EC 1.1.1.49). By these criteria we consider PAO in oat leaves to be totally apoplastic and PAO found in the isolated protoplast to be contamination. The degree of protoplast contamination by PAO depends on the pH and ionic strength of the isolating and washing medium. It can be eliminated by washing protoplasts in 0.6 M sorbitol with 100 mM KPO,, pH 6.5. Pellets of lysed protoplasts incubated with dialyzed apoplastic enzymes in 5 mM KPO4, pH 5.5 adsorb about 87% ofthe added PAO activity hut only about 25% ofthe added peroxidase (EC 1.11.1.7) activity. The adsorbed activity can be solubilized from the pellet by extraction with 1 M NaCl. The results demonstrate that weakly ionicaily bound cell w,ai enzymes may contaminate protoplasts isolated and purified by conventional techniques.

Apoplastic Sugars, Fructans, Fructan Exohydrolase, and Invertase in Winter Oat: Responses to Second-Phase Cold Hardening

“…Although the location of FEH is generally accepted to be vacuolar (Pollock and Chatterton, 1988), we considered that it may be present in the apoplast for several reasons: (a) we found extracellular fructans present in oat crowns of all three hardening treatments, (b) numerous carbohydratehydrolyzing enzymes (acid invertase, ␣-amylase, ␤-amylase, ␣-glucosidase, and ␤-glucosidases) are known to be present in the apoplast of many plant species (Hatch et al, 1963;Hawker and Hatch, 1965;Beers and Duke, 1988;Li and McClure, 1989;Beers et al, 1990), and (c) previous localizations of FEH activity relied upon isolation of vacuoles from protoplasts using a protocol that did not account for extracellular enzyme activities (Wagner and Wiemken, 1986). The levels of FEH activities that we found in apoplastic fluid from crowns of NH and 1PH oat were not significantly different from each other and were about 2% of the total activity in the tissue.…”