Effect of cell-wall-digesting enzymes on physiological state and competence of maize coleoptile cells

Protoplasts are frequently isolated from maize coleoptiles with cell-wall-degrading enzymes such as pectolyase (PEC), mazerozyme, and cellulase. Incubation of coleoptiles with these enzymes caused rapid depolarizations of the membrane voltage (V-M). The depolarizing effect of 0.5% (w/v) mazerozyme or 1.5% (w/v) cellulase was unaffected by denaturation of the enzymes. In the case of pectolyase (0.1%, w/v), however, the active enzyme was significantly more potent than the denaturated enzyme in depolarizing coleoptile cells. Exposure to 0.1% active PEC but not to inactive PEC also caused an oxidative burst in coleoptiles and enhanced KC efflux. Together this suggests that pectic breakdown products of the cell wall act as signal for wounding. Typically addition of 10 mu M 1-naphthylene acetic acid (NAA) to coleoptiles causes a transient depolarization followed by a slow hyperpolarization of V-M. However, in the presence of PEG. V-M only depolarized in NAA. After PEG-treated coleoptiles were washed free of the enzyme, NAA caused only small fluctuations of V-M. A similarly small V-M response to NAA appeared in coleoptiles pretreated with heat-denaturated supernatant (SUP) from a protoplast isolation buffer, the latter suspected to contain the PEG-generated wounding signal. Comparable pretreatment of coleoptiles with PEC or SUP had no significant effect on the spontaneous and NAA-evoked acidification of the incubation medium. Pretreatment with SUP also had no significant effect on the NAA-stimulated elongation of coleoptile segment. Hence, PEC treatment of coleoptile tissue affects the membrane transport properties of the cells. This effect is partly maintained after removal of the enzyme from the incubation medium, an effect not significant for NAA-generated acidification and cell elongation.