Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth

Hydroxyl radicals ((OH)-O-.), produced in the cell wall, are capable of cleaving wall polymers and can thus mediate cell wall loosening and extension growth. It has recently been proposed that the biochemical mechanism responsible for (OH)-O-. generation in the cell walls of growing plant organs represents an enzymatic reaction catalyzed by apoplastic peroxidase (POD). This hypothesis was investigated by supplying cell walls of maize (Zea mays L.) coleoptiles and sunflower (Helianthus annuus L.) hypocotyls with external NADH, an artificial substrate known to cause (OH)-O-. generation by POD in vitro. The effects of NADH on wall loosening, growth, and (OH)-O-. production in vivo were determined. NADH mediates cell wall extension in vitro and in vivo in an H2O2-dependent reaction that shows the characteristic features of POD. NADH-mediated production of (OH)-O-. in vivo was demonstrated in maize coleoptiles using electron paramagnetic resonance spectroscopy in combination with a specific spin-trapping reaction. Kinetic properties and inhibitor/activator sensitivities of the (OH)-O-.-producing reaction in the cell walls of coleoptiles resembled the properties of horseradish POD. Apoplastic consumption of external NADH by living coleoptiles can be traced back to the superimposed action of two enzymatic reactions, a KCN-sensitive reaction mediated by POD operating in the (OH)-O-.-forming mode, and a KCN-insensitive reaction with the kinetic properties of a superoxide-producing plasma-membrane NADH oxidase the activity of which can be promoted by auxin. Under natural conditions, i.e. in the absence of external NADH, this enzyme may provide superoxide (O-2(.-)) (and H2O2 utilized by POD for) (OH)-O-. production in the cell wall.