Reexamining Polyphenol Oxidase, Peroxidase, and Leaf-blackening Activity in Protea

Premature leaf blackening in Protea severely reduces vase life and market value. The current hypothesis suggests that leaf blackening is induced by a sequence of events related to metabolic reactions associated with senescence, beginning with total depletion of leaf carbohydrates. It is thought that this carbohydrate depletion may induce hydrolysis of intercellular membranes to supply respiratory substrate, and subsequently allow vacuole-sequestered phenols to be oxidized by polyphenol oxidase (PPO) and peroxidase (POD) (Whitehead and de Swardt, 1982). To more thoroughly examine this hypothesis, leaf carbohydrate depletion and the activities of PPO and POD in cut flower Protea susannae x P. compacta stems held under light and dark conditions were examined in relationship to postharvest leaf blackening. Leaf blackening proceeded rapidly on dark-held stems, approaching 100% by day 8, and was temporally coincident with a rapid decline in starch concentration. Blackening of leaves on light-held stems did not occur until after day 7, and a higher concentration of starch was maintained earlier in the postharvest period for stems held in light than those held in dark. A large concentration of the sugar alcohol, polygalatol, was maintained in dark- and light-held stems over the postharvest period, suggesting that it is not involved in growth or maintenance metabolism. Polyphenol oxidase activity in light- and dark-held stems was not related to appearance of blackening symptoms. Activity of PPO at pH 7.2 in light-held stems resulted in a 10-fold increase over the 8-day period. Activity in dark-held stems increased initially, but declined at the onset of leaf blackening. There was no significant difference in POD activity for dark- or light-held stems during the postharvest period. Total chlorophyll and protein concentrations did not decline over the 8-day period or differ between light- and dark-held stems. Total phenolics in the dark-held stems increased to concentrations ≈30% higher than light-held stems. Consequently, the lack of association between membrane collapse, leaf senescence, or activities of oxidative enzymes (PPO or POD) with leaf blackening does not support the hypothesis currently accepted by many Protea researchers. An alternative scenario may be that the rapid rate of leaf starch hydrolysis imposes an osmotic stress resulting in cleavage of glycosylated phenolic compounds to release glucose for carbohydrate metabolism and coincidentally increase the pool of free phenolics available for nonenzymatic oxidation. The physiology of such a carbohydrate-related cellular stress and its manifestation in cellular blackening remains to be elucidated. Premature leaf blackening in many cut flower Protea species seriously reduces vase life and market value. When stems are held under dark postharvest conditions, blackening symptoms can appear within 3 days after harvest (Bieleski et al., 1992; McConchie et al., 1991). The current hypothesis suggests that postharvest leaf blackening in Protea is induced by a sequence of metabolic events associated closely with senescence, beginning with the total deple- tion of leaf carbohydrates and followed by subsequent hydrolysis of intercellular membranes to supply respiratory substrate for the developing inflorescence (Ferreria, 1986). Membrane hydrolysis, or other reactions associated with senescence, may allow vacuole- sequestered phenols to come in contact with oxidative enzymes (Jones and Clayton-Greene, 1992). Polyphenol oxidase (PPO) and peroxidase (POD) have been proposed as the enzymes responsible for leaf blackening symptoms (Whitehead and de Swardt, 1982). In fact, the general acceptance of PPO and POD involvement in Protea leaf blackening has focused recent research efforts on modification of their postharvest activity, as by inhibition with