Extraction and activity of polyphenoloxidase and peroxidase from senescing leaves of Protea neriifoiia

Whitehead, C.S.; Swardt, G. H. de

doi:10.1016/s0022-4618(16)30161-9

Cited by 17 publications

(20 citation statements)

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“…Leaves of susceptible species begin to turn black within 3 to 7 days after harvest, severely reducing the market value of a flower stem that otherwise has a potential vase life of 3 to 4 weeks. Leaf blackening appears to be caused by oxidation of the polyphenols and leuco-anthocyanins (Whitehead and de Swardt, 1982) that leak into the cyfosol from the vacuole after rupture of intracellular membranes (Brink and de Swardt, 1986;Ferreira, 1986;Newman et al, 1989). The physiological mechanisms or stresses that initiate rupture of the intracellular membrane and subsequent leaf blackening have not been clearly established.…”

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confidence: 99%

Carbohydrate Depletion and Leaf Blackening in Protea neriljiolia

McConchie¹,

Lang²,

Gross³

1991

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Leaf blackening on cut flower Protea nerii[olia R. Br. stems was dramatically reduced under a 12-hour photosynthetic light period (120 μmol·m-2·s-1) at 25C for 15 days compared with stems kept in the dark. In the light, addition of 0.5% exogenous sugar to the vase solution resulted in a maximum of 2.5% leaf blackening, while stems with no exogenous sugar had a maximum of 16.5%. Continuous darkness resulted in 94% leaf blackening by day 7, irrespective of sugar treatment. Starch and sucrose concentrations were markedly lower in leaves on dark-held stems than in leaves on stems held in the light; thus, carbohydrate depletion could be the primary stress that initiates leaf blackening. In the light, rates of carbon exchange and assimilate export were similar, indicating that the amount of carbon fixed maybe regulated by sink demand. The pattern of carbon partitioning changed in light-held leaves of the 0% sugar treatment during rapid floral expansion and senescence. Inflorescence expansion appears to influence partitioning of photoassimilates and storage reserves into transport carbohydrates; under decreased sink demand, the assimilate export rate decreases and photoassimilates are partitioned into starch. The data suggest that sink strength of inflorescences held in darkness may be responsible for the depletion of leaf carbohydrates and. consequently, blackening.

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Carbohydrate Depletion and Leaf Blackening in Protea neriljiolia

McConchie¹,

Lang²,

Gross³

1991

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“…The pellet was resuspended in 200 µl solubilization buffer (Webber et al, 1988) containing Triton X-100 or sodium dodecyl sulfate (SDS) at 0%, 0.5%, 1%, 2%, or 5% each and incubated on ice in the dark for 1 h with periodic mixing, followed by microfuge centrifugation (Microfuge E, Beckman Instruments, San Ramon, Calif.) for 1 min. Polyphenol oxidase activity was assayed as described above at pH 7.2 (Vaughn and Duke, 1981) and at pH 4.5, for which PPO activity has been reported in P. neriifolia (Whitehead and de Swardt, 1982). Optimum activity was obtained using SDS at 2% at pH 7.2 and pH 4.5 (data not shown); higher concentrations of SDS released not only PPO, but also chlorophylls, which interfered with the assay procedure.…”

Section: Methodsmentioning

confidence: 92%

“…Membrane hydrolysis, or other reactions associated with senescence, may allow vacuolesequestered 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 anti-oxidant dips (Jones and Clayton-Greene, 1992).…”

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confidence: 99%

“…Low levels of lipid peroxidation, maintenance of the ascorbate-glutathione antioxidant system, and continued respiration in dark-held stems of P. neriifolia leaves after the occurrence of substantial leaf blackening suggest that membrane integrity is maintained. In addition, the potential association of leaf blackening with PPO and POD activity must be examined further since Whitehead and de Swardt (1982) did not demonstrate an actual relationship between the enzyme activity they reported and subsequent leaf blackening (which was not investigated experimentally). Their assumption that leaf senescence occurred during the postharvest interval was not based on measured parameters.…”

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confidence: 99%

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

McConchie¹,

Lang²,

Lax³

et al. 1994

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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 × 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.

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“…Isto ocorre devido à oxidação de compostos fenólicos pelas enzimas Peroxidase (PER) e Polifenoloxidase (PFO), resultando na formação de pigmentos escuros e causando o endurecimento do grão (WHITEHEAD & SWARDT, 1982). Esse processo é gradativo, acumulativo e irreversível.…”

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