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Mosbah M. Kushad, Mohammed Guidera, and Anthony D. Bratsch

Thirty horseradish (Armoracia rusticana Gaertn., Mey., & Scherb.) cultivars from eight countries in Europe and North America and from advanced lines developed at the Univ. of Illinois were evaluated for horseradish peroxidase (HRP; electrical conductivity 1.11.1.7, donor: hydrogen peroxide oxido-reductase) activity. Nearly 86% of the activity was present in the taproot and lateral roots and 14% in the leaf petiole, but there was no activity in the leaf blade. The 30 cultivars were divided into three groups with high (eight cultivars), medium (13 cultivars), and low (nine cultivars) activities [11.58 to 16.97, 7.19 to 9.79, and 2.88 to 6.91 μmol·min-1·g-1 fresh weight (FW), respectively]. The cultivars with the highest activity were 819-A from the Illinois and 810-A from Switzerland with 16.97 and 16.67 μmol·min-1·g-1 FW, respectively. The cultivar with the lowest HRP activity was 244-A from the United States with 2.88 μmol·min-1·g-1 FW. Cultivar 819-A also had the highest protein concentration (4.92 mg·g-1 FW). When HRP activity was expressed per milligrams of protein, cultivar 167-A, also known as `Bohemian', had the highest activity and cultivar 244-A had the lowest (5.35 and 0.83 μmol·min-1·mg-1 protein, respectively).

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Robyn McConchie, N. Suzanne Lang, Alan R. Lax, and Gregory A. Lang

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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Yuehe Huang and Gregory A. Lang

Five-year-old `Sharpblue' southern highbush (Vaccinium corymbosum) plants were self- and cross-pollinated (`O'Neal') to study peroxidase activities and isozyme patterns during fruit development. Both soluble and bound peroxidase activities were present throughout development. Activities were very high during early fruit development, with peaks at 10 and 20 days after self- and cross-pollination, respectively. Activity was much higher for cross-pollinations. During rapid fruit development, peroxidase activities were low. During ripening, the activity of soluble peroxidases increased, then declined in both treatments. Bound peroxidase activity increased during the color transition from blue to dark blue, with the increase being much greater in self-pollinated fruits. Banding patterns of both soluble and bound isoperoxidases varied by pollination treatment as well as fruit developmental stage. Pollen sources alter peroxidase isozymes and activities in developing fruits. During fruit ripening, soluble peroxidase activity appears to be associate with the color transition from light blue to blue, while bound peroxidase activity appears to be associated with the color transition from blue to dark blue.

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Antonio A. Calderón, Jose M. Zapata, Romualdo Muñoz, and A. Ros Barceló

A technique has been developed to study the histochemical localization of peroxidase in Vitis vinifera by blotting freezing/thawing tissue sections on nitrocellulose membranes. After being stained with 4-methoxy- α -naphthol and H2O2, peroxidase-mediated reaction products in mature `Gamay' grapes were seen principally in the skin and, to a lesser extent, the pericarp, where discrete areas of reaction products were located in the vascular bundles. However, for immature `Gamay' and `Grenache' grapes, peroxidase activity in the skin was low and similar to that found in the pericarp. With this technique, fruit vascular bundle structure was preserved. The reliability of the technique in the histochemical localization of peroxidase in grapes was confirmed by fractionation and determining the peroxidase activity in the various tissues.

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G. Préstamo and P. Manzano

The various isozymes of peroxidase of a range of vegetables and kiwifruit were compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by specific activity staining. Peroxidase isozymes were determined in potato (Solanum tuberosum L.), carrot (Daucus carota L.), tomato (Lycopersicon esculentum Mill.), kiwifruit [Actinidia deliciosa (A. Chev.) C.F. Liang et A.R. Ferguson], cauliflower [Brassica oleracea (Botrytis group)], green beans (Phaseolus vulgaris L.), and horseradish (Armoracia rusticana Gaertn, Mey Scherb.). There was only one isozyme in cauliflower (70 kDa), two in kiwifruit (45-43 kDa), and a range of isozymes (120-36 kDa) in horseradish. Ascorbic acid inhibited peroxidase activity in the extracts.

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MadhuraBabu Kunta and Eliezer Louzada*

Ascorbate Peroxidase (APX) is a heme-containing, non-glycosylated enzyme that destroys harmful hydrogen peroxide via the ascorbate glutathione pathway in plants. This enzyme is considered to be an indispensable part of the electron-scavenging pathway and is involved in preventing oxidative damage in plants. Using differential display RT-PCR and 5' RACE a full length c-DNA clone was isolated, from citrus, with very high similarity at the nucleotide and amino acid level, to ascorbate peroxidases from several plant species. It is well known that APXs have highly conserved motifs like the Arg-38, Ars-71, Glu-65 and Asp-208 residues around the distal Hist-42 and proximal His-163. These residues are essential for binding the ligand heme. Additionally, Trp-179 is conserved in most APXs and is the third participant in hydrogen bonding network, together with His-163 and Asp-208. All these conserved motifs were present in the putative APX from citrus in addition of the presence of the peroxidase active site motif residues (APITLRLAWHSA) and the peroxidase heme-ligand motif (DIVVLSGGHTL). Expression analysis in E. Coli reviewed a recombinant protein of 27 Kda.

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Charles L. Rohwer and John E. Erwin

Jasmonates are a class of plant hormones involved in plant defense and stress responses. For example, jasmonate-induced defense responses in Lycopersicon esculentum include increases in activity of proteinase inhibitors, polyphenol oxidases, and peroxidases. As part of our efforts to reduce or control greenhouse pest infestations, we hypothesized that methyl jasmonate (MeJA) could induce these biochemical changes in common greenhouse crops. We studied Impatiens wallerana `Super Elfin Pink', L. esculentum `Big Boy', Petunia ×hybrida `Bravo Lavendar', Viola ×wittrockiana `Imperial Beaconsfield', Coleus ×hybridus `Wizard Jade', Nicotiana alata `Saratoga Lime', Pelargonium ×hortorum `Pinto Pink', and Tagetes erecta `Antigua Primrose'. Polyphenol oxidase and peroxidase activity was studied in the first four species, and proteinase inhibitors were studied in all eight. We sprayed plants with 0, 5 × 10-6, or 10-4 molar MeJA and made measurements after 24 hours. We detected a small increase in polyphenol oxidase activity of plants treated with 10-4 molar MeJA; 5 × 10-6 molar had no effect, and L. esculentum had the highest polyphenol oxidase activity. Peroxidase activity was not affected by MeJA. I. wallerana had the highest peroxidase activity, L. esculentum and V. ×wittrockiana had the lowest. 5 × 10-6 molar MeJA increased proteinase inhibitor activity in most species, and 10-4 molar increased activity in every species except P. ×hortorum.

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M. López-Serrano and A. Ros Barceló

Levels and histochemical localization of peroxidase and polyphenol oxidase, and levels of anthocyanins and (+)-catechin, were studied in fruit of two strawberry (Fragaria ×ananassa Duch.) cultivars (`Oso Grande' and `Chandler'), which show different degrees of susceptibility to enzymatic browning after processing. Although the levels of anthocyanins at the processing-ripe stage may be important in determining pigment stability, and therefore market suitability, the color stability of `Chandler' is apparently determined by the lower endogenous levels of peroxidase and polyphenol oxidase in the processing-ripe stage, which are also accompanied by a lower (+)-catechin content. Polyphenol oxidase was localized almost exclusively in the cortex and to a lesser extent in the pith, showing a complementary pattern to that shown by peroxidase, which was localized in the vascular bundles. Since peroxidase and polyphenol oxidase showed a complementary localization pattern in the fruit, these results strongly suggest a synergic role for these two oxidative enzymes in pigment decay and the associated browning reaction, which occurs in processed strawberry fruit and their derived foods.

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Naoki Yamauchi, Xiao-Ming Xia, and Fumio Hashinaga

Effects of flavonoid pigments on chlorophyll (Chl) degradation by Chl peroxidase in the flavedo of Wase satsuma mandarin (Citrus unshiu Marc. var. praecox Tanaka) fruits were studied. Chl was degraded when hydrogen peroxide was added in a reaction mixture containing Chl and a phosphate buffer extract from the flavedo. Chlorophyllide, which was formed by the action of chlorophyllase in the extract, was also degraded. The flavonoid contents decreased with the Chl degradation in the reaction mixture. Analysis of the flavonoid with HPLC showed that hesperidin and narirutin were contained in the flavedo as a major flavonoid, and that the former decreased significantly and the latter showed almost no change with the Chl degradation in the reaction mixture. In the ethylene-treated fruits, the hesperidin content in the flavedo also decreased with the degreening of stored fruits, suggesting that the flavonoid oxidation by Chl peroxidase could be involved in the Chl degradation.

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P. Perkins-Veazie, J.K. Collins, V. Russo, and B. Cartwright

Individually, green melon aphids (Aphis gossypi) and anthracnose (Colletotricum lagenarium) can cause serious economic damage to watermelons by reducing stands and marketable yields. Greenhouse-grown watermelon seedlings at the third true leaf stage were infected with anthracnose (106 spores/mL) and/or infested with 30 aphids per plant. At the 5th leaf stage (about 7 days after inoculation/infestation), leaf disks were harvested from plants and indicators of stress measured. Peroxidase activity increased from 0.03 to 0.28 absorbance units/mg protein-minute in leaves with anthracnose. When plants were infested with aphids after anthracnose inoculation, peroxidase activity was 0.40 absorbance units/mg protein-minute. Plants having both aphids and anthracnose had more anthracnose lesions when leaves were infested with aphids prior to anthracnose inoculation. The presence of aphids and/or anthracnose stimulated 1-aminocyclopropane-1-caroxylic acid (ACC) oxidase activity from 28 to 44 nL/g-h, indicating enhanced ethylene production. However, aphids had to be present on plants at least 5 days before ACC oxidase activity was stimulated above control levels. Aphids combined with anthracnose failed to elevate ACC oxidase levels higher than either aphids or anthracnose alone. Both peroxidase activity and ACC oxidase activity in watermelon plants increased with anthracnose infection. Thus, watermelon plants stressed by aphids and anthracnose responded differently from plants stressed individually by aphids or anthracnose.