Cucurbita pepo (Pumpkin)

2015

Pumpkins (Cucurbita spp.), which belong to the family Cucurbitaceae, were originally found in Central and South America, and have long been cultivated throughout the world. The fruits, which contain a large amount of starch, proteins, free amino acids, and vitamins A, B, and C, have popularly been used as a vegetable, for pies, and for livestock feed. In Japan, the custom prevails of eating pumpkin fruits in the winter solstice, as they are thought to prevent a cold or palsy. The young vines and leafstalks have also been eaten as a vegetable. Dried ripe seeds (Cucurbitae semen) of cultivated varieties of Cucurbita pepo contain fixed oil, myosin, vitellin, and sugar, and have been used as food. Furthermore, the dried seeds have been used as a teniacide, since they contain resin (peporesin).

Aseorbate oxidase (EC 1.10.3.3) is a copper-containing enzyme that occurs in cucurbitaceous plants such as pumpkin () and cucumber () and plays a role in secondary metabolism (). The precise biological function of aseorbate oxidase has not been clarified, although it has been reported that the enzyme may participate in redox system involving ascorbic acid (). However, the enzyme has been recently used as a valuable reagent for clinical and food analyses of L-ascorbic acid ().

Recently, much attention has been focused on the induction mechanisms of enzymes responsible for secondary metabolism in cultured plant cells, with a view to higher productivity of useful metabolites ().

Ascorbate oxidase in cultured pumpkin cells was studied. Its activity rapidly increased during callus formation from fruit tissue. The activity reached a maximum at 5 days after transfer and then declined. In callus which had been subcultured at about 4-week intervals for more than 1 year, the activity also increased after transfer of fresh medium and reached a maximum in the early logarithmic phase of growth. Light had little effect on the appearance of ascorbate oxidase activity in callus. In the callus grown in the presence of 10 µM CuS04, the activity was about 10 times that in the presence of 0.1 µM CuS04, suggesting that the formation of ascorbate oxidase in callus is stimulated by copper, a prosthetic metal of the enzyme. The study with a specific antibody against purified ascorbate oxidase showed that the increase in ascorbate oxidase activity during the growth of callus was accompanied by an equal increase in enzyme protein. Furthermore, the marked increase in the enzyme activity by adding copper to the culture medium was also accompanied by an increase in enzyme protein. It remains to be established whether copper enhances the de novo biosynthesis of the enzyme or prevents its degradation.

Ascorbate oxidase was found to be released into the medium in pumpkin-cell suspension cultures. Ascorbate oxidase activity in the medium, as well as that in the cells, increased soon after transfer to fresh medium, and reached a maximum at about 5 days. Alcohol dehydrogenase and glucose-6-phosphate dehydrogenase (cytosolic enzyme) and catalase (microbody enzyme) were not detected in the medium, whereas peroxidase, which is known to be a secretory protein, was slightly detectable. Purified ascorbate oxidase on acrylamide gel was stained by the periodic acid-Schiff method. Thus, ascorbate oxidase is a secretory glycoprotein. Perhaps ascorbate oxidase is actively secreted during the growth of cultured pumpkin cells. Ca2+ markedly stimulated the accumulation of ascorbate oxidase in the culture medium. Furthermore, the specific activity of the enzyme in the culture medium was markedly increased by adding CaCl2. On the other hand, Ca2+ had little effect on the accumulation of peroxidase in the medium. Mg2+, but not K + was shown to be effective in the stimulation of ascorbate oxidase secretion, suggesting that Mg2+ can be substituted for Ca2+ in stimulating the secretion of ascorbate oxidase. Further studies will be required to elucidate the process of extracellular secretion of ascorbate oxidase by cultured pumpkin cells and to investigate why divalent cations stimulate ascorbate oxidase secretion. Recently, we succeeded in cloning ascorbate oxidase cDNA in pumpkin callus ().

 

Selections from the book: “Medicinal and Aromatic Plants IV”, 1993.