Formation of Ascorbate Oxidase in Cultured Pumpkin Cells

Ascorbate oxidase activity rapidly increased during callus formationfrom pumpkin fruit tissue. The activity reached a maximum at5 days after transfer and then declined. In callus which hadbeen subcultured at about 4-week intervals for more than oneyear, the activity also increased after transfer to fresh mediumand reached a maximum in the early logarithmic phase of growth.Light had little effect on the appearance of ascorbate oxidaseactivity in pumpkin callus. In the callus grown in the presenceof 10µM CuSO₄, the activity was about 10 times that inthe presence of 0.1 µM CuSO₄, suggesting that the formatonof ascorbate oxidase in pumpkin callus is stimulated by copper,a prosthetic metal of the enzyme. From 45 to 75% of the totalascorbate oxidase activity in pumpkin cell suspension cultureswas found in the medium. Ascorbate oxidase activity in the medium,as well as that in the cells, increased soon after transferto fresh medium, and reached a maximum at about 5 days. (Received July 2, 1987; Accepted November 21, 1987)     

A diminution in ascorbate oxidase activity affects carbon allocation and improves yield in tomato under water deficit

The regulation of carbon allocation between photosynthetic source leaves and sink tissues in response to stress is an important factor controlling plant yield. Ascorbate oxidase is an apoplastic enzyme, which controls the redox state of the apoplastic ascorbate pool. RNA interference was used to decrease ascorbate oxidase activity in tomato (Solanum lycopersicum L.). Fruit yield was increased in these lines under three conditions where assimilate became limiting for wild‐type plants: when fruit trusses were left unpruned, when leaves were removed or when water supply was limited. Several alterations in the transgenic lines could contribute to the improved yield and favour transport of assimilate from leaves to fruits in the ascorbate oxidase lines. Ascorbate oxidase plants showed increases in stomatal conductance and leaf and fruit sugar content, as well as an altered apoplastic hexose : sucrose ratio. Modifications in gene expression, enzyme activity and the fruit metabolome were coherent with the notion of the ascorbate oxidase RNAi lines showing altered sink strength. Ascorbate oxidase may therefore be a target for strategies aimed at improving water productivity in crop species.     

Ascorbate oxidase of Cucumis melo L. var. reticulatus: purification, characterization and antibody production

Ascorbate oxidase activity and ascorbic acid content were followedduring the development of muskmelon (Cucumis melo L. var. reticulatus)fruits. The enzyme was highly expressed in ovaries and veryyoung fruit tissues, followed by a decrease in 10- and 20-d-oldfruits and an increase in 30- and 35-d-old fruits which coincidedwith early events of fruit ripening. Ascorbic acid content wasnegatively correlated with ascorbate oxidase activity. The enzymewas purified to homogeneity following ion exchange, affinityand gel filtration chromatographic trials. The purified enzymewas a glycoprotein of molecular weight 137 000 composed of twosubunits of molecular weight 68000, and formed by six isoenzymeswith isoelectric points in the range of pH 7.7 to 8.3. Its electronparamagnetic resonance and optical spectra were in agreementwith other copper proteins and the enzyme contained eight copperatoms per dimeric molecule. The K_m of the enzyme for ascorbicacid was 50 µM. Ascorbate oxidase activity was inhibitedby azide and by EDTA, two inhibitors of copper proteins. Optimalconditions for enzyme activity was pH 5.5, and a temperatureof 37 C. Polyclonal antibodies were produced against the purifiedprotein and immunoprecipitated ascorbate oxidase activity. Key words: Cucumis melo, muskmelon, ascorbate oxidase, fruit ripening     

Marked increase in ascorbate oxidase protein in pumpkin callus by adding copper

Ascorbate oxidase from pumpkin (Cucurbita sp.) was purified from a commercially available preparation. A single polypeptide band with M_r 64,000 was detected after sodium dodecylsulfate-polyacrylamide gel electrophoresis of the purified enzyme. In double immunodiffusion tests, antiserum against the purified preparation formed a single precipitin line with the crude extract from pumpkin fruit tissue or the callus as well as with the purified preparation. Immunological blotting method showed that mol wt of ascorbate oxidase subunit in pumpkin callus was the same as that of the purified preparation. Analysis with the single radial immunodiffusion method showed that the increase in ascorbate oxidase activity during the growth of pumpkin callus correlated with an increase in the enzyme protein. Furthermore, enzyme protein in the callus grown in the presence of 10 micromolar CuSO₄ for 2 weeks was about eight times that grown in the presence of 0.1 micromolar CuSO₄. The synthesis of ascorbate oxidase in pumpkin callus may be induced by copper, a prosthetic metal of the enzyme, or copper may help stabilize the enzyme against proteolytic breakdown.     

cDNA cloning and differential gene expression of three catalases in pumpkin

Three cDNA clones (cat1, cat2, cat3) for catalase (EC 1.11.1.6) were isolated from a cDNA library of pumpkin (Cucurbita sp.) cotyledons. In northern blotting using the cDNA-specific probe, the cat1 mRNA levels were high in seeds and early seedlings of pumpkin. The expression pattern of cat1 was similar to that of malate synthase, a characteristic enzyme of glyoxysomes. These data suggest that cat1 might encode a catalase associated with glyoxysomal functions. Furthermore, immunocytochemical analysis using cat1-specific anti-peptide antibody directly showed that cat1 encoding catalase is located in glyoxysomes. The cat2 mRNA was present at high levels in green cotyledons, mature leaf, stem and green hypocotyl of light-grown pumpkin plant, and correlated with chlorophyll content in the tissues. The tissue-specific expression of cat2 had a strong resemblance to that of glycolate oxidase, a characteristic enzyme of leaf peroxisomes. During germination of pumpkin seeds, cat2 mRNA levels increased in response to light, although the increase in cat2 mRNA by light was less than that of glycolate oxidase. cat3 mRNA was abundant in green cotyledons, etiolated cotyledons, green hypocotyl and root, but not in young leaf. cat3 mRNA expression was not dependent on light, but was constitutive in mature tissues. Interestingly, cat1 mRNA levels increased during senescence of pumpkin cotyledons, whereas cat2 and cat3 mRNAs disappeared during senescence, suggesting that cat1 encoding catalase may be involved in the senescence process. Thus, in pumpkin, three catalase genes are differentially regulated and may exhibit different functions.     

The control of ascorbic acid synthesis and turnover in pea seedlings

The rate of ascorbate synthesis and turnover in pea seedling embryonic axes was investigated in relation to its pool size. Ascorbate accumulated in embryonic axes of germinating pea seeds which has been supplied with ascorbate. Incorporation of [U-14C]glucose into ascorbate after a 2 h labelling period was reduced by ascorbate loading for 3 h and 20 h, providing evidence that ascorbate biosynthesis is inhibited by endogenous ascorbate. Ascorbate turnover was estimated by following the metabolism of [1-14C]ascorbate over 2 h after ascorbate loading and by the rate of decrease of the ascorbate pool size after ascorbate loading. Ascorbate turnover rate, determined by [1-14C]ascorbate metabolism, increased as a linear function of pool size. The absolute turnover rate was higher in ascorbate-loaded embryonic axes but was always about 13% of the pool per hour. The initial rate of ascorbate turnover, estimated from the net decrease in pool size after ascorbate loading, also showed a similar turnover rate to that estimated from [1-14C]ascorbate metabolism. Ascorbate loading had no effect on ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase or glutathione reductase activity. Ascorbate oxidase activity decreased after ascorbate loading.     

Role of peroxisomes in the oxidative injury induced by 2,4-dichlorophenoxyacetic acid in leaves of pea plants

The role of peroxisomes in the oxidative injury induced by the auxin herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) in leaves of pea (Pisum sativum L.) plants was studied. Applications of (2,4-D) on leaves or to root substrate increased the superoxide radical production in leaf peroxisomes. Foliar application also increased H₂O₂ contents in leaf peroxisomes. Reactive oxygen species (ROS) overproduction was accompanied by oxidative stress, as shown by the changes in lipid peroxidation, protein carbonyls, total and protein thiols, and by the up-regulation of the activities of superoxide dismutase, ascorbate peroxidase, glutathione reductase, catalase, glucose 6-phosphate dehydrogenase and NADP⁺-dependent isocitrate dehydrogenase. Foliar or root 2,4-D applications also induced senescence symptoms in pea leaf peroxisomes, as shown by the decrease of protein content and glycolate oxidase and hydroxypyruvate reductase activities, and by the increase of endopeptidase, xanthine oxidase, isocitrate lyase and acyl-CoA oxidase activities as well as of 3-ketoacyl-CoA thiolase and thiol-protease protein contents. 2,4-D did not induce proliferation of pea leaf peroxisomes but induced senescence-like morphological changes in these organelles. Results suggest that peroxisomes might contribute to 2,4-D toxicity in pea leaves by overproducing cell-damaging ROS and by participating actively in 2,4-D-induced leaf senescence.     

Ascorbate oxidase from Cucurbita maxima

Ascorbate oxidase is present in homogenates of the flesh of Cucurbita maxima fruits. Its activity is independent of ascorbate concentration over th     

Changes in ascorbate oxidase gene expression and ascorbate levels in cell division and cell elongation in tobacco cells

The biological function of ascorbate oxidase (AAO) was not yet clarified, although it was suggested that AAO may be involved in cell growth. We investigated AAO expression and ascorbate metabolism during non-synchronous, synchronous, and elongation cultures of tobacco BY-2 cells. In non-synchronous culture, AAO mRNA was abundant in logarithmic growth phase. Ascorbate content greatly increased during the growth, whereas dehydroascorbate content was slightly increased. In synchronous division culture, AAO mRNA was detected in all phases, but the levels were quite low in G1 phase. Ascorbate content was high in all phases, whereas dehydroascorbate content was low, especially in G1 phase. In elongation culture, the levels of AAO mRNA increased during elongation of the cells. AAO activity in the culture medium, as well as ascorbate and dehydroascorbate contents in the cells, also increased during the elongation. We propose that AAO expression and production of dehydroascorbate are under the control of the cell cycle and that AAO may function apoplastically as an ascorbate oxidizer in the process of cell elongation.     

Expression of Ascorbic Acid Oxidase in Zucchini Squash (Cucurbita pepo L.)

The expression of ascorbic acid oxidase was studied in zucchini squash (Cucurbita pepo L.), one of the most abundant natural sources of the enzyme. In the developing fruit, specific activity of ascorbic acid oxidase was highest between 4 and 6 days after anthesis. Protein and mRNA levels followed the same trend as enzyme activity. Highest growth rate of the fruit occurred before 6 days after anthesis. Within a given fruit, ascorbic acid oxidase activity and mRNA level were highest in the epidermis, and lowest in the central placental region. In leaf tissue, ascorbic acid oxidase activity was higher in young leaves, and very low in old leaves. Within a given leaf, enzyme activity was highest in the fast-growing region (approximately the lower third of the blade), and lowest in the slow-growing region (near leaf apex). High expression of ascorbic acid oxidase at a stage when rapid growth is occurring (in both fruits and leaves), and localization of the enzyme in the fruit epidermis, where cells are under greatest tension during rapid growth in girth, suggest that ascorbic acid oxidase might be involved in reorganization of the cell wall to allow for expansion. Based on the known chemistry of dehydroascorbic acid, the end product of the ascorbic acid oxidase-catalyzed reaction, we have proposed several hypotheses to explain how dehydroascorbic acid might cause cell wall “loosening.”     

Studies on plant growth-regulating substances XXVII. The growth-regulating activity and metabolism of 2,4-dichlorophenoxyethylamine and related compounds in higher plants

2-(2,4-Dichlorophenoxy)ethylamine (2,4-D ethylamine) was converted to 2,4-dichlorophenoxyacetaldehyde (2,4-D acetaldehyde) by extracts of pea cotyledons. The 2,4-D acetaldehyde was further converted to 2,4-dichloro-phenol and 2,4-dichlorophenoxyacetic acid (2,4-D). Under the same conditions, 2-(2,6-dichlorophenoxy)ethylamine was converted to 2,6-dichloro-phenoxyacetaldehyde and 2,6-dichlorophenol, although at a relatively slow rate. In pea stem segments and wheat coleoptiles the main products of 2,4-D ethylamine metabolism were 2,4-dichlorophenol, 2,4-D acetaldehyde and 2,4-D. In comparison with the wheat coleoptiles, larger amounts of these products were found in the pea stem segments. Metabolism of 2,4-D acetaldehyde gave 2-(2,4-dichlorophenoxy)ethanol (2,4-D ethanol) and 2,4-D in both pea and wheat tissues. Pretreatment with the amine oxidase inhibitor, 2-hydroxyethylhydrazine (HEH) completely prevented the extension of pea stem segments and substantially prevented the extension of wheat coleoptiles on subsequent treatment with 2,4-D ethylamine. No such protection was found against 2,4-D acetaldehyde or 2,4-D after pretreating the tissues with HEH. In pea, radish, and tomato plants, epinasty resulted from treatment with 2,4-D ethylamine, 2,4-D acetaldehyde and 2,4-D. Prior treatment with HEH prevented the epinasty due to the 2,4-D ethylamine, but no protection was given by HEH against 2,4-D acetaldehyde or 2,4-D.     

Changes in the Ascorbate System during Seed Development of Vicia faba L

Large changes occur in the ascorbate system during the development of Vicia faba seed and these appear closely related to what are generally considered to be the three stages of embryogenesis. During the first stage, characterized by embryonic cells with high mitotic activity, the ascorbic acid/dehydroascorbic acid ratio is about 7, whereas in the following stage, characterized by rapid cell elongation (stage 2), it is lower than 1. The different ascorbic/dehydroascorbic ratio may be correlated with the level of ascorbate free radical reductase activity, which is high in stage 1 and lower in stage 2. Ascorbate peroxidase activity is high and remains constant throughout stages 1 and 2, but it decreases when the water content of the seed begins to decline (stage 3). In the dry seed, the enzyme disappears together with ascorbic acid. Ascorbate peroxidase activity is observed to be 10 times higher than that of catalase, suggesting that ascorbate peroxidase, rather than catalase, is utilized in scavenging the H₂O₂ produced in the cell metabolism. There is no ascorbate oxidase in the seed of V. faba. V. faba seeds acquire the capability to synthesize ascorbic acid only after 30 days from anthesis, i.e. shortly before the onset of seed desiccation. This suggests that (a) the young seed is furnished with ascorbic acid by the parent plant throughout the period of intense growth, and (b) it is necessary for the seed to be endowed with the ascorbic acid biosynthetic system before entering the resting state so that the seed can promptly synthesize the ascorbic acid needed to reestablish metabolic activity when germination starts.     

The Role of Cellulase and Polygalacturonase in Abscission of Young and Mature Shamouti Orange Fruits

During the first eight weeks after setting young citrus fruits gradually lose their ability to abscise at the abscission zone between the stem and the pedicel; in fruits older than eight weeks abscission occurs at the calyx area. The activity of cellulase and polygalacturonase in the two abscission zones was markedly increased before and during abscission, and was localized mainly in the abscission zone. Ethylene accelerated the increase in enzymic activity after an 8- to 10-h lag period; 2,4-D delayed abscission and enzymic activity when applied during the first 24 h after excision. During this period 2,4-D also partly suppressed the enhancing effect of ethylene. Early application of cyclo-heximide inhibited the formation of the enzymes and thus abscission was delayed to a certain extent. Although there are some indications that the relationship between enzymic activity and abscission is a complex one, the data presented indicate that cellulase and polygalacturonase play a significant role in abscission of citrus fruits at various developmental stages. Both enzymes act almost simultaneously and are equally controlled by ethylene and 2,4-D.     

Is uronic acid oxidase involved in the hormonal regulation of abscission in explants of citrus leaves and fruits?

The role of uronic acid oxidase in abscission was studied in explants of citrus ( Citrus sinensis L. Osbeck; var. Shamouti) leaves and fruits. In leaf explants, activity of uronic acid oxidase prior to onset of abscission and the rate of abscission were markedly accelerated by ethylene and delayed by 2,4-dichlorophenoxyacetie acid. Similar results were obtained for uronic acid oxidase activity in the exocellular fraction of young fruit explants. In mature fruit explants, treated with ethylene, an immediate increase in activity was evidenty in the non-active shoot/peduncle abscission zone, whereas in the calyx abscission zone the rise in activity occurred after a prolonged exposure to ethylene, when most of the fruits had already abscised. Whenever ethylene enhanced uronic acid oxidase activity, 2,4-dichlorophenoxyacetic acid delayed it. A gradient of decreasing activity or uronic acid oxidase was recorded from both sides of the abscission zone in leaves and fruits toward the separation line, where activity was the lowest as compared with the activity found in adjacent tissues. It is suggested that uronic acid oxidase is involved in senescence and cell wall degradation. However, it is yet questionable whether this enzyme is directly related to the control mechanism of abscission.     

Induction and peroxisomal appearance of gulonolactone oxidase upon clofibrate treatment in mouse liver

Various antihyperlipemic peroxisome proliferators are known to be carcinogenic in rodents but not in human, other primates and guinea pig, which species lost their ability to synthesize ascorbate due to mutations in the gulonolactone oxidase gene. Ascorbate synthesis is accompanied by H2O2 production, consequently its induction can be potentially harmful; therefore, the in vivo effect of the peroxisome proliferator clofibrate was investigated on gulonolactone oxidase expression in mouse liver. Liver weights and peroxisomal protein contents were increased upon clofibrate treatment. Elevated plasma ascorbate concentrations were found in clofibrate-treated mice due to the higher microsomal gulonolactone oxidase activities. Remarkable gulonolactone oxidase activity appeared in the peroxisomal fraction upon the treatment. Increased activity of the enzyme was associated with an elevation of its mRNA level. According to the present results the evolutionary loss of gulonolactone oxidase may contribute to the explanation of the missing carcinogenic effect of peroxisome proliferators in humans.     

cDNA cloning and gene expression of ascorbate oxidase in tobacco

A cDNA clone for ascorbate oxidase (AAO) has been isolated from a cDNA library of tobacco (Nicotiana tabacum) cells. The identity of the amino acid sequence deduced from tobacco AAO cDNA to that from pumpkin AAO cDNA was 68%, which was much lower than the identity (80%) between pumpkin and cucumber AAO. AAO activity in tobacco cells was much lower than that in pumpkin cells, whereas the immunoreactive protein in tobacco cells was more abundant than that in pumpkin cells. We suppose that AAO protein in tobacco cells may be less active than that in pumpkin cells. Genomic Southern blotting suggested that AAO in tobacco was encoded by a single-copy gene. Northern blotting revealed that mRNA of AAO was highly expressed in young and growing tissues of tobacco plant.     

In vitro development of parthenocarpic fruits of Crocus sativus L.

In vitro development of parthenocarpic fruits of Crocus sativus L. was induced by culturing ovaries on MS agar medium supplemented with growth-regulators (2,4-D, GA₃ and BAP). Amongh these, 2,4-D was the most effective in promoting fructification. The fructigenic activity was independent of both the stage at which the ovaries were excised (before, during or after anthesis) and pollination of the stigmas. Unlike the above compounds, abscisic acid inhibited fructification.     

The Assimilation of Ureides in Shoot Tissues of Soybeans : 1. CHANGES IN ALLANTOINASE ACTIVITY AND UREIDE CONTENTS OF LEAVES AND FRUITS

The ureides, allantoin and allantoic acid, are major forms of N transported from nodules to shoots in soybeans (Merr.). Little is known about the occurrence, localization, or properties of the enzymes involved in the assimilation of ureides in shoot tissues. We have examined the capacity of the shoot tissues to assimilate allantoin via allantoinase (EC 3.5.2.5) during leaf and fruit development in nodulated soybeans. Specific activity of allantoinase in leaves peaked during pod formation and early seed filling. In developing fruits allantoinase activity in the seeds was 2 to 4 times that in the pods when expressed on a fresh weight or organ basis. In seeds, the embryos contained the highest specific allantoinase activity. Stems and petioles also had appreciable allantoinase activity. With development, peaks in the amounts of allantoic acid, but not allantoin, were measured in both leaves and fruits suggesting that the assimilation of allantoic acid may be a limiting factor in ureide assimilation. Highest amounts of ureides were measured in the pith and xylem of stem tissues and in developing pod walls.