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.     

Regulation of ascorbate oxidase expression in pumpkin by auxin and copper

Ascorbate oxidase expression in pumpkin (Cucurbita spp.) tissues was studied. Specific ascorbate oxidase activities in pumpkin leaf and stem tissues were about 2 and 1.5 times that in the fruit tissues, respectively. In seeds, little ascorbate oxidase activity was detected. Northern blot analyses showed an abundant ascorbate oxidase mRNA in leaf and stem tissues. Fruit tissues had lower levels of ascorbate oxidase mRNA than leaf and stem tissues. Ascorbate oxidase mRNA was not detected in seeds. Specific ascorbate oxidase activity gradually increased during early seedling growth of pumpkin seeds. The increase was accompanied by an increase in ascorbate oxidase mRNA. When ascorbate oxidase activity in developing pumpkin fruits was investigated, the activities in immature fruits that are rapidly growing at 0, 2, 4, and 7 d after anthesis were much higher than those in mature fruits at 14 and 30 d after anthesis. The specific activity and mRNA of ascorbate oxidase markedly increased after inoculation of pumpkin fruit tissues into Murashige and Skoog's culture medium in the presence of an auxin such as 2,4-dichlorophenoxyacetic acid (2,4-D) but not in the absence of 2,4-D. In the presence of 10 mg/L of 2,4-D, ascorbate oxidase mRNA was the most abundant. Thus, ascorbate oxidase is induced by 2,4-D. These results indicate that ascorbate oxidase is involved in cell growth. In pumpkin callus, ascorbate oxidase activity could be markedly increased by adding copper. Furthermore, immunological blotting showed that the amount of ascorbate oxidase protein was also increased by adding copper. However, northern blot analyses showed that ascorbate oxidase mRNA was not increased by adding copper. We suggest that copper may control ascorbate oxidase expression at translation or at a site after translation.     

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     

Ascorbate enhancement of H1 histamine receptor sensitivity coincides with ascorbate oxidation inhibition by histamine receptors

Ascorbate has previously been shown to enhance both ₁- and ₂-adrenergic activity. This activity is mediated by ascorbate binding to the extracellular domain of the adrenergic receptor, which also decreases the oxidation rate of ascorbate. H1 histamine receptors have extracellular agonist or ascorbate binding sites with strong similarities to _1- and ₂-adrenergic receptors. Physiological concentrations of ascorbate (50 µM) significantly enhanced histamine contractions of rabbit aorta on the lower half of the histamine dose-response curve, increasing contractions of 0.1, 0.2, and 0.3 µM histamine by two- to threefold. Increases in ascorbate concentration significantly enhanced 0.2 µM histamine (5–500 µM ascorbate) and 0.3 µM histamine (15–500 µM ascorbate) in a dose-dependent manner. Histamine does not measurably oxidize over 20 h in oxygenated PSS at 37°C. Thus the ascorbate enhancement is independent of ascorbate's antioxidant effects. Ascorbate in solution oxidizes rapidly. Transfected histamine receptor membrane suspension with protein concentration at >3.1 µg/ml (56 nM maximum histamine receptor) decreases the oxidation rate of 392 µM ascorbate, and virtually no ascorbate oxidation occurs at >0.0004 mol histamine receptor/mol ascorbate. Histamine receptor membrane had an initial ascorbate oxidation inhibition rate of 0.094 min·µg protein^–1·ml^–1, compared with rates for transfected ANG II membrane (0.055 min·µg protein^–1·ml^–1), untransfected membrane (0.052 min·µg protein^–1·ml^–1), creatine kinase (0.0082 min·µg protein^–1·ml^–1), keyhole limpet hemocyanin (0.00092 min·µg protein^–1·ml^–1), and osmotically lysed aortic rings (0.00057 min·µg wet weight^–1·ml^–1). Ascorbate enhancement of seven-transmembrane-spanning membrane receptor activity occurs in both adrenergic and histaminergic receptors. These receptors may play a significant role in maintaining extracellular ascorbate in a reduced state. molecular complementarity; vitamin C; seven-transmembrane-spanning membrane receptors     

Changes in respiratory metabolism in tissue cultures of carrot root

Changes in respiratory metabolism accompanied by callus formationin cultured explants of carrot root were followed and the followingresults were obtained. 1) When the explant was cultured on amedium containing kinetin and 2,4-D, active cell division occurredand resulted in callus formation by the 9th–12th days.2) Fresh weight remarkably increased after a lag-time of about5 days. Changes in protein content on fresh weight basis weresimilar to changes in fresh weight. 3) Respiration rate increasedduring the first few days, when growth could not be distinctlymeasured. Accompanying the rise in respiration, the C₆/C₁ ratioalso increased. As callus developed, the respiratory rate andC₆/C₁ ratio gradually decreased and RQ, became higher than unity.4) Alcohol dehydrogenase activity increased between the 4thand 9th days after culture. 5) When sub-cultured callus tissuewas fed with G-U-¹⁴C, some radioactivity was detected in thealcohol of the tissues. 6) These results suggest that duringthe first 4–6 days after culture the activity of the EMBDEN-MEYERHOF-PARNAS-TCApathway was remarkably increased and, as callus developed, therelative participation of the pentose phosphate pathway graduallyincreased and simultaneously alcohol fermentation occurred. (Received December 13, 1968; )     

Effects of some SH-inhibitors and EDTA on flowering in Lemna perpusilla 6746

Lemna perpusilla 6746, a short-day duckweed, flowered undercontinuous illumination on M-sucrose medium containing CuSO₄,AgNO₃ and HgCl₂, which are SH-inhibitors. The optimum concentrationsof CuSO₄, AgNO₃ and HgCl₂ were 5, 1 and 20 µM, respectively.Other metal ions tested were ineffective, but at least two otherSHinhibitors, potassium ferricyanide and iodoacetamide, alsoinduced long-day flowering at the concentrations of 0.1-1 µM. Adding 50 µM EDTA to the medium prevented the effect ofcupric ion, but not that of other SH-inhibitors. EDTA at 200µM induced some long-day flowering when added to a mediumwith no SH-inhibitors. It also permitted some flowering whenadded together with cupric ion, and accelerated flowering inthe presence of the other SHinhibitors listed above. EDTA andSH-inhibitor effects appeared to be additive. (Received May 25, 1973; )     

Establishment and physiological analyses of photoautotrophic callus cultures of the fern Platycerium coronarium (Koenig) Desv. under CO2 enrichmen

Gametophyte-derived callus cultures of Platycerium coronariumcould be maintained under photoautotrophic conditions on Murashigeand Skoog medium supplemented with 2µM 2,4-dichlorophenoxyaceticacid (2,4-D) and with CO₂ enrichment. Progressive reductionof sucrose from the medium resulted in a reduction in growth,but an increase in total chlorophyll content. When subculturingwas delayed beyond 2 weeks, callus cells differentiated intogametophytes on the medium with 0.2 sucrose and no CO₂ enrichment.Enriching the photoautotrophic cultures on 2µM 2, 4-Dwith 1% CO₂ resulted in about 1.7-fold increase in fresh weightwithin 42 d. Total chlorophyll content was generally higherwith 1% CO₂ enrichment than with 10%. F_v/F_m ratio was higherfor callus on low levels of sucrose (>0.5%) than that onsucrose 1.0%. An increase in autofluorescence of chloroplasts,but not the size, was observed with decreasing sucrose levelsin the medium. Autofluorescence decreased with increase in CO₂from 0.03%. Our data are in agreement with the view that long-termexposure to high levels of decrease in photosynthetic capacity. Key words: Platycerium coronarium, stag's horn fern, autofluorescence of chloroplasts, confocal laser scanning microscope, F_v/F_m ratio, photoautotrophic callus     

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.     

Increase in Enzyme Activities Related to Ascorbate Metabolism during Cold Acclimation in Poplar Twigs

Activities of ascorbate free radical reductase, ascorbate peroxidase,dehydroascorbate reductase, ascorbate oxidase and catalase inthe twigs of poplar, Populus gelrica, were measured throughouta year. Activity levels of the first three enzymes were highduring the wintering period of the life cycle, and the changesin the three enzyme activities occurred simultaneously. In fall,when the growth and enlargement of the tissues began to cease,the activities began to increase. In contrast to the activitiesof the above three enzymes, catalase activity began to increaseas the growth and enlargement proceeded, and the activity droppedby early November. Ascorbate levels in the twigs were measured,and the living bark and the xylem tissue were found to containsimilar levels of ascorbate (8 to 20 µmol and 2 to 14µmol per gram dry weight, respectively). From these results,it was suggested that in the growing period, deleterious H₂O₂produced in such organelles as the endoplasmic reticulum isdecomposed by catalase, and that in winter, oxidation and reductionreactions of ascorbate function not only to detoxify the peroxideproduced in the tissues, but also serve as a respiratory processto regenerate NADPH in the non-photosynthetic tissues of perennials. ¹Contribution No. 2624 from the Institute of Low TemperatureScience, Hokkaido University, Sapporo 060, Japan. (Received January 25, 1984; Accepted June 1, 1984)     

Increased Copper Content of the Medium Improves Plant Regeneration from Immature Embryo Derived Callus of Wheat (Triticum aestivum)

The effect of various concentrations of CuSO₄ on the induction and regeneration of embryogenic callus from immature embryos of wheat was investigated. Immature embryos of wheat cvs C-306 and R-3777 were cultured on MS medium supplemented with 2,4-D (11.3 µM) and different levels of cupric sulphate, i.e. 0, 0.1 (MS level), 0.5, 1 and 5 µM. Relatively high induction frequency of callus was obtained on MS medium supplemented with 2,4-D (11.3 µM) and 0.5 µM CuSO₄. The compact, nodular, embryogenic callus was maintained on the medium having 2,4-D (11.3 µM) and proline (86.8 µM) by regular subculturing. Plant regeneration from the embryogenic callus occurred on MS medium supplemented with NAA (1.07 µM) and BAP (44.4 µM). Regenerated plantlets were rooted on MSmedium supplemented with IAA (2.85 µM). The average number of regenerated plantlets produced from primary callus induced on 2,4-D (11.3 µM) and 5x CuSO₄ was significantly higher.     

Characterization of Sulfate Transport in the Green Alga Chlorella ellipsoidea

A rapid induction of sulfate transport was observed in the greenalga Chlorella ellipsoidea during sulfur-limited growth. Bothaffinity and V_max increased about five-fold within 6 h of transferringcells from Bold's basal medium with 350 µM MgSO₄ to sulfur-deficientBold's medium. High affinity sulfate transport was induced within15 min and reached maximum rate within 3 h of transferring cellsto sulfur-deficient condition, indicating that a new, high-affinity-sulfatetransport system is induced by sulfur starvation in C. ellipsoidea.Eadie-Hofstee plots of initial rates of sulfate uptake indicatedthat the K of sulfur-starved cells was about 17 µM. Bothsulfur-starved and unstarved cells grown in air had a V_max of1.5 times higher than that of high-CO₂ grown cells. Sulfatetransport was completely inhibited by 30 µM CCCP or 800µMKCN both in the light and the dark but transport in the lightwas not inhibited by 20 µM DCMU. Treatment with 50 µMor 500 µM vanadate caused 50% inhibition of uptake. Therate of sulfate uptake in the dark was twice that in the lightand was stimulated by low pH. These results suggest that thesulfate transport system in C. ellipsoidea is operated by protonsymport across the plasmamembrane which is partially mediatedby P-type ATPase and that these systems depend exclusively onenergy derived from oxidative phosphorylation in the mitochondria. (Received June 28, 1995; Accepted August 8, 1995)     

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.     

2,4-D-Sustained Photomixotrophic Growth of a Chlorophyllous Cell Suspension Culture of Nicotiana tabacum

A chlorophyllous, photomixotrophic cell suspension culture oftobacco (Nicotiana tabacum L.) was established using mediumcontaining 30 g/liter of sucrose and 1.5 µM 2,4-D. The2,4-D-sustained photomixotrophic line was able to show rapidregreening in the light after bleaching in the dark and characterizedwith a much slower and longer growth cycle than a heterotrophicline derived from the same original callus (cell doubling timeof 100 h vs. 40 h and duration of logarithmic phase of 17 daysvs. 7 days). The photomixotrophic line took up sucrose morerapidly than the heterotrophic line and accumulated starch duringthe early logarithmic phase when it showed a maximum photosyntheticcapacity on a chlorophyll basis (6.3µmol O₂/min/mg Chl).Chlorophyll content and photosynthetic capacity on a per cellbasis and on a cell fresh weight basis, on the other hand, decreasedduring this phase and reincreased later to reach maximum levels(310 µg Chl/g fr wt; 1.4 µmol O₂/min/g fr wt) whenthe line exhibited the highest activities of dark respiration(1.0 µmol; O₂/min/g fr wt) and cell division (mitoticindex of 3.0%). These characteristics of the photomixotrophicline were lost if it was grown in the dark to become non-chlorophyllous.Although net O₂ evolution could not be detected in the photomixotrophicline throughout the growth cycle when assayed under suboptimumlight intensity, reaccumulation of starch and a marked increasein cell fresh weight upon addition of minerals, vitamins and2,4-D without sucrose at the late logarithmic phase indicatedthe development of photosynthetic activity under the cultureconditions. ¹The investigations reported were included in the thesis submittedto the Graduate School, Faculty of Agriculture, Kobe University,in partial fulfillment of the requirement for M. Agr. degree. (Received May 30, 1988; Accepted October 5, 1988)     

Changes in Respiratory Metabolism during Callus Growth and Adventitious Root Formation in Jerusalem Artichoke Tuber Tissues

The CN-resistant alternative oxidase pathway was examined inrelation to the formation of callus and adventitious roots ofJerusalem artichoke tuber tissues. During the early stage ofincubation at 28?C, respiration was significantly activatedand the O₂-uptake rate via the Cyt pathway increased 2.5–2.6times by the second day of culture regardless of the presenceof 2,4-D. However, further increases and preservation of highlevels of Cyt pathway activities were observed only in the callusformingtissues. The capacity of the alternative pathway also increased,but the actually operating fraction was very small (0–4%of total respiration). On the other hand, during the later stagesof culture in which adventitious roots emerged from the callus,activation of the alternative pathway was observed. These resultssuggested that the alternative pathway was not involved in callusgrowth but was related to root formation in callus tissues ofJerusalem artichoke tubers. (Received October 29, 1986; Accepted April 20, 1987)     

Specific Inhibition of Electron Transfer for Nitrate Reduction by a Low Concentration of Cyanide in Rhodobacter sphaeroides f. s. denitrificans

The effects of cyanide on the electron flow in NO₃^– andNO₂^– reductions and photosynthetic electron transfer wereinvestigated with intact cells of a photodenitrifier, Rhodobactersphaeroides f. s. denitrificans. In the presence of 30 µMKCN, electron transfer for NO₃^– reduction was inhibitedby about 70% and the concomitant H translocation was completelyinhibited. However, neither NO₂^– reduction nor photosyntheticcyclic electron transfer was affected at 30 µM. Theseresults suggested that the electron transfer pathway to NO₃^–has, in addition to a b-type cytochrome and the nitratereductase,a component sensitive to a low concenration of cyanide whichis not involvedin the cytochrome bc₁ complex. (Received April 13, 1987; Accepted July 23, 1987)     

Effect of tunicamycin on the activity and immunoreactivity of ascorbate oxidase (Cucurbita pepo medullosa) expressed in cultured green zucchini cells

Ascorbate oxidase activity and immunoreactivity were evaluated in crude tissue extracts obtained from callus cell cultures induced by green zucchini sarcocarp and grown in the presence of tunicamycin, a powerful N-glycosylation inhibitor. Tunicamycin at 2 or 4 g ml–1 blocked cell growth within a couple of weeks, although a sustained cell viability was observed in the same period. A significant inhibition of total protein synthesis was observed at 10 and 15 days of culture time, with a decrease of 30% and 43% respectively when cells were grown in the presence of 2 g ml–1 tunicamycin, and of 48% and 57% respectively when the tunicamycin concentration was 4 g ml–1. After the same culture times ascorbate oxidase specific activity assayed in crude tissue extracts showed increases of about 1.9-fold and 3.5-fold (10 days) and 1.7-fold and 3.1-fold (15 days) at 2 and 4 g ml–1 tunicamycin, respectively. Ascorbate oxidase mRNA levels, however, did not appreciably differ between control and treated samples, measured at the same growing times. Lectin-blot, based on the use of concanavalin A, indicated a marked decrease of glycosylated proteins in tunicamycin-treated cultures. As judged by immunoblot, anti-native ascorbate oxidase antibodies scarcely recognized the enzyme expressed in tunicamycin-treated cells; on the contrary, anti-deglycosylated ascorbate oxidase antibodies were more reactive to the enzyme expressed in tunicamycin-treated cultures.     

Preliminary characterization of 1-aminocyclopropane-1-carboxylate oxidase properties from embryonic axes of chick-pea (Cicer arietinum L.) seeds

For a deeper understanding of the germination of chick–pea(Cicer arietinum) seeds, which is dependent upon ethylene synthesis,a crude extract containing authentic ACC oxidase (ACCO) activitywas isolated in soluble form from the embryonic axes of seedsgerminated for 24 h. Under our optimal assay conditions (200mM HEPES at pH 7.0, 4µM FeS0₄, 6 mM Na–ascorbate,1 mM ACC, 20% 0₂, 3% CO₂ , and 10%glycerol) this enzyme was5–fold more active than under the conditions we used initiallyin the present work. The enzyme has the following K_m: 28 µMfor ACC (approximately 4–fold less than in vivo), 1.2%for O₂ (in the presence of an optimal CO₂ concentration of 3%),and 1% for CO₂ in the presence of O₂ (20%). The enzyme is inhibitedby phenanthroline (PNT) (specific chelating agent of ferrousion), and competitively inhibited (K₁, =0.5 mM) by 2–aminoisobutyricacid (AIB), and the enzymatic activity was not detectable inthe absence of CO₂. Under optimal assay conditions, the enzymehas two optimum temperatures (28 C and 35 C) and is inhibitedby divalent metal cations (Zn²⁺> CO²⁺>Ni²⁺>Cu²⁺>Mn²⁺>Mg²⁺) and by salicylic acid, propylgallate, carbonyl cyanidem–chlorophenyl hydrazone (CCCP), dinitrophenol (DNP),and Na–benzoate. The in vitro ACCO activity which we recoveredin soluble form is equivalent to approximately 80–85%of the apparent activity evaluated in vivo. Key words: ACC oxidase, Cicer arietinum, ethylene, germination, seeds     

The ascorbate system and lipid peroxidation in stored potato (Solanum tuberosum L.) tubers

The changes in the components of the ascorbateglutathione systemduring the storage of potato (Solanum tuberosum L. cv. Spunta)tubers for 40 weeks at both 3C and 9C were studied in relationto lipid peroxidation. The level of malondialdehyde was foundto be higher at 3C than at 9C throughout storage. Thus, lipidperoxidation, which is the main cause of membrane deterioration,was accelerated at the lower temperature. Catalase activityincreased throughout storage independently of temperature. Theascorbate content of tubers decreased during storage both at3C and at 9C, as in other ageing processes. However, ascorbateperoxidase activity reached a maximum after about 8 weeks ofstorage, then declined at 9C, but held a higher level at 3C.The dehydroascorbic content also reached a maximum after about8 weeks and was significantly higher in tubers stored at 3C.These findings indicate a greater utilization of ascorbate byascorbate peroxidase at the lower temperature. Ascorbate freeradical reductase, dehydroascorbate reductase and glutathionereductase, the enzymes involved in the regeneration of ascorbate,were not affected by temperature and remained quite unchangedthroughout storage. It can be concluded that the ascorbate systemis involved in the scavenging of the free-radicals responsiblefor lipid peroxidation in stored potato tubers, at least atlow temperatures and in the first period of storage. Key words: Ascorbate, lipid peroxidation, potato tubers, Solanum tuberosum L