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PubMed1986

Binding of Butyl Gallate to Plant Mitochondria : II. Relationship to the Presence or Absence of the Alternative Pathway.

[(14)C]butyl gallate was used in binding studies to investigate the cyanide-resistant respiratory pathway in mitochondria isolated from a variety of sources displaying varying levels of cyanide resistance. Highly cyanide-resistant mitochondria were isolated from aroid spadices, while moderately cyanide-resistant mitochondria were isolated from either mung bean (Vigna radiata L.) hypocotyls or carbon dioxide/oxygen/ethylene-treated tubers. Totally cyanide-sensitive mitochondria were isolated from untreated tubers and rat liver. With one exception, all the plant mitochondria showed a reversible butyl gallate binding site which saturated at a level of 1.0 to 2.0 nanomoles per milligram protein. The exception, freshly harvested white potato tubers (<1 month from harvest), showed little specific butyl gallate binding, and also showed no appreciable induction of the cyanide-resistant pathway following carbon dioxide/oxygen/ethylene treatment. Only a low level, linear binding, well below that seen with plant mitochondria, was observed with rat liver mitochondria. Taken together, these results suggest a model for the interaction of the alternative pathway with the cytochrome pathway. In this model, the butyl gallate binding site (alternative oxidase) is a constitutive component in those mitochondria that are capable of developing the alternative pathway, and the binding sites associated with a second, inducible component that functions to couple the oxidase to the cytochrome pathway.

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PubMed1986

Alternative Respiratory Path Capacity in Plant Mitochondria: Effect of Growth Temperature, the Electrochemical Gradient, and Assay pH.

Influence of growth temperature on the capacity of the mitochondrial alternative pathway of electron transport was investigated using etiolated corn (Zea mays L.) seedlings. These seedlings were grown to comparable size in either a warm (30 degrees C) or a cold (13 degrees C) temperature regime, and then their respiration rates were measured as O(2) uptake at 25 degrees C. The capacity of the alternative pathway (KCN-insensitive O(2) uptake) was found essentially to double in shoots of cold-grown seedlings. This increased capacity slowly developed over several days growth in the cold, but was lost within 1 day when the seedlings were exposed to a warm regime. When mitochondria were isolated from the shoots of these seedlings, a greater potential for flow through the alternative path was observed in mitochondria from the cold-grown seedlings with all substrates used (an average increase of 84%). Using exogenous NADH as the substrate, the effect of the electrochemical gradient on measurable capacities of the cytochrome and alternative pathways was investigated in mitochondria from both etiolated seedlings and thermogenic spadices. The uncoupler FCCP (p-trifluoromethoxycarbonylcyanide phenylhydrazone) was used to diminish the electrochemical gradient when desired. In corn (Zea mays L.) shoot and mung bean (Vigna radiata L.) hypocotyl mitochondria, which have relatively low capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the electrochemical gradient was found not to influence the potential for flow through the alternative path. However, in mitochondria from skunk cabbage (Symplocarpus foetidus L.) and voodoo lily (Sauromatum guttatum Schott) spadices, which have high capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the gradient occurred at the expense of flow through the alternative pathway. These results suggest that in mitochondria of thermogenic spadices, the combined capacities of the cytochrome and alternative paths exceed the capacity of the exogenous NADH dehydrogenase. The effect of assay pH on measurable capacities of the cytochrome and alternative paths was determined over a pH range of 5.6 to 8.8 using exogenous NADH as the mitochondrial substrate. When the electrochemical gradient was present, it limited the electron transport rate and little effect of assay pH was observed. However, when formation of the gradient was prevented through inclusion of FCCP, measurable capacities of the cytochrome and alternative paths were found to be greatly influenced by pH. This experiment also revealed that the potential for respiratory control is largely dependent upon the assay pH.

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PubMed1986

Phase transitions in thylakoid polar lipids of chilling-sensitive plants: a comparison of detection methods.

The phase behavior of thylakoid polar lipids from plants sensitive to chilling injury was investigated by calorimetry, electron spin resonance spectroscopy of spin labels, and fluorescence intensity after labeling with trans-parinaric acid. The plants used were oleander (Nerium oleander), mung bean (Vigna radiata L. var Mungo), and tomato (Lycopersicon esculentum cv Grosse Lisse). For all plants the initiation temperature for the calorimetric exotherm was coincident (+/-1 degrees C) with the transition determined by the increase in the temperature coefficient of spin label motion and fluorescence intensity of trans-parinaric acid. For oleander plants, grown at 45 degrees C, the transition was at 7 degrees C while for plants from the same clone, grown at 20 degrees C, it was at -2 degrees C. For mung bean and tomato the transition was between 9 and 12 degrees C. The similarity in the transition detected by spin labeling and fluorescence intensity suggest that spin labels, like the fluorescent label trans-parinaric acid, preferentially partition into domains of ordered lipid. The coincidence of the temperature for initiation of the transition, determined by the three techniques, shows that each is a valid method of assessing a phase transition in membrane polar lipids.

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PubMed1986

Phase transitions in liposomes formed from the polar lipids of mitochondria from chilling-sensitive plants.

The thermal response of mitochondrial polar lipids from a variety of chilling-sensitive and chilling-insensitive plants was determined by differential scanning calorimetry. A phase transition was observed at 15 degrees C for mitochondria from soybeam (Glycine max. cv Davis) hypocotyl, at 16 degrees C for tomato (Lycopersicon esculentum cv Flora-Dade and cv Grosse Lisse) fruit, at 15 degrees C for cucumber (Cucumus sativus L.) fruit, at 14 degrees C for mung bean (Vigna radiata var Berken) hypocotyl, and at 15 degrees C for sweet potato (Ipomea batatas L.) roots. The transition temperature was not significantly altered by the scan rate and was reversible. Changes in the temperature coefficient of motion for a spin label, intercalated with the polar lipids, occurred at a temperature slightly below that of the phase transition, indicating that the polar lipids phase separate below the transition. No phase transition was observed for mitochondrial polar lipids from barley (Hordeum vulgare) roots, wheat (Triticum aestivum L. cv Falcon) roots, and Jerusalem artichoke (Helianthus tuberosus L.) tubers. The results show that a phase change occurs in the membrane lipids of mitochondria a few degrees above the temperature below which chilling injury is evident in the sensitive species. Thus they are consistent with the hypothesis that sensitivity to chilling injury is related to a temperature-induced alteration in the structure of cell membranes.

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PubMed1986

Preparation and properties of mitochondria from cowpea nodules.

Mitochondria were isolated from nodules of cowpea (Vigna unguiculata (L). Walp.) and purified on a Percoll gradient. They were only slightly contaminated by bacteroids (an average of 3.5%), and had low lipoxygenase activity. Compared to mitochondria from hypocotyls the nodule mitochondria had similar O(2) uptake rates and respiratory control ratios. The ADP/O ratios for both preparations were 1.4 to 1.7 and 2.3 to 2.6 with succinate and malate, respectively. Whereas mitochondria isolated from etiolated cowpea hypocotyls had 14 to 18% of their respiration insensitive to KCN, the respiration of nodule mitochondria was completely inhibited by KCN. Enzyme activities of nodule mitochondria were similar to those found in hypocotyl mitochondria, except for NAD(+)-malic enzyme which was 12-fold lower in the mitochondria from nodules.

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PubMed1986

Metabolism under Microaerobic Conditions of Mitochondria from Cowpea Nodules.

A method is described for isolating mitochondria from nodules of cowpea (Vigna unguiculata [L.] Walp.) under completely anaerobic conditions. The mitochondria were immediately active when incubated aerobically with substrates, and their respiration rates were higher than mitochondria prepared in air. The mitochondria lacked fumarate reductase and were not inhibited by 5% CO(2). When incubated under microaerobic conditions, their respiration could be measured by leghemoglobin spectroscopy. Microaerobic respiration was inhibited approximately 50% by 1 millimolar malonate, and was completely inhibited by cyanide. O(2) uptake and the ATP/O ratio declined under microaerobic conditions, and therefore ATP production may be low in the environment of infected nodule cells.

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PubMed1986

Root respiration associated with nitrate assimilation by cowpea.

Nitrate uptake by roots of cowpea (Vigna unguiculata) was measured using (15)NO(3) (-), and the energy cost to the root was estimated by respirometry. Roots of 8-day-old cowpea seedlings respired 0.6 to 0.8 milligram CO(2) per plant per hour for growth and maintenance. Adding 10 millimolar NO(3) (-) to the root medium increased respiration by 20 to 30% during the following 6 hours. This increase was not observed if the shoots were in the dark. Removal of NO(3) (-) from the root medium slowed the increase of root respiration. The ratios of additional respiration to the total nitrogen uptake and reduced nitrogen content in roots were 0.4 gram C per gram N and 2.3 grams C per gram N, respectively. The latter value is close to theoretical estimates of nitrate assimilation, and is similar to estimates of 1 to 4 grams C per gram N for the respiratory cost of symbiotic N(2) fixation.

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PubMed1986

Presence and identification of polyamines in xylem and Phloem exudates of plants.

Polyamines were identified by high performance liquid chromatography (benzoylation) and by thin layer chromatography (dansylation) in xylem exudates from stems of sunflowers (Helianthus annuus [L.]), mung bean (Vigna radiata [L.] Wilczek), grapevine (Vitis vinifera [L.] cv Grenache), and orange (Citrus sinensis [L.] Osbeck, cv Valencia), as well as in phloem sap (using elution into EDTA) of sunflower and mung bean plants. Putrescine was the major polyamine detected, ranging in concentrations of 150 to 9200 picomoles per milliliter exudate, whereas only trace amounts of spermine were detected. High amounts of putrescine and spermidine were found in EDTA eluates (possibly phloem sap) as compared with elution into water. Concentrations of putrescine and spermidine in xylem exudates were related to the physiological conditions of the plants prior to exudate collection. More putrescine was found in exudates of older than in younger sunflower plants, and salt stress applied to sunflower plants resulted in a higher concentration of putrescine and spermidine in the exudate. The presence and abundance of putrescine and spermidine in xylem and phloem exudates indicate that polyamines may be translocated in plants. This long-distance translocation further supports the hypothesis that polyamines have a regulatory role in plant growth and response to stress.

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PubMed1986

Species variation in the predawn inhibition of ribulose-1,5-bisphosphate carboxylase/oxygenase.

The activity of ribulose-1,5-bisphosphate carboxylase/oxygenase was measured in extracts of leaves collected before dawn (predawn activity, pa) and at midday (midday activity, ma). Twenty-three of the 37 species examined showed a pa/ma ratio (</=0.75, while only Capsicum frutescens, Cucumis sativa, Glycine max, Nicotiana tabacum, Vigna unguiculata, and 3 Solanum species showed a pa/ma ratio </=0.5. Phaseolus vulgaris consistently showed a pa/ma ratio of </=0.1. Activities and pa/ma ratios of the same species grown in the United States and the United Kingdom were very similar. Gel filtration of extracts before assay had no effect on the observed activities and the pa/ma ratios. These data are consistent with the hypothesis that in a number of species the enzyme is partially inhibited following the night period by the presence of a tight-binding inhibitor.

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PubMed1987

UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product.

A re-examination of the kinetic properties of UDP-glucose: (1-->3)-beta-glucan (callose) synthases from mung bean seedlings (Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca(2+) and millimolar concentrations of beta-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V(max) of the enzyme, and by lowering the apparent K(m) for UDP-glucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg(2+) markedly enhances the affinity of the mung bean enzyme for Ca(2+) but not for beta-glucoside; with saturating Ca(2+), Mg(2+) only slightly stimulates further production of glucan. However, the presence of Mg(2+) during synthesis, or NaBH(4) treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca(2+), beta-glucoside-activated cotton fiber enzyme, with or without Mg(2+), is very similar in size to callose isolated from cotton fibers, but is a linear (1-->3)-beta-glucan lacking the small amount of branches at C-0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg(2+)in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg(2+) on the conformation of the enzyme. Rate-zonal centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.

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PubMed1987

Abscission: ethylene and light control.

The role of ethylene in light control of leaf abscission im mung bean, Vigna radiata (L.) Wilczek cv Jumbo, cuttings was examined. While red light inhibits and far-red light promotes loss of break strength in abscission zones as compared with dark controls, changes in the rate of abscission could not be associated with changes in the rate of ethylene production. Reducing ethylene synthesis in tissue with aminoethoxyvinylglycine did not alter the effects of red or far-red light on abscission. Far-red light appeared to increase and red light appeared to decrease tissue sensitivity to ethylene.

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PubMed1987

Synthesis of only two heat shock proteins is required for thermoadaptation in cultured cowpea cells.

Cell cultures of a heat sensitive genotype of cowpea (Vigna unguiculata) were adapted to tolerate moderate levels of heat by maintaining cells at 32, 36, and 38 degrees C over many cell generations. Cells adapted to 32 and to 36 degrees C did not produce the typical heat shock proteins (HSP). Cells adapted to 38 degrees C synthesized two new proteins, which appear to be a subset of the HSP. In many temperature sensitive organisms it is thought that HSP confer thermotolerance. However, we hypothesize that specific proteins are associated with heat tolerance in cowpea, other heat tolerant plants (species such as sorghum and millet), and adapted cells which provide them with enhanced heat tolerance. From present data we suggest two proteins (70 and 80 kilodaltons) are strongly associated with heat tolerance and heat adaptation.

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PubMed1987

Chloroplast as a Locale of L-myo-Inositol-1-Phosphate Synthase.

Chloroplasts from 5 to 7 day old Vigna radiata seedling, grown under alternate light/dark conditions or from green Euglena gracilis Z. cells have been found to harbor L-myo-inositol-1-phosphate synthase (EC 5.5.1.4) activity. In contrast, dark-grown V. radiata seedlings, or streptomycin-bleached Euglena cells exhibit either reduced or no enzyme activity. An apparent enhancement of the chloroplastic inositol synthase by growth in presence of light is observed.

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PubMed1987

Inhibition of Mung Bean UDP-Glucose: (1-->3)-beta-Glucan Synthase by UDP-Pyridoxal: Evidence for an Active-Site Amino Group.

UDP-pyridoxal competitively inhibits the Ca(2+)-, cellobiose-activated (1-->3)-beta-glucan synthase activity of unfractionated mung bean (Vigna radiata) membranes, with a K(i) of 3.8 +/- 0.7 micromolar, when added simultaneously with the substrate UDP-glucose in brief (3 minute) assays. Preincubation of membranes with UDP-pyridoxal and no UDP-glucose, however, causes progressive reduction of the V(max) of subsequently assayed enzyme and, after equilibrium is reached, 50% inhibition occurs with 0.84 +/- 0.05 micromolar UDP-pyridoxal. This progressive inhibition is reversible provided that the UDP-pyridoxylated membranes are not treated with borohydride, indicating formation of a Schiff's base between the inhibitor and an enzyme amino group. Consistent with this, UDP-pyridoxine is not an inhibitor. The reaction of (1-->3)-beta-glucan synthase with UDP-pyridoxal is stimulated strongly by Ca(2+) and, less effectively, by cellobiose or sucrose, and the enzyme is protected against UDP-pyridoxal by UDP-glucose or by other competitive inhibitors, implying that modification is occurring at the active site. Pyridoxal phosphate is a less potent and less specific inhibitor. Latent (1-->3)-beta-glucan synthase activity inside membrane vesicles can be unmasked and rendered sensitive to UDP-pyridoxal by the addition of digitonin. Treatment of membrane proteins with UDP-[(3)H]pyridoxal and borohydride labels a number of polypeptides but labeling of none of these specifically requires Ca(2+) and sucrose; however, a polypeptide of molecular weight 42,000 is labeled by UDP-[(3)H]pyridoxal in the presence of Mg(2+) and copurifies with (1-->3)-beta-glucan synthase activity.

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PubMed1987

Carbon isotope ratios demonstrate carbon flux from c(4) host to c(3) parasite.

Carbon isotope ratios of mature leaves from the C(3) angiosperm root hemiparasites Striga hermonthica (Del.) Benth (-26.7 per thousand) and S. asiatica (L.) Kuntze (-25.6 per thousand) were more negative than their C(4) host, sorghum (Sorghum bicolor [L.] Moench cv CSH1), (-13.5 per thousand). However, in young photosynthetically incompetent plants of S. hermonthica this difference was reduced to less than 1 per thousand. Differences between the carbon isotope ratios of two C(3)-C(3) associations, S. gesnerioides (Willd.) Vatke-Vigna unguiculata (L.) Walp. and Oryza sativa L.-Rhamphicarpa fistulosa (Hochst.) Benth differed by less than 1 per thousand. Theoretical carbon isotope ratios for mature leaves of S. hermonthica and S. asiatica, calculated from foliar gas exchange measurements, were -31.8 and -32.0 per thousand, respectively. This difference between the measured and theoretical delta(13)C-values of 5 to 6 per thousand suggests that even in mature, photosynthetically active plants, there is substantial input of carbon from the C(4) host. We estimate this to be approximately 28% of the total carbon in S. hermonthica and 35% in S. asiatica. This level of carbon transfer contributes to the host's growth reductions observed in Striga-infected sorghum.

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PubMed1987

Conversion of xanthoxin to abscisic Acid by cell-free preparations from bean leaves.

Cell-free extracts from the leaves of Phaseolus vulgaris L. convert xanthoxin to abscisic acid. The enzyme activity in dialyzed or acetone-precipitated extracts shows a strong dependence on either NAD or NADP. The enzyme activity appears to be cytosolic with no significant activity observed in chloroplasts. The activity was observed in extracts from roots of Phaseolus vulgaris, and also in extracts prepared from the leaves of Pisum sativum L., Zea mays L., Cucurbita maxima Duchesne, and Vigna radiata L. Neither water stress nor cycloheximide appear to significantly affect the level of enzyme activity in leaves. No intermediates between xanthoxin and abscisic acid were detected.

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PubMed1987

Carbon Dioxide and Light Responses of Photosynthesis in Cowpea and Pigeonpea during Water Deficit and Recovery.

Greenhouse-grown pigeonpea (Cajanus cajan, [L.] Millsp.; cultivar UW-10) and cowpea (Vigna unguiculata, [L.] Walp.; cultivar California No. 5) were well-watered (control) or subjected to low water potential by withholding water to compare their modes of adaptation to water-limited conditions. Leaf CO(2) exchange rate (CER), leaf diffusive conductance to CO(2) (g(l)), and CO(2) concentration in the leaf intercellular air space (C(i)) were determined at various CO(2) concentrations and photon flux densities (PFD) of photosynthetically active radiation (400 to 700 nanometer). In cowpea, g(l) declined to less than 15% of controls and total water potential (psi(w)) at midafternoon declined to -0.8 megapascal after 5 days of withholding water, whereas g(l) in pigeonpea was about 40% of controls even though midafternoon psi(w) was -1.9 megapascal. After 8 days of withholding water, psi(w) at midafternoon declined to -0.9 and -2.4 megapascals in cowpea and pigeonpea, respectively. The solute component of water potential (psi(s)) decreased substantially less in cowpea than pigeonpea. Photosynthetic CER at saturation photon flux density (PFD) and ambient external CO(2) concentration (360 microliters per liter) on day 5 of withholding decreased by 83 and 55% in cowpea and pigeonpea, respectively. When measured at external, CO(2) concentration in bulk air of 360 microliters per liter, the CER of cowpea had fully recovered to control levels 3 days after rewatering; however, at 970 microliters per liter the PFD-saturated CERs of both species were substantially lower than in controls, indicating residual impairment. In stressed plants of both species the CER responses to C(i) from 250 to 600 microliters per liter indicated that a substantial nonstomatal inhibition of CER had occurred. Although the sensitivity of g(l) to water limitation in cowpea suggested a dehydration avoidance response, parallel measurements of CER at various C(i) and PFD indicated that photosynthetic activity of cowpea mesophyll was substantially inhibited by the water-limited treatment.

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PubMed1988

Pathways of Nitrogen Assimilation in Cowpea Nodules Studied using N(2) and Allopurinol.

In the presence of 0.5 millimolar allopurinol (4-hydroxypyrazolo [3,4-d]pyrimidine), an inhibitor of NAD:xanthine oxidoreductase (EC 1.2.3.2), intact attached nodules of cowpea (Vigna unguiculata L. Walp. cv Vita 3) formed [(15)N]xanthine from (15)N(2) at rates equivalent to those of ureide synthesis, confirming the direct assimilation of fixed nitrogen into purines. Xanthine accumulated in nodules and was exported in increasing amounts in xylem of allopurinol-treated plants. Other intermediates of purine oxidation, de novo purine synthesis, and ammonia assimilation did not increase and, over the time course of experiments (4 hours), allopurinol had no effect on nitrogenase (EC 1.7.99.2) activity. Negligible (15)N-labeling of asparagine from (15)N(2) was observed, suggesting that the significant pool (up to 14 micromoles per gram of nodule fresh weight) of this amide in cowpea nodules was not formed directly from fixation but may have accumulated as a consequence of phloem delivery.

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