Effect of Ethylene and Ethylene Precursors on Protein Phosphorylation and Xylogenesis in Tuber Explants of Helianthus tuberosus (L.)

Koritsas, V. M. 1988. Effect of ethylene and ethylene precursorson protein phosphorylation and xylogenesis in tuber explantsof Helianthus tuberosus (L.).J. exp. Bot. 39: 375–386. The role of ethylene in protein phosphorylation and xylem-celldifferentiation was studied in explant cultures of Jerusalemartichoke, Helianthus tuberosus (L.). Explants on xylem-inducingmedium growing at 25 °C developed more xylem elements andproduced more ethylene gas within 3 d of culture than controlcultures growing and developing at similar rates. L-methionineand 1-aminocdopropane-1-carboxylic acid (ACC) incorporated intoboth xylem-inducing and control media enhanced xylem differentiation,increased protein levels and stimulated protein kinase activityin the explants. Inhibitors of the ethylene biosynthetic pathway,cobalt nitrate and aminoethoxyinyl glycine (AVG), depressedxylem differentiation, protein phosphorylation and polypeptidesynthesis. Ethylene gas or ACC reversed the effects of the inhibitors.Ethylene production, protein phosphoryilation and xylem-celldifferentiation were all linked. Ethylene thus promotes xylem-celldifferentiation in Jerusalem artichoke explants, the processprobably being regulated by protein phosphorylation. Key words: Ethylene, phosphorylation, xylem differentiation, Jerusalem artichoke explants     

Effect of ATP on the EPR spectrum at 20°K of phosphorylating sub-mitochondrial particles

By measurements of the EPR spectrum of substrate-reduced anaerobic phosphorylating sub-mitochondrial particles at 20°K, ATP was found to bring about the oxidation of four components (two iron-sulphur proteins associated with NADH dehydrogenase, and two unidentified iron proteins) and the reduction of one component. Thus energization of the particles lowers the effective redox potential of four components and raises that of a fifth.     

Studies on the mechanism of inhibition of the mitochondrial electron transport by antimycin. III. Binding of antimycin to sub-mitochondrial particles and to complex III

1. Extraction by ether removes only about one-half of antimycin added to sub-mitochondrial particles, independently of the amount of antimycin added up to that necessary for 100% inhibition.

2. The amount of antimycin extractable with ether remains the same even when the antimycin is redistributed between an antimycin-inhibited preparation and an untreated. The antimycin remaining after ether extraction is redistributed between ether-accessible and ether-inaccessible sites when the preparation is incubated on its own or with an untreated preparation.

3. Low concentrations of cholate increase the extractability of the antimycin by ether.

4. Complex III binds antimycin more firmly than sub-mitochondrial particles. However, antimycin is readily extracted by ether, leading to restoration of enzymic activity and cleavage of the complex by bile salts.

5. The results are consistent with an explanation of the sigmoidal inhibition curve with antimycin and preparations of the intact respiratory chain in terms of an allosteric model.     

Studies of energy-linked reactions: stimulation of the mitochondrial Pi-ATP exchange reaction by oleoyl lipoate, oleoyl CoA and oleoyl phosphate.

Oleoyl lipoate, oleoyl CoA, oleoyl phosphate and dihydrolipoate have been shown to stimulate Pi-ATP exchange activity in sub-mitochondrial particles. A different sensitivity to uncouplers and inhibitors of oxidative phosphorylation is observed, indicating that the various oleoyl derivatives interact at different levels of a multistep reaction sequence, a postulated ‘oleoyl cycle.’     

Characterization of Mitotic Cycles Preceding Xylogenesis in Cultured Explants of Helianthus tuberosus L.

The number of mitotic cycles intervening between the transferof dormant Helianthus tuberosus (Jerusalem artichoke) tuberexplants to culture medium and the differentiation of the firsttracheary elements at 48 h was investigated by a pulse-labellingtechnique employing quantitative autoradiography. Silver-graincounts indicated that differentiation was preceded by threemitotic cycles Duration of the cell cycle phases were estimatedby a pulse-labelling method. From calculation of the phase durationsit was estimated that the first visible signs of tracheary elementdifferentiation occurred from 7–10 h after the last mitosis. Helianthus tuberosus L., Jerusalem artichoke, tracheary elements, cultured explants, tissue culture, mitotic cycles, cell cycle, tritiated thymidine, autoradiography     

YEAST SPORULATION AND RNA AS AFFECTED BY GIBBERELLIC ACID

Comparison with respect to the promoting effect on the yeastsporulation was made between RNA fractions prepared by a phenolmethod from two strains of yeast (Saccharomyces ellipsoideus),the one (KV2) which needs GA-treatment to be responsive to thecell-elongating action of auxin and the other (P 602) whichdoes not need it. For obtainting RNA preparation active in promotingthe sporulation, cells of the former strain had to be pretreatedwith GA, but the GA-treatment was unnecessary in the latterstrain. An RNA fraction that remarkably promoted sporulation in yeastwas obtained from the GA-treated tuber tissue of Jerusalem artichoke.The tuber tissue is known to become highly responsive to thecell expansion-promoting action of auxin through GA-treatment. In any case, the sporulation-promoting effect was found onlyin "phenol RNA" but not in "water RNA", and the former "RNA"was inactivated by the treatment with RNase. (Received November 1, 1966; )     

Discrimination between duroquinol oxidase activity and the terminal oxidation step of the cyanide-resistant electron transport pathway of plant mitochondria

Comparison of the cyanide-resistant duroquinol oxidase activity of sub-mitochondrial particles from Arum maculatum L. with their ability to carry out a cyanide-resistant oxidation of NADH and succinate shows that heat-inactivation of the duroquinol oxidase activity does not proportionally affect NADH and succinate oxidation. Moreover, 1 microM antimycin inhibits duroquinol oxidase activity by 50% while not decreasing the rates of NADH and succinate oxidation. Therefore, the cyanide-resistant electron transport does not appear to be mediated by a "duroquinol oxidase", and a convincing proof of the existence of a specific protein acting as a cyanide-resistant oxidase in plant mitochondria is still lacking.     

Resolution and reconstitution of the succinoxidase pathway of Mycobacterium phlei

Alkaline treatment of the electron transport particles of Mycobacterium phlei resulted in a loss of oxidation and coupled phosphorylation with succinate and NAD⁺-linked substrates but not with ascorbate-TPD as the electron donor. Furthermore, alkaline treatment of the electron transport particles resulted in dissociation of succinic dehydrogenase from the membrane vesicles. However, the membrane retained the menaquinone MK₉(II-H), cytochromes b, c₁ + c, and a + a₃. Restoration of oxidation and coupled phosphorylation with succinate was found to occur on addition of a succinic dehydrogenase preparation to the resolved particles. Silicotungstate treatment of ETP yielded particles deficient in succinie dehydrogenase. Furthermore, membrane-bound or solubilized-latent ATPase was inactivated in the presence of low concentration of silicotungstate. The addition of a soluble succinic dehydrogenase to the silicotungstate-treated particles resulted in the restoration of only oxidation.     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Properties of the oxidation of exogenous NADH and NADPH by plant mitochondria. Evidence against a phosphatase or a nicotinamide nucleotide transhydrogenase being responsible for NADPH oxidation

(1) The optimum pH for the oxidation of exogenous NADH by mitochondria from both Jerusalem artichoke (Helianthus tuberosus) tubers and Arum maculatum spadices was 7.0–7.1. NADPH oxidation had a lower optimum pH of 6.6 in Arum and 6.0 in Jerusalem artichoke mitochondria. In both types of mitochondria the rates of NADH and NADPH oxidation were identical below pH 6.0–5.5. (2) It is shown conclusively that neither a phosphatase converting NADPH to NADH nor a nicotinamide nucleotide transhydrogenase was involved in the oxidation of NADPH by these mitochondria. (3) Palmitoyl-CoA, an inhibitor of transhydrogenase activity in mammalian mitochondria, inhibits both NADH and NADPH oxidation by plant mitochondria with a K_i of about 10 μM. (4) It is concluded that the known properties of NAD(P)H oxidation are best explained by assuming the presence of a second dehydrogenase specific for NADPH. At low pH, electron flow from the two dehydrogenases to oxygen shares a common rate-limiting step.     

Partial Characterization of Artichoke Virus M

A virus with filamentous particles 697 nm in length was isolated from artichoke plants in Southern Italy and identified as a new possible member of Carlavirus group, for which the name artichoke virus M (AVM) is suggested. AVM could not be transmitted by sap inoculation to herbaceous hosts and was always present in artichoke in mixed infections with other viruses. Virus particles had a buoyant density in CsCl of 1.31 g × cm^?3 and contained a single species of nucleic acid with an apparent size of 7.5 Kb and a single coat protein species with a mol. wt of 31,000. The virus was distantly related serologically to carnation latent and poplar mosaic carlaviruses but not to other members of the group including the recently described artichoke latent S carlavirus. Cytological alterations consisted of complex cytoplasmic inclusions composed of deranged organelles, lipid droplets and accumulations of membranes.     

Binding of HQNO to beef-heart sub-mitochondrial particles

1. The fluorescence spectra of HQNO (2-n-heptyl-4-hydroxyquinoline-N-oxide) in water at pH 7.5 show an emission maximum at 480 nm and an excitation maximum at 355 nm.2. The fluorescence is enhanced by binding to bovine serum albumin, and is completely quenched by binding to sub-mitochondrial particles of beef heart.3. Binding experiments reveal specific binding of HQNO to sub-mitochondrial particles with a dissociation constant of 64 nM and, depending on the protein concentration, a considerable amount of aspecific binding.4. The concentration of specific binding sites for HQNO is identical with that of antimycin-binding sites. Furthermore, the presence of antimycin prevents the binding of HQNO and antimycin releases HQNO from its binding site.5. The binding of HQNO is not sensitive to the redox state of the respiratorychain components.6. Inhibition of electron transfer by HQNO is caused by binding to the specific binding site.7. The relation between inhibition of NADH or succinate oxidation and saturation of the binding site is hyperbolic.8. The increase in the reduction level of cytochrome b on addition of HQNO in the presence of succinate and oxygen, either in the presence or absence of cyanide, does not parallel the inhibition of overall electron transfer.9. All data can be quantitatively described and analysed using the model for electron transfer proposed by Wikström and Berden in 1972 (Wikström, M. K. F. and Berden, J. A. (1972) Biochim. Biophys. Acta 283, 403–420).     

Polyamines in Relation to Cell Division and Xylogenesis in Cultured Explants of Helianthus tuberosus: Lack of Evidence for Growth-Regulatory Action

Phillips, R., Press, M. C. and Eason, A. 1987. Polyamines inrelation to cell division and xylogenesis in cultured explantsof Helianthus tuberosus: lack of evidence for growth-regulatoryaction.—J. exp. Bot. 38: 164–172. The polyamines spermidine, diaminopropane, and cadaverine werefound to accumulate in cultured tuber explants of H. tuberosus(Jerusalem artichoke). Rapid increases in all amines occurredduring the initial 24 h corresponding to the period of activationand the onset of mitosis. Levels then declined during the followingphases of rapid cell proliferation and xylem differentiation.The type and distribution of polyamines was not markedly affectedby changes in medium or culture conditions, and the inhibitorMGBG did not alter cell division rates or polyamine contentmarkedly although xylem differentiation was substantially depressed.Exogenously supplied spermidine and putrescine did not substantiallyalter the cellular responses of explants cultured in the presenceof auxin. In the absence of supplied auxin, spermidine at 1?0mol m^–3 produced an increase in cell division, althoughthis was small in comparison with auxin-stimulated responses.The implications of these findings on the possibility that polyaminesact as growth regulators in plants is discussed. Key words: Polyamines, Jerusalem artichoke, cultured explants, cell division, xylem differentiation     

Rapid Phytochrome-Controlled Protein Phosphorylation and Dephosphorylation in Avena sativa L.

The time course for in vivo changes in the protein phosphorylationpattern was measured after red and red/far-red light. Avenacoleoptile tips were incubated in ³²P-labeled phosphate andirradiated. The supernatant fractions of homogenates were subjectedto SDS-poly-acrylamide gel electrophoresis and then autoradiographed.Within seconds, the radioactive label of two proteins decreasedand the radioactive label of one protein increased. These datasuggest that the phosphorylation states for these proteins maybe under phytochrome control. (Received July 20, 1987; Accepted July 20, 1988)     

ATP induces dephosphorylation of myosin light chain in endothelial cells

In cultured porcine aortic endothelial monolayers, theeffect of ATP on myosin light chain (MLC) phosphorylation, whichcontrols the endothelial contractile machinery, was studied. ATP (10 µM) reduced MLC phosphorylation but increased cytosolicCa²⁺ concentration ([Ca²⁺]_i).Inhibition of the ATP-evoked [Ca²⁺]_i rise byxestospongin C (10 µM), an inhibitor of the inositol trisphosphate-dependent Ca²⁺ release from endoplasmicreticulum, did not affect the ATP-induced dephosphorylation of MLC. MLCdephosphorylation was prevented in the presence of calyculin A (10 nM),an inhibitor of protein phosphatases PP-1 and PP-2A. Thus ATP activatesMLC dephosphorylation in a Ca²⁺-independent manner. In thepresence of calyculin A, MLC phosphorylation was incremented afteraddition of ATP, an effect that could be abolished when cellswere loaded with the Ca²⁺ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acidacetoxymethyl ester (10 µM). Thus ATP also activates aCa²⁺-dependent kinase acting on MLC. In summary, ATPsimultaneously stimulates a functional antagonism toward bothphosphorylation and dephosphorylation of MLC in which thedephosphorylation prevails. In endothelial cells, ATP is the firstphysiological mediator identified to activate MLC dephosphorylation bya Ca²⁺-independent mechanism.

     

Regulation of malate oxidation in plant mitochondria. Response to rotenone and exogenous NAD+.

Exogenous NAD+ stimulated the rotenone-resistant oxidation of all the NAD+-linked tricarboxylic acid-cycle substrates in mitochondria from Jerusalem artichoke (Helianthus tuberosus L.) tubers. The stimulation was not removed by the addition of EGTA, which is known to inhibit the oxidation of exogenous NADH. It is therefore concluded that added NAD+ gains access to the matrix space and stimulates oxidation by the rotenone-resistant NADH dehydrogenase located on the matrix surface of the inner membrane. Added NAD+ stimulated the activity of malic enzyme and displaced the equilibrium of malate dehydrogenase; both observations are consistent with entry of NAD+ into the matrix space. Analysis of products of malate oxidation showed that rotenone-resistant oxygen uptake only occurred when the concentration of oxaloacetate was low and that of NADH was high. Thus it is proposed that the concentration of NADH regulates the activity of the two internal NADH dehydrogenases. Evidence is presented to suggest that the rotenone-resistant NADH dehydrogenase is engaged under conditions of high phosphorylation potential, which restricts electron flux through the rotenone-sensitive dehydrogenase (coupled to ATP synthesis).     

Phloretin - an uncoupler and an inhibitor of mitochondrial oxidative phosphorylation

The effect of phloretin on respiration by isolated mitochondria and submitochondrial particles was studied. In submitochondrial particles, both NADH- and succinate-dependent respiration was inhibited by phloretin. 50% maximum inhibition was reached at phloretin concentrations of 0.1 mM (NADH oxidation) and 0.7 mM (succinate oxidation). In isolated mitochondria, phloretin inhibited glutamate oxidation in both State 3 and State 4; 50% maximum inhibition occurred at about 30 microM. Succinate oxidation is inhibited in State 3 by phloretin, inhibition being half its maximum value at 0.5 mM, but in State 4 it is stimulated about 2-fold by phloretin at a concentration of 0.6 mM. Ascorbate oxidation is stimulated in both State 3 and State 4, maximum stimulation being equal to that obtained with an uncoupler of oxidative phosphorylation. Under all circumstances, phloretin lowered the transmembrane electrical potential difference in isolated mitochondria. These results are discussed in terms of mosaic non-equilibrium thermodynamics. We conclude that phloretin is both an uncoupler and an inhibitor of oxidative phosphorylation.     

NAD (P)H-Duroquinone Reductase in the Plant Plasma Membrane

The intracellular distribution of NADPH- and NADH-dependentduroquinone reductase (NAD (P)H-DQR) from etiolated zucchinihypocotyls (Cucurbita pepo L.) was investigated. About 80% ofthis enzyme is in the supernatant fraction and is probably cytosolic.Particulate NAD (P)H-DQR was largely (42%) found in associationwith the plasma membrane and was strongly stimulated by TX100.Another 33% of NAD (P)H-DQR was associated with mitochondria,and minor fractions with the endoplasmic reticulum (8%) andother particles. All these fractions were little or not stimulatedby TX100. The distribution of detergent-activated NAD (P)H-DQRis thus distinct from microsomal NADH- and NADPH-CCR. The plasma membrane was purified from microsomal fractions bymetrizamide plus sucrose density gradient centrifugation orby PEG/dextran phase partitioning. Both types of particle preparationspeaked at a density (d) of 1.165 g cm^–3 in sucrose gradientsand contained substantial TX100-sensitive NADH-DQR, TX100-stimulatedNAD (P)H-DQR, together with traces of NADH-CCR and trapped ‘soluble’enzyme (MDH, NADP-malic enzyme) activities. In isopycnic gradientsof unfractionated microsomes, however, trapped enzymes peakedat d 1.155 whereas NAD (P)H-DQR peaked at d 1.165 and GSII atd 1.170, probably revealing plasma membrane heterogeneity. Furtherevidence of heterogeneity was provided by fractionation of plasmamembrane vesicles on dextran step-gradients. Most of the trapped MDH was released to the supernatant by sonicationor treatment with 0.0125% TX100. Under these conditions mostof the NAD (P)H-DQR sedimented with the membranes. It is concludedthat NAD (P)H-DQR is bound to the inside of plasma membranevesicles, but a fraction (7 to 31%) may be ‘soluble’and sequestered within the vesicle lumen. Part of the detergent-sensitiveNADH-DQR may be externally bound and accessible to non-permeatingsubstrates. Key words: Cucurbita, NAD (P)H-quinone reductase, plasma membrane     

The Intramitochondrial Localization of Superoxide Dismutase from Hypocotyl of Etiolated Soybean Seedlings

In the KCN inhibition experiment, it was found that superoxide dismutase from the hypo- cotyl of etiolated soybean seedlings which is composed of 88% Mn-SOD and 12% Cu-Zn-SOD. Latent activity of SOD was evidently liberated after the mitochondria were treated by supersonication. Most of SOD activity was present in supernatant fraction after the supersonicated mitochondria were centrifuged at 150000 g for 60 min. The method of separation and praparation of sub-mitochondrial fraction is described in this paper, and the results indicate that the 80% of mitochondrial SOD present in mitochondrial matrix fraction and a small amount SOD (16%) present in the mitochondrial intermembrane space. The results indicate that distribution and localization of intramitochondrial SOD from higher plant tissues are similar to that from animal tissues.     

On the Mechanism of Respiratory Control

Control of oxidation is the key mechanism in the regulation of energy metabolism. In glycolysis the oxidation of glyceraldehyde-3-phosphate is controlled by DPNH, which inhibits glyceraldehyde-3-phosphate dehydrogenase. In oxidative phosphorylation the inhibition of electron flow from DPNH to oxygen, called "respiratory control," is the subject of this paper. After a discussion of the physiological significance of the "tight coupling" between phosphorylation and oxidation, studies on "loosely coupled" submitochondrial particles are reported. These particles are capable of oxidative phosphorylation in the presence of a suitable phosphate acceptor system, but in contrast to controlled, intact mitochondria they oxidize DPNH in the absence of phosphate and ADP. The addition of o-phenanthroline to submitochondrial particles gives rise to an inhibition of respiration, which is partly reversed by phosphate and ADP or by dinitrophenol. The properties of this model system of respiratory control will be described.