Phosphoenolpyruvate metabolism in Jerusalem artichoke mitochondria

We report here initial studies on phosphoenolpyruvate metabolism in coupled mitochondria isolated from Jerusalem artichoke tubers. It was found that:

(1)

phosphoenolpyruvate can be metabolized by Jerusalem artichoke mitochondria by virtue of the presence of the mitochondrial pyruvate kinase, shown both immunologically and functionally, located in the inner mitochondrial compartments and distinct from the cytosolic pyruvate kinase as shown by the different pH and inhibition profiles.

(2)

Jerusalem artichoke mitochondria can take up externally added phosphoenolpyruvate in a proton compensated manner, in a carrier-mediated process which was investigated by measuring fluorimetrically the oxidation of intramitochondrial pyridine nucleotide which occurs as a result of phosphoenolpyruvate uptake and alternative oxidase activation.

(3)

The addition of phosphoenolpyruvate causes pyruvate and ATP production, as monitored via HPLC, with their efflux into the extramitochondrial phase investigated fluorimetrically. Such an efflux occurs via the putative phosphoenolpyruvate/pyruvate and phosphoenolpyruvate/ATP antiporters, which differ from each other and from the pyruvate and the adenine nucleotide carriers, in the light of the different sensitivity to non-penetrant compounds. These carriers were shown to regulate the rate of efflux of both pyruvate and ATP. The appearance of citrate and oxaloacetate outside mitochondria was also found as a result of phosphoenolpyruvate addition.

     

Transport and metabolism of D-lactate in Jerusalem artichoke mitochondria

We report here initial studies on D-lactate metabolism in Jerusalem artichoke. It was found that: 1) D-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-D-lactoyl-glutathione. 2) Externally added D-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) D-lactate was metabolized inside mitochondria by a flavoprotein, a putative D-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added D-lactate by means of a D-lactate/H(+) symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial D-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke.     

Some effects of tetrazolium salt on the metabolism of mitochondria isolated from Jerusalem artichoke tubers

Summary A series of tetrazolium salts were found to accept electrons more readily from succinate than malate even though the rate of oxygen uptake was similar with both substrates. This difference was explained by showing that all the tetrazolium salts tested caused a reduction in electron flow between NAD⁺ and Cyt.b. The tetrazolium salts were also found to be able to uncouple phosphorylation from electron transport. The monotetrazolium salts causing complete uncoupling around 100 moles/litre and the ditetrazolium salts causing complete uncoupling around 20 moles/litre.     

Transport and metabolism of d-lactate in Jerusalem artichoke mitochondria

We report here initial studies on d-lactate metabolism in Jerusalem artichoke. It was found that: 1) d-lactate can be synthesized by Jerusalem artichoke by virtue of the presence of glyoxalase II, the activity of which was measured photometrically in both isolated Jerusalem artichoke mitochondria and cytosolic fraction after the addition of S-d-lactoyl-glutathione. 2) Externally added d-lactate caused oxygen consumption by mitochondria, mitochondrial membrane potential increase and proton release, in processes that were insensitive to rotenone, but inhibited by both antimycin A and cyanide. 3) d-lactate was metabolized inside mitochondria by a flavoprotein, a putative d-lactate dehydrogenase, the activity of which could be measured photometrically in mitochondria treated with Triton X-100. 4) Jerusalem artichoke mitochondria can take up externally added d-lactate by means of a d-lactate/H⁺ symporter investigated by measuring the rate of reduction of endogenous flavins. The action of the d-lactate translocator and of the mitochondrial d-lactate dehydrogenase could be responsible for the subsequent metabolism of d-lactate formed from methylglyoxal in the cytosol of Jerusalem artichoke.     

Jerusalem artichoke mitochondria can export reducing equivalents in the form of malate as a result of D-lactate uptake and metabolism

We found that as a result of d-lactate uptake and metabolism by Jerusalem artichoke mitochondria, reducing equivalents were exported from the mitochondrial matrix to the exterior in the form of malate. The rate of malate efflux, as measured photometrically using NADP+ and malic enzyme, depended on the rate of transport across the mitochondrial membrane. It showed saturation characteristics (K(m) = 5 mM; V(max) = 9 nmol/min mg of mitochondrial protein) and was inhibited by non-penetrant compounds. We conclude that reducing equivalent export from mitochondria is due to the occurrence of a putative d-lactate/malate antiporter which differs from other mitochondrial carriers, as shown by the different inhibitor sensitivity.     

The increase of hydroxyproline-containing proteins in Jerusalem artichoke mitochondria during the development of cyanide-insensitive respiration

Mitochondria purified from freshly cut and from aged slices of Jerusalem artichoke tubers contain hydroxyproline proteins. These proteins are present in much higher amounts in mitochondria with KCN-insensitive respiration, i.e. in those from aged slices. It is suggested that the dependence of KCN-insensitive respiration on Vit. C is due to the fact that the development of this alternate oxidase requires the ascorbic acid-dependent synthesis of a hydroxyproline-containing mitochondrial protein.     

The purine nucleosidases of Jerusalem artichoke shoots

Extracts from Jerusalem artichoke shoots exhibited adenosine and inosine—guanosine nucleosidase activities. The results suggest the existence of two     

High-yield ethanol production from Jerusalem artichoke tubers

Fermentation conditions were optimized for the production of ethanol from Jerusalem artichoke with a strain of Saccharomyces cerevisiae able to use high-concentration juice and undiluted pulp. Yields (95 to 125 g ethanol/l=85 to 98% of the theoretical value) exceeded those obtained with strain of Kluyveromyces used classically.The authors are with the Laboratoire de Pharmacognosie et Biotechnologie, UFR Pharmacie, 28 Place Henri-Dunant, 63001 Clermont-Ferrand Cédex, France. H. Pourrat is the corresponding author.     

Ribosome metabolism in hormone-treated Jerusalem artichoke tuber slices in the absence and presence of 5-fluorouracil

In order to examine the relation of protein synthesis to the onset of growth, changes in ribosome content and activity were compared in aged, metabolically active Jerusalem artichoke (Helianthus tuberosus L.) slices incubated in water or 2,4-dichlorophenoxyacetic acid+kinetin. In water, cells do not grow or divide and rRNA and protein levels remain constant. The percentage membrane-bound (mb) ribosomes drops from 25% to 16% during 24h. At the same time the proportion of ribosomes active in protein synthesis in both free and mb populations declines from about 69% to 54%. In auxin+kinetin, cell expansion occurs and is accompanied by a 3-fold increase in rRNA and a 50% increase in total protein content. The percentage mb ribosomes remains at 25% throughout 48 h of growth. During the first 24h of growth 70% of ribosomes in both free and mb populations are active; this value declines to near water levels at 48 h. Considering the large increase in total ribosomes the number of synthetically active ribosomes is substantially increased during growth. 5-Fluorouracil (5-FU) does not inhibit hormone induced growth but does depress total rRNA content by about one-third. It also reduces [³H]uridine incorporation into ribosomes by 70% and the newly made ribosomes are mostly inactive in protein synthesis. On the other hand, the inhibitor does not significantly affect the proportion of total ribosomes active in protein synthesis and only partially reduces protein accumulation during the second 24 h of growth. It is suggested that while ribosome production is reduced in 5-FU, ribosome turnover is also retarded resulting in retention of near normal capacity for protein synthesis and growth.     

Localized coupling in oxidative phosphorylation by mitochondria from Jerusalem artichoke (Helianthus tuberosus)

Delocalized chemiosmotic coupling of oxidative phosphorylation requires that a single-value correlation exists between the extent of and the kinetic parameters of respiration and ATP synthesis. This expectation was tested experimentally in nigericin-treated plant mitochondria in single combined experiments, in which simultaneously respiration (in State 3 and in State 4) was measured polarographically, FΔψ (which under these conditions was equivalent to ) was evaluated potentiometrically from the uptake of tetraphenylphosphonium⁺ and the rate of phosphorylation was estimated from the transient depolarization of mitochondria during State 4-State 3-State 4 transitions. The steady-state rates of the different biochemical reactions were progressively inhibited by specific inhibitors active with different modalities on various steps of the energy-transducing process: succinate respiration was inhibited competitively with malonate or noncompetitively with antimycin A, or by limiting the rate of transport into the mitochondria of the respiratory substrate with phenylsuccinate; was dissipated by uncoupling with increasing concentrations of valinomycin; ADP phosphorylation was limited with oligomycin. The results indicate generally that when the rate of respiratory electron flow is decreased, a parallel inhibition of the rate of phosphorylation is also observed, while very limited effects can be detected on the extent of . This behavior is in marked contrast to the effect of uncoupling where the decreased rate of ATP synthesis is clearly due to energy limitation. Extending previous observations in bacterial photosynthesis and in respiration by animal mitochondria and submitochondrial particles the results indicate, therefore, that respiration tightly controls the rate of ATP synthesis, with a mechanism largely independent of . These data cannot be reconciled with a delocalized chemiosmotic coupling model.     

Biorefinery products from the inulin-containing crop Jerusalem artichoke

The polysaccharides in Jerusalem artichoke (JA) carry a substantial amount of energy that can be partly accessed through bioconversion into storable fuels. We review the potential for converting inulin into a variety of high value-added biorefinery products, including biofuels and biochemicals, and consider the feasibility of regarding JA as a model species of an inulin-rich crop. We discuss feedstock pretreatment, microorganisms used during fermentation, biorefinery products derived from JA, and how to enhance the economic competitiveness of JA as an energy crop.     

Photosynthetic characterization of Jerusalem artichoke during leaf expansion

Gas exchange, chlorophyll a fluorescence and modulated 820 nm reflection were investigated to explore the development of photosynthesis in Jerusalem artichoke (Helianthus tuberosus L.) leaves from initiation to full expansion. During leaf expansion, photosynthetic rate (Pn) increased and reached the maximal level when leaves were fully expanded. The same change pattern was also found in the stomatal conductance and chlorophyll content. Lower Pn could not be ascribed to the higher stomatal resistance in developing leaves, as intercellular CO₂ concentration was not significantly lower in these leaves. Lower Pn partly resulted from the lower actual photochemical efficiency of PSII in developing leaves, as more excited energy was dissipated through non-photochemical quenching. The development of primary photochemical reaction and electron transport in the donor side of PSII was completed in the initiating leaves. However, the development of electron transport in the acceptor side of PSII was not accomplished until leaves were fully expanded, indicated by the change in probability that an electron moves further than primary quinone (ψo). PSI activity changed in parallel with ψo suggesting that PSI cooperated well with PSII during leaf expansion. It should be stressed that the development of carbon fixation process was later than primary photochemical reaction but earlier than photosynthetic electron transport during leaf expansion. The later development of photosynthetic electron transport may reduce the production of reactive oxygen species from Mehler reaction, particularly under low carbon fixation.     

Microtuber formation in micropropagated Jerusalem artichoke (Helianthus tuberosus)

Clonal micropropagation of Jerusalem artichoke (Helianthus tuberosus L.) was initiated from axillary meristems of lateral shoots of field-grown plants on medium with MS salts, 2% sucrose, 1 mg l-1 thiamine-HCl, 1 mg l-1 IAA and 0.6% agar. Plantlets were cut into nodal sections and used for subsequent subcultures and for microtuber induction. Microtubers were induced from axillary meristems on medium with half-strength MS salts, 8% sucrose and 0.5 mg l-1 BA in darkness at 18 °C. They had near to 30% of dry matter. Microtubers resumed growth in light room at 23 °C after 4–6 months of cold storage. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tissue Differentiation of the Regeneration Rind in Jerusalem artichoke Stem

In trees, after removal of the bark, the vascular tissues of the newly-formed bark usually developed as a continuous layer. However, the stem of the herbaceous Jerusalem artichoke, after girdled, gives rise to regeneration of many irregularly arranged vascular bundles. Early July is the best time for girdling as the vascular bundles are well-developed, One week after girdling, some small groups of vascular tissues appeared in callus. Later on the vascular bundles eventually grew close together sooner or later, yet there were some wide pith rays which separated the various sized vascular bundles and exhibited irregularly contours. From these experiments, it is further evidenced that tile stem of herbaceous plants can also be girdled and regenerates a new rind. Furthermore, the girdled portion of this plant regenerates the vascular tissues which in a rather different way from all the plants that previously studied.