Plant growth shapes the effects of elevation on the content and variability of flavonoids in subalpine bilberry stands.

The study of morphological and physiological responses of shrubs to climate is crucial for the understanding of future scenarios regarding climate change. In this light, studying shrub growth and physiological acclimation along an elevation gradient might be insightful. The phenolic metabolic pathway represents a powerful tool to interpret such processes. In the South-Eastern Alps, we investigated the relationships between elevation, plant traits (i.e. age, xylem ring width, annual shoot length), plant-plant interaction (i.e. shrub cover) and flavonoids in Vaccinium myrtillus L. (leaves, berries) in stands above the treeline. The relationships were parsed within causal networks using a confirmatory path analysis. Elevation was the main driver of V. myrtillus growth, having both direct and indirect effects on the leaf flavonoid content, but this was less evident for berries. In particular, the content of foliar flavonoids showed a peak at mid-elevation and where the growth of xylem rings was intermediate, while it decreased in stands with higher shoot length. Flavonoid content variability of both leaves and berries was affected by elevation and shoot length. In berries, flavonoid variability was further related to all growth traits and shrub cover. These findings evidence that flavonoid content is influenced by both elevation and growth traits of V. myrtillus, often showing non-linear relationships. These results suggest a trait-mediated response of this plant to climate conditions as a result of trade-offs between plant growth, plant defence, environmental stress and nutrient/resource availability.

'Last In-First Out': seasonal variations of non-structural carbohydrates, glucose-6-phosphate and ATP in tubers of two Arum species.

Knowledge on the metabolism of polysaccharide reserves in wild species is still scarce. In natural sites we collected tubers of Arum italicum Mill. and A. maculatum L. - two geophytes with different apparent phenological timing, ecology and chorology - during five stages of the annual cycle in order to understand patterns of reserve accumulation and degradation. Both the entire tuber and its proximal and distal to shoot portion were utilised. Pools of non-structural carbohydrates (glucose, sucrose and starch), glucose-6-phosphate and ATP were analysed as important markers of carbohydrate metabolism. In both species, starch and glucose content of the whole tuber significantly increased from sprouting to the maturation/senescence stages, whereas sucrose showed an opposite trend; ATP and glucose-6-phosphate were almost stable and dropped only at the end of the annual cycle. Considering the two different portions of the tuber, both ATP and glucose-6-phosphate concentrations were higher in proximity to the shoot in all seasonal stages, except the flowering stage. Our findings suggest that seasonal carbon partitioning in the underground organ is driven by phenology and occurs independently of seasonal climate conditions. Moreover, our results show that starch degradation, sustained by elevated ATP and glucose-6-phosphate pools, starts in the peripheral, proximal-to-shoot portion of the tuber, consuming starch accumulated in the previous season, as a 'Last In-First Out' mechanism of carbohydrate storage.

Evidence for a putative flavonoid translocator similar to mammalian bilitranslocase in grape berries (Vitis vinifera L.) during ripening.

During maturation, Vitis vinifera berries accumulate a large amount of several anthocyanins in the epidermal tissue, whereas their precursors and intermediates are ubiquitously synthesized within the fruit. Up to date, several mechanisms of flavonoid transport at subcellular level have been hypothesized, but it is not possible to identify a general model applicable in every plant tissue and organ. Recently, a putative anthocyanin carrier, homologue to mammalian bilitranslocase (BTL) (TC 2.A.65.1.1), was found in Dianthus caryophyllus petal microsomes. In the present paper, an immunohistochemical and immunochemical analysis, using an antibody raised against a BTL epitope, evidences the expression and function of such a transporter in V. vinifera berries (cv. Merlot). Specific localisations of the putative carrier within berry tissues together with expression changes during different developmental stages are shown. Water stress induces an increase in protein expression in both skin and pulp samples. A bromosulfalein (BSP) uptake activity, inhibitable by the BTL antibody, is detected in berry mesocarp microsomes, with K (m) = 2.39 microM BSP and V (max) = 0.29 micromol BSP min(-1) mg(-1) protein. This BSP uptake is also competitively inhibited by quercetin (K (i) = 4 microM). A putative role for this carrier is discussed in relation to the membrane transport of secondary metabolites.

Cyclosporin A induces the opening of a potassium-selective channel in higher plant mitochondria.

The immunosuppressive drug, cyclosporin A (CsA), induces the generation of a transmembrane electrical potential difference (deltapsi) in deenergized plant mitochondria incubated in sucrose-based media. Build up of deltapsi is prevented by external monovalent cations in the order K+ > Rb+ = Li+ > Na+, or by the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone, which also collapses the deltapsi generated by CsA. Entry of K+ into mitochondria can be monitored as swelling by incubating the organelles in a medium containing KCl to maintain constant osmolarity. This swelling is inhibited by ATP and stimulated by CsA or valinomycin. In addition, in mitochondria energized by succinate, KCl causes a dissipation of deltapsi, with sigmoidal kinetics, which is favored by CsA. Therefore, plant mitochondria appear to possess a K+ selective, voltage-dependent channel, which is opened by CsA, regulated by the redox state, and inhibited by nucleotides. The hypothetical roles of this new K+ATP channel are discussed in relation to its potential involvement in mitochondrial volume regulation, thermogenesis, apoptosis, and/or prevention of reactive oxygen species formation in plants.

Hydrogen peroxide generation by higher plant mitochondria oxidizing complex I or complex II substrates.

The generation of H2O2 by isolated pea stem mitochondria, oxidizing either malate plus glutamate or succinate, was examined. The level of H2O2 was almost one order of magnitude higher when mitochondria were energized by succinate. The succinate-dependent H2O2 formation was abolished by malonate, but unaffected by rotenone. The lack of effect of the latter suggests that pea mitochondria were working with a proton motive force below the threshold value required for reverse electron transfer. The activation by pyruvate of the alternative oxidase was reflected in an inhibition of H2O2 formation. This effect was stronger when pea mitochondria oxidized malate plus glutamate. Succinate-dependent H2O2 formation was ca. four times lower in Arum sp. mitochondria (known to have a high alternative oxidase) than in pea mitochondria. An uncoupler (FCCP) completely prevented succinate-dependent H2O2 generation, while it only partially (40-50%) inhibited that linked to malate plus glutamate. ADP plus inorganic phosphate (transition from state 4 to state 3) also inhibited the succinate-dependent H2O2 formation. Conversely, that dependent on malate plus glutamate oxidation was unaffected by low and stimulated by high concentrations of ADP. These results show that the main bulk of H2O2 is formed during substrate oxidation at the level of complex II and that this generation may be prevented by either dissipation of the electrochemical proton gradient (uncoupling and transition state 4-state 3), or preventing its formation (alternative oxidase). Conversely, H2O2 production, dependent on oxidation of complex I substrate, is mainly lowered by the activation of the alternative oxidase.

Zearalenone-induced uncoupling in plant mitochondria is sensitive to 6-ketocholestanol.

Zearalenone (F-2) is a mycotoxin which acts as a protonophoric uncoupler in plant mitochondria [Macri, F. and Vianello, A., (1990) J. Plant Physiol. 136, 754-757]. In the present paper, the mechanism of F-2-induced uncoupling in pea mitochondria was studied. The uncoupling by F-2 was partially reversed by 6-ketocholestanol (kCh) under conditions in which kCh completely reversed the FCCP-induced uncoupling and almost did not affect the palmitate-induced uncoupling. Recoupling effects of carboxyatractylate, ADP and cyclosporin A were small and could not essentially decrease the kCh-insensitive part of F-2-induced uncoupling. It is suggested that a protein, mediating kCh-sensitive uncoupling, is involved in the F-2 effect in plant mitochondria.

Lipoxygenase activity associated to isolated soybean plasma membranes.

Highly purified soybean (Glycine max L. Merr.) plasma membranes exhibit a lipoxygenase activity with a pH optimum in the acidic (5.5-6.0) range and with a Km value of 200 microM for both linolenic and linoleic acids. This activity is inhibited by nordihydroguaiaretic acid (NDGA), salicylhydroxamic acid (SHAM) and propyl gallate, stimulated by CaCl2 up to 0.25 mM, H2O2 (5 to 10 nM range) and by some nucleotide triphosphates (125 to 1000 nM range) in the following order ATP > GTP = UTP > CTP. The enzyme is not released by treatment of the membranes with 0.05% Brij 58 and its activity is approx. 65% inhibited by the impermeant p-chloromercuryphenyl-sulfonate only in 0.01% Triton X-100-treated membrane vesicles. These results indicate that soybean cells have an acid lipoxygenase, associated to the plasmalemma, with the catalytic site on the cytoplasmic surface. It may be distinguished from the soluble counterpart, because the latter is not stimulated by nucleotide triphosphates. The plasma membrane vesicles also show a lipoxygenase, active in the alkaline (9.0-9.5) range, inhibited by NDGA, SHAM and propyl gallate, stimulated by H2O2, but with a lower Km value (60 microM) and less sensitive to calcium stimulation than the acidic one. The possible involvement of acid lipoxygenase in senescence and in the response of plant cells to wounding and pathogen infection is discussed.

Effect of carboxyatractylate on transmembrane electrical potential of plant mitochondria in different metabolic states.

The effects of carboxyatractylate (CAtr) on delta psi in sunflower hypocotyl and pea stem mitochondria were compared. In sunflower mitochondria, (1) CAtr at higher concentration increased delta psi in the presence of palmitate and delta psi in metabolic state 3; (2) ]1 microM CAtr did not prevent delta psi decrease, induced by ADP addition (in contrast to pea mitochondria); (3) The ATP-generated delta psi was small and was insensitive to 40 microM CAtr. Under the same conditions, in pea mitochondria generation of delta psi by ATP was inhibited by 1 microM CAtr.

ATP/ADP antiporter is involved in uncoupling of plant mitochondria induced by low concentrations of palmitate.

Carboxyatractyloside partially restored the transmembrane electrical potential difference (delta psi) dissipated by low concentrations of palmitate in pea stem mitochondria. This effect was more marked when mitochondria from sunflower were assayed. It is suggested that the ATP/ADP translocator is involved in the free fatty acid-induced uncoupling of oxidative phosphorylation in plant mitochondria, only when its level is sufficiently high and the concentration of the fatty acid is low to collapse only partially the delta psi.

ATP/ADP antiporter is involved in uncoupling of plant mitochondria induced by low concentrations of palmitate.

Carboxyatractyloside partially restored the transmembrane electrical potential difference (delta psi) dissipated by low concentrations of palmitate in pea stem mitochondria. This effect was more marked when mitochondria from sunflower were assayed. It is suggested that the ATP/ADP translocator is involved in the free fatty acid-induced uncoupling of oxidative phosphorylation in plant mitochondria, only when its level is sufficiently high and the concentration of the fatty acid is low to collapse only partially delta psi.

Electrical potential dissipation induced by free fatty acids in pea stem mitochondria.

Linolenic, linoleic, oleic, palmitic and stearic acids (FFA) collapse the electrical potential of pea stem mitochondria in the absence or in the presence of 0.5 mM Mg2+. Higher concentrations of this cation (5 mM) lower the rate of dissipation caused by linoleic, oleic and palmitic acids, while abolishing that induced by stearic acid. Carboxyatractyloside and ADP do not reverse the FFA-induced collapse both in the presence or absence of Mg2+. EDTA, EGTA or BHT do not influence the dissipation caused by FFA that, in addition, is not linked to lipid peroxidation evaluated as malondialdehyde or conjugated diene formation. Only linolenic acid sustains a peroxidation which, however, appears to be caused by its own oxidation catalysed by lipoxygenases rather than by membrane lipoperoxidation induced by this free fatty acid. These results suggest that neither the ATP/ADP exchanger nor lipid peroxidation appear to be involved in FFA-induced uncoupling in pea stem mitochondria.