Energy conservation and dissipation in mitochondria isolated from developing tomato fruit of ethylene-defective mutants failing normal ripening: the effect of ethephon,a chemical precursor of ethylene

Alternative oxidase (AOX) and uncoupling protein (UCP) are present simultaneously in tomato fruit mitochondria. In a previous work, it has been shown that protein expression and activity of these two energy-dissipating systems exhibit large variations during tomato fruit development and ripening on the vine. It has been suggested that AOX and UCP could be responsible for the respiration increase at the end of ripening and that the cytochrome pathway could be implicated in the climacteric respiratory burst before the onset of ripening. In this study, the use of tomato mutants that fail normal ripening because of deficiencies in ethylene perception or production as well as the treatment of one selected mutant with a chemical precursor of ethylene have revealed that the bioenergetics of tomato fruit development and ripening is under the control of this plant hormone. Indeed, the evolution pattern of bioenergetic features changes with the type of mutation and with the introduction of ethylene into an ethylene-synthesis-deficient tomato fruit mutant during its induced ripening.     

Internal carbon dioxide and ethylene levels in ripening tomato fruit attached to or detached from the plant

The carbon dioxide and ethylene concentrations in tomato fruit ( Lycopersicon esculentum cv. Castelmart) and their stage of ripeness (characteristic external color changes) were periodically measured in fruit attached to and detached from the plant. An external collection apparatus was attached to the surface of individual tomato fruit to permit non-destructive sampling of internal gases. The concentration of carbon dioxide and ethylene in the collection apparatus reached 95% of the concentration in the fruit after 8 h. Gas samples were collected every 24 h. A characteristic climacteric surge in carbon dioxide (2-fold) and ethylene (10-fold) concentration occurred coincident with ripening of detached tomato fruit. Fruit attached to the plant exhibited a climacteric rise in ethylene (20-fold) concentration during ripening, but only a linear increase in carbon dioxide concentration. The carbon dioxide concentration increases in attached fruit during ripening, but the increase is a continuation of the linear increase seen in both attached and detached fruit before ripening and does not exhibit the characteristic pattern normally associated with ripening climacteric fruit. In tomato fruit, it appears that a respiratory climacteric per se, which has been considered intrinsic to the ripening of certain fruit, may not be necessary for the ripening of "climacteric" fruit at all, but instead may be an artifact of using harvested fruit.     

地菍果实成熟过程中品质及种子油脂肪酸组分的变化

对地菍果实不同成熟期品质的研究表明,成熟时的地菍果实,维生素C、总糖含量达最高值,酸度则降至最低。此期风味最好,软硬适宜,色泽甚佳,为地菍鲜食的最佳采收期。对不同成熟期的种子油脂肪酸分析表明,种子油以不饱和脂肪酸为主,尤其是亚油酸含量很高,达84%;果实成熟过程中脂肪酸各级分的含量变化趋势不明显。     

Mechanisms and functions of nonphosphorylating electron transport in respiratory chain of plant mitochondria

Data are raeviewed on mitochondrial systems whose functioning in plants diminishes the efficiency of oxidative phosphorylation. The involvement in this process of alternative oxidase, thermogenin-like uncoupling proteins, a 310 kD stress protein, free fatty acids, and the ADP/ATP antiporter is considered. The role of these systems is discussed with regard to thermogenesis, controlled production of reactive oxygen species, and regulation of bioenergetics and metabolism.     

Ribonucleic acid metabolism during the development and ripening of tomato fruits

The accumulation of RNA in the outer locule tissue of tomato fruits was measured during development and ripening. Labelling studies suggest two peaks of synthesis, the first during early development and the second just before the onset of ripening (colour change). During the second period of increased RNA labelling the amount of total RNA per fruit either remains constant or starts to decline. Synthesis of rRNA and soluble RNA occurred at all stages. Polydisperse RNA containing polyadenylic acid was isolated and shown to direct the synthesis of protein in vitro. No significant changes in the amount of polyadenylic acid, relative to total RNA were detectable during the ripening period.     

A novel tomato F‐box protein,SlEBF3, is involved in tuning ethylene signaling during plant development and climacteric fruit ripening

Ethylene is instrumental to climacteric fruit ripening and EIN3 BINDING F‐BOX (EBF) proteins have been assigned a central role in mediating ethylene responses by regulating EIN3/EIL degradation in Arabidopsis. However, the role and mode of action of tomato EBFs in ethylene‐dependent processes like fruit ripening remains unclear. Two novel EBF genes, SlEBF3 and SlEBF4, were identified in the tomato genome, and SlEBF3 displayed a ripening‐associated expression pattern suggesting its potential involvement in controlling ethylene response during fruit ripening. SlEBF3 downregulated tomato lines failed to show obvious ripening‐related phenotypes likely due to functional redundancy among SlEBF family members. By contrast, SlEBF3 overexpression lines exhibited pleiotropic ethylene‐related alterations, including inhibition of fruit ripening, attenuated triple‐response and delayed petal abscission. Yeast‐two‐hybrid system and bimolecular fluorescence complementation approaches indicated that SlEBF3 interacts with all known tomato SlEIL proteins and, consistently, total SlEIL protein levels were decreased in SlEBF3 overexpression fruits, supporting the idea that the reduced ethylene sensitivity and defects in fruit ripening are due to the SlEBF3‐mediated degradation of EIL proteins. Moreover, SlEBF3 expression is regulated by EIL1 via a feedback loop, which supposes its role in tuning ethylene signaling and responses. Overall, the study reveals the role of a novel EBF tomato gene in climacteric ripening, thus providing a new target for modulating fleshy fruit ripening.     

Cyanide-and rotenone-resistant respiration in mitochondria of sugar beet taproots during plant growth and development

Effects of cyanide and rotenone were examined on respiration (oxygen uptake) in mitochondria isolated from sugar beet (Beta vulgaris L.) taproots at various stages of plant growth and development. In mitochondria from growing and cool-stored taproots, the ability of cyanide-resistant, salicylhydroxamic acid-sensitive alternative oxidase (AO) to oxidize malate, succinate, and other substrates of tricarboxylic acid cycle (TCA) was low and constituted less than 10% compared to predominant activity of the cytochrome oxidase pathway during State 3 respiration. Artificial aging of storage tissue (2-day incubation of tissue sections under high humidity at 20°C) substantially activated AO, but the highest capacity (V _alt) of this pathway of mitochondrial oxidation was only observed in the presence of pyruvate and a reducing agent dithiothreitol. At the same time, mitochondria from growing taproots exhibited high rates of rotenone-resistant respiration, and these rates gradually declined during plant growth and development. The slowest rates of this respiration were observed during oxidation of NAD-dependent TCA substrates in mitochondria from dormant storage organ. The results are discussed in relation to significance of alternative electron transport pathways during growth and storage of sugar beet taproots.     

The possible involvement of polyamines in the development of tomato fruits in vitro

The apparent involvement of ornithine decarboxylase (ODC) and putrescine in the early stages of fruit growth in tomato (Lycopersicon esculentum Mill.) has been previously described. Further evidence presented here supports the direct involvement of ODC and putrescine in the cell division process in tomato fruits. In tomato fruits grown in vitro, in which basic growth processes are inhibited, the activity of ODC and arginine decarboxylase (ADC) and the level of free polyamines were reduced. While ODC and ADC activity was correlated with the period of cell division in the tomato fruit, the free polyamine content was correlated with the DNA content, cell size, and fruit fresh weight. The addition of exogenous putrescine, however, did not restore the basic growth processes in the fruits grown in vitro.     

Effect of ethanol on the palmitate-induced uncoupling of oxidative phosphorylation in liver mitochondria

The effect of ethanol on the uncoupling activity of palmitate and recoupling activities of carboxyatractylate and glutamate was studied in liver mitochondria at various Mg²⁺ concentrations and medium pH values (7.0, 7.4, and 7.8). Ethanol taken at concentration of 0.25 M had no effect on the uncoupling activity of palmitic acid in the presence of 2 mM MgCl₂ and decreased the recoupling effects of carboxyatractylate and glutamate added to mitochondria either just before or after the fatty acid. However, ethanol did not modify the overall recoupling effect of carboxyatractylate and glutamate taken in combination. The effect of ethanol decreased as medium pH was decreased to 7.0. Elevated concentration of Mg²⁺ (up to 8 mM) inhibits the uncoupling effect of palmitate. Ethanol eliminates substantially the recoupling effect of Mg²⁺ under these conditions, but does not influence the recoupling effects of carboxyatractylate and glutamate. It is inferred that ADP/ATP and aspartate/glutamate antiporters are involved in uncoupling function as single uncoupling complex with the common fatty acid pool. Fatty acid molecules gain the ability to migrate under the action of ethanol: from ADP/ATP antiporter to aspartate/glutamate antiporter on addition of carboxyatractylate and in opposite direction on addition of glutamate. Possible mechanisms of fatty acid translocation from one transporter to another are discussed.     

The role of fatty acid binding protein on the metabolism of fatty acids in isolated rat hepatocytes

In isolated rat hepatocytes flavaspidic acid, a competitor with free fatty acids for the fatty-acid-binding-protein, decreased the uptake of oleic acid and triglyceride synthesis but stimulated the formation of CO₂ and ketone bodies from oleic acid. Flavaspidic acid had no effect on the utilization of octanoic acid. Stimulation of the microsomal fatty-acid-activating enzyme by the fatty-acid-binding protein was reversed by flavaspidic acid. In contrast, the binding protein inhibited the mitochondrial fatty-acid-activating enzyme. Flavaspidic acid not only prevented this inhibition but actually stimulated the enzyme activity. The results indicate that the cytosol fatty-acid-binding protein directs the metabolism of long chain fatty acids toward esterification as well as enhancing their cellular uptake.     

Determination of hydroxylated fatty acids from the biopolymer of tomato cutin and their fate during incubation in soil

Introduction – The plant cuticle is a thin, predominantly lipid layer that covers all primary aerial surfaces of vascular plants. The monomeric building blocks of the cutin biopolymer are mainly ω‐hydroxy fatty acids. Objective – Analysis of ω‐hydroxy fatty acids from cutin isolated from tomato fruits at different stages of decomposition in soil. Different derivatives and mass spectrometric techniques were used for peak identification and evaluation. Methodology – Preparation of purified cutin involving dewaxing and HCl treatment. Incubation of purified cutin for 20 months in soil. Pentafluorobenzoyl derivatives were used for GC/MS operated in the electron capture negative ion (ECNI) mode and trimethylsilyl ethers for GC/MS operated in the electron ionisation (EI) mode for analysis of ω‐hydroxy fatty acids. Results – Six ω‐hydroxy fatty acids were detected in the purified cutin, three of which were identified as degradation products of 9,16‐dihydroxyhexadecanoic acid as a consequence of the HCl treatment involved in the purification step. Incubation of the isolated cutin in soil was accompanied with decrease in concentration of all hydroxyl fatty acids. Conclusion – We produced evidence that the HCl treatment only affected free hydroxyl groups and thus could be used for proportioning free and bound OH‐groups on cutin fatty acids. The method enabled a direct quantification of the ω‐hydroxy fatty acids throughout the incubation phase. Copyright © 2010 John Wiley & Sons, Ltd.     

The appearance of polygalacturonase mRNA in tomatoes: one of a series of changes in gene expression during development and ripening

Tomato mRNA was extracted from individual fruits at different stages of development and ripening, translated in a rabbit reticulocyte lysate and the protein products analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The results indicate that there are at least two classes of mRNA under separate developmental control. One group of approximately six mRNAs is present during fruit growth and then declines at the mature-green stage. Another group of between four and eight mRNAs increases substantially in amount at the onset of ripening, after the start of enhanced ethylene synthesis by the fruit, and continues to accumulate as ripening progresses. Studies of protein synthesis in vivo show that several new proteins are synthesised by ripening fruits including the fruit-softening enzyme polygalacturonase. One of the ripening-related mRNAs is shown to code for polygalacturonase, by immunoprecipitation with serum from rabbits immunised against the purified tomato enzyme. Polygalacturonase mRNA is not detectable in green fruit but accumulates during ripening. It is proposed that the ripening-related mRNAs are the products of a group of genes that code for enzymes important in the ripening process.Abbreviation SDS sodium dodecyl sulfate     

The role of different plant seedling shoots mitochondrial uncoupling systems in thermogenesis during low-temperature stress

A difference was found between the temperature of control and heat-treated winter wheat and pea seedlings shoots during low temperature stress. Functioning of three thermogenic mitochondrial systems was established: (i) alternative cyanide-resistant oxidase, (ii) plant uncoupling mitochondrial protein and (iii) stress protein CSP 310 and these three caused the higher temperature of winter wheat control shoots. In peas only two thermogenic systems, the alternative cyanide-resistant oxidase and plant uncoupling mitochondrial proteins were found.     

Involvement of oxygen free radicals in the respiratory uncoupling induced by free calcium and ADP-magnesium in isolated cardiac mitochondria: Comparing reoxygenation in cultured cardiomyocytes

Recently, we have observed that the simultaneous application of free calcium (fCa) and ADP-magnesium (Mg) reduced the ADP:O ratio in isolated cardiac mitochondria. The uncoupling was prevented by cyclosporin A, an inhibitor of the permeability transition pore. The purpose of this study was to know if the generation of oxygen free radicals (OFR) is involved in this phenomenon and if it occurs during reoxygenation (Reox) of cultured cardiomyocytes. Cardiac mitochondria were harvested from male Wistar rats. Respiration was assessed in two media with different fCa concentrations (0 or 0.6 M) with palmitoylcarnitine and ADP-Mg as respiration substrates. The production of Krebs cycle intermediates (KCI) was determined. Without fCa in the medium, the mitochondria displayed a large production of citrate + isocitrate + -ketoglutarate. fCa drastically reduced these KCI and promoted the accumulation of succinate. To know if OFR are involved in the respiratory uncoupling, the effect of 4OH-TEMPO (250 M), a hydrosoluble scavenger of OFR, was tested. 4OH-TEMPO completely abolished the fCa- and ADP-Mg-induced uncoupling. Conversely, vitamin E contributed to further decreasing the ADP:O ratio. Since no hydrosoluble electron acceptor was added in our experiment, the oxygen free radical-induced oxidized vitamin E was confined near the mitochondrial membranes, which should reduce the ADP:O ratio by opening the permeability transition pore. The generation of OFR could result from the matrix accumulation of succinate. Taken together, these results indicate that mitochondrial Ca uptake induces a slight increase in membrane permeability. Thereafter, Mg enters the matrix and, in combination with Ca, stimulates the isocitrate and/or -ketoglutarate dehydrogenases. Matrix succinate favors oxygen free radical generation that further increases membrane permeability and allows respiratory uncoupling through proton leakage. To determine whether the phenomenon takes place during Reox, cultured cardiomyocytes were subjected to hypoxia and Reox. ¹⁴C-palmitate was added during Reox to determine the KCI profile. Succinate had not increased during Reox. In conclusion, calcium- and ADP-Mg-induced respiratory uncoupling is due to oxygen free radical generation through excess matrix accumulation of succinate. The phenomenon does not occur during reoxygenation because of a total restoration of mitochondrial magnesium and/or ADP concentration.     

The codA transgene for glycinebetaine synthesis increases the size of flowers and fruits in tomato

The tolerance of various species of plant to abiotic stress has been enhanced by genetic engineering with certain genes. However, the use of such transgenes is often associated with negative effects on growth and productivity under non-stress conditions. The codA gene from Arthrobacter globiformis is of particular interest with respect to the engineering of desirable productive traits in crop plants. The expression of this gene in tomato plants resulted in significantly enlarged flowers and fruits under non-stress conditions. The enlargement of flowers and fruits was associated with high levels of glycinebetaine that accumulated in reproductive organs, such as flower buds and fruits. The enlargement of flowers was related to an increase in the size and number of cells, and reflected the pleiotropic effect of the codA transgene on the expression of genes involved in the regulation of cell division.     

Effects of Ca2+ on flavin-linked complex enzymes in mitochondria isolated from eggs and embryos of sea urchin

Mitochondria isolated from sea urchin embryos in early development show almost the same activities of cytochrome c oxidase and flavin-linked complex enzymes, which are estimated by cytochrome c reductases as in those isolated from unfertilized eggs. The activities of these cytochrome c reductases are inhibited by Ca2+ at above 10-5 M more strongly than cytochrome c oxidase. To investigate the changes in intramitochondrial Ca2+ concentration at fertilization, the activity of pyruvate dehydrogenase, another mitochondrial enzyme, was measured. The activity of this enzyme was controlled by phosphorylation and Ca2+-dependent dephosphorylation of the catalytic unit. The enzyme activity increased for 30 min after fertilization, decreased and became close to zero within ~60 min. Then, the activity appreciably increased again after hatching. This seems to reflect changes in the intramitochondrial Ca2+ concentration. The enzyme activity was enhanced by pre-incubation with Ca2+ at concentrations up to 10-5 M but was made quite low at above 10-4 M Ca2+ and 10-3 M adenosine triphosphate. Although the changes in pyruvate dehydrogenase activity observed at fertilization will reflect the changes in the intramitochondrial calcium concentration, the intramitochondrial Ca2+ concentration of unfertilized eggs cannot be estimated from these results because high (> 10-4 M) or low (10-6 M) Ca2+ can inhibit the enzyme. Measurement of respiration of a single egg showed that injection of ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid released the mitochondrial electron transport in the unfertilized egg. The possibility that changes in intramitochondrial calcium concentration occur at fertilization is discussed in relation to activation of both mitochondrial respiration and pyruvate dehydrogenase.     

Patterns of Dwarf expression and brassinosteroid accumulation in tomato reveal the importance of brassinosteroid synthesis during fruit development

Brassinosteroids (BRs) are essential for many physiological functions in plants, however little is known concerning where and when they are synthesized. This is especially true during flower and fruit production. To address this we have used a promoter-GUS reporter fusion and RT-PCR to determine the relative expression levels of the tomato Dwarf (D) gene that encodes a BR C-6 oxidase. In young seedlings GUS reporter activity was observed mainly in apical and root tissues undergoing expansion. In flowers GUS activity was observed in the pedicel joints and ovaries, whereas in fruits it was strongest during early seed development and was associated with the locular jelly and seeds. RT-PCR analysis showed that tissue-specific expression of Dwarf mRNA was consistent with that of the Dwarf:GUS fusion. In good correlation with the high local Dwarf activity, quantitative measurements of endogenous BRs indicated intense biosynthesis in developing tomato fruits, which were also found to contain high amounts of brassinolide. Grafting experiments showed the lack of BR transport indicating that BR action occurs at the site of synthesis.