Plant Uncoupling Protein Research Articles

DIA-Based Quantitative Proteomics Reveals the Protein Regulatory Networks of Floral Thermogenesis in Nelumbo nucifera.

The sacred lotus (Nelumbo nucifera) can maintain a stable floral chamber temperature between 30 and 35 °C when blooming despite fluctuations in ambient temperatures between about 8 and 45 °C, but the regulatory mechanism of floral thermogenesis remains unclear. Here, we obtained comprehensive protein profiles from receptacle tissue at five developmental stages using data-independent acquisition (DIA)-based quantitative proteomics technology to reveal the molecular basis of floral thermogenesis of N. nucifera. A total of 6913 proteins were identified and quantified, of which 3513 differentially abundant proteins (DAPs) were screened. Among them, 640 highly abundant proteins during the thermogenic stages were mainly involved in carbon metabolism processes such as the tricarboxylic acid (TCA) cycle. Citrate synthase was identified as the most connected protein in the protein-protein interaction (PPI) network. Next, the content of alternative oxidase (AOX) and plant uncoupling protein (pUCP) in different tissues indicated that AOX was specifically abundant in the receptacles. Subsequently, a protein module highly related to the thermogenic phenotype was identified by the weighted gene co-expression network analysis (WGCNA). In summary, the regulation mechanism of floral thermogenesis in N. nucifera involves complex regulatory networks, including TCA cycle metabolism, starch and sucrose metabolism, fatty acid degradation, and ubiquinone synthesis, etc.

Modulation of Potassium Channel Activity in the Balance of ROS and ATP Production by Durum Wheat Mitochondria-An Amazing Defense Tool Against Hyperosmotic Stress.

In plants, the existence of a mitochondrial potassium channel was firstly demonstrated about 15 years ago in durum wheat as an ATP-dependent potassium channel (PmitoKATP). Since then, both properties of the original PmitoKATP and occurrence of different mitochondrial potassium channels in a number of plant species (monocotyledonous and dicotyledonous) and tissues/organs (etiolated and green) have been shown. Here, an overview of the current knowledge is reported; in particular, the issue of PmitoKATP physiological modulation is addressed. Similarities and differences with other potassium channels, as well as possible cross-regulation with other mitochondrial proteins (Plant Uncoupling Protein, Alternative Oxidase, Plant Inner Membrane Anion Channel) are also described. PmitoKATP is inhibited by ATP and activated by superoxide anion, as well as by free fatty acids (FFAs) and acyl-CoAs. Interestingly, channel activation increases electrophoretic potassium uptake across the inner membrane toward the matrix, so collapsing membrane potential (ΔΨ), the main component of the protonmotive force (Δp) in plant mitochondria; moreover, cooperation between PmitoKATP and the K+/H+ antiporter allows a potassium cycle able to dissipate also ΔpH. Interestingly, ΔΨ collapse matches with an active control of mitochondrial reactive oxygen species (ROS) production. Fully open channel is able to lower superoxide anion up to 35-fold compared to a condition of ATP-inhibited channel. On the other hand, ΔΨ collapse by PmitoKATP was unexpectedly found to not affect ATP synthesis via oxidative phosphorylation. This may probably occur by means of a controlled collapse due to ATP inhibition of PmitoKATP; this brake to the channel activity may allow a loss of the bulk phase Δp, but may preserve a non-classically detectable localized driving force for ATP synthesis. This ability may become crucial under environmental/oxidative stress. In particular, under moderate hyperosmotic stress (mannitol or NaCl), PmitoKATP was found to be activated by ROS, so inhibiting further large-scale ROS production according to a feedback mechanism; moreover, a stress-activated phospholipase A2 may generate FFAs, further activating the channel. In conclusion, a main property of PmitoKATP is the ability to keep in balance the control of harmful ROS with the mitochondrial/cellular bioenergetics, thus preserving ATP for energetic needs of cell defense under stress.

Transport pathways--proton motive force interrelationship in durum wheat mitochondria.

In durum wheat mitochondria (DWM) the ATP-inhibited plant mitochondrial potassium channel (PmitoKATP) and the plant uncoupling protein (PUCP) are able to strongly reduce the proton motive force (pmf) to control mitochondrial production of reactive oxygen species; under these conditions, mitochondrial carriers lack the driving force for transport and should be inactive. However, unexpectedly, DWM uncoupling by PmitoKATP neither impairs the exchange of ADP for ATP nor blocks the inward transport of Pi and succinate. This uptake may occur via the plant inner membrane anion channel (PIMAC), which is physiologically inhibited by membrane potential, but unlocks its activity in de-energized mitochondria. Probably, cooperation between PIMAC and carriers may accomplish metabolite movement across the inner membrane under both energized and de-energized conditions. PIMAC may also cooperate with PmitoKATP to transport ammonium salts in DWM. Interestingly, this finding may trouble classical interpretation of in vitro mitochondrial swelling; instead of free passage of ammonia through the inner membrane and proton symport with Pi, that trigger metabolite movements via carriers, transport of ammonium via PmitoKATP and that of the counteranion via PIMAC may occur. Here, we review properties, modulation and function of the above reported DWM channels and carriers to shed new light on the control that they exert on pmf and vice-versa.

Sensitivity of the aldehyde-induced and free fatty acid-induced activities of plant uncoupling protein to GTP is regulated by the ubiquinone reduction level

Using isolated potato tuber mitochondria possessing uncoupling protein (StUCP), we found that, under non-phosphorylating conditions, the sensitivity of aldehyde (all trans-retinal or 4-hydroxy-2-nonenal)-induced and fatty acid (linoleic acid)-induced StUCP-mediated proton leaks to GTP is controlled by the endogenous ubiquinone (Q) reduction level. The action of StUCP activators was abolished by GTP only when Q was sufficiently oxidized, but no inhibitory effect was observed when Q was highly reduced. Thus, the Q reduction level-dependent regulation of StUCP inhibition functions independently of the type of UCP activation and could be an important physiological factor affecting the efficiency of UCP-catalyzed uncoupling in plant mitochondria.

Activation of the plant mitochondrial potassium channel by free fatty acids and acyl-CoA esters: a possible defence mechanism in the response to hyperosmotic stress

The effect of free fatty acids (FFAs) and acyl-CoA esters on K(+) uptake was studied in mitochondria isolated from durum wheat (Triticum durum Desf.), a species that has adapted well to the semi-arid Mediterranean area and possessing a highly active mitochondrial ATP-sensitive K(+) channel (PmitoK(ATP)), that may confer resistance to environmental stresses. This was made by swelling experiments in KCl solution under experimental conditions in which PmitoK(ATP) activity was monitored. Linoleate and other FFAs (laurate, palmitate, stearate, palmitoleate, oleate, arachidonate, and the non-physiological 1-undecanesulphonate and 5-phenylvalerate), used at a concentration (10 μM) unable to damage membranes of isolated mitochondria, stimulated K(+) uptake by about 2-4-fold. Acyl-CoAs also promoted K(+) transport to a much larger extent with respect to FFAs (about 5-12-fold). In a different experimental system based on safranin O fluorescence measurements, the dissipation of electrical membrane potential induced by K(+) uptake via PmitoK(ATP) was found to increase in the presence of 5-phenylvalerate and palmitoyl-CoA, both unable to elicit the activity of the Plant Uncoupling Protein. This result suggests a direct activation of PmitoK(ATP). Stimulation of K(+) transport by FFAs/acyl-CoAs resulted in a widespread phenomenon in plant mitochondria from different mono/dicotyledonous species (bread wheat, barley, triticale, maize, lentil, pea, and topinambur) and from different organs (root, tuber, leaf, and shoot). Finally, an increase in mitochondrial FFAs up to a content of 50 nmol mg(-1) protein, which was able to activate PmitoK(ATP) strongly, was observed under hyperosmotic stress conditions. Since PmitoK(ATP) may act against environmental/oxidative stress, its activation by FFAs/acyl-CoAs is proposed to represent a physiological defence mechanism.

The role of free fatty acids in mitochondrial energetic metabolism in winter wheat seedlings

The effect of fatty acids (FAs) (C12–C24) on the functioning of winter wheat (Triticum aestivum L.) mitochondria was studied. Such fatty acids as C12:0, C16:0, and C18:0 and unsaturated FAs, such as C18:1 (n-9 cis), C18:1 (n-12 cis), C18:2 (n-9, 12), (18:3, n-3), and C22:1 (n-9 cis) caused efficient uncoupling of oxidative phosphorylation in mitochondria, i.e., an increase in the nonphosphorylating respiration rate and a decrease in the respiratory control value. It was established that C16:0 had the strongest uncoupling effect among all saturated FAs, and C18:3, among unsaturated FAs. The uncoupling effect of saturated FAs is provided by the ADP/ATP-antiporter, while plant uncoupling proteins play an important role in the uncoupling effect of unsaturated FAs. In addition, unsaturated, as well as saturated FAs might serve as oxidative substrates for mitochondria. It was concluded that the role of FAs in energetic metabolism of winter wheat seedlings consisted of uncoupling of oxidative phosphorylation and of serving as substrates for oxidation.

Characterization of the plant uncoupling protein, SrUCPA, expressed in spadix mitochondria of the thermogenic skunk cabbage

In mammalian brown adipose tissue, uncoupling protein 1 (UCP1), an integral inner mitochondrial membrane protein, triggers a proton leak and converts the energy generated by the resulting electron flow into heat. Although the recent finding of plant UCPs in non-thermogenic tissues has questioned their involvement in thermogenesis, there are few studies of plant UCPs in thermogenic tissues. Therefore, in this work, two cloned UCP cDNAs, SrUCPA and SrUCPB, isolated from the thermogenic spadix of skunk cabbage, were analysed. SrUCPA, not SrUCPB, was identified as the major uncoupling protein, and it was found to be integrated into the inner mitochondrial membrane. Topological analyses indicate that the 1st and 2nd intra-matrix loops are sensitive to trypsin treatment, but the 3rd intra-matrix loop is resistant to it. Using spadix mitochondria, the uncoupling activity of SrUCPA was examined. Although SrUCPA transcripts were constitutively expressed in various tissues irrespective of thermogenic stage, the SrUCPA protein was detected only in the thermogenic tissue or stage. On the other hand, both gene and protein expression for another heat-generating protein, SrAOX, were increased specifically in the thermogenic tissue or stage. Quantitative immunoblot analysis revealed that SrUCPA was an abundant protein in spadix mitochondria, accounting for about 3% of the total mitochondrial protein in the spadix. The results suggest that specific co-expression of SrUCPA and SrAOX protein in the thermogenic tissue or stage, as well as the high expression of SrUCPA protein in spadix mitochondria, may play a role in thermogenesis of skunk cabbage.

Mutational analysis of Arabidopsis thaliana plant uncoupling mitochondrial protein

In this study, point mutations were introduced in plant uncoupling mitochondrial protein AtUCP1, a typical member of the plant uncoupling protein (UCP) gene subfamily, in amino acid residues Lys147, Arg155 and Tyr269, located inside the so-called UCP-signatures, and in two more residues, Cys28 and His83, specific for plant UCPs. The effects of amino acid replacements on AtUCP1 biochemical properties were examined using reconstituted proteoliposomes. Residue Arg155 appears to be crucial for AtUCP1 affinity to linoleic acid (LA) whereas His83 plays an important role in AtUCP1 transport activity. Residues Cys28, Lys147, and also Tyr269 are probably essential for correct protein function, as their substitutions affected either the AtUCP1 affinity to LA and its transport activity, or sensitivity to inhibitors (purine nucleotides). Interestingly, Cys28 substitution reduced ATP inhibitory effect on AtUCP1, while Tyr269Phe mutant exhibited 2.8-fold increase in sensitivity to ATP, in accordance with the reverse mutation Phe267Tyr of mammalian UCP1.

Possible plant mitochondria involvement in cell adaptation to drought stress: A case study: durum wheat mitochondria

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.

The Transcript Levels of two Plant Mitochondrial Uncoupling Protein (pUCP)-Related Genes are not Affected by Hyperosmotic Stress in Durum Wheat Seedlings Showing an Increased Level of pUCP Activity

Etiolated early seedlings of durum wheat submitted to moderate and severe salt (NaCl) and osmotic (mannitol) stress showed no relevant increase of both transcript levels of two plant uncoupling protein (pUCP)-related genes and maximal pUCP activity in purified mitochondria (which estimates protein level); contrarily, pUCP functioning due to endogenous free fatty acids strongly increased. These results show that pUCP activation under hyperosmotic stress may be due to modulation of pUCP reaction rather than to an increased protein synthesis. Finally, a properly developed method, based on a single membrane potential measurement, to evaluate both pUCP maximal activity and functioning, is reported.

PLANT UNCOUPLING MITOCHONDRIAL PROTEINS

Uncoupling proteins (UCPs) are membrane proteins that mediate purine nucleotide-sensitive free fatty acid-activated H(+) flux through the inner mitochondrial membrane. After the discovery of UCP in higher plants in 1995, it was acknowledged that these proteins are widely distributed in eukaryotic organisms. The widespread presence of UCPs in eukaryotes implies that these proteins may have functions other than thermogenesis. In this review, we describe the current knowledge of plant UCPs, including their discovery, biochemical properties, distribution, gene family, gene expression profiles, regulation of gene expression, and evolutionary aspects. Expression analyses and functional studies on the plant UCPs under normal and stressful conditions suggest that UCPs regulate energy metabolism in the cellular responses to stress through regulation of the electrochemical proton potential (Deltamu(H)+) and production of reactive oxygen species.

ZmPUMP encodes a fully functional monocot plant uncoupling mitochondrial protein whose affinity to fatty acid is increased with the introduction of a His pair at the second matrix loop

Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins that uncouple respiration from ATP synthesis. In this study, cDNA encoding maize uncoupling protein (ZmPUMP) was expressed in Escherichia coli and recombinant ZmPUMP reconstituted in liposomes. ZmPUMP activity was associated with a linoleic acid (LA)-mediated H + efflux with K m of 56.36 ± 0.27 μM and V max of 66.9 μmol H + min −1 (mg prot) −1. LA-mediated H + fluxes were sensitive to ATP inhibition with K i of 2.61 ± 0.36 mM (at pH 7.2), a value similar to those for dicot UCPs. ZmPUMP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in plant UCPs. ZmPUMP with introduced His pair (Lys155His and Ala157His) displayed a 1.55-fold increase in LA-affinity while its activity remained unchanged. Our data indicate conserved properties of plant UCPs and suggest an enhancing but not essential role of the histidine pair in proton transport mechanism.

The plant energy-dissipating mitochondrial systems: depicting the genomic structure and the expression profiles of the gene families of uncoupling protein and alternative oxidase in monocots and dicots

The simultaneous existence of alternative oxidases and uncoupling proteins in plants has raised the question as to why plants need two energy-dissipating systems with apparently similar physiological functions. A probably complete plant uncoupling protein gene family is described and the expression profiles of this family compared with the multigene family of alternative oxidases in Arabidopsis thaliana and sugarcane (Saccharum sp.) employed as dicot and monocot models, respectively. In total, six uncoupling protein genes, AtPUMP1-6, were recognized within the Arabidopsis genome and five (SsPUMP1-5) in a sugarcane EST database. The recombinant AtPUMP5 protein displayed similar biochemical properties as AtPUMP1. Sugarcane possessed four Arabidopsis AOx1-type orthologues (SsAOx1a-1d); no sugarcane orthologue corresponding to Arabidopsis AOx2-type genes was identified. Phylogenetic and expression analyses suggested that AtAOx1d does not belong to the AOx1-type family but forms a new (AOx3-type) family. Tissue-enriched expression profiling revealed that uncoupling protein genes were expressed more ubiquitously than the alternative oxidase genes. Distinct expression patterns among gene family members were observed between monocots and dicots and during chilling stress. These findings suggest that the members of each energy-dissipating system are subject to different cell or tissue/organ transcriptional regulation. As a result, plants may respond more flexibly to adverse biotic and abiotic conditions, in which oxidative stress is involved.

Plant uncoupling protein in mitochondria from aged-dehydrated slices of Jerusalem artichoke tubers becomes sensitive to superoxide and to hydrogen peroxide without increase in protein level

We investigated the occurrence of the plant Uncoupling Protein (UCP) in mitochondria isolated from both fresh (f-JAM) and aged-dehydrated (a-d-JAM) slices of Jerusalem artichoke tubers ( Helianthus tuberosus L.). The presence of UCP was shown by immunological analysis and its function was investigated by measuring the decrease of the mitochondrial membrane potential due to linoleic acid (LA) and its inhibition by purine nucleotides under conditions in which the adenine nucleotide translocator (ANT) was inhibited by atractyloside (Atr). f-JAM and a-d-JAM had the same protein content, but differed from one another with respect to purine nucleotide inhibition, substrate specificity, and sensitivity to ROS. Hydrogen peroxide and superoxide anion, generated in situ by xanthine plus xanthine oxidase, caused a significant increase in the UCP function in a-d-JAM, but not in f-JAM. This occurred in a manner sensitive to ATP, but not to Atr, thus showing that ANT has no role in the process. The dependence of the rate of membrane potential decrease on increasing LA concentrations, either in the absence or the presence of ROS, showed a sigmoidal saturation both in f-JAM and a-d-JAM. However, addition of ROS in a-d-JAM resulted in about 40% increase of the V max value, with no change in the K 0.5 (about 20 μM), whereas in f-JAM no effect on either the V max or K 0.5 (about 28 μM) was found. Furthermore, a decreased ROS production as a result of LA addition was found in both f-JAM and a-d-JAM, the effect being more marked in a-d-JAM.

Genomic Structure and Regulation of Mitochondrial Uncoupling Protein Genes in Mammals and Plants

Uncoupling mitochondrial proteins (UCPs) belong to a discrete family within the mitochondrial anion carrier superfamily. Several uncoupling protein types have been found in mitochondria from mammals and plants, as well as in fishes, fungi, and protozoa. Mammalian UCPs and plant uncoupling proteins (PUMPs) form five distinct subfamilies. Only subfamily III contains both plant and animal uncoupling proteins, as well as UCPs from primitive eukaryotic organisms, which suggest that this group may represent an ancestral cluster from which other UCPs/PUMPs may have evolved. Genetic data indicate that UCPs/PUMPs are regulated at the transcriptional, post-transcriptional, and translational levels. Tissue/organ- and stress-specific gene expression suggests that UCPs/PUMPs are involved in the general balance of basic energy expenditure, protection against reactive oxygen species, and thermogenesis. Finally, the simultaneous occurrence of PUMP and alternative oxidase, another energy-dissipating system in plant mitochondria, raises the question of their response to biotic and abiotic stress at the transcriptional and functional levels.

The distribution of electron transport between the main cytochrome and alternative pathways in plant mitochondria during short-term cold stress and cold hardening

The function of the main cytochrome and alternative (alternative cyanide-resistant oxidase and plant uncoupling proteins) electron transport pathways in plant mitochondria during short-term (cold shock) and long-term (hardening) cold exposure was studied. It was shown that influence of cold shock and hardening on the mitochondria respiration are different. The increase of cyanide-resistant respiration in the mitochondria of cereals under both exposures is detected. It is shown that cereals have an alternative pathway associated with activities of uncoupling protein CSP 310.

The plant uncoupling protein homologues: a new family of energy-dissipating proteins in plant mitochondria

Uncoupling proteins (UCPs) form a subfamily within the mitochondrial carrier protein family, which catalyze a free fatty acid-mediated proton recycling and can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis. As in mammalian tissues, UCPs are rather ubiquitous in the plant kingdom and widespread in plant tissues in which they could have various physiological roles, such as heat production or protection against free oxygen radicals. The simultaneous occurrence in plant mitochondria of two putative energy-dissipating systems, namely UCP which dissipates the proton motive force, and alternative oxidase (AOX) which dissipates the redox potential, raises the question of their functional interactions.

Stress CSP310 protein uncouples oxidative phosphorylation otherwise than other plant uncoupling proteins do

The addition of cold shock CSP310 protein to mitochondria isolated from both monocotyledonous (rye, wheat, and maize) and dicotyledonous (pea) plants uncoupled oxidation from phosphorylation. This uncoupling was caused neither by the damage to mitochondrial membranes nor by the activation of alternative cyanide-resistant oxidase. As distinct from the classical plant uncoupling mitochondrial protein (PUMP), CSP310 uncoupling effect was insensitive to BSA. Therefore, we believe that the mechanism of CSP310 action differs from that of known plant uncoupling proteins.

Alteration of Plant Mitochondrial Proton Conductance by Free Fatty Acids: UNCOUPLING PROTEIN INVOLVEMENT

We characterized the uncoupling activity of the plant uncoupling protein from Solanum tuberosum (StUCP) using mitochondria from intact potato tubers or from yeast (Saccharomyces cerevisiae) expressing the StUCP gene. Compared with mitochondria from transfected yeast, StUCP is present at very low levels in intact potato mitochondrial membranes (at least thirty times lower) as shown by immunodetection with anti-UCP1 antibodies. Under conditions that ruled out undesirable effects of nucleotides and free fatty acids on uncoupling activity measurement in plant mitochondria, the linoleic acid-induced depolarization in potato mitochondria was insensitive to the nucleotides ATP, GTP, or GDP. In addition, sensitivity to linoleic acid was similar in potato and in control yeast mitochondria, suggesting that uncoupling occurring in potato mitochondria was because of a UCP-independent proton diffusion process. By contrast, yeast mitochondria expressing StUCP exhibited a higher sensitivity to free fatty acids than those from the control yeast and especially a marked proton conductance in the presence of low amounts of linoleic acid. However, this fatty acid-induced uncoupling was also insensitive to nucleotides. Altogether, these results suggest that uncoupling of oxidative phosphorylation and heat production cannot be the dominant feature of StUCP expressed in native potato tissues. However, it could play a role in preventing reactive oxygen species production as proposed for mammalian UCP2 and UCP3.

Mitochondrial protein expression in tomato fruit during on-vine ripening and cold storage.

We have investigated the activity and abundance of a number of respiratory chain components in ripening and cold-treated tomato fruits (Lycopersicon esculentum L. Mill cvv. Moneymaker and Sweetie). Expression of the alternative oxidase (AOX) protein increased dramatically in both situations. Levels of the plant uncoupling protein (UCP) initially fell, but increased substantially in the later stages of ripening. In contrast, ATP synthase subunits and the COXII subunit of cytochrome oxidase decreased during ripening and increased slightly in response to cold stress. Other proteins involved in electron transport, tricarboxylic acid cycle function, chaperonin function, and membrane transport were also studied. These showed varying degrees of enhanced and depressed expression patterns. There were modest changes in whole fruit respiratory activities, and electron transport capacity of isolated mitochondria in response to these stimuli. However, respiratory control by ADP in the isolated mitochondria decreased as AOX capacity and abundance increased, indicating that although total respiration rates changed little, flux between the coupled and uncoupled pathways altered. The changes observed in AOX and UCP accumulation in tomato fruit that were vine-ripened were significantly different from post-harvest ripening patterns previously reported. The altered protein profiles are discussed in the context of on- and off-vine ripening and the potentially different roles of uncoupled respiration in each situation.