Alternative Respiration Pathways and Secondary Metabolism in Plants with Different Adaptive Strategies under Mineral Deficiency

The costs of adaptation respiration, the contribution of different biochemical pathways of respiration and alternative oxidases to the adaptation to mineral deficiency were compared in plant species representing different types of adaptive strategies: ruderal (Ru) (Amaranthus retroflexus L. and Leonurus quinquelobatus Gilib.) and predominantly stress-tolerant type (St) (Dactylis glomerata L. and Medicago sativa L.). The adaptation component of the total dark respiration was calculated on the basis of the relative growth rate and the ratio of dark respiration to gross photosynthesis. Adaptation costs were greater in less tolerant species. The inhibitory analysis showed that the contribution of glycolytic pathway decreased and the proportion of oxidative pentose phosphate pathway and residual respiration increased in the intolerant Ru-species under the conditions of abiotic stress. Conversely, in the tolerant St-species, the contribution of glycolysis increased. In all species studied, the contribution of the cytochrome pathway of substrate oxidation was reduced and the contribution of the alternative cyanide-insensitive and residual respiration increased under conditions of mineral deficiency; this phenomenon was particularly pronounced in a less tolerant species L. quinquelobatus. In the species characterized by Ru-strategy, stress increased the content of phenolic compounds and shikimic acid. We suppose that the adaptation respiration costs are associated with alternative biochemical pathways and alternative cyanide-resistant oxidase. The role of these pathways in the plants with St-strategy consists in maintaining the active state of oxidative pathways, whereas in the plants with Ru-strategy, they serve to burn excess metabolites and enhance the synthesis of secondary metabolites, which perform protective functions.     

Reoxidation of cytosolic NADPH in Kluyveromyces lactis

Saccharomyces cerevisiae and Kluyveromyces lactis are considered to be the prototypes of two distinct metabolic models of facultatively-aerobic yeasts: Crabtree-positive/fermentative and Crabtree-negative/respiratory, respectively. Our group had previously proposed that one of the molecular keys supporting this difference lies in the mechanisms involved in the reoxidation of the NADPH produced as a consequence of the activity of the pentose phosphate pathway. It has been demonstrated that a significant part of this reoxidation is carried out in K. lactis by mitochondrial external alternative dehydrogenases which use NADPH, the enzymes of S. cerevisiae being NADH-specific. Moreover, the NADPH-dependent pathways of response to oxidative stress appear as a feasible alternative that might co-exist with direct mitochondrial reoxidation.     

Respiratory metabolism in the embryonic axis of germinating pea seed exposed to cadmium

Seeds of pea (Pisum sativum L.) were germinated for 5d by soaking in distilled water or 5mM cadmium nitrate. The relationships among cadmium stress, germination rate, changes in respiratory enzyme activities and carbohydrates mobilization were studied. Two cell fractions were obtained from embryonic axis: (1) mitochondria, used to determine enzyme activities of citric acid cycle and electron transport chain, and (2) soluble, to measure some enzyme activities involved in fermentation and pentose phosphate pathway. Activities of malate- and succinate-dehydrogenases (MDH, SDH) and NADH- and succinate-cytochrome c reductases (NCCR, SCCR) were rapidly inhibited, while cytochrome c oxidase (CCO) was unaltered by cadmium treatment. However, this stimulated the NADPH-generating enzyme activities of the pentose phosphate pathway, glucose-6-phosphate- and 6-phosphogluconate-dehydrogenases (G6PDH, 6PGDH), as well as enzyme activity of fermentation, alcohol dehydrogenase (ADH), with concomitant inhibition in the capacity of enzyme inactivator (INADH). Moreover, Cd restricted carbohydrate mobilization in the embryonic axis. Almost no glucose and less than 7% of control fructose and total soluble sugars were available in the embryo tissues after 5d of exposure to cadmium. Cotyledonary invertase isoenzyme activity was also inhibited by Cd. The results indicate that cadmium induces disorder in the resumption of respiration in germinating pea seeds. The contribution of Cd-stimulated alternative metabolic pathways to compensate for the failure in mitochondrial respiration is discussed in relation to the delay in seed germination and embryonic axis growth.     

Copper-induced oxidative stress in Saccharomyces cerevisiae targets enzymes of the glycolytic pathway

Increased cellular levels of reactive oxygen species are known to arise during exposure of organisms to elevated metal concentrations, but the consequences for cells in the context of metal toxicity are poorly characterized. Using two-dimensional gel electrophoresis, combined with immunodetection of protein carbonyls, we report here that exposure of the yeast Saccharomyces cerevisiae to copper causes a marked increase in cellular protein carbonyl levels, indicative of oxidative protein damage. The response was time dependent, with total-protein oxidation peaking approximately 15 min after the onset of copper treatment. Moreover, this oxidative damage was not evenly distributed among the expressed proteins of the cell. Rather, in a similar manner to peroxide-induced oxidative stress, copper-dependent protein carbonylation appeared to target glycolytic pathway and related enzymes, as well as heat shock proteins. Oxidative targeting of these and other enzymes was isoform-specific and, in most cases, was also associated with a decline in the proteins' relative abundance. Our results are consistent with a model in which copper-induced oxidative stress disables the flow of carbon through the preferred glycolytic pathway, and promotes the production of glucose-equivalents within the pentose phosphate pathway. Such re-routing of the metabolic flux may serve as a rapid-response mechanism to help cells counter the damaging effects of copper-induced oxidative stress.     

The oxidative pentose phosphate pathway: structure and organisation

The oxidative pentose phosphate pathway is a major source of reducing power and metabolic intermediates for biosynthetic processes. Some, if not all, of the enzymes of the pathway are found in both the cytosol and plastids, although the precise distribution of their activities varies. The apparent absence of sections of the pathway from the cytosol potentially complicates metabolism. These complications are partly offset, however, by exchange of intermediates between the cytosol and the plastids through the activities of a family of plastid phosphate translocators. Molecular analysis is confirming the widespread presence of multiple genes encoding each of the enzymes of the oxidative pentose phosphate pathway. Differential expression of these isozymes may ensure that the kinetic properties of the activity that catalyses a specific reaction match the metabolic requirements of a particular tissue. This hypothesis can be tested thanks to recent developments in the application of 13C-steady-state labelling strategies. These strategies make it possible to quantify flux through metabolic networks and to discriminate between pathways of carbohydrate oxidation in the cytosol and plastids.     

Evolution of metabolic pathways by chance assembly of enzyme proteins generated from sense and antisense strands of pre-existing genes.

In order to get an insight into the evolutionary aspect of metabolic pathways, especially of the ubiquitous glycolytic pathway, we have carried out an extensive search of sense-sense and sense-antisense similarities for enzyme proteins in the glycolytic pathway, the pentose phosphate cycle, alcohol and lactate fermentation pathways and the TCA cycle. This investigation of amino acid sequences reveals a curious pattern of similarity relations; no similarity can be found between the enzyme proteins in a section of the glycolytic pathway where the glyceraldehyde-3-phosphate or even glycerol-3-phosphate is converted into the pyruvate while many examples of sense-sense and sense-antisense similarities are found even between enzyme proteins in distant blocks, e.g. between the proteins in the TCA cycle and those in the pentose phosphate cycle, as well as between the functionally associated proteins in each of these blocks. Complementary to this characteristic pattern of amino acid sequence similarity, the search for similarities of nucleotide sequences also finds that the similarities of glycolytic enzyme genes, some sense-sense and others sense-antisense similarities, are concentrated on the nucleotide sequences of prokaryotic 16S or eukaryotic 18S ribosomal RNA gene with its flanks, although some of the copy sequences are also found in transfer RNA genes as well as in 23S or 26S ribosomal RNA gene. These results strongly suggest that the metabolic pathways have been developed by the chance assembly of enzyme proteins generated from the sense and antisense strands of pre-existing genes, e.g. the fermentation pathways and pentose phosphate cycle by the proteins from the genes of enzymes in the glycolytic pathway and the TCA cycle from all these successively increased genes, ascribing the origin of metabolic enzyme genes to the close relation between the glycolytic enzyme protein genes and the RNA gene cluster.     

Kernel abortion in maize : I. Carbohydrate concentration patterns and Acid invertase activity of maize kernels induced to abort in vitro

The distribution of the glycolytic enzymes, phosphofructokinase, aldolase, triosephosphate isomerase, phosphoglycerate kinase, pyruvate kinase, and the oxidative pentose phosphate pathway enzymes, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase, was determined in the leaf tissues of two C₃-plants, pea and leek, and two C₄-plants, maize and sorghum. All enzymes examined were found in epidermal tissue. In pea, maize, and sorghum leaves, the specific activities of these enzymes were usually higher in the nonphotosynthetic epidermal tissue than in the photosynthetic tissues of the leaves. In leek leaves, which were etiolated, specific activities were similar in both epidermal and mesophyll tissue. The distribution of the rate limiting enzymes of glycolysis and the oxidative pentose phosphate pathways probably reflects the capacity of each tissue to generate NADH, NADPH, and ATP from the oxidation of glucose. This capacity appears to be greater in leaf tissues unable to generate reducing equivalents and ATP by photosynthesis, that is, in epidermal tissues and etiolated mesophyll tissue.     

Functional significance of the pentose phosphate pathway and glutathione reductase in the antioxidant defenses of human sperm

Glutathione peroxidase is one of the principal antioxidant defense enzymes in human spermatozoa, but it requires oxidized glutathione to be reduced by glutathione reductase using NADPH generated in the pentose phosphate pathway. We investigated whether flux through the pentose phosphate pathway would increase in response to oxidative stress and whether glutathione reductase was required to protect sperm from oxidative damage. Isotopic measurements of the pentose phosphate pathway and glycolytic flux, thiobarbituric acid assay of malondialdehyde for lipid peroxidation, and computer-assisted sperm analysis for sperm motility were assessed in a group of normal, healthy semen donors. Applying moderate oxidative stress to human spermatozoa by adding cumene hydroperoxide, H(2)O(2), or xanthine plus xanthine oxidase or by promoting lipid peroxidation with ascorbate increased flux through the pentose phosphate pathway without changing the glycolytic rate. However, adding higher concentrations of oxidants inhibited both the pentose phosphate pathway and glycolytic flux. At concentrations of 50 microg/ml or greater, the glutathione reductase-inhibitor 1,3-bis-(2-chloroethyl) 1-nitrosourea decreased flux through the pentose phosphate pathway and blocked the response to cumene hydroperoxide. It also increased lipid peroxidation and impaired the survival of motility in sperm incubated under 95% O(2). These data show that the pentose phosphate pathway in human spermatozoa can respond dynamically to oxidative stress and that inhibiting glutathione reductase impairs the ability of sperm to resist lipid peroxidation. We conclude that the glutathione peroxidase-glutathione reductase-pentose phosphate pathway system is functional and provides an effective antioxidant defense in normal human spermatozoa.     

Physiology of Sporeforming Bacteria Associated with Insects IV. Glucose Catabolism in Bacillus larvae

Bacillus larvae appears to be unique among related bacilli in that it contains enzymes of the Embden-Meyerhof-Parnas, pentose phosphate, and Entner-Doudoroff pathways. Simultaneous occurrence of enzymes of all three metabolic pathways has not until now been reported in other Bacillus species. Radiorespirometric analyses of specifically labeled glucose catabolism reveal that vegetative cells of B. larvae dissimilate glucose predominately via a direct oxidative route and to a lesser extent by a nonoxidative scheme although specific activities of enzymes of all three pathways are comparable. Predominance of an oxidative pathway is unusual and also has not been reported for other bacilli. Studies on the oxidation of pyruvic, acetic, succinic, and alpha-ketoglutaric acids show that terminal respiration of cells in transition from vegetative growth to sporulation involves both the tricarboxylic acid and glyoxylic acid cycles. The relationship of these findings to the fastidiousness and oligosporogeny of B. larvae is discussed.     

Pathways of carbohydrate metabolism in peripheral nervous tissue. I. The contribution of alternative routes of glucose utilization in peripheral nerve and brain

The activities of enzymes of the glycolytic route, the pentose phosphate pathway, the tricarboxylic acid cycle and lipogenesis have been measured in rat sciatic nerve and brain. Parallel studies have been made of the utilization of 14 C-labelled glucose and pyruvate in these two tissues. Comparison of the enzyme profiles and flux through alternative routes was based on activity relative to the rate of glucose phosphorylation as measured by the rate of formation of 3H2O from [2-3H]glucose. The contributions of the pentose phosphate pathway and lipogenesis to glucose utilization were substantially higher in sciatic nerve than brain. The relatively high activity of transketolase (EC 2.2.1.1) and transaldolase (EC 2.2.1.2) suggested a special role for these enzymes in sciatic nerve.     

Variazioni di attività della clorogenico ossidasi e di altri enzimi interessati all'ossidoriduzione del TPN durante l'attivazione di fettine di tubero di patata

Abstract

CHANGES IN THE ACTIVITY OF CHLOROGENIC ACID OXIDASE AND OTHER ENZYMES INVOLVED IN OXIDATION AND REDUCTION OF TPN IN AGEING POTATO TUBER SLICES. — The activation of respiration, and in particular of the pentose phosphate pathway, during incubation of potato tuber slices could depend on the increase of activity of oxidative enzymes mediating electron transfer from Gl. 6-P to oxygen.

The present report deals with the activity changes, in the first period of incubation, of the following enzymes: Gl. 6-P-dehydrogenase, TPNH-glutathione reductase, gluta-thione-dehydroascorbate reductase, chlorogenic acid oxidase and a TPNH diaphorase utilizing tetrazolium salts as electron acceptors.

The activity of all of these enzymes, with the exception of TPNH diaphorase, was found to bs, at all stages of incubation, in large excess respect that required to account for the estimated contribution of the pentose phosphate pathway to respiration.

Gl. 6-P dehydrogenase, glutathione reductase and chlorogenic oxidase activities markedly incresed during incubation; but their increase appeared to be clearly delayed (of some hours) respect that of oxygen uptake. This seems to indicate that the increase in activity of these anzymes is rather a consequence than a cause of the respiratory activation.

TPNH diaphorase showed a very low activity in the fresh slices, and it increased quite significantly already in the very first period (5 hours) of incubation. This behaviour suggests the possibility that this enzyme could limit TPNH oxidation, and thus the pentose phosphate pathway activity, and that its activation could be correlated with that of oxidative metabolism in the ageing slices. Further investigation of this hypothesis requires the identification of the natural electron acceptor of this enzyme.     

A dual role of tobacco hexokinase 1 in primary metabolism and sugar sensing

Hexokinase (HXK) is present in all virtually living organisms and is central to carbohydrate metabolism catalysing the ATP‐dependent phosphorylation of hexoses. In plants, HXKs are supposed to act as sugar sensors and/or to interact with other enzymes directly supplying metabolic pathways such as glycolysis, the nucleotide phosphate monosaccharide (NDP‐glucose) pathway and the pentose phosphate pathway. We identified nine members of the tobacco HXK gene family and observed that among RNAi lines of these nine NtHXKs, only RNAi lines of NtHXK1 showed an altered phenotype, namely stunted growth and leaf chlorosis. NtHXK1 was also the isoform with highest relative expression levels among all NtHXKs. GFP‐tagging and immunolocalization indicated that NtHXK1 is associated with mitochondrial membranes. Overexpression of NtHXK1 resulted in elevated glucose phosphorylation activity in leaf extracts or chloroplasts. Moreover, NtHXK1 was able to complement the glucose‐insensitive Arabidopsis mutant gin2‐1 suggesting that NtHXK1 can take over glucose sensing functions. RNAi lines of NtHXK1 showed severely damaged leaf and chloroplast structure, coinciding with an excess accumulation of starch. We conclude that NtHXK1 is not only essential for maintaining glycolytic activity during respiration but also for regulating starch turnover, especially during the night.     

Carbohydrate metabolism in the liver of rats in food deprivation and experimental diabetes

In experiments with starving rats (Rattus rattus L.) and rats with alloxan-induced diabetes, the activity of enzymes and the contents of metabolites of the main metabolic pathways have been studied. It has been shown that adaptation of carbohydrate metabolism to these stress conditions has common trends: intensification of gluconeogenetic processes and glyoxylate cycle induction are observed against the background of decrease in the activities of glycolytic enzymes and the pentose phosphate pathway. A hypothetical mechanism of adaptation of rat liver cell metabolism to a deficiency of glucose as the main energy substrate is proposed.     

Glycolytic enzymes associate dynamically with mitochondria in response to respiratory demand and support substrate channeling

In Arabidopsis thaliana, enzymes of glycolysis are present on the surface of mitochondria and free in the cytosol. The functional significance of this dual localization has now been established by demonstrating that the extent of mitochondrial association is dependent on respiration rate in both Arabidopsis cells and potato (Solanum tuberosum) tubers. Thus, inhibition of respiration with KCN led to a proportional decrease in the degree of association, whereas stimulation of respiration by uncoupling, tissue ageing, or overexpression of invertase led to increased mitochondrial association. In all treatments, the total activity of the glycolytic enzymes in the cell was unaltered, indicating that the existing pools of each enzyme repartitioned between the cytosol and the mitochondria. Isotope dilution experiments on isolated mitochondria, using (13)C nuclear magnetic resonance spectroscopy to monitor the impact of unlabeled glycolytic intermediates on the production of downstream intermediates derived from (13)C-labeled precursors, provided direct evidence for the occurrence of variable levels of substrate channeling. Pull-down experiments suggest that interaction with the outer mitochondrial membrane protein, VDAC, anchors glycolytic enzymes to the mitochondrial surface. It appears that glycolytic enzymes associate dynamically with mitochondria to support respiration and that substrate channeling restricts the use of intermediates by competing metabolic pathways.     

Are hyperhydric shoots of Prunus avium L. energy deficient?

The content of oxidized and reduced pyridine nucleotides and some enzymatic activities of the oxidative pentose phosphate and glycolytic pathways were compared in normal (NS, growing on agar) and hyperhydric (HS, growing on gelrite) shoots of Prunus aviumL. after 4 weeks of in vitro culture. The chlorophyll fluorescence from leaves and the redox capacity of the plasma membrane (reduction of exogenously added ferricyanide) of both types of shoots were recorded. The pool of oxidized and reduced pyridine nucleotides was lower in HS than in NS. These results suggested a reduced metabolism of HS in comparison to normal ones. This hypothesis was also supported by other observations. First, chlorophyll fluorescence measurements showed a lower chlorophyll content and a slight reduction of the photosynthetic capacity in HS. Second, the low activity of some enzymes of oxidative pentose phosphate pathway (OPP) and glycolysis indicated a decline of these biochemical pathways in HS with the consequence of a reduced production of chemical energy in the form of NAD(P)H and ATP. Finally, the lower reduction of ferricyanide by HS suggested a lower rate of redox reactions at the level of the plasma membrane of these shoots in comparison to NS.     

REGULATION OF ALTERNATIVE PATHWAY RESPIRATION IN CHLAMYDOMONAS REINHARDTH (CHLOROPHYCEAE)1

The inhibitor propyl gallate was used to estimate partitioning of respiratory electron flow between the cytochrome amd alternative pathways in Chlamydomonas reinhardtii Dangeard. Nutrient limitation (nitrogen or phosphorus resulted in a large increase in alternative pathway capacity relative to cytochrome pathway activity, without regulating in engagement of the alternative pathway. High rates of respiration, which could be induced in phosphate-starved cells by a combination of phosphate addition and uncoupler, resulted in alternative pathway activity. Osmotic stress resulted in decreased electron flow through the cytochrome pathway and increased flow through the alternative pathway, while high temperature also resulted in alternative pathway engagement. Incubation with exogenous carbon sources could increase the rate of respiratory O₂ consumption; the increase was mediated entirely by the alternative pathway. We suggest that the alternative pathway functions in these cells both to maintain respiration during environmentally induced stress and as on energy overflow.     

Analysis of growth of Gluconobacter oxydans in glucose containing media

Gluconobacter oxydans oxidizes glucose via alternative pathways: one involves the non-phosphorylative, direct oxidation route to gluconic acid and ketogluconic acids, and the second requires an initial phosphorylation and then oxidation via the pentose phosphate pathway enzymes. During growth of G. oxydans in glucose-containing media, the activity of this pathway is strongly influenced by (1) the pH value of the environment and (2) the actual concentration of glucose present in the culture. At pH values below 3.5 the activity of the pentose phosphate pathway was completely inhibited resulting in an increased requirement of the organism for nutrient substances, and a poor cell yield. At pH 5.5 a triphasic growth response was observed when G. oxydans was grown in a defined medium. Above a threshold value of 5–15 mM glucose, oxidation of both glucose and gluconate by the pentose phosphate pathway enzymes was repressed, causing a rapid accumulation of gluconic acid in the culture medium. When growing under these conditions, a low affinity for the oxidation of glucose was found (K _s=13 mM). Below this threshold glucose concentration, pentose phosphate pathway enzymes were synthesized and glucose was actively assimilated via this pathway. It was shown that de novo enzyme synthesis was necessary for increased pentose phosphate pathway activity and that assimilation of gluconate by washed cell suspensions was inhibited by glucose.     

The NADPH-dependent electron flow in chloroplasts of the higher plants

The NADPH-dependent reduction of some photosynthetic electron carriers in the dark, and the reduction of NADP+ associated with the glycolytic sequence and the oxidative pentose phosphate pathway in chloroplasts are reviewed. The postulated pathways of electron transports sensitive and insensitive to antimycin A are also evaluated. It is proposed that the electron flow, predominantly through cytochrome bf complex, may be also involved in the pathway of NADPH-dependent and antimycin A-insensitive back electron transport. An information on the chlororespiration in higher plants is also included.