Cyanide-resistant respiration in photosynthetic organs of freshwater aquatic plants

The rate and sensitivity to inhibitors (KCN and salicylhydroxamic acid[SHAM]) of respiratory oxygen uptake has been investigated in photosynthetic organs of several freshwater aquatic plant species: six angiosperms, two bryophytes, and an alga. The oxygen uptake rates on a dry weight basis of angiosperm leaves were generally higher than those of the corresponding stems. Leaves also had a higher chlorophyll content than stems. Respiration of leaves and stems of aquatic angiosperms was generally cyanide-resistant, the percentage of resistance being higher than 50% with very few exceptions. The cyanide resistance of respiration of whole shoots of two aquatic bryophytes and an alga was lower and ranged between 25 and 50%. These results suggested that the photosynthetic tissues of aquatic plants have a considerable alternative pathway capacity. The angiosperm leaves generally showed the largest alternative path capacity. In all cases, the respiration rate of the aquatic plants studied was inhibited by SHAM alone by about 13 to 31%. These results were used for calculating the actual activities of the cytochrome and alternative pathways. These activities were generally higher in the leaves of angiosperms. The basal oxygen uptake rate of Myriophyllum spicatum leaves was not stimulated by sucrose, malate or glycine in the absence of the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP), but was greatly increased by CCCP, either in the presence or in the absence of substrates. These results suggest that respiration was limited by the adenylate system, and not by substrate availability. The increase in the respiratory rate by CCCP was due to a large increase in the activities of both the cytochrome and alternative pathways. The respiration rate of M. spicatum leaves in the presence of substrates was little inhibited by SHAM alone, but the SHAM-resistant rate (that is, the cytochrome path) was greatly stimulated by the further addition of CCCP. Similarly, the cyanide-resistant rate of O₂ uptake was also increased by the uncoupler.     

Short-term effects of carbon dioxide on carnation callus cell respiration

The addition of potassium bicarbonate to the electrode cuvette immediately stimulated the rate of dark O₂ uptake of photomixotrophic and heterotrophic carnation (Dianthus caryophyllus L.) callus, of Elodea canadensis (Michx) leaves, and of other plant tissues. This phenomenon occurred at pH values lower than 7.2 to 7.8, and the stimulation depended on the concentration of gaseous CO₂ in the solution. These stimulatory responses lasted several minutes and then decreased, but additional bicarbonate or gaseous CO₂ again stimulated respiration, suggesting a reversible effect. Carbonic anhydrase in the solution increased the stimulatory effect of potassium bicarbonate. The CO₂/bicarbonate dependent stimulation of respiration did not occur in animal tissues such as rat diaphragm and isolated hepatocytes, and was inhibited by salicylhydroxamic acid in carnation callus cells and E. canadensis leaves. This suggested that the alternative oxidase was engaged during the stimulation in plant tissues. The cytochrome pathway was severely inhibited by CO₂/bicarbonate either in the absence or in the presence of the uncoupler carbonylcyanide m-chlorophenyl hydrazone. The activity of cytochrome c oxidase of callus tissue homogenates was also inhibited by CO₂/bicarbonate. The results suggested that high carbon dioxide levels (mainly free CO₂) partially inhibited the cytochrome pathway (apparently at the oxidase level), and this block in electron transport elicited a large transient engagement of the alternative oxidase when present uninhibited.     

The regulation of glycolysis and electron transport in roots

The respiration of roots and isolated root mitochondria was investigated in Phaseolus vulgaris L., Spinacea oleracea L.; Triticum aestivum L., and Zea mays L. Although the respiration of both intact roots and isolated mitochondria displayed resistance to cyanide and sensitivity to SHAM, the percentage resistance and inhibition in roots was not the same as that in the mitochondria, with the exception of wheat. Adding FCCP to roots stimulated oxygen uptake and equalized the effects of SHAM and cyanide on roots and mitochondria. In spinach and maize roots, FCCP stimulated both the cytochrome and alternative pathways, while in bean roots, only the alternative pathway was stimulated. FCCP had little effect on wheat root respiration rates. Potential in vivo rates of oxygen uptake were estimated by expressing rates obtained with isolated mitochondria on a fumarase activity basis, and fumarase activity on a root weight basis. In wheat roots the potential rate was approximately equal to the measured in vivo rate; in the other species the potential rates were substantially greater than measured rates, but approximately equal to uncoupled in vivo rates. Key glycolytic intermediates in roots were measured, and it was found that the phosphofructokinase and pyruvate kinase reactions were displaced far from equilibrium, the degree of displacement being approximately equal in roots with little, and roots with substantial, alternative path engagement. Thus, although glycolysis is controlled, the regulation of this pathway appears to be quite flexible. The results are discussed in terms of possible regulatory mechanisms.     

Cyanide-resistant respiration in roots and leaves. Measurements with intact tissues and isolated mitochondria

A comparison was made between the oxygen uptake of roots and leaves and of mitochondria isolated from the same tissues. Ten species were included in this study: three legumes, one C3-monocotyledon, one C4-monocotyledon, the rest non-leguminous C3-dicotyledons. Root and leaf respiration in all species examined displayed substantial resistance to KCN (0.1–1.0 mM) and the cyanide-resistant respiration was completely inhibited by salicylhydroxamic acid (SHAM; 10–20 mM). SHAM alone inhibited oxygen uptake to varying degrees, depending on the species. Mitochondria were isolated from roots and leaves of many of the species examined and also displayed cyanide-resistant oxygen uptake, which was sensitive to both SHAM and tetraethylthiuram disulfide (disulfiram). Concentrations of SHAM greater than 2 mM caused inhibition of the cytochrome path as well as of the alternative path in isolated mitochondria. Respiration rates of intact roots and leaves in the presence of varying concentrations of SHAM alone were plotted against those obtained in the presence of both SHAM and KCN. This plot showed that in vivo the cytochrome pathway was not affected by 10 or 20 mM SHAM in the external solution. We conclude that the activity of the alternative pathway in intact roots and leaves can be reliably estimated by comparing SHAM-sensitivity and cyanide-resistance of respiration.     

Leaf Photosynthesis and Respiration of High CO(2)-Grown Tobacco Plants Selected for Survival under CO(2) Compensation Point Conditions

Four self-pollinated, doubled-haploid tobacco, (Nicotiana tabacum L.) lines (SP422, SP432, SP435, and SP451), selected as haploids by survival in a low CO₂ atmosphere, and the parental cv Wisconsin-38 were grown from seed in a growth room kept at high CO₂ levels (600-700 parts per million). The selected plants were much larger (especially SP422, SP432, and SP451) than Wisconsin-38 nine weeks after planting. The specific leaf dry weight and the carbon (but not nitrogen and sulfur) content per unit area were also higher in the selected plants. However, the chlorophyll, carotenoid, and alkaloid contents and the chlorophyll a/b ratio varied little. The net CO₂ assimilation rate per unit area measured in the growth room at high CO₂ was not higher in the selected plants. The CO₂ assimilation rate versus intercellular CO₂ curve and the CO₂ compensation point showed no substantial differences among the different lines, even though these plants were selected for survival under CO₂ compensation point conditions. Adult leaf respiration rates were similar when expressed per unit area but were lower in the selected lines when expressed per unit dry weight. Leaf respiration rates were negatively correlated with specific leaf dry weight and with the carbon content per unit area and were positively correlated with nitrogen and sulfur content of the dry matter. The alternative pathway was not involved in respiration in the dark in these leaves. The better carbon economy of tobacco lines selected for low CO₂ survival was not apparently related to an improvement of photosynthesis rate but could be related, at least partially, to a significantly reduced respiration (mainly cytochrome pathway) rate per unit carbon.     

Tomato Fruit Chromoplasts Behave as Respiratory Bioenergetic Organelles during Ripening

During tomato (Solanum lycopersicum) fruit ripening, chloroplasts differentiate into photosynthetically inactive chromoplasts. It was recently reported that tomato chromoplasts can synthesize ATP through a respiratory process called chromorespiration. Here we show that chromoplast oxygen consumption is stimulated by the electron donors NADH and NADPH and is sensitive to octyl gallate (Ogal), a plastidial terminal oxidase inhibitor. The ATP synthesis rate of isolated chromoplasts was dependent on the supply of NAD(P)H and was fully inhibited by Ogal. It was also inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone, suggesting the involvement of a chemiosmotic gradient. In addition, ATP synthesis was sensitive to 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, a cytochrome b₆f complex inhibitor. The possible participation of this complex in chromorespiration was supported by the detection of one of its components (cytochrome f) in chromoplasts using immunoblot and immunocytochemical techniques. The observed increased expression of cytochrome c₆ during ripening suggests that it could act as electron acceptor of the cytochrome b₆f complex in chromorespiration. The effects of Ogal on respiration and ATP levels were also studied in tissue samples. Oxygen uptake of mature green fruit and leaf tissues was not affected by Ogal, but was inhibited increasingly in fruit pericarp throughout ripening (up to 26% in red fruit). Similarly, Ogal caused a significant decrease in ATP content of red fruit pericarp. The number of energized mitochondria, as determined by confocal microscopy, strongly decreased in fruit tissue during ripening. Therefore, the contribution of chromoplasts to total fruit respiration appears to increase in late ripening stages.Chromoplasts are plastids specialized in the production and accumulation of carotenoids, conferring color to many fruits and flowers. During tomato (Solanum lycopersicum) fruit ripening, chloroplasts differentiate into chromoplasts in a process that involves the dismantling of the photosynthetic apparatus and a massive synthesis and deposition of lycopene (Camara et al., 1995). Chromoplasts show a barely studied respiratory process, first reported for daffodil (Narcissus pseudonarcissus) chromoplasts and called chromorespiration, which consists of a membrane-bound redox pathway associated with carotenoid desaturation and results in oxygen uptake activity (Nievelstein et al., 1995). The most likely oxidase involved in this respiratory activity is the plastidial terminal oxidase (PTOX), a plastoquinol oxidase homologous to the mitochondrial alternative oxidase (AOX; Carol et al., 1999; Wu et al., 1999). According to its role in chromorespiration and in carotenoid biosynthesis, the expression of PTOX increases during the ripening process of tomato and bell pepper (Capsicum annuum) fruits (Josse et al., 2003), in parallel to chromoplast differentiation. PTOX has been characterized in vitro and it has been reported to be inhibited by pyrogallol analogs, specially by octyl gallate (Ogal; Josse et al., 2000). In vivo, PTOX has been studied mainly in chloroplasts. PTOX not only participates in carotenoid biosynthesis in chloroplasts but is also involved in chlororespiration, an electron transport chain present in thylakoids that shares plastoquinone with the photosynthetic electron transport chain (Carol and Kuntz, 2001; McDonald et al., 2011).In daffodil chromoplast homogenates (Nievelstein et al., 1995) as well as in isolated tomato fruit chromoplasts (Pateraki et al., 2013), NAD(P)H acts as an electron donor for chromorespiration, indicating the participation of NAD(P)H plastoquinone oxidoreductase activity. Considering that tomato fruit chromoplasts derive from chloroplasts, it is possible that some components of the chromoplastic redox pathway could originate from chlororespiration, such as the NAD(P)H:plastoquinone-reductase complex (NDH), which could act as the electron entrance. However, the enzymes involved in chromorespiration are not well known. It was also reported that the oxygen uptake activity of daffodil chromoplast homogenates was sensitive to the classic uncoupler 2,4-dinitrophenol (Nievelstein et al., 1995), and this observation led to the proposal that chromorespiration could be linked to membrane energization. Morstadt et al. (2002) found that liposomes containing daffodil chromoplast proteins and energized by an acid-base transition were able to produce ATP through a chemiosmotic mechanism, demonstrating that daffodil chromoplasts contain a functional H⁺-ATP synthase complex. We recently reported that isolated chromoplasts from tomato fruits can synthesize ATP de novo (Pateraki et al., 2013). This process is dependent on an ATP synthase complex containing an atypical γ-subunit without the regulatory dithiol domain, which may be active using lower proton gradients than those present in the chloroplast (Pateraki et al., 2013). This finding is consistent with proteomic analyses that reveal that several proteins related to electron transport and ATP production are present in chromoplasts of ripe fruits, like ATP synthase, some subunits of the NDH complex, and the cytochrome b₆f complex (Barsan et al., 2012; Wang et al., 2013).Several anabolic pathways that require ATP and reducing agents are active in ripe fruit chromoplasts, such as synthesis of carotenoids, lipids (glycolipids, phospholipids, and sterols), and the shikimate pathway (Bian et al., 2011; Angaman et al., 2012). On the other hand, the ATP synthesis capacity of mitochondria in ripe fruit is low, because its membrane potential diminishes during ripening as a result of the increasing activity of the mitochondrial uncoupling protein (Almeida et al., 1999; Costa et al., 1999). This fact raised the question of whether chromorespiration could play a significant role in the production of ATP at the last stages of ripening. To our knowledge, the ATP synthesis rates of chromoplasts have not been quantified; therefore, it was uncertain whether the endogenous production could provide ATP in significant amounts to address the energy requirements of the chromoplasts. Moreover, there was no information about the quantitative contribution of chromorespiration to total fruit tissue respiration. This work aimed to deepen the study of the chromorespiratory process in isolated tomato fruit chromoplasts and to analyze the relative participation of this pathway in the overall respiration and ATP levels of fruit pericarp in vivo.     

Effects of temperature,oxygen concentration,leaf age and seasonal variations on the CO2 compensation point of Lolium perenne L.

Various factors affect the CO₂ compensation point of detached leaves of Lolium perenne L. These include oxygen concentration, temperature, leaf age, and season (spring and summer). Analysis of the results using the model of G.D. Farquhar, S. von Caemmerer and J.A. Berry (1980) Planta 149, 78–90, indicates that some of the CO₂ evolved by leaves in the light is derived from sources other than photorespiration. It is suggested that the operation of the tricarboxylic acid in the light can account for most of this CO₂.Azcón-Bieto—experimental work was done in Barcelona, the further analyses white at the A.N.U.     

Actual and potential dark respiration rates and different electron transport pathways in freshwater aquatic plants

The rates of respiratory O₂ uptake have been studied in leaves, stems and whole shoots of several freshwater plants: 6 angiosperms, 2 bryophytes and one alga. For angiosperm leaves, rates varied widely with species (30–142 μmol O₂ (gDW)⁻¹ h⁻¹), were correlated with chlorophyll content and were higher than those of the stems (13–71 μmol O₂ (gDQ)⁻¹ h⁻¹). The rates for the shoots of bryophytes (53–66 μmol O₂ (gDW)⁻¹ h⁻¹) and for the alga Cladophora glomerata (L.) Kütz. (96 μmol O₂ (gDW)⁻¹ h⁻¹) were slightly higher than those of most angiosperm stems, but lower than those for most leaves.

These plants had a significant cyanide-resistant respiration, suggesting the existence of an alternative pathway to the “classic” cytochrome system. This pathway was found to be active in all the species studied, as judged by responses to a specific inhibitor, SHAM (salicylhydroxamic acid). Measurement of electron-transport system (ETS) activity showed that there is a large electron-transport capacity which is not normally used by respiration in vivo.     

Effect of photosynthesis and carbohydrate status on respiratory rates and the involvement of the alternative pathway in leaf respiration

In spinach (Spinacia oleracea Hybrid 102 [New World seeds]) and wheat (Triticum aestivum L. cv Gabo) leaves, O₂ uptake rates in the dark were faster after the plants had been allowed to photosynthesize for a period of several hours. Alternative path activity also increased following a period of photosynthesis in these leaves. No such effects were observed with isolated mitochondria. In spinach and wheat leaves, the level of fructose plus glucose decreased during a period of darkness. In pea (Pisum sativum cv Alaska) leaves, the level of these sugars did not vary significantly during the day, and respiratory rates were also constant. In slices cut from wheat leaves harvested at the end of the night, addition of sugars increased the rate of respiration and engaged the previously latent alternative oxidase. In pea leaves, O₂ uptake in the first few minutes following illumination was faster than that observed before illumination, but declined during the next 15 to 20 minutes. Adding the alternative oxidase inhibitor salicylhydroxamic acid, or imposing high bicarbonate concentrations during the period of photosynthesis, prevented the rise in O₂ uptake rate during the immediate post illumination period.

We conclude that the level of respiratory substrate in leaves determines their rate of O₂ uptake, and the degree to which the alternative path contributes to that O₂ uptake.

     

The metabolic environment of cancer

The tumor cell has a very distinctive metabolism. It acts as a metabolic trap for host nutrients thus taking vital compounds for the metabolism of the host. Depending on the particular tumor growing pattern, cancer cells use preferentially glucose or amino acids for their energetic or biosynthetic needs. Lipids, fatty acids in particular, can also be taken up by the tumor cell. In addition, it can also release some compounds into the host circulation which are not normally produced by the original cell before neoplastic transformation. Some of these compounds affect the metabolism of the host in an unfavorable way since they can oppose the host's metabolic responses, which sustain homeostasis. The final product is that the metabolic machinery of these cells allows them to grow continuously in an uncontrolled manner. The consequences of tumor invasion on the host's metabolism are varied. They have, however, one thing in common: the reduction of the metabolic efficiency of the host. Muscular protein depletion, increased gluconeogenesis, uncoupling of oxidative phosphorylation constitute the main metabolic responses of the host as a result of tumor invasion. The net result of all these metabolic changes is profound energy imbalance which normally ends with cachexia and, eventually, death.