Improving respiration measurements with gas exchange analyzers.

Dark respiration measurements with open-flow gas exchange analyzers are often questioned for their low accuracy as their low values often reach the precision limit of the instrument. Respiration was measured in five species, two hypostomatous (Vitis Vinifera L. and Acanthus mollis) and three amphistomatous, one with similar amount of stomata in both sides (Eucalyptus citriodora) and two with different stomata density (Brassica oleracea and Vicia faba). CO2 differential (ΔCO2) increased two-fold with no change in apparent Rd, when the two leaves with higher stomatal density faced outside. These results showed a clear effect of the position of stomata on ΔCO2. Therefore, it can be concluded that leaf position is important to guarantee the improvement of respiration measurements increasing ΔCO2 without affecting the respiration results by leaf or mass units. This method will help to increase the accuracy of leaf respiration measurements using gas exchange analyzers.

Mesophyll conductance to CO2 and Rubisco as targets for improving intrinsic water use efficiency in C3 plants.

Water limitation is a major global constraint for plant productivity that is likely to be exacerbated by climate change. Hence, improving plant water use efficiency (WUE) has become a major goal for the near future. At the leaf level, WUE is the ratio between photosynthesis and transpiration. Maintaining high photosynthesis under water stress, while improving WUE requires either increasing mesophyll conductance (gm ) and/or improving the biochemical capacity for CO2 assimilation-in which Rubisco properties play a key role, especially in C3 plants at current atmospheric CO2 . The goals of the present analysis are: (1) to summarize the evidence that improving gm and/or Rubisco can result in increased WUE; (2) to review the degree of success of early attempts to genetically manipulate gm or Rubisco; (3) to analyse how gm , gsw and the Rubisco's maximum velocity (Vcmax ) co-vary across different plant species in well-watered and drought-stressed conditions; (4) to examine how these variations cause differences in WUE and what is the overall extent of variation in individual determinants of WUE; and finally, (5) to use simulation analysis to provide a theoretical framework for the possible control of WUE by gm and Rubisco catalytic constants vis-à-vis gsw under water limitations.

Diffusional limitations explain the lower photosynthetic capacity of ferns as compared with angiosperms in a common garden study.

Ferns are thought to have lower photosynthetic rates than angiosperms and they lack fine stomatal regulation. However, no study has directly compared photosynthesis in plants of both groups grown under optimal conditions in a common environment. We present a common garden comparison of seven angiosperms and seven ferns paired by habitat preference, with the aims of (1) confirming that ferns do have lower photosynthesis capacity than angiosperms and quantifying these differences; (2) determining the importance of diffusional versus biochemical limitations; and (3) analysing the potential implication of leaf anatomical traits in setting the photosynthesis capacity in both groups. On average, the photosynthetic rate of ferns was about half that of angiosperms, and they exhibited lower stomatal and mesophyll conductance to CO2 (gm ), maximum velocity of carboxylation and electron transport rate. A quantitative limitation analysis revealed that stomatal and mesophyll conductances were co-responsible for the lower photosynthesis of ferns as compared with angiosperms. However, gm alone was the most constraining factor for photosynthesis in ferns. Consistently, leaf anatomy showed important differences between angiosperms and ferns, especially in cell wall thickness and the surface of chloroplasts exposed to intercellular air spaces.

Stomatal and mesophyll conductances to CO2 are the main limitations to photosynthesis in sugar beet (Beta vulgaris) plants grown with excess zinc.

*The effects of zinc (Zn) toxicity on photosynthesis and respiration were investigated in sugar beet (Beta vulgaris) plants grown hydroponically with 1.2, 100 and 300 microM Zn. *A photosynthesis limitation analysis was used to assess the stomatal, mesophyll, photochemical and biochemical contributions to the reduced photosynthesis observed under Zn toxicity. *The main limitation to photosynthesis was attributable to stomata, with stomatal conductances decreasing by 76% under Zn excess and stomata being unable to respond to physiological and chemical stimuli. The effects of excess Zn on photochemistry were minor. Scanning electron microscopy showed morphological changes in stomata and mesophyll tissue. Stomatal size and density were smaller, and stomatal slits were sealed in plants grown under high Zn. Moreover, the mesophyll conductance to CO(2) decreased by 48% under Zn excess, despite a marked increase in carbonic anhydrase activity. Respiration, including that through both cytochrome and alternative pathways, was doubled by high Zn. *It can be concluded that, in sugar beet plants grown in the presence of excess Zn, photosynthesis is impaired due to a depletion of CO(2) at the Rubisco carboxylation site, as a consequence of major decreases in stomatal and mesophyll conductances to CO(2).

Interactive effects of soil water deficit and air vapour pressure deficit on mesophyll conductance to CO2 in Vitis vinifera and Olea europaea.

The present work aims to study the interactive effect of drought stress and high vapour pressure deficit (VPD) on leaf gas exchange, and especially on mesophyll conductance to CO(2) (g(m)), in two woody species of great agronomical importance in the Mediterranean basin: Vitis vinifera L. cv. Tempranillo and Olea europaea L. cv. Manzanilla. Plants were grown in specially designed outdoor chambers with ambient and below ambient VPD, under both well-irrigated and drought conditions. g(m) was estimated by the variable J method from simultaneous measurements of gas exchange and fluorescence. In both species, the response to soil water deficit was larger in g(s) than in g(m), and more important than the response to VPD. Olea europaea was apparently more sensitive to VPD, so that plants growing in more humid chambers showed higher g(s) and g(m). In V. vinifera, in contrast, soil water deficit dominated the response of g(s) and g(m). Consequently, changes in g(m)/g(s) were more related to VPD in O. europaea and to soil water deficit in V. vinifera. Most of the limitations of photosynthesis were diffusional and especially due to stomatal closure. No biochemical limitation was detected. The results showed that structural parameters played an important role in determining g(m) during the acclimation process. Although the relationship between leaf mass per unit area (M(A)) with g(m) was scattered, it imposed a limitation to the maximum g(m) achievable, with higher values of M(A) in O. europaea at lower g(m) values. M(A) decreased under water stress in O. europaea but it increased in V. vinifera. This resulted in a negative relationship between M(A) and the CO(2) draw-down between substomatal cavities and chloroplasts in O. europaea, while being positive in V. vinifera.

Phytochrome-driven changes in respiratory electron transport partitioning in soybean (Glycine max. L.) cotyledons.

After it was observed that light induces changes in electron partitioning between the cytochrome and the alternative pathway, the focus interest was directed to assessing what type of photoreceptors are involved and the extent of such modifications. Studies on 5-day-old soybean (Glycine max L.) cotyledons using an oxygen isotope fractionation technique showed that phytochrome is involved in changes in electron partitioning between the cytochrome and the alternative respiratory pathway. A follow-up of a previous study, showing that 5 min of white light caused changes in mitochondrial electron partitioning, demonstrated that while blue light was not involved in any such changes, red light caused a significant shift of electrons toward the alternative pathway. The major shift, observed after 24 h of light, is mainly due to both a decrease in the activity of the cytochrome pathway and an increase in the activity of the alternative pathway. The involvement of a phytochrome receptor was confirmed by demonstration of reversibility by far-red light. The implications of the possible involvement of phytochrome in the regulation of mitochondrial electron transport are discussed.

Mesophyll conductance to CO2 in Arabidopsis thaliana.

The close rosette growth form, short petioles and small leaves of Arabidopsis thaliana make measurements with commercial gas exchange cuvettes difficult. This difficulty can be overcome by growing A. thaliana plants in 'ice-cream cone-like' soil pots. This design permitted simultaneous gas exchange and chlorophyll fluorescence measurements from which the first estimates of mesophyll conductance to CO(2) (g(m)) in Arabidopsis were obtained and used to determine photosynthetic limitations during plant ageing from c. 30-45 d. Estimations of g(m) showed maximum values of 0.2 mol CO(2) m(-2) s(-1) bar(-1), lower than expected for a thin-leaved annual species. The parameterization of the response of net photosynthesis (A(N)) to chloroplast CO(2) concentrations (C(c)) yielded estimations of the maximum velocity of carboxylation (V(c,max_Cc)) which were also lower than those reported for other annual species. As A. thaliana plants aged from 30 to 45 d, there was a 40% decline of A(N) that was entirely the result of increased diffusional limitations to CO(2) transfer, with g(m) being the largest. The results suggest that in A. thaliana A(N) is limited by low g(m) and low capacity for carboxylation. Decreased g(m) is the main factor involved in early age-induced photosynthetic decline.

Analysis of leakage in IRGA's leaf chambers of open gas exchange systems: quantification and its effects in photosynthesis parameterization.

The measurement of the response of net photosynthesis to leaf internal CO2 (i.e. A-Ci curves) is widely used for ecophysiological studies. Most studies did not consider CO2 exchange between the chamber and the surrounding air, especially at the two extremes of A-Ci curves, where large CO2 gradients are created, leading to erroneous estimations of A and Ci. A quantitative analysis of CO2 leakage in the chamber of a portable open gas exchange system (Li-6400, LI-COR Inc., NE, USA) was performed. In an empty chamber, the measured CO2 leakage was similar to that calculated using the manufacturer's equations. However, in the presence of a photosynthetically inactive leaf, the magnitude of leakage was substantially decreased, although still significant. These results, together with the analysis of the effects of chamber size, tightness, flow rate, and gasket material, suggest that the leakage is larger at the interface between the gaskets than through the gaskets. This differential leakage rate affects the parameterization by photosynthesis models. The magnitude of these errors was assessed in tobacco plants. The results showed that leakage results in a 10% overestimation of the leaf maximum capacity for carboxylation (Vc,max) and a 40% overestimation of day respiration (Rl). Using the manufacturer's equations resulted in larger, non-realistic corrections of the true values. The photosynthetic response to CO2 concentrations at the chloroplast (i.e. A-Cc curves) was significantly less affected by leakage than A-Ci curves. Therefore, photosynthetic parameterization can be improved by: (i) correcting A and Ci values for chamber leakage estimated using a photosynthetically inactive leaf; and (ii) using A-Cc instead of A-Ci curves.

Decreased Rubisco activity during water stress is not induced by decreased relative water content but related to conditions of low stomatal conductance and chloroplast CO2 concentration.

Rubisco activity decreases under water stress, for reasons as yet unclear. Here, the covariation of stomatal conductance (gs) and relative water content (RWC), often observed during water stress, was impaired to assess the separate effects of these factors on Rubisco activity. Three different treatments were applied to soybean (Glycine max) and tobacco (Nicotiana tabacum): leaf desiccation (LD), in which stomatal closure was accompanied by large decreases of RWC; water stress (WS), in which minor decreases of RWC were observed along with stomatal closure; and exogenous application of abscisic acid (ABA), which triggered stomatal closure without changing RWC. Decreased RWC did not induce decreased initial Rubisco activity, which was impaired only in soybean by 40% when the gs dropped below 50 mmol m(-2) s(-1), regardless of the treatment. The mechanism for decreased activity differed among treatments, owing to decreased activation in LD and to total activity and protein content in WS and ABA. Despite the occurrence of Rubisco regulation, CO2 availability in the chloroplast, not impairment of Rubisco activity, limits photosynthesis during WS.

Regulation of alternative oxidase activity in six wild monocotyledonous species. An in vivo study at the whole root level.

The activity of the alternative pathway is affected by a number of factors, including the level and reduction state of the alternative oxidase (AOX) protein, and the reduction state of the ubiquinone pool. To investigate the significance of these factors for the rate of alternative respiration in vivo, we studied root respiration of six wild monocotyledonous grass species that were grown under identical controlled conditions. The activity of the alternative pathway was determined using the oxygen isotope fractionation technique. In all species, the AOX protein was invariably in its reduced (high activity) state. There was no correlation between AOX activity and AOX protein concentration, ubiquinone (total, reduced, or oxidized) concentration, or the reduction state of the ubiquinone pool. However, when some of these factors are combined in a linear regression model, a good fit to AOX activity is obtained. The function of the AOX is still not fully understood. It is interesting that we found a positive correlation between the activity of the alternative pathway and relative growth rate; a possible explanation for this correlation is discussed. Inhibition of the AOX (with salicylhydroxamic acid) decreases respiration rates less than the activity present before inhibition (i.e. measured with the 18O-fractionation technique).

The electron partitioning between the cytochrome and alternative respiratory pathways during chilling recovery in two cultivars of maize differing in chilling sensitivity.

Chilling effects on respiration during the recovery period were studied in two maize (Zea mays L.) cultivars differing in their tolerance to chilling: Penjalinan, a chilling-sensitive cultivar, and Z7, a chilling-tolerant cultivar. Both cultivars were exposed to 5 degrees C for 5 d, after which measurements were taken at 25 degrees C. Chlorophyll fluorescence analysis in dark-adapted leaves showed less damage in cv Z7 than in cv Penjalinan during recovery from the chilling treatment. Studies of the electron partitioning between the cytochrome and the alternative respiratory pathways during chilling recovery using the oxygen isotope fractionation technique showed that, although total leaf respiration was not affected by the chilling treatment in either of the two cultivars, electron partitioning to the alternative pathway was significantly increased in the more stressed chilling-sensitive cv Penjalinan, suggesting that increased activity of the alternative pathway is not related to the plant tolerance to chilling. These results suggest a possible role of the alternative pathway in plants under stress rather than specifically contributing to plant resistance to chilling.

The effect of growth and measurement temperature on the activity of the alternative respiratory pathway

A postulated role of the CN-resistant alternative respiratory pathway in plants is the maintenance of mitochondrial electron transport at low temperatures that would otherwise inhibit the main phosphorylating pathway and prevent the formation of toxic reactive oxygen species. This role is supported by the observation that alternative oxidase protein levels often increase when plants are subjected to growth at low temperatures. We used oxygen isotope fractionation to measure the distribution of electrons between the main and alternative pathways in mung bean (Vigna radiata) and soybean (Glycine max) following growth at low temperature. The amount of alternative oxidase protein in mung bean grown at 19 degrees C increased over 2-fold in both hypocotyls and leaves compared with plants grown at 28 degrees C but was unchanged in soybean cotyledons grown at 14 degrees C compared with plants grown at 28 degrees C. When the short-term response of tissue respiration was measured over the temperature range of 35 degrees C to 9 degrees C, decreases in the activities of both main and alternative pathway respiration were observed regardless of the growth temperature, and the relative partitioning of electrons to the alternative pathway generally decreased as the temperature was lowered. However, cold-grown mung bean plants that up-regulated the level of alternative oxidase protein maintained a greater electron partitioning to the alternative oxidase when measured at temperatures below 19 degrees C supporting a role for the alternative pathway in response to low temperatures in mung bean. This response was not observed in soybean cotyledons, in which high levels of alternative pathway activity were seen at both high and low temperatures.

The Regulation of Electron Partitioning between the Cytochrome and Alternative Pathways in Soybean Cotyledon and Root Mitochondria.

The regulation of electron partitioning between the cytochrome (Cyt) and alternative pathways in soybean (Glycine max L. cv Ransom) mitochondria in the absence of added inhibitors has been studied using the oxygen isotope fractionation technique. This regulation can depend on several factors, including the amount of alternative oxidase protein, the redox status of the alternative oxidase regulatory sulfhydryl-disulfide system, the degree of activation by [alpha]-keto acids, and the concentration and redox state of the ubiquinone pool. We studied electron partitioning onto the alternative pathway in mitochondria isolated from etiolated and light-grown cotyledons and roots to ascertain how these factors interact in different tissues. In light-grown cotyledon mitochondria there is some partitioning to the alternative pathway in state 4, which is increased dramatically by either pyruvate or dithiothreitol. In etiolated cotyledon mitochondria, the alternative pathway shows little ability to compete for electrons with the Cyt pathway under any circumstances. In root mitochondria, control of alternative pathway activity is exercised by both the ubiquinone pool and the regulatory sulfhydryl-disulfide system. In addition, oxygen isotope fractionation by the Cyt and alternative pathways in mitochondria were identical to the fractionation for the respective pathways seen in intact tissue, suggesting that residual respiration is not present in the absence of inhibitors.

The Effects of Salicylic Acid and Tobacco Mosaic Virus Infection on the Alternative Oxidase of Tobacco.

Salicylic acid (SA) is a signal in systemic acquired resistance and an inducer of the alternative oxidase protein in tobacco (Nicotiana tabacum cv Xanthi nc) cell suspensions and during thermogenesis in aroid spadices. The effects of SA on the levels of alternative oxidase protein and the pathogenesis-related 1a mRNA (a marker for systemic acquired resistance), and on the partitioning of electrons between the Cyt and alternative pathways were investigated in tobacco. Leaves were treated with 1.0 mM SA and mitochondria isolated at times between 1 h and 3 d after treatment. Alternative oxidase protein increased 2.5-fold within 5 h, reached a maximum (9-fold) after 12 h, and remained at twice the level of control plants after 3 d. Measurements of isotope fractionation of 18O by intact leaf tissue gave a value of 23% at all times, identical to that of control plants, indicating a constant 27 to 30% of electron-flow partitioning to the alternative oxidase independent of treatment with SA. Transgenic NahG tobacco plants that express bacterial salicylate hydroxylase and possess very low levels of SA gave a fractionation of 23% and showed control levels of alternative oxidase protein, suggesting that steady-state alternative oxidase accumulates in an SA-independent manner. Infection of plants with tobacco mosaic virus resulted in an increase in alternative oxidase protein in both infected and systemic leaves, but no increase was observed in comparably infected NahG plants. Total respiration rate and partitioning of electrons to the alternative pathway in virus-infected plants was comparable to that in uninfected controls.

Direct Inhibition of Plant Mitochondrial Respiration by Elevated CO2.

Doubling the concentration of atmospheric CO2 often inhibits plant respiration, but the mechanistic basis of this effect is unknown. We investigated the direct effects of increasing the concentration of CO2 by 360 [mu]L L-1 above ambient on O2 uptake in isolated mitochondria from soybean (Glycine max L. cv Ransom) cotyledons. Increasing the CO2 concentration inhibited the oxidation of succinate, external NADH, and succinate and external NADH combined. The inhibition was greater when mitochondria were preincubated for 10 min in the presence of the elevated CO2 concentration prior to the measurement of O2 uptake. Elevated CO2 concentration inhibited the salicylhydroxamic acid-resistant cytochrome pathway, but had no direct effect on the cyanide-resistant alternative pathway. We also investigated the direct effects of elevated CO2 concentration on the activities of cytochrome c oxidase and succinate dehydrogenase (SDH) and found that the activity of both enzymes was inhibited. The kinetics of inhibition of cytochrome c oxidase were time-dependent. The level of SDH inhibition depended on the concentration of succinate in the reaction mixture. Direct inhibition of respiration by elevated CO2 in plants and intact tissues may be due at least in part to the inhibition of cytochrome c oxidase and SDH.

Effects of allelochemicals on plant respiration and oxygen isotope fractionation by the alternative oxidase.

The goal of this investigation was to determine the effects of allelochemicals on plant respiration that thereby may be responsible for their role in growth inhibition. We have tested the effects of juglone, quercetin, cinnamic acid, andα-pinene on respiration rates, and electron partitioning through the cytochrome and alternative respiratory pathways, by measuring on-line oxygen consumption and oxygen isotope fractionation in soybean cotyledon tissue. Cinnamic acid andα-pinene decreased the oxygen consumption rate and increased the relative partitioning of electron transport to the alternative pathway. Possible biochemical mechanisms of these effects are discussed.

Electron Partitioning between the Cytochrome and Alternative Pathways in Plant Mitochondria.

The contribution of the cyanide-resistant, alternative pathway to plant mitochondrial electron transport has been studied using a modified aqueous phase on-line mass spectrometry-gas chromatography system. This technique permits direct measurement of the partitioning of electrons between the cytochrome and alternative pathways in the absence of added inhibitors. We demonstrate that in mitochondria isolated from soybean (Glycine max L. cv Ransom) cotyledons, the alternative pathway contributes significantly to oxygen uptake under state 4 conditions, when succinate is used as a substrate. However, when NADH is the substrate, addition of pyruvate, an allosteric activator of the alternative pathway, is required to achieve the same level of alternative pathway activity. Under state 3 conditions, when the reduction state of the ubiquinone pool is low, the addition of pyruvate allows the alternative pathway to compete with the cytochrome pathway for electrons from the ubiquinone pool when the cytochrome pathway is not saturated. These results provide direct experimental verification of the kinetics consequences of pyruvate addition on the partitioning of electron flow between the two respiratory pathways. This distribution of electrons between the two unsaturated pathways could not be measured using conventional oxygen electrode methods and illustrates a clear advantage of the mass spectrometry technique. These results have significant ramifications for studies of plant respiration using the oxygen electrode, particularly those studies involving intact tissues.

Ubiquinone redox behavior in plant mitochondria during electron transport.

The redox poise of the ubiquinone pool during electron transfer in isolated soybean mitochondria has been compared using two different procedures: the rapid organic extraction of ubiquinone followed by quantification of the oxidized and reduced forms using high-pressure liquid chromatography and an electrochemical technique that measures ubiquinone reduction voltametrically. The goal of these studies was to rigorously test the use of the voltametric technique to monitor the redox status of ubiquinone during the course of mitochondrial electron transfer. The linear relationship between the two methods confirms the reliability of the data obtained with the voltametric technique; however, redox inactive pools of ubiquinone were detected with the HPLC technique. We also quantified the absolute amounts of the ubiquinone homologues ubiquinone-9 and ubiquinone-10 in mitochondria isolated from different soybean tissues and compared their behavior during electron transfer in the presence and absence of pyruvate, an allosteric effector of the cyanide-resistant electron transfer pathway. Both homologues belong to a common redox-active pool in the inner mitochondrial membrane. The results indicate that ubiquinone can be a limiting component of electron transfer through the cyanide-resistant pathway, particularly in roots where its concentration is much lower than in cotyledons.

Measurements of the Engagement of Cyanide-Resistant Respiration in the Crassulacean Acid Metabolism Plant Kalanchoë daigremontiana with the Use of On-Line Oxygen Isotope Discrimination.

Discrimination against (18)O during dark respiration in tissues of Kalanchoë daigremontiana, Medicago sativa, and Glycine max was measured using an on-line system that enabled direct measurements of the oxygen fractionation of samples in a gas-phase leaf disk electrode unit. Discrimination factors for cytochrome pathway respiration were 18.6 to 19.8%(o) for all tissues. However, discrimination in cyanide-resistant respiration was significantly higher in green tissues (30.4-31.2%(o)) compared with nongreen tissues (25.3-25.9%(o)). Using these discrimination factors, the partitioning of electron transport to these pathways was calculated from measurements of discrimination in the absence of inhibitors. Changes in flux through the alternative pathway were measured during the light and dark phases of Crassulacean acid metabolism in leaf disks of K. daigremontiana. The flux of electrons through the alternative pathway was higher during deacidification than during the other phases of Crassulacean acid metabolism. The increase in alternative pathway electron flux accounted for all of the increased respiration in the light phase. Despite this increase, simultaneous measurements of malate concentration and respiratory flux confirm that only a small proportion of the total malate decarboxylation occurs in the mitochondria.

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 O(2) 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 CO(2) in the solution. These stimulatory responses lasted several minutes and then decreased, but additional bicarbonate or gaseous CO(2) again stimulated respiration, suggesting a reversible effect. Carbonic anhydrase in the solution increased the stimulatory effect of potassium bicarbonate. The CO(2)/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 CO(2)/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 CO(2)/bicarbonate. The results suggested that high carbon dioxide levels (mainly free CO(2)) 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.