Larry Giles

Effects of Water Stress on Respiration in Soybean Leaves

The effect of water stress on respiration and mitochondrial electron transport has been studied in soybean (Glycine max) leaves, using the oxygen-isotope-fractionation technique. Treatments with three levels of water stress were applied by irrigation to replace 100%, 50%, and 0% of daily water use by transpiration. The levels of water stress were characterized in terms of light-saturated stomatal conductance (gs): well irrigated (gs > 0.2 mol H2O m−2 s−1), mildly water stressed (gs between 0.1 and 0.2 mol H2O m−2 s−1), and severely water stressed (gs < 0.1 mol H2O m−2 s−1). Although net photosynthesis decreased by 40% and 70% under mild and severe water stress, respectively, the total respiratory oxygen uptake (Vt) was not significantly different at any water-stress level. However, severe water stress caused a significant shift of electrons from the cytochrome to the alternative pathway. The electron partitioning through the alternative pathway increased from 10% to 12% under well-watered or mild water-stress conditions to near 40% under severe water stress. Consequently, the calculated rate of mitochondrial ATP synthesis decreased by 32% under severe water stress. Unlike many other stresses, water stress did not affect the levels of mitochondrial alternative oxidase protein. This suggests a biochemical regulation (other than protein synthesis) that causes this mitochondrial electron shift.

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.

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.

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.

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.

The Application of the Oxygen-Isotope Technique to Assess Respiratory Pathway Partitioning

The oxygen isotope technique is currently the only reliable method for studying relative electron partitioning between the cytochrome and alternative plant respiratory pathways. The theoretical background to this technique is described, as well as some of the difficulties that can complicate measurements. This chapter describes the development of systems over the last 15 years that currently allow measurement of respiration in both intact tissues and in the aqueous phase. Initially, the focus was on developing on-line systems for both gas and liquid phase measurements, but in recent years attention has shifted to the development of portable off-line systems which will allow measurements of respiratory electron partitioning in field studies. Measurements can now be made much more rapidly and accurately than a decade ago, however, the application of this technique is still limited by the availability of dedicated systems. Finally, a summary of data obtained with this technique is presented.

In the heat of the night – alternative pathway respiration drives thermogenesis in Philodendron bipinnatifidum

• Philodendron bipinnatifidum inflorescences heat up to 42 °C and thermoregulate. We investigated whether they generate heat via the cytochrome oxidase pathway uncoupled by uncoupling proteins (pUCPs), or the alternative oxidase (AOX). • Contribution of AOX and pUCPs to heating in fertile (FM) and sterile (SM) male florets was determined using a combination of oxygen isotope discrimination, protein and substrate analyses. • Both FM and SM florets thermoregulated independently for up to 30 h ex planta. In both floret types, AOX contributed > 90% of respiratory flux during peak heating. The AOX protein increased fivefold with the onset of thermogenesis in both floret types, whereas pUCP remained low throughout development. These data indicate that AOX is primarily responsible for heating, despite FM and SM florets potentially using different substrates, carbohydrates or lipids, respectively. Measurements of discrimination between O₂ isotopes in strongly respiring SM florets were affected by diffusion; however, this diffusional limitation was largely overcome using elevated O₂. • The first in vivo respiratory flux measurements in an arum show AOX contributes the bulk of heating in P. bipinnatifidum. Fine-scale regulation of AOX activity is post-translational. We also demonstrate that elevated O₂ can aid measurement of respiratory pathway fluxes in dense tissues.

Interpretations of gradients in δ13C value in thick photosynthetic tissues of plants with Crassulacean acid metabolism

In Ceropegia dichotoma, Crassula argentea, Esheveria colorata, Kalanchoë beharensis, Opuntia ficus-indica, Sansveria stuckyi and Opuntia inermis the carbon-isotope ratio (δ13C) of tissues close to the epidermis is 2–4.3‰ more negative than those in the centre of the leaf or cladode. The greatest change in δ13C value occurs between the epidermal layer and the layer of mesophyll tissue immediately underneath. Analysis of major metabolic and structural components in successive layers of Crassula argentea grown under controlled environmental conditions conducive to Crassulacean acid metabolism confirmed that much of the variation in δ13C values of bulk carbon is caused by differences in chemical composition. Thus the steep gradient in δ13C value at the epidermis reflects, in part, the contribution of more-negative δ13C values of lipids in these tissues. Moreover, during nocturnal CO2 fixation the amount of malic acid synthesised decreases with depth and the δ13C value of the methanol-soluble fraction is less negative with distance away from the upper epidermis. These results are consistent with diffusion limitation to CO2 uptake in these thick leaf tissues, which also contributes to the observed gradients in δ13C value.

The 18O of water in the metabolic compartment of transpiring leaves.

Publisher Summary Photosynthesis in terrestrial plants is invariably associated with transpiration of water with associated isotopic fractionation. Water at the site of photosynthetic metabolism is enriched in 18 O relative to the water that is transpired. Diffusion of water is the principal mechanism of mixing operating in a leaf, and this relatively slow process is opposed by a large net flux of liquid water to the evaporating surfaces. This chapter discusses a set of experiments done in the phytotron. The chapter delineates a series of corrections to earlier data sets. The corrections do not alter the conclusion that leaf metabolism in transpiring leaves of sunflower—Helianthus annuus cv. giant mammoth—occurs in a water fraction the oxygen isotopic composition of which is distinctly less positive than that predicted for water at the sites of evaporation. The isotopic exchange of CO 2 between the leaves and the atmosphere is dynamic and beset with species-dependent, capacitance-related effects on isotopic gradients within leaves.

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.