Plant Respiration: From Cell to Ecosystem: 18 (Advances in Photosynthesis and Respiration)
Learn more. Variations in rates of plant respiration e. However, compared with the relatively comprehensive understanding of photosynthetic metabolism, we lack basic information on key determinants of respiratory rates in photosynthetic and nonphotosynthetic plant organs. Moreover, our ability to predict the scale and magnitude of future rates of respiration remains limited.
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Dealing with these issues requires a dialogue between researchers working over a wide range of spatial and temporal scales in order to better integrate their combined knowledge and to help reconcile differences in perspectives, approaches and facts. Over participants from highly diverse backgrounds were attracted to the meeting, with the research interests of participants spanning 16 orders of magnitude Fig.
A total of 25 talks were given over five sessions: Respiratory carbon release over large spatial and temporal scales; Mitochondrial composition and respiratory function; Regulation of respiration in plants and fungal partners; Heterogeneity of respiration in contrasting cell types and tissues; and Respiratory responses to environmental gradients. To facilitate integration, each session comprised talks covering a range of scales and disciplines, and speakers were strongly encouraged to make their talks accessible to the diverse backgrounds of the audience.
A feature of the meeting was the opportunity for participants to discuss and debate the issues raised in individual talks. The presentation of over 70 posters and associated discussion periods further extended the opportunity to share ideas. Here, we present some of the emerging and unresolved issues that formed the basis of many lively debates.
From a metabolic perspective, several speakers focussed on the catabolic aspects of respiration and the use of respiratory products for biosynthesis and cellular maintenance e. ATP, reducing equivalents and carbon skeletons. Where CO 2 and O 2 exchange exhibit an asynchronous response e. Finally, there was considerable debate about the carbon sources used by respiration. In many predictive models, an implicit assumption is made that there is a direct coupling between respiration and the use of recently fixed carbon from photosynthesis.
However, as reported by Susan Trumbore Max Planck Institute, Jena, Germany and others, respiration in leaves, stems and roots often uses a variety of carbon sources, with older pools of stored carbon contributing substantially to respiration in some tissues particularly roots Trumbore, If we are to predict, more effectively, the effects of climate change on rates of respiration, the international research community needs to formulate a better understanding of the key determinants of respiration in aboveground and belowground organs.
In both cases, integrating our molecular—biochemical—physiological understanding of mitochondrial responses to their environment is likely to be vital. Achieving this integration, however, remains a major challenge. The discussion periods also identified the difficulties in integrating existing and emerging subcellular knowledge with an understanding of the processes taking place at higher scales whole plant, ecosystem and global.
Experiments are in progress to evaluate whether different factors, such as elicitor dose, plant age, and environmental conditions, might differentially modulate the response of both genotypes to harpin. Except otherwise stated, plants were grown in the Institut de Biotechnologie des Plantes greenhouses under a h photoperiod.
Plants were fed with Hydrokani C2 nutritive solution. For oxygen discrimination measurements see below , plants were grown in the greenhouses of the University de les Illes Balears under natural light and watered every 2 d with tap water.
Organization and Regulation of Mitochondrial Respiration in Plants
Half-strength Hoagland solution Epstein, was applied every 5 d. Harpin was produced as described by Gaudriault et al. Mock infiltration of buffer was performed as a control by the same procedure on different plants. Samples were punched out with a core-borer 1. The second fully developed leaf of 6-week-old wild-type and 2-month-old CMSII plants were used to compare plants at the same developmental stage. In all experiments, careful controls were performed to ensure that the infiltrated zone was actually undergoing cell death.
To determine this point, we measured the following features at the h time point: 1 the immediate area outside the harvested zone Figure 5 ; 2 a comparable infiltration site on the same leaf was not harvested, ensuring that the whole area was fully necrotic. Experiments that did not meet these two criteria were dismissed. Electrolyte leakage was measured as described by Pike et al.
At different times after harpin or buffer infiltration, eight discs 2. Total RNA was extracted by the Trizol Gibco BRL procedure from mature leaf pieces harvested from 2-month-old plants after 10 h of illumination, as described by Dutilleul et al. Mitochondrial preparations were as described by Sabar et al. Briefly, freshly harvested leaves of 3-month-old plants were homogenized in 0. The supernatant was centrifuged at 10, g for 15 min and the pellet resuspended in 0. In some experiments, 5 mM pyruvate was added to all buffers during the mitochondrial isolation process.
The extract was centrifuged for 10 min at 20, g to eliminate insoluble material, and protein was determined according to Bradford Leaf disks were ground in liquid nitrogen and extraction buffer 0. Extracts were cleared by centrifugation for 5 min at 20, g. The supernatant was kept, and debris was pelleted by centrifugation for 30 min at , g. SOD activities were visualized using the in situ staining technique of Beauchamp and Fridovitch After 30 min of dark incubation with a mixture containing Nitro Blue Tetrazolium, gels were placed in the light for 30 min.
SOD activity caused achromatic zones on otherwise uniformly blue-stained gels. Horseradish peroxidase—conjugated anti-mouse IgG was used as a secondary antibody at a dilution of , and immune complex was visualized by ECL according to manufacturer's instructions Roche Diagnostics. The pH of the clarified supernatant was adjusted to 5.
Ascorbate and glutathione were measured in the same supernatant. Total and reduced ascorbate contents were measured as the ascorbate oxidase—dependent decrease in A , before reduced ascorbate and after total ascorbate treatment of the sample for 15 min with 0. The 1-mL reaction mixture contained 0.
Total and oxidized glutathione contents were measured using the enzymatic recycling assay involving the NADPH-driven glutathione-dependent reduction of 5,5-dithiobis 2-nitro-benzoic acid at nm Noctor and Foyer, V t , v cyt , and v alt in leaves were determined using a closed gas phase system connected to a dual-inlet mass spectrometer as previously described Gaston et al. The isotope ratio mass spectrometer Thermo Scientific simultaneously analyzed mass-to-charge ratios of 32 16 O 2 , 34 18 O 16 O , and 28 N 2 operating in dual-inlet mode and by comparison with a standard air sample.
The stainless steel cuvette was equipped with two inlets: one connected to a 5-mL air-tight syringe and the other to the mass spectrometer sample bellows through a capillary tube with a pneumatically controlled on—off microneedle valve. The sampled air went trough a liquid N 2 trap for water and CO 2 removal.
To avoid any drop in cuvette pressure during the experiment, the air was mixed using an air-tight syringe, which was initially left with 1 mL of air. During the experiment, the syringe was used to mix the air. The system was regularly tested for leaks by filling the cuvette with He and measuring the sample over three times the experimental time span.
No oxygen signals were observed. The time between successive samples was 12 min, and the length of each full experiment varied between 60 and 90 min. One or two leaf samples 5. For inhibitory treatments used to measure fractionation values through each pathway, leaf discs were kept in the dark in 5 mM KCN in water for 30 min with constant shaking. No recovery from inhibitor treatment was observed as respiratory rates and oxygen isotope fractionation remained constant throughout the experiment.
All stocks were freshly prepared before measurement. Calculations of the oxygen isotopic fractionation were made as described by Guy et al.
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The electron partitioning between the two pathways in the absence of inhibitors was calculated as described by Guy et al. The individual activities of the COX v cyt and the AOX alternative pathway v alt were obtained from multiplying the total oxygen uptake V tot by the partitioning to each pathway as follows:.
The stoichiometry of proton pumping by the pathway from external NADH via AOX was derived theoretically, but it has also been observed in organello Glaser et al. For malate, the factor is 3. Relative signal intensities were quantified using Scion Image Beta 4. We also thank T. Asahi for the gift of the anti-COX antibody, T.
Elthon and A. Millar for the anti-AOX antibodies, and J. Grienenberger for the anti-NAD9 antibodies. We thank R. Boyer for photographic art, P. Priault for help in statistical analyses, D. Geisler for technical help, and M. Hodges and J. Vidal for critically reading the manuscript. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors www.
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Skip to main content. Research Article Research Article. You have access Restricted Access. Rasmusson , Chantal Mathieu , Christine H.
Foyer , Rosine De Paepe. Guillaume Vidal. American Society of Plant Biologists Abstract Alternative oxidase AOX functions in stress resistance by preventing accumulation of reactive oxygen species ROS , but little is known about in vivo partitioning of electron flow between AOX and the cytochrome pathway. View this table: View inline View popup. Table 1. Figure 1. Figure 2.