Direct Inhibition of Plant Mitochondrial Respiration by Elevated CO2

In situ measurements of alternative respiratory pathway activity are needed to provide insight into the energy efficiency of plant metabolism under various conditions in the field. The only reliable method at present to measure alternative oxidase (AOX) activity is through measurement of changes in ␦ 18 O(O 2 ), which to date has only been used in laboratory environments. We have developed a cuvette system to measure partitioning of electrons to AOX that is suitable for off-line use and for field experiments. Plant samples are enclosed in airtight cuvettes and O 2 consumption is monitored. Gas samples from the cuvette are stored in evacuated gas containers until measurement of ␦ 18 O(O 2 ). We have validated this method using differing plant material to assess AOX activity. Fractionation factors were calculated from ␦ 18 O(O 2 ) measurements, which could be measured with an accuracy and precision to 0.1‰ and 0.3‰, respectively. Potential sources of error are discussed and quantified. Our method provides results similar to those obtained with laboratory incubations on-line to a mass spectrometer but greatly increases the potential for adoption of the stable isotope method.Measurements of partitioning of respiratory electron flow between the energy-conserving cytochrome oxidase (COX) pathway and the energy-inefficient alternative oxidase (AOX) pathway were initially quantified by assessing the effect on O 2 consumption of titrations with specific inhibitors of both respiratory pathways (see Møller et al., 1988). However, titration with inhibitors is associated with a number of problems. The most important is the assumption that inhibition of the alternative pathway would not affect the cytochrome pathway; this is not necessarily true (Wilson, 1988;Millar et al., 1995;. Therefore, AOX activity may be underestimated by titration. This problem was overcome by the noninvasive stable isotope method, which was initially developed by Guy et al. (1989) and has been perfected during the last decade (Robinson et al., 1992;Ribas-Carbo et al., 1997;Gonzàlez-Meler et al., 1999;Henry et al., 1999). This method relies on the different discrimination against the 18 O isotope by COX and AOX. Although inhibitors are used to measure the discrimination factor (D) of either oxidase, the actual measurement does not depend on inhibitors.The present on-line method (Robinson et al., 1992;Gonzàlez-Meler et al., 1999) uses a cuvette for incubation of plant material that is directly connected to a gas chromatograph and an isotope ratio mass spectrometer (IRMS), operating in continuous flow mode. This approach allows rapid and accurate measurements but has a few limitations. In particular, it is restricted to the very few laboratories worldwide that can acquire and maintain a dedicated on-line system. There is a great need to extend the use of the stable isotope approach to assess the role of AOX in the dissipation of excess reducing power under unfavorable growth conditions, such as low temperatures (Stewart et al., 1990;Vanlerberghe and McI...

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“…Gonzàlez-Meler et al, 1996;Drake et al, 1999) or low concentrations of O 2 (e.g. Gale et al, 1992;Ribas-Carbo et al, 1994).…”

Section: Effects Of Buildup Of Co 2 and Depletion Of O 2 On Respiratimentioning

confidence: 99%

An Off-Line Implementation of the Stable Isotope Technique for Measurements of Alternative Respiratory Pathway Activities

2001

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“…While a part of this trend can be explained by the inereased proportion of leaf dry matter remaining after defoliation at elevated p^^^ with low N supply (Table 1, Experiment II), it nevertheless supports our previous conclusion that the increase in carbohydrate availability exceeded any stimulation of sink demand due to elevated Pcoo (e-g-in the root system). Such an imbalance could be exacerbated by a potential /^co^-dependent reduction of respiration (Gonzalez-Meier et al 1996 and references therein).…”

Section: Comparison Of Sources: Leaves and Pseudostemsmentioning

confidence: 99%

Source‐sink relations in Lolium perenne L. as reflected by carbohydrate concentrations in leaves and pseudo‐stems during regrowth in a free air carbon dioxide enrichment (FACE) experiment^*

Fischer

Frehner

Hebeisen

et al. 1997

Plant Cell & Environment

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The effect of an elevated partial pressure of CO2 (pCO2) on carbohydrate concentrations in source leaves and pseudo‐stems (stubble) of Lolium perenne L. (perennial ryegrass) during regrowth was studied in a regularly defoliated grass sward in the field. The free air carbon dioxide enrichment (FACE) technology enabled natural environmental conditions to be provided. Two levels of nitrogen (N) supply were used to modulate potential plant growth. Carbohydrate concentrations in source leaves were increased at elevated pCO2, particularly at low N supply. Elevated leaf carbohydrate concentrations were related to an increased structural carbon (C) to N ratio and thus reflected an increased C availability together with a N‐dependent sink limitation. Immediately after defoliation, apparent assimilate export rates (differences in the carbohydrate concentrations of young source leaves measured in the evening and on the following morning) showed a greater increase at elevated pCO2 than at ambient pCO2; however, replenishment of carbohydrate reserves was not accelerated. Distinct, treatment‐dependent carbohydrate concentrations in pseudo‐stems suggested an increasing degree of C‐sink limitation from the treatment at ambient pCO2 with high N supply to that at elevated pCO2 with low N supply. During two growing seasons, no evidence of a substantial change in the response of the carbohydrate source in L. perenne to elevated pCO2 was found. Our results support the view that the response of L. perenne to elevated pCO2 is restricted by a C‐sink limitation, which is particularly severe at low N supply.

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“…Samples of fully developed leaves (0·9-1·2 g fresh weight) were kept in the dark for 25 min at 23°C before gas-phase respiratory measurements were taken in a closed 4·96 mL stainless-steel temperature-controlled cuvette at 23°C . A CO 2 absorber (ascarite II) was present during measurements to avoid inhibition of respiration as a consequence of the build up of CO 2 in the closed cuvette during the course of the experiment (Gonzàlez-Meler et al 1996). The CO 2 absorber was replaced after each measurement.…”

Section: Respiratory Measurements and Oxygen Isotope Analysismentioning

confidence: 99%

Metabolic regulation of leaf respiration and alternative pathway activity in response to phosphate supply

et al. 2001

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In this study the question whether the alternative respiratory pathway acts as an electron bypass for the cytochrome pathway under conditions of growth on limited phosphorus in leaves of bean (Phaseolus vulgaris L.), tobacco (Nicotiana tabacum L.) and Gliricidia sepium Walp was investigated. The oxygen isotope fractionation technique was used to assess the in vivo activities of the cytochrome and alternative respiratory pathways in the absence of added inhibitors. The response of respiration to low phosphorus supply varied among species. Growth at low phosphorus reduced cytochrome pathway activity in bean and tobacco. Alternative pathway activity increased only in bean leaves in response to low phosphorus and not in tobacco. In the case of G. sepium, cytochrome pathway activity remained unchanged whereas the alternative pathway activity increased with low nutritional phosphorus. At low phosphorus, alternative oxidase protein levels increased in the leaves of bean and G. sepium but not in tobacco, suggesting a dependence of alternative pathway activity on protein level. Alternative pathway activity was also not correlated with soluble carbohydrate concentration in bean or tobacco at any phosphorus level. These results show that the alternative pathway does not always act as an electron bypass in response to the downstream restriction of the cytochrome pathway imposed by low phosphorus supply. These results suggest that factors in addition to cellular carbohydrate level and adenylate control can act to regulate alternative pathway activity.

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Direct Inhibition of Plant Mitochondrial Respiration by Elevated CO2

Cited by 148 publications

References 31 publications

An Off-Line Implementation of the Stable Isotope Technique for Measurements of Alternative Respiratory Pathway Activities

An Off-Line Implementation of the Stable Isotope Technique for Measurements of Alternative Respiratory Pathway Activities

Source‐sink relations in Lolium perenne L. as reflected by carbohydrate concentrations in leaves and pseudo‐stems during regrowth in a free air carbon dioxide enrichment (FACE) experiment^*

Metabolic regulation of leaf respiration and alternative pathway activity in response to phosphate supply

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Direct Inhibition of Plant Mitochondrial Respiration by Elevated CO2

Cited by 148 publications

References 31 publications

An Off-Line Implementation of the Stable Isotope Technique for Measurements of Alternative Respiratory Pathway Activities

An Off-Line Implementation of the Stable Isotope Technique for Measurements of Alternative Respiratory Pathway Activities

Source‐sink relations in Lolium perenne L. as reflected by carbohydrate concentrations in leaves and pseudo‐stems during regrowth in a free air carbon dioxide enrichment (FACE) experiment*

Metabolic regulation of leaf respiration and alternative pathway activity in response to phosphate supply

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Source‐sink relations in Lolium perenne L. as reflected by carbohydrate concentrations in leaves and pseudo‐stems during regrowth in a free air carbon dioxide enrichment (FACE) experiment^*