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

Abstract

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...

Thermal acclimation of respiration but not photosynthesis in Pinus radiata

“…This was especially evident in cold transferred plants, where COX activity declined whilst AOX activity increased significantly. This switching between the AOX and COX pathways is well documented in a range of species such as Alocasia odora, Spinacia oleracea, Zea mays and Phaseolus vulgaris (Ribas-Carbo et al 2000;Nagel et al 2001;Nogochi et al 2001) and have been confirmed in our recent study involving poplar (Ow et al 2008). Furthermore, the AOX pathway is highly sensitive to changes in temperature and engagement of this pathway may have advantages for plant acclimation to colder temperature, whereby energy that should otherwise be conserved as ATP is converted to heat (Ribas-Carbo et al 2000).…”

Seasonal variation of leaf respiration and the alternative pathway in field‐grown Populus×canadensis

“…Changes in electron partitioning through the two terminal oxidase pathways in respiration were assessed in vivo based on the premise that the CP and the AP discriminate against the heavier isotope of oxygen ( 18 O) to different extents (Guy et al 1989, Ribas‐Carbo et al 2005a). We adopted an ‘off‐line’ method modified from that of Nagel et al (2001) that has been described previously (Searle et al in press). Leaves were cut in half before being placed in 12 ml Exetainers (Labco, High Wycombe, Buckinghamshire, UK).…”

Respiratory alternative oxidase responds to both low- and high-temperature stress in Quercus rubra leaves along an urban-rural gradient in New York

“…1989; Ribas‐Carbo, Robinson & Giles 2005a). We adopted an ‘off‐line’ method modified from that of Nagel, Waldron & Jones (2001) that has been previously described (Searle et al. 2011a).…”