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- Materials and Methods
- Supporting Information
Rubisco (ribulose-1,5-bisphosphate carboxylase/oxygenase) is an enzyme in plants whose original function was to fix carbon in an atmosphere of high CO2 concentration and negligible O2 concentration > 2 billion yr ago (Broda, 1975). This carboxylase activity, termed photosynthesis, converts atmospheric CO2 into carbon-rich compounds and leads to the release of O2. When plants appeared on Earth, the oxygen content of the atmosphere increased. Rubisco now operates under a CO2 concentration of c. 385 ppm (by volume) and an O2 concentration of 21% (210 000 ppm).
Atmospheric CO2 concentrations are anticipated to increase to c. 600 ppm in < 40 yr (Prentice, 2001). This will be the highest CO2 concentration on Earth in millions of years. Some analysts are convinced that a benefit of high CO2 will be increased photosynthetic activity. However, the expected theoretical gain in productivity has not been realized for soybean (Glycine max) plants grown in the open field under 550-ppm free-air CO2 enhanced (FACE) conditions (Rogers et al., 2004). These experiments used computer-controlled CO2 release from pipes surrounding 25-m-diameter rings; there were no artificial canopies, no root constraints, and no nitrogen limitations. Soybean plants appear to respond to high CO2 concentrations with fully increased photosynthetic rates and biomass accumulations (relative to those under ambient CO2 concentrations) in the absence of water stress in open-top chamber experiments (Ziska et al., 2001), but not in the presence of periodic water stress, either in open-top chamber experiments (Huber et al., 1984) or FACE experiments (Ziska & Bunce, 2007).
Because water stress appears to be a factor for plant productivity at high CO2, photorespiration may also be involved (see next paragraph). Photorespiration refers to the oxygenation of ribulose-1,5-bisphosphate (RuBP) by Rubisco (Berry et al., 1978) and the events that are stimulated by the immediate production and subsequent rescue of a two-carbon molecule that is not compatible with the Calvin cycle (Bowes et al., 1971; Ogren, 1984). Carbon salvage requires machinery distributed over three organelles to convert a two-carbon molecule (phosphoglycolate) into a useful three-carbon intermediate (3-phosphoglycerate) via the condensation of two glycines to form a serine with the concomitant release of CO2. This process is known as the photorespiratory C2 cycle. Photorespiration is generally considered a wasteful process, an unavoidable side-reaction of Rubisco. The notion that a reduction in photorespiration in C3 plants may lead to increased plant productivity has been expressed frequently over the years (for recent references, see e.g. Zhu et al., 2004; Blankenship et al., 2011).
Using solid-state nuclear magnetic resonance (NMR), we have found that a fraction of the glycine from the photorespiratory C2 cycle is not decarboxylated or returned to the Calvin cycle (Noctor et al., 1999), but is inserted instead as glycyl residues in proteins (Cegelski & Schaefer, 2005; Gullion et al., 2010). This result is consistent with earlier experiments using 14CO2 labeling and detection of label in both amino acids and proteins (Ongun & Stocking, 1965; Dickson & Larson, 1975). Glycyl-13C incorporation in leaf protein or protein precursors occurs as soon as 2 min after the start of 13CO2 labeling and is most pronounced at low external CO2 concentrations (Cegelski & Schaefer, 2006). Normally, low CO2 concentrations within a leaf result from the increased stomatal resistance and decreased gas diffusivity that accompany water stress (Bunce, 1998). These conditions deplete CO2 within the leaf, but have little effect on the much more abundant O2. Thus, the carboxylase activity of Rubisco is reduced but not the oxygenase activity (Sharkey, 1988). Under these subambient CO2 concentrations, some of the glycine from oxygenase activity has been detected by solid-state NMR in Gly-Gly peptide sequences (Yu et al., 2010), presumably part of glycine-rich proteins (GRPs) or their precursors (Cassab, 1998). In soybean leaves under 600 ppm external CO2, this routing of glycine to GRPs is less pronounced and the decarboxylation pathway is active (Yu et al., 2010), a result that suggests that a part of the soybean response to water stress may be overridden by high external CO2 concentrations.
The NMR experiments described above were performed on single intact leaves. However, leaves frequently exhibit spatial and temporal heterogeneity of their stomatal openings during active photosynthesis. Collections or ‘patches’ of stomata may close while neighbors in adjacent areas of the leaf remain open. This behavior is sometimes called ‘patchy stomatal conductance’, or ‘stomatal patchiness’ (Mott & Buckley, 2000; Mott & Peak, 2007). Patches are usually bounded by leaf veins and each patch has a uniform local conductance. Patches sometimes appear and disappear transiently on a 20-min time-scale in what appears to be a largely unpredictable way. Thus, NMR results based on whole-leaf averages may not represent accurately the metabolism in any single region of a leaf.
Patches have been observed using a variety of techniques such as iodine staining (Terashima et al., 1988), 14C autoradiography (Downton et al., 1988; Gunasekera & Berkowitz, 1992), leaf thermograms (Hashimoto et al., 1984; Jones, 1999), and leaf fluorescence (Daley et al., 1989; Genty & Meyer, 1995; Beyschlag & Eckstein, 2001). When stomata are closed, photosynthesis and electron transport from light-harvesting quinones are inhibited. The net result is that chlorophyll fluorescence as excess energy is dissipated by photon emission. Thus, the red fluorescing parts of a leaf are those for which the stomata are closed and photosynthesis has shut down.
The first two patch-detection methods mentioned above are destructive techniques, the third is most sensitive at low relative humidity, and the leaf fluorescence measurement, while the most versatile of the four, is sensitive to oxygen quenching and so works best at low concentrations of O2 (Mott & Peak, 2007). We believe that leaf patchiness and metabolic activity can be measured under high humidity and 21% O2, conditions under which photorespiration may be active, by the direct detection of 11C decay (Thorpe et al., 2007). The resulting images would provide useful space and time information about leaf metabolism and so identify regions of the leaf to be examined subsequently by NMR.
11Carbon is a positron-emitting isotope with a 20-min half life that is routinely made for use in positron (positive electron or β+ particle) emission tomography (PET). The positron emitted by 11C has an average range of c. 1 mm (in condensed matter) and then annihilates with an electron generating two 511-keV photons. The back-to-back photons serve as the signal carrier for PET imaging (Phelps et al., 1975). Coincidence detection of the annihilation photons is suitable for imaging humans, animals, and the bulk of a plant, but not for a leaf, the thickness of which can be substantially less than the 1-mm positron range. That is, the positron will often get out of the leaf, and will probably not annihilate and generate photons until it stops in some other condensed matter. Imaging the positron itself is perhaps the more appropriate tool for leaf imaging. In our work, images of 11C assimilated by the leaf are made by direct positron imaging. This is accomplished by the detection of the visible photons emitted by a plastic scintillator when the positrons generated by 11C decay are stopped within the scintillator. After imaging, the leaf is frozen in liquid nitrogen to suspend all metabolism, and lyophilized after sufficient time has been allowed for radioactive decay and safe handling of the leaf, typically a few hours. Solid-state NMR of the same intact lyophilized leaf is then possible.
In this report, we describe our efforts to answer the following two questions. Can we combine 11CO2 and 13CO2 labeling with detection of the labels by 11C positron imaging and solid-state 13C NMR to measure variations in soybean leaf metabolism for photosynthetically active and inactive regions within a single leaf? Can we use these leaf-fragment measurements to gain insight into the source(s) of the apparent shortfall in productivity (carbon assimilation) for periodically water-stressed soybean plants grown under elevated CO2 conditions?