Abstract
Carbon isotope discrimination (Δ) during C3 photosynthesis is dominated by the fractionation occurring during CO2-fixation by the enzyme Rubisco. While knowing the fractionation by enzymes is pivotal to fully understanding plant carbon metabolism, little is known about variation in the discrimination factor of Rubisco (b) as it is difficult to measure using existing in vitro methodologies. Tuneable diode laser absorption spectroscopy has improved the ability to make rapid measurements of Δ concurrently with photosynthetic gas exchange. This study used this technique to estimate b in vivo in five tobacco (Nicotiana tabacum L. cv Petit Havana [N,N]) genotypes expressing alternative Rubisco isoforms. For transplastomic tobacco producing Rhodospirillum rubrum Rubisco b was 23.8±0.7‰, while Rubisco containing the large subunit Leu-335-Val mutation had a b-value of 13.9±0.7‰. These values were significantly less than that for Rubisco from wild-type tobacco (b=29‰), a C3 species. Transplastomic tobacco producing chimeric Rubisco comprising tobacco Rubisco small subunits and the catalytic large subunits from either the C4 species Flaveria bidentis or the C3-C4 species Flaveria floridana had b-values of 27.8±0.8 and 28.6±0.6‰, respectively. These values were not significantly different from tobacco Rubisco.
Highlights
Carbon isotope discrimination occurring during C3 photosynthesis is determined by CO2-diffusion processes from the atmosphere to the chloroplast and the biochemical fractionation occurring during CO2 fixation by Rubisco and during respiratory and photorespiratory CO2 release (Farquhar et al, 1989a)
Gas exchange and carbon isotope discrimination measurements were made as described by Tazoe et al (2011) using either a 6-cm2 chamber of the LI-6400 with a red-blue light-emitting diode (LED) light source (Li-Cor, Lincoln, Nebraska, USA) or a laboratory-constructed whole-leaf chamber (115 × 110 × 25 mm depth, boundary layer conductance 4 mol m–2 s–1) together with a red-green-blue LED light source (6400–18 RGB Light source, Li-Cor) and the LI-6400
All gas exchange measurements were made at low O2 partial pressure (19 mbar, ~2% atmospheric pO2) to ensure adequate CO2-assimilation rates could be measured at intercellular CO2 pressures between 100 and 800 μbar for all tobacco genotypes and to minimize photorespiratory fractionation
Summary
Carbon isotope discrimination occurring during C3 photosynthesis is determined by CO2-diffusion processes from the atmosphere to the chloroplast and the biochemical fractionation occurring during CO2 fixation by Rubisco and during respiratory and photorespiratory CO2 release (Farquhar et al, 1989a). The strong 13CO2 discrimination by Rubisco is the primary cause of depleted 13C levels in plant biomass. This effect has proved experimentally versatile by allowing photosynthetic carbon isotope discrimination to be used as a tool to elucidate CO2-diffusion processes through stomata and from the leaf intercellular airspace to the sites of Rubisco carboxylation in the chloroplast stroma of C3 plant species (Evans et al, 1986, 2009; Farquhar et al, 1989b). Interpreting 13CO2 discrimination in C4 plants has proved more challenging as a CO2-concentrating mechanism (CCM) operates that spatially localizes Rubisco in bundle sheath compartments with reduced access to atmospheric CO2. C4 acids diffuse into the bundle sheath where decarboxylation supplies
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