Exploiting transplastomically modified Rubisco to rapidly measure natural diversity in its carbon isotope discrimination using tuneable diode laser spectroscopy

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 signifi-cantly 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.