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

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.

Mesophyll conductance to CO2 and Rubisco as targets for improving intrinsic water use efficiency in C3 plants

“…; von Caemmerer et al . ). Although the large subunit of Rubisco hosts the catalytic site, and some of the foreign Rubiscos were selected because they presented different kinetic traits, problems in the assembly and folding between the foreign large subunits and the native small subunits provoked a decrease in the concentration of Rubisco in most of these transplantomic lines (Table ).…”

“…All remaining Rubisco transgenic lines for which the main photosynthetic parameters have been reported showed decreased A N /g sw . These include Nicotiana tabacum and Nicotiana benthamiana transplantomic plants expressing functional Rubisco large subunits from different species (Whitney and Andrews 2001;Sharwood et al 2008;Whitney et al 2009;Galmés et al 2013;Li et al 2013;von Caemmerer et al 2014). Although the large subunit of Rubisco hosts the catalytic site, and some of the foreign Rubiscos were selected because they presented different kinetic traits, problems in the assembly and folding between the foreign large subunits and the native small subunits provoked a decrease in the concentration of Rubisco in most of these transplantomic lines (Table 2).…”

Evolving Methanococcoides burtonii archaeal Rubisco for improved photosynthesis and plant growth

“…4b ). As this rate is more than 10-fold slower than the 26–30 μmol CO 2 fixed.m 2 .s −1 rates measured in high CO 2 grown wild type leaves 33 the tob MbRK332E grew ~5-fold slower than wild-type under high CO 2 ( Fig. 5b,c ).…”

Internal transport of CO2 from the root‐zone to plant shoot is pH dependent

“…To directly detect CO 2 efflux from the leaf surface, gas exchange measurements were conducted at the Australian National University, Canberra, Australia (35°16′S, 149°7′E). Plants and plant organs were placed in a temperature‐controlled growth cabinet together with the measuring heads (2 cm × 3 cm) of two LI‐6400XT (Li‐Cor, Lincoln, NE) that were coupled to a TDL (TGA100, Campbell Scientific, Logan, UT) that enabled on‐line carbon isotope measurement (Von Caemmerer et al ) from December 2013 to January 2014. The flow rate through each leaf chamber was 200 μmol s −1 and block temperature was set at 25°C, measurements conditions were based on preliminary experiments.…”