C4 photosynthesis at low temperature. A study using transgenic plants with reduced amounts of Rubisco

C4 plants are rare in the cool climates characteristic of high latitudes and elevations, but the reasons for this are unclear. We tested the hypothesis that CO2 fixation by Rubisco is the rate-limiting step during C4 photosynthesis at cool temperatures. We measured photosynthesis and chlorophyll fluorescence from 6°C to 40°C, and in vitro Rubisco and phosphoenolpyruvate carboxylase activity from 0°C to 42°C, in Flaveria bidentis modified by an antisense construct (targeted to the nuclear-encoded small subunit of Rubisco, anti-RbcS) to have 49% and 32% of the wild-type Rubisco content. Photosynthesis was reduced at all temperatures in the anti-Rbcs plants, but the thermal optimum for photosynthesis (35°C) did not differ. The in vitro turnover rate (kcat) of fully carbamylated Rubisco was 3.8 mol mol-1 s-1 at 24°C, regardless of genotype. The in vitro kcat (Rubisco Vcmax per catalytic site) and in vivo kcat (gross photosynthesis per Rubisco catalytic site) were the same below 20°C, but at warmer temperatures, the in vitro capacity of the enzyme exceeded the realized rate of photosynthesis. The quantum requirement of CO2 assimilation increased below 25°C in all genotypes, suggesting greater leakage of CO2 from the bundle sheath. The Rubisco flux control coefficient was 0.68 at the thermal optimum and increased to 0.99 at 6°C. Our results thus demonstrate that Rubisco capacity is a principle control over the rate of C4 photosynthesis at low temperatures. On the basis of these results, we propose that the lack of C4 success in cool climates reflects a constraint imposed by having less Rubisco than their C3 competitors.