Antisense Reduction of NADP-Malic Enzyme in Flaveria bidentis Reduces Flow of CO2 through the C4 Cycle

An antisense construct targeting the C-4 isoform of NADP-malic enzyme (ME), the primary enzyme decarboxylating malate in bundle sheath cells to supply CO2 to Rubisco, was used to transform the dicot Flaveria bidentis. Transgenic plants (alpha-NADP-ME) exhibited a 34% to 75% reduction in NADP-ME activity relative to the wild type with no visible growth phenotype. We characterized the effect of reducing NADP-ME on photosynthesis by measuring in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (D). In a-NADP-ME plants with less than 40% of wild-type NADP-ME activity, CO2 assimilation rates at high intercellular CO2 were significantly reduced, whereas the in vitro activities of both phosphoenolpyruvate carboxylase and Rubisco were increased. D measured concurrently with gas exchange in these plants showed a lower D and thus a lower calculated leakiness of CO2 (the ratio of CO2 leak rate from the bundle sheath to the rate of CO2 supply). Comparative measurements on antisense Rubisco small subunit F. bidentis plants showed the opposite effect of increased D and leakiness. We use these measurements to estimate the C-4 cycle rate, bundle sheath leak rate, and bundle sheath CO2 concentration. The comparison of a-NADP-ME and antisense Rubisco small subunit demonstrates that the coordination of the C-3 and C-4 cycles that exist during environmental perturbations by light and CO2 can be disrupted through transgenic manipulations. Furthermore, our results suggest that the efficiency of the C-4 pathway could potentially be improved through a reduction in C-4 cycle activity or increased C-3 cycle activity.