Antisense reduction of NADP-malic enzyme in Flaveria bidentis reduces flow of CO₂ through the C₄ cycle

An antisense construct targeting the C₄ 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 (a-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 (Δ). In α-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. Δ measured concurrently with gas exchange in these plants showed a lower Δ 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 Δ and leakiness. We use these measurements to estimate the C₄ 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 C₃ and C₄ 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 C₄ pathway could potentially be improved through a reduction in C₄ cycle activity or increased C₃ cycle activity.