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 (NADP-ME), the primary enzyme decarboxylating malate in bundle sheath cells to supply CO 2 to Rubisco, was used to transform the dicot Flaveria bidentis . Transgenic plants (α-NADP-ME), exhibited a 34-75% reduction in NADP-ME activity relative to wild-type with no visible growth phenotype. We characterised the effect of reducing NADP-ME on photosynthesis by measuring in vitro photosynthetic enzyme activity, gas exchange and real-time carbon isotope discrimination. In α-NADP-ME plants with less than 40% of wild-type NADP-ME activity, CO 2 assimilation rates at high intercellular CO 2 (Ci) were significantly reduced, whereas the in vitro activity of both PEP carboxylase and Rubisco were increased. Carbon isotope discrimination (Δ) measured concurrently with gas exchange in these plants showed a lower Δ and thus a lower calculated leakiness of CO 2 (the ratio of CO 2 leak rate from the bundle sheath to the rate of CO 2 supply). Comparative measurements on antisense Rubisco small subunit (α-SSu) F. bidentis plants showed the opposite effect of increased Δ and leakiness. We use these measurements to estimate the C 4 cycle rate, bundle sheath leak rate and bundle sheath CO 2 concentration (Cs). The comparison of α-NADP-ME and α-SSu demonstrates that the coordination of the C 4 and C 3 cycle that exists during environmental perturbations by light and CO 2 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.