Abstract
An antisense construct targeting the C(4) isoform of NADP-malic enzyme (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% 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, CO(2) assimilation rates at high intercellular CO(2) 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 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 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. The comparison of α-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 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.
Highlights
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
In most C4 plants, CO2 is fixed by phosphoenolpyruvate carboxylase (PEPC) in the mesophyll cells into four-carbon acids, which diffuse to an inner ring of bundle sheath cells, where they are decarboxylated and the CO2 is refixed by Rubisco
The comparison of the performance of a-NADP-ME and antisense Rubisco small subunit (a-SSu) plants examined here suggests that the coordination of the C3 and C4 cycles that is apparent during environmental perturbations by light and CO2 can be disrupted through transgenic manipulations
Summary
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. We have designed experiments to simultaneously look at in vitro photosynthetic enzyme activity, gas exchange, and real-time carbon isotope discrimination (D), facilitating estimates of f, C4 cycle rate, and the possible range of Cs within transgenic a-NADPME and antisense Rubisco small subunit (a-SSu) F. bidentis plants (Furbank et al, 1996) These measurements aim to show the impact of our perturbations of the C3/C4 balance, highlighting possible communication pathways between the cycles and other possible targets for future genetic manipulation to improve the rate and/or efficiency of photosynthesis in C4 plants
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