Expression of Tobacco Carbonic Anhydrase in the C(4) Dicot Flaveria bidentis Leads to Increased Leakiness of the Bundle Sheath and a Defective CO(2)-Concentrating Mechanism

Flaveria bidentis (L.) Kuntze, a C(4) dicot, was genetically transformed with a construct encoding the mature form of tobacco (Nicotiana tabacum L.) carbonic anhydrase (CA) under the control of a strong constitutive promoter. Expression of the tobacco CA was detected in transformant whole-leaf and bundle-sheath cell (bsc) extracts by immunoblot analysis. Whole-leaf extracts from two CA-transformed lines demonstrated 10% to 50% more CA activity on a ribulose-1,5-bisphosphate carboxylase/oxygenase-site basis than the extracts from transformed, nonexpressing control plants, whereas 3 to 5 times more activity was measured in CA transformant bsc extracts. This increased CA activity resulted in plants with moderately reduced rates of CO(2) assimilation (A) and an appreciable increase in C isotope discrimination compared with the controls. With increasing O(2) concentrations up to 40% (v/v), a greater inhibition of A was found for transformants than for wild-type plants; however, the quantum yield of photosystem II did not differ appreciably between these two groups over the O(2) levels tested. The quantum yield of photosystem II-to-A ratio suggested that at higher O(2) concentrations, the transformants had increased rates of photorespiration. Thus, the expression of active tobacco CA in the cytosol of F. bidentis bsc and mesophyll cells perturbed the C(4) CO(2)-concentrating mechanism by increasing the permeability of the bsc to inorganic C and, thereby, decreasing the availability of CO(2) for photosynthetic assimilation by ribulose-1,5-bisphosphate carboxylase/oxygenase.