Victoria C. Clarke, Annamaria De Rosa, Baxter Massey, Aleu Mani George, John R. Evans, Susanne von Caemmerer, Michael Groszmann

In plants with C₃ photosynthesis, increasing the diffusion conductance for CO₂ from the substomatal cavity to chloroplast stroma (mesophyll conductance) can improve the efficiencies of both CO₂ assimilation and photosynthetic water use. In the diffusion pathway from substomatal cavity to chloroplast stroma, the plasmalemma and chloroplast envelope membranes impose a considerable barrier to CO₂ diffusion, limiting photosynthetic efficiency. In an attempt to improve membrane permeability to CO₂, and increase photosynthesis in tobacco, we generated transgenic lines in Nicotiana tabacum L. cv Petite Havana carrying either the Arabidopsis PIP1;2 (AtPIP1;2) or PIP1;4 (AtPIP1;4) gene driven by the constitutive dual 2x35S CMV promoter. From a collection of independent T₀ transgenics, two T₂ lines from each gene were characterized, with western blots confirming increased total aquaporin protein abundance in the AtPIP1;2 tobacco lines. Transient expression of AtPIP1;2-mGFP6 and AtPIP1;4-mGFP6 fusions in Nicotiana benthamiana identified that both AtPIP1;2 and AtPIP1;4 localize to the plasmalemma. Despite achieving ectopic production and correct localization, gas exchange measurements combined with carbon isotope discrimination measurements detected no increase in mesophyll conductance or CO₂ assimilation rate in the tobacco lines expressing AtPIP. We discuss the complexities associated with trying to enhance g_m through modified aquaporin activity.