Mesophyll conductance is unaffected by expression of Arabidopsis PIP1 aquaporins in the plasmalemma of Nicotiana

In plants with C(3) photosynthesis, increasing the diffusion conductance for CO(2) from the substomatal cavity to chloroplast stroma (mesophyll conductance) can improve the efficiencies of both CO(2) 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 CO(2) diffusion, limiting photosynthetic efficiency. In an attempt to improve membrane permeability to CO(2), 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 T(0) transgenics, two T(2) 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 CO(2) assimilation rate in the tobacco lines expressing AtPIP. We discuss the complexities associated with trying to enhance g(m) through modified aquaporin activity.