Understanding CO₂ diffusion in C₄ plants: An investigation of CO₂ permeable aquaporins and carbonic anhydrase in the C₄ grass Setaria viridis

Field: Agricultural and Biological Sciences (Life Sciences)

The productive yield of key C₄ crops must increase in the future to meet the demands of an increasing global population. We are therefore endeavouring to improve the availability of CO₂ for photosynthesis, one of the fundamental limitations to photosynthetic carbon fixation. The initial steps of CO₂ assimilation in leaf mesophyll cells involve the diffusion of CO₂ from the intercellular airspace to the mesophyll cytosol (mesophyll conductance). This involves CO₂ passing through the liquid phase and the plasma membrane, a process believed to be both passive and possibly facilitated by protein pores, known as aquaporins. Within the cytosol of mesophyll cells, carbonic anhydrase (CA) catalyses the hydration of CO₂ to HCO₃- which PEP Carboxylase uses in the first CO₂ fixation step of C₄ photosynthesis. Here, I have examined the role of CO₂ permeable aquaporins and CA from a C₄ photosynthesis perspective using the model monocot species Setaria viridis (Foxtail millet). CO₂ permeable aquaporins have been demonstrated to increase CO₂ diffusion in C3 plants. However, to date very little is known about the role of CO₂ permeable aquaporins in the highly efficient and specialised C₄ photosynthetic pathway. After bioinformatic identification of all twelve Setaria PIPs (plasma membrane intrinsic proteins) I first used yeast as a heterologous expression system to confirm plasma membrane localisation and determine CO₂ permeability of the plasma membrane using CO₂ triggered intracellular acidification on a stopped flow spectrophotometry. This in vitro approach identified SiPIP2;7 as a putative CO₂ permeable aquaporin, adding a third CO₂ pore to the list of C₄ plant aquaporins characterised to date. I also examined the effect of PIP1 and PIP2 co-expression and found improved localisation to the plasma membrane but no improvement to CO₂ permeability compared to the single PIP1s. The effects of modifying CA activity in C₄ photosynthesis was examined in planta. I silenced the major leaf CA in Setaria viridis in three independent, stably transformed lines. At low CO₂ a strong correlation between photosynthetic assimilation rate and CA hydration rates was observed in the transformed lines, which have as little as 13% of wild type CA activity. Significantly, no visual phenotype or photosynthetic effect was observed in the transformed lines at ambient CO₂. C¹⁸O¹⁶O isotope discrimination was used to estimate the mesophyll conductance to CO₂ diffusion from the intercellular air space to the mesophyll cytosol in control plants, which allowed us to calculate CA activities in the mesophyll cytosol. These results indicated that CA is not rate limiting for C₄ photosynthesis in S. viridis under current atmospheric conditions. We conclude that CO₂ permeable aquaporins and CA activity are factors with variable importance to CO₂ diffusion in C₄ photosynthesis, with both factors becoming rate limiting under extreme environmental conditions that result in low intercellular CO₂ such as drought stress.