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

Aquaporins are channel proteins that function to increase the permeability of biological membranes. In plants, aquaporins are encoded by multigene families that have undergone substantial diversification in land plants. The plasma membrane intrinsic proteins (PIPs) subfamily of aquaporins is of particular interest given their potential to improve plant water relations and photosynthesis. Flowering plants have between 7 and 28 PIP genes. Their expression varies with tissue and cell type, through development and in response to a variety of factors, contributing to the dynamic and tissue specific control of permeability. There are a growing number of PIPs shown to act as water channels, but those alteringmembrane permeability to CO2 are more limited. The structural basis for selective substrate specificities has not yet been resolved, althougha fewkey amino acid positions have been identified. Several regions important for dimerization, gating and trafficking are also known. PIP aquaporins assemble as tetramers and their properties depend on the monomeric composition. PIPs control water flux into and out of veins and stomatal guard cells and also increase membrane permeability to CO2 in mesophyll and stomatal guard cells. The latter increases the effectiveness of Rubisco and can potentially influence transpiration efficiency. INTRODUCTION During photosynthesis, CO2 assimilation reduces the partial pressure of CO2 inside chloroplasts relative to that in the surrounding atmosphere. As a result, there is a net diffusion of CO2 into the leaf. At the same time, water diffuses from wet mesophyll cell wall surfaces out to the drier atmosphere. Plants control this diffusive exchange of water and CO2 by enclosing leaves with an impermeable barrier perforated by dynamic pores called stomata. CO2 uptake from the atmosphere is inevitably linked to transpirational water loss because water and CO2 share the same diffusional path across the epidermis through stomata. Awall surrounds each plant cell that defines its shape and allows hydraulic pressure to build up within it. The porous matrix of the wall contains an aqueous solution that allows water and gases to pass between the inside of the cell and the atmosphere. Behind each cell wall lies the plasma membrane. This lipid bilayer contains almost equal proportions of phospholipids and sterols while proteins account for about 40% of themass (Yoshida &Uemura 1986). The plasma membrane provides cells with a barrier that limits the exchange of water, solutes and gases between the external and internal solution. Exchange across the membrane is then controlled by protein pores and transporters that are integrated into it. Inmammalian red blood cells, there is an abundant 28kD integral membrane protein (CHIP28, now called AQP1) which was firstly cloned in 1991 (Preston & Agre 1991). The function of this protein as a water channel was discovered by expressing it in Xenopus oocytes (Preston et al. 1992). Oocyte membrane water permeability containing CHIP28 could be inhibited by HgCl2. Using the same expression system, two other proteins homologous to CHIP28 were found to be water channels, one from the collecting duct of the rat kidney (Fushimi et al. 1993) and the other a tonoplast intrinsic protein (TIP) from Arabidopsis thaliana (Maurel et al. 1993). Given that these proteins came from a large family of integral membrane proteins identified in diverse organisms and the demonstration that all three acted as water channels, the term aquaporin (AQP) was coined (Agre et al. 1993). Since then, there has been an explosion of research on AQPs and numerous reviews on their role in plants (Javot & Maurel 2002; Tyerman et al. 2002; Kaldenhoff & Fischer 2006; Katsuhara et al. 2008; Maurel et al. 2008; Heinen et al. 2009; Prado&Maurel 2013; Chaumont & Tyerman 2014; Maurel et al. 2015; Moshelion et al. 2015). This review focuses on AQPs in the plasma membrane that alter permeability to water and CO2. We firstly introduce the phylogenetic origins of major intrinsic proteins (MIPs) before considering the evidence that they are water channels. The role of AQPs varies depending on the location in the plant. Evidence for AQPs altering the membrane permeability to CO2 is more limited than for water, but there is evidence that CO2 permeable AQPs function in leaves both in mesophyll cells and stomatal guard cells. We consider the functionally significant amino acids and end with a brief summary of attempts to engineer improved plants by manipulating AQPs. PHYLOGENETIC BACKGROUND TO MAJOR INTRINSIC PROTEINS MIPs are an ancient superfamily of proteins found essentially throughout all taxonomic kingdoms. Their classic six transmembrane structure consisting of two repeated halves evolved through a tandem intragenic duplication of a three transmembrane domain protein that may have functioned as a homodimer (Park & Saier 1996). The first major phylogenetic division of MIPs is considered to be the separation into water channel AQPs and glycerol transporting aquaglyceroporins Correspondence: J. R. Evans. e-mail: john.evans@anu.edu. au © 2016 John Wiley & Sons Ltd 1 doi: 10.1111/pce.12844 Plant, Cell and Environment (2016)