1 | INTRODUCTION
Macrophytes form the base of the freshwater food web and are major contributors to primary production, especially in shallow systems (Silva et al., 2013; Maberly & Gontero, 2018). However, the supply of CO2 for photosynthesis in water is potentially limited by the approximately 10,000 lower rate of diffusion compared to that in air (Raven, 1970). This imposes a large external transport resistance through the boundary layer (Black et al., 1981), that results in the K½ for CO2 uptake by macrophytes to be 100-200 μM, roughly 6-11 times air-equilibrium concentrations (Maberly & Madsen, 1998). Furthermore, in productive systems the concentration of CO2 can be depleted close to zero (Maberly & Gontero, 2017). Freshwater plants have evolved diverse strategies to minimize inorganic carbon (Ci) limitation (Klavsen et al., 2011) including the active concentration of CO2 at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), collectively known as CO2 concentrating mechanisms (CCMs). The most frequent CCM in freshwater plants is based on the biophysical uptake of bicarbonate (HCO3-), which is present in ~50% of the species tested (Maberly & Gontero, 2017; Iversen et al., 2019). While CO2 can diffuse through the cell membrane passively, HCO3- use requires active transport because the plasmalemma is impermeable to HCO3- and the negative internal membrane potential (Denny & Weeks, 1970) produces a large electrochemical gradient resisting passive HCO3- entry (Maberly & Gontero, 2018).
Detailed studies of the mechanisms of HCO3- use have been carried out in microalgae, marine macroalgae, seagrasses and to a lesser extent, freshwater macrophytes (Giordano et al., 2005). Direct uptake/transport of HCO3- can occur via an anion exchanger (AE) located at the plasmalemma (Sharkia et al., 1994). Inhibition of this protein by the membrane impermeable and highly specific chemical, 4,4’-diisothiocyanatostilbene-2,2’-disulfonate (DIDS), has confirmed its effect in a range of marine macroalgae and seagrasses (Drechsler et al., 1993; Björk et al., 1997; Fernández et al., 2014). Genomic studies have found probable AE proteins, from the solute carrier 4 (SLC4) family bicarbonate transporters (Romero et al., 2013), in marine microalgae (Nakajima et al., 2013; Poliner et al., 2015).
Carbonic anhydrase (CA) is a ubiquitous enzyme and is present in photosynthetic organisms. It interconverts CO2 and HCO3-, maintaining equilibrium concentrations when rates of carbon transformation are high (Moroney et al., 2001; Dimario et al., 2018). External carbonic anhydrase (CAext) is inhibited by the impermeable inhibitor acetazolamide (AZ). The widespread nature of CAext is demonstrated by the inhibition of rates of photosynthesis in a range of aquatic photoautotrophs (James & Larkum, 1996; Larsson & Axelsson, 1999; Moroney et al., 2011; Tachibana et al., 2011; van Hille et al., 2014; Fernández et al., 2018). In many marine species, both CAext and an AE protein are implicated in the uptake of HCO3- but very little is known about freshwater macrophytes (Millhouse & Strother, 1986; Beer & Rehnberg, 1997; Björk et al., 1997; Gravot et al., 2010; Tsuji et al., 2017).
Ottelia alismoides (L.) Pers., a member of the monocot family Hydrocharitaceae, possesses two biochemical CCMs: constitutive C4 photosynthesis and facultative Crassulacean Acid Metabolism (CAM; Zhang et al., 2014; Shao et al., 2017; Huang et al., 2018). The leaves ofO. alismoides comprise epidermal and mesophyll cells that contain chloroplasts and large air spaces but lack Kranz anatomy (Han et al., 2020). Although it is known that it can use HCO3- in addition to CO2, little is known about the mechanisms responsible for HCO3- uptake. We have addressed this issue, with Ci uptake measurements using the pH-drift technique, experiments with inhibitors of CA and AE and analysis of transcriptomic data.