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.