Energizing the plasmalemma of marine photosynthetic organisms: the role of primary active transport

Abstract

Generation of ion electrochemical potential differences by primary active transport can involve energy inputs from light, from exergonic redox reactions and from exergonic ATP hydrolysis. These electrochemical potential differences are important for homoeostasis, for signalling, and for energizing nutrient influx. The three main ions involved are H⁺, Na⁺ (efflux) and Cl⁻ (influx). In prokaryotes, fluxes of all three of these ions are energized by ion-pumping rhodopsins, with one archaeal rhodopsin pumping H⁺into the cells; among eukaryotes there is also an H⁺ influx rhodopsin in Acetabularia and (probably) H⁺ efflux in diatoms. Bacteriochlorophyll-based photoreactions export H⁺ from the cytosol in some anoxygenic photosynthetic bacteria, but chlorophyll-based photoreactions in marine cyanobacteria do not lead to export of H⁺. Exergonic redox reactions export H⁺ and Na⁺ in photosynthetic bacteria, and possibly H⁺ in eukaryotic algae. P-type H⁺- and/or Na⁺-ATPases occur in almost all of the photosynthetic marine organisms examined. P-type H⁺-efflux ATPases occur in charophycean marine algae and flowering plants whereas P-type Na⁺-ATPases predominate in other marine green algae and non-green algae, possibly with H⁺-ATPases in some cases. An F-type Cl⁻-ATPase is known to occur in Acetabularia. Some assignments, on the basis of genomic evidence, of P-type ATPases to H⁺ or Na⁺ as the pumped ion are inconclusive.

Introduction

Transport of solutes and water across the plasmalemma is vital for homoeostasis and growth of all organisms. For many of these solutes, and perhaps water in some cases, the movement of the ions and molecules is in the direction opposite to that dictated by the free energy difference across the plasmalemma for that ion or compound: this is the strict definition of active transport. For neutral solutes and water, the free energy difference is the difference in chemical activity (concentration times activity coefficient). For ions there is the additional component of electrical potential difference in the overall free energy difference. Active transport necessitates an input of free energy per molecule or ion transported in excess of the free energy difference per molecule or ion. We follow here the distinction made by Mitchell (1979) between primary and secondary active transport (see also Saier, 2000; Saier et al., 2015). In primary active transport the energy input to the transporter is an exergonic scalar chemical reaction (e.g. oxidation of NAD(P)H by O₂; hydrolysis of ATP to produce ADP and Pi) or photons absorbed by a chromophore component of the transporter (e.g. the retinal component of ion-pumping rhodopsins). In secondary active transport the energy input to the transporter is from the energetically downhill transmembrane flux of a driving ion or ions, with re-energization by primary active transport of those driving ions.

The three major ions involved in primary active transport at the plasmalemma of Archaea, Bacteria and Eukarya are H⁺, Na⁺ and Cl⁻. Table 1 shows what is known of the distribution of these pumps among the three higher taxa and the immediate energy sources. In marine photosynthetic (including photoheterotrophic) organisms there are examples of all of the pumps in Table 1 located in the plasmalemma. It is these ions that are the focus of the subsequent discussion in this paper. Other important ions subject to primary active transport are Ca²⁺ and Mg²⁺, transported out of cells by P-type ATPases, that also pump, in various organisms, Na⁺ out:K⁺ in, K⁺ in, Na⁺ out, H⁺ out and H⁺ out:K⁺ in (Kuhlbrandt, 2004; Bublitz et al., 2011; Chan et al., 2011, 2012; Palmgren & Nissen, 2011; Søndergaard & Pedersen, 2015; Palmgren et al., 2020). As well as variants pumping H⁺, Na⁺, K⁺, Mg²⁺ and Ca²⁺ (topological type II: Thever & Saier, 2009, in prokaryotes and eukaryotes), there are also P-type ATPases (topological type I, Thever & Saier, 2009, not considered further here) that pump out metal cations such as Cu(I), Ag(I), Zn(II), Cd(II) and Pb(II) (Kuhlbrandt, 2004; Thever & Saier, 2009; Bublitz et al., 2011; Palmgren & Nissen, 2011; Søndergaard & Pedersen, 2015). There is also an F-type Cl⁻ influx ATPase in the plasmalemma of some marine algae, as well as P-type Cl⁻-ATPases as found in metazoans (Gerencser & Zhang, 2003; Raven, 2017). There are also ATP (adenosine triphosphate) binding cassette proteins (sometimes referred to as ABC transporters) involved in active solute influx at the plasmalemma, although there is little evidence of these in marine photosynthetic organisms (Chan et al., 2011, 2012; but see Badger & Price, 2003 for HCO₃⁻ accumulation in freshwater and marine cyanobacteria).

Table 1. Energy sources for primary active transporters of H⁺, Na⁺ and Cl⁻ in the Archaea, Bacteria and Eukarya

See also Kuhlbrandt (2004), Bublitz et al. (2011), Palmgren & Nissen (2011), Søndergaard & Pedersen (2015).

Possible original functions of primary active H⁺, Na⁺ and Cl⁻ pumps at the plasmalemma

An argument for the early origin and functions of primary active H⁺ pumps (ATP-driven; light-driven via rhodopsins) is intracellular acid-base regulation in early cells related, for example, to fermentation of neutral substrates (e.g. sugars) to organic acids (e.g. lactic acid) (Raven & Smith, 1981, 1982) in anoxic environments. Occurrence of the redox-driven or light-driven H⁺ pumps and ATP-driven H⁺ pumps in the plasmalemma of the same cell permits the use of redox or light energy to phosphorylate ADP to ATP, granted appropriate free energy differences and stoichiometries (Raven & Smith, 1981, 1982). Early chemolithotrophy could generate the H⁺ free energy difference across the plasmalemma, and thence phosphorylate ADP (Russell & Hall, 1997; Martin & Russell, 2007; Mulkidjanian et al., 2008; Duchuzeau et al., 2014; but see Jackson, 2016). In seawater at the present pH, and with intracellular (cytosolic) pH about 0.5 units lower than seawater (Raven & Smith, 1981, 1982), the electrical potential difference across the membrane must be more negative than is found in most marine eukaryotes to definitely need active H⁺ efflux (see below). The requirement for intracellular pH regulation in oxygenic photolithotrophic marine cells involves several metabolic reactions assimilating inorganic nutrient solutes and is shown in Table 2. Volume regulation of wall-less marine cells by active Na⁺ efflux would, in the case of primary active H⁺ transport, involve H⁺:Na⁺ antiport (Raven & Smith, 1982; Katz et al., 1991; Gimmler, 2000).

Table 2. Intracellular H⁺ production and consumption in redox reactions, and calcification, in marine phytoplankton

Elemental atomic ratios based on the Redfield ratio, with a upper limit on reduced S from Ksionzek et al. (2016) since values are for total S and not all S in the cell is reduced to the −SH level. For intracellular calcification in coccolithophores (Taylor et al., 2017) and some dinoflagellates (Van de Waal et al., 2013), a particulate inorganic C: particulate organic C ratio of 1.0 is assumed. Other references for H⁺:N are assimilated from Brewer & Goldman (1976), Smith & Raven (1979) and Raven (2013).

Well characterized in the, mainly freshwater, green algal macrophytes of the Characeae (Walker et al., 1980), localized active efflux of H⁺ across the plasmalemma causes a localized acidification in the cell wall and diffusion boundary layer. This shifts the CO₂:HCO₃^– equilibrium in favour of CO₂, increasing the rate of uncatalysed HCO₃⁻ conversion to CO₂ and thereby improving cellular CO₂ supply (Raven & Beardall, 2016). A similar mechanism is thought to occur in many marine macrophytes as a mechanism of using external HCO₃⁻ (Raven & Hurd, 2012). However, the evidence for this in marine macroalgae is indirect and based on effects of buffers on HCO₃⁻ use, inhibition of external carbonic anhydrase using membrane-impermeant inhibitors or a lack of effects of inhibitors of HCO₃⁻ transport (Raven & Hurd, 2012; Raven & Beardall, 2016). It is considered unlikely that this process occurs in microalgae, however, as their size would mean they have a thinner diffusive boundary layer and greater proton leakage (Flynn et al., 2012; Raven & Beardall, 2016).

For primary active Na⁺ efflux at the plasmalemma of wall-less cells, the higher intracellular K⁺:Na⁺ ratio in the cytosol than in a high-salinity medium such as seawater, driven by the primary active Na⁺ efflux, could be involved in cell volume regulation, countering the Donnan effect of negative charge in the cytosol (Raven & Smith, 1982). The high K⁺:Na⁺ in the cytosol is attributed by Dibrova et al. (2015) to the origin of life at inland hot springs with high K⁺:Na⁺, resulting in a requirement for high (about 100 mol m⁻³) K⁺ for activity of many enzymes. When life invaded the much more widespread freshwater and marine habitats with low K⁺:Na⁺ ratios, active Na⁺ efflux maintained high internal K⁺:Na⁺ ratios despite a finite Na⁺ permeability of the plasmalemma and, in some bacteria, significant energy storage as a Na⁺ electrochemical potential gradient (Na⁺ motive force) (Skulachev, 1984; Skulachev, 1989; Mulkidjanian et al., 2008; Dibrova et al., 2015). Primary active Na⁺ efflux in the marine species of the wall-less chlorophycean Dunaliella spp. is involved in osmoregulation with varying external osmolarities (Ehrenfeld & Cousin, 1984; Gimmler, 2000; Popova et al., 2005; Popova & Balnokin, 2013). The Na⁺ electrochemical potential gradient is widely used in marine (and some other) photosynthetic organisms to energize the influx of nutrients and osmolytes by Na⁺ co-transport (Raven, 1984; Chan et al., 2011, 2012). A further function of the Na⁺ electrochemical potential difference is in the action potentials in marine diatoms (Taylor, 2009; Helliwell et al., 2019) and, from genomic evidence, the marine phaeophycean Ectocarpus and the marine prasinophyceans Micromonas and Ostreococcus, as well as freshwater chlorophyceans (Fux et al., 2018). Recovery of the resting state after an action potential requires active Na⁺ efflux, either by primary active Na⁺ efflux, or by Na⁺:H⁺ antiport following primary active H⁺ efflux.

Primary active transport of Cl⁻ at the plasmalemma of walled marine ulvophycean algal cells is involved in turgor generation, and in action potentials (Bisson et al., 2006; Raven, 2017). At least the turgor generation role could be performed by Cl⁻ influx coupled to H⁺ or Na⁺ influx and primary active H⁺ or Na⁺ efflux. The roles of Cl⁻ as an essential micronutrient in photosynthesis (Raven, 2017) can be satisfied by passive Cl⁻ distribution between seawater and the cytosol even with an inside-negative electrical potential of 150 mV, but not the role established in terrestrial flowering plants as a beneficial nutrient (Raven, 2017).

Not considered in detail here is active Ca²⁺ efflux at the plasmalemma that maintains the very low free Ca²⁺ concentration in the cytosol (100–200 μmol m⁻³) used in preventing damage to proteins and in signalling (Roberts et al., 1994; Thompson et al., 2007; Wheeler et al., 2019). Ca²⁺-H⁺ antiporters and Ca²⁺ P-ATPases that are involved in Ca²⁺ efflux from the cytosol are known, on genomic evidence, to be very widespread among algae (Emery et al., 2012; Palmgren et al., 2020).

Energetics of primary active transport

An important aspect of analysing primary active ion transport at the plasmalemma is the electrochemical potential difference for ions across that membrane. The electrochemical potential difference is defined by equation (1) (Mitchell, 1979; Raven, 1984; Nobel, 2009; Nichols & Ferguson, 2013).

(1)$$\Delta \lpar \bar{\mu }\rpar _{\,j^{ +{/}-}{\rm NP}} = zF\Psi _{{\rm NP}} + {\rm RT}\ln \displaystyle{{{\lsqb {\,j^{ +{/}-}} \rsqb }_{\rm N}} \over {{\lsqb {\,j^{ +{/}-}} \rsqb }_{\rm P}}}$$

where $\Delta \lpar \bar{\mu }\rpar _{j^{ +{/}-}{\rm NP}}$ = electrochemical potential of ion j ^+/− in phase N relative to that in phase P; N = electrically negative phase (cytosol for the plasmalemma); P = electrically positive phase (aqueous medium for the plasmalemma); j ^+/− = ion under consideration, either a cation (j ⁺) or anion (j ⁻); z = numerical charge on the ion (+1 for H⁺ or Na⁺, +2 for Ca²⁺, −1 for Cl⁻); F = Faraday constant (96,485 Joule V⁻¹ mol⁻¹); Ψ _NP = electrical potential of phase N relative to phase P; R = gas constant (8.314 Joule mol⁻¹ °K); T = temperature (°K); ln = natural logarithm; [j ⁺]_N = concentration of j ⁺ in phase N (mol m⁻³); [j ⁻]_P = concentration of j ⁻ in phase P (mol m⁻³).

The sign of the electrochemical potential difference defines the direction of active transport of the ion, i.e. in the energetically uphill direction, requiring an input of energy from coupling to photons, exergonic redox reactions, or ATP conversion to ADP and phosphate in primary active transport, or coupling to exergonic ion fluxes in secondary active transport. The magnitude of the electrochemical potential difference defines the minimum energy input to primary or secondary active transport. As will be seen in the rest of the paper, Na⁺ invariably, and H⁺ very widely, are actively transported by marine photosynthetic organisms from the cytosol to the medium. If only one of these ion species, e.g. H⁺, is subject to primary active transport, then antiport coupling exergonic H⁺ re-entry to secondary active Na⁺ efflux must have a H⁺:Na⁺ stoichiometry consistent with the antiport being overall exergonic.

Hereinafter, the electrical potential of the cytosol (N phase) relative to the outside medium (P phase) is represented as Ψ _CO.

Organisms with ion-pumping rhodopsins

Ion-pumping rhodopsins are light energy transducers that bring about active ion transport as the sole product of the photochemical reaction usable in cell metabolism (Oesterhelt & Stoeckenius, 1973). The photoreaction and ion transport occurs using a single retinol-binding opsin protein, and brings about positive charge (H⁺ or Na⁺) flux from the N to the P side of the membrane, or negative charge (Cl⁻) flux from the P to the N side of the membrane, where ‘N’ and ‘P’ are as defined by Mitchell (1979). For the plasmalemma, the cytosol is the N side and the outside medium is the P side (see equation (1)).

Some marine bacteria have rhodopsins that pump H⁺ or Na⁺ out of the cells, or Cl⁻ into the cells; some marine Archaea have been shown to have H⁺ efflux or Cl⁻ influx rhodopsins, but there are no reports of Na⁺ efflux rhodopsins in Archaea (Oesterhelt & Stoeckenius, 1973; Bejá & Lanyi, 2014; Yoshizawa et al., 2014; Larkum et al., 2018). Unexpectedly, there is an inwardly directed H⁺ pump driven by xenorhodopsin in Nanosalina (Shevchenko et al., 2017); the significance of this energized, but energetically downhill, flux is not clear. Some marine Archaea and bacteria have autotrophic CO₂ assimilation pathways, but none of these are energized entirely by ion-pumping rhodopsins despite the possibility of such energization (Raven & Smith, 1981; Raven, 2009a, 2009b; Berg et al., 2010; Bejá & Lanyi, 2014; Larkum et al., 2018). A marine aerobic anoxygenic photoheterotrophic bacterium, with bacteriochlorophylls, also has an H⁺-pumping rhodopsin, presumably in the plasmalemma (Larkum et al., 2018). There seem to be no reports of ion-pumping rhodopsins in marine aerobic or anaerobic anoxygenic photosynthetic bacteria, or in marine photosynthetic cyanobacteria, although a freshwater/terrestrial cyanobacterium (Gloeobacter) has a H⁺ efflux rhodopsin, with respiratory and photosynthetic H⁺ pumps, in the plasmalemma (Larkum et al., 2018). In an analysis of the energetics of phototrophic marine picoplankton, ion-pumping rhodopsins are major energy-transducing pigments in the ocean in organisms that are small enough to pass through a 2 μm filter (Gómez-Consarnau et al., 2019; see also Kirchman & Hanson, 2013).

Ion-pumping rhodopsins also occur in eukaryotes. Among marine photosynthetic eukaryotes, the best-characterized case of ion-pumping rhodopsin in the plasmalemma is in Acetabularia, which contains an H⁺-pumping rhodopsin. Paradoxically, this rhodopsin moves H⁺into the cytosol, rather than the expected direction of acting as a H⁺ efflux pump (Raven, 2009a; Wada et al., 2011; Bejá & Lanyi, 2014; Tamogami et al., 2017; Larkum et al., 2018), i.e. in the same direction as the inwardly directed H⁺ pump driven by xenorhodopsin in the bacterium Nanosalina (Shevchenko et al., 2017). The Cl⁻ ATPase in the plasmalemma of Acetabularia generates a Ψ _CO of −180 mV, inside negative, in the light that, with a probable cytosol pH between 7 and 8, means an inwardly directed H⁺ electrochemical difference. This means that the H⁺-transporting rhodopsin ‘pumps’ H⁺ downhill. Other cases, where it is thought that the H⁺-pumping rhodopsin is in the plasmalemma and functions in H⁺ efflux, are found in some diatoms where it is thought to be an Fe-sparing way of energizing the plasmalemma (Raven, 2009a; Slamovits et al., 2011; Marchetti et al., 2015; Cohen et al., 2017; Larkum et al., 2018). In at least one case (the dinoflagellate Noctiluca which lacks oxygenic photosynthesis, except by symbiosis) the H⁺-pumping rhodopsin is found in a digestive vacuole; H⁺-pumping rhodopsins also occur in dinoflagellates with oxygenic photosynthesis (Slamovits et al., 2011; Vader et al., 2018).

It is clear that there is a significant variation in the phylogenetic analysis, and the richness of relevant data available among taxa. This is also the case for data considered below for organisms with bacteriochlorophylls and chlorophylls.

Organisms with bacteriochlorophylls

The anoxygenic photosynthetic bacteria function as photolithotrophs with reductants other than water in anaerobic environments or photoheterotrophically by assimilating CO₂ with an organic reductant more oxidizing than H₂ in anaerobic environments. In both of these cases autotrophic CO₂ assimilation pathways are used (Larkum et al., 2018). These photolithotrophic organisms occupy geographically limited benthic marine anoxic illuminated habitats. The other marine bacteria with bacteriochlorophyll are the planktonic aerobic anoxygenic bacteria (Kolber et al., 2001) that lack autotrophic CO₂ assimilation pathways, and so are photoheterotrophs (Larkum et al., 2018; Gómez-Consarnau et al., 2019). Bacteriochlorophylls are less significant in terms of photon absorption than ion-pumping rhodopsins or than chlorophylls as energy-transducing pigments in the ocean in organisms capable of passing through a 2 μm filter (Gómez-Consarnau et al., 2019; see also Kirchman & Hanson, 2013).

Bacteriochlorophylls, like chlorophylls but unlike ion-pumping rhodopsins, are light energy transducers that indirectly bring about active transport of H⁺. The primary photochemistry moves an electron from a high potential electron donor on the P side of the membrane (sensu Mitchell, 1979) to a low potential acceptor on the N side of the membrane (sensu Mitchell, 1979) (equation (1)). H⁺ active transport from the P to the N side of the membrane is a result of secondary, thermodynamically downhill, redox reactions.

The freshwater and marine Chlorobi and Chloroflexi and, in inland hot springs, Acidobacteria, are photolithotrophic bacteria with light-harvesting bacteriochlorophyll c in chlorosomes. These organisms have no intracellular membranes, and have either Type 1 (Chlorobi, Acidobacteria) or Type 2 (Chloroflexi) reaction centres and associated redox catalysts, and the (photo-)redox H⁺ pumps and H⁺ electrochemical difference-driven F_OF₁ ATP synthases, are in their plasmalemma (Adams et al., 2013; Larkum et al., 2018). This plasmalemma location of photochemistry is also the case for the firmicute Heliobacterium without chlorosomes and with Type 1 reaction centres. H⁺ efflux across the plasmalemma is driven by light energy in the photoperiod, and by non-photochemical redox reactions in the scotophase. The H⁺ free energy difference across the plasmalemma in the light is large enough to synthesize ATP, granted the H⁺:ATP ratio of the bacterial F_OF₁ ATP synthase (Larkum et al., 2018).

The marine photosynthetic Proteobacteria with Type 2 reaction centres are the anaerobic photolithotrophic purple sulphur bacteria, and the anaerobic or aerobic photoheterotrophic purple non-sulphur bacteria. These organisms have their Type 2 reaction centres in plasmalemma invaginations and/or intracellular vesicles or flattened thylakoids (Larkum et al., 2018). These structural features mean that most, or all, of the photochemistry and associated H⁺ pumping is in membranes other than the plasmalemma that directly exchanges solutes with the bulk medium. It is, however, unclear what primary ion pumps occur in those parts of the plasmalemma that are not invaginated, and so are involved in nutrient uptake.

The marine anaerobic anoxygenic photosynthetic bacteria of the Chlorobi, Chloroflexi and Proteobacteria make a very small contribution (less than 0.1%) to global marine primary productivity (Johnson et al., 2009; Raven, 2009b). Photons absorbed by marine aerobic anoxygenic photoheterotrophic bacteria probably make a larger contribution (0.5–5%) to global marine primary productivity than the anaerobic anoxygenic photolithotrophic bacteria (Kolber et al., 2000, 2001; Goericke, 2002; Johnson et al., 2009; Raven, 2009b; Kirchman & Hanson, 2013; Gómez-Consarnau et al., 2019).

Organisms with chlorophylls

Cyanobacteria

Some phylogenetic evidence is consistent with a freshwater/terrestrial origin of cyanobacteria (Blank & Sánchez-Baracaldo, 2010; Blank, 2013b; Sánchez-Baracaldo et al., 2014). Data from freshwater cyanobacteria show that respiratory and photosynthetic redox and proton pumping reactions, and the associated CF_OCF₁ ATP synthase, occur in thylakoids. The exception is in Gloeobacter, where there are no thylakoids and photosynthesis and respiration, and ion-pumping rhodopsin, as well as nutrient transporters, occur in the plasmalemma (Mullineaux, 2014; Lea-Smith et al., 2016). The plasmalemma of thylakoid-containing freshwater Cyanobacteria has vanadate-sensitive (presumably ATP-driven) H⁺ efflux (Scherer & Böger, 1984; Kaplan et al., 1989; Schultze et al., 2009), and can oxidize NAD(P)H and reduce O₂; and contains plastoquinone (PQ) and ATP synthase (Mullineaux, 2014; Lea-Smith et al., 2016), and moves protons out of the cell (Scherer et al., 1984). While this is consistent with primary active H⁺ efflux at the plasmalemma in freshwater cyanobacteria, the work of Ritchie (1992) shows that there is an electrogenic Na⁺ efflux pump at the plasmalemma of Synechococcus R2, and that the H⁺ efflux involves a Na⁺:H⁺ antiporter. This Na⁺ active efflux occurs over a wide range of external pH (5–10), K⁺ (0.1–300 mol m⁻³) and Na⁺ (0.1–300 mol m⁻³). The halophilic, alkalophilic cyanobacterium Aphanothece halophytica has a Na⁺-dependent F_OF₁ ATP synthase which, working as an ATPase in the plasmalemma, could act in active Na⁺ efflux (Wiangno et al., 2007; Soontharapirakkul & Incharoensakdi, 2010; Soontharapirakkul et al., 2011; see prediction by Mitchell, 1979). Gabbay-Azaria et al. (2000) demonstrated cytochrome oxidase activity in the plasmalemma of the marine cyanobacterium Spirulina subsalsa (now Arthrospira subsalsa) that could be involved in active H⁺ efflux (Gabbay-Azaria et al., 1992). Bergman et al. (1993) found cytochrome oxidase in the plasmalemma as well as the thylakoid membranes of the marine diazotrophic cyanobacterium Trichodesmium thiebaultii. A P-type Ca²⁺ ATPase has been found in a marine cyanobacterium that can excavate solid CaCO₃ (Garcia-Pichel et al., 2010).

Na⁺ symport of HCO₃⁻ is one of the means of concentrating inorganic C in freshwater cyanobacterial cells, although these organisms also have other HCO₃⁻ transporters, one of which is an ABC transporter (Omata et al., 1999; Badger & Price, 2003). Freshwater cyanobacteria also use ABC transporters for NO₃⁻ (Maeda & Omata, 1997). However, the HCO₃⁻ and NO₃⁻ transporters in marine cyanobacteria are not ABC transporters such as occur in their freshwater counterparts (Wang et al., 2000; Badger & Price, 2003; Maeda et al., 2015).

Eukaryotic algae: relation to the supergroups in the New Tree of Life

Almost all eukaryotic photolithotrophs have a photosynthetic apparatus most of whose core genes were derived from endosymbiosis of a freshwater β-cyanobacterium with a non-photosynthetic unicellular eukaryote to comprise the Archaeplastida (Burki et al., 2020; Palmgren et al., 2020). ‘Green’ Archaeplastida with chlorophyll b produced, by endosymbiosis in an Excavate cell, the secondarily photosynthetic Euglenophyta and, by endosymbiosis in a cell of the Rhizaria in the TSAR (Telonemia, Stramenopila, Alveolata, Rhizaria) supergroup, the secondarily photosynthetic Chlorarachniophyta. In a further set of secondary endosymbioses, ‘Red’ Archaeplastida with phycobilins produced, in cells of the Cryptista, the phycobilin-containing Cryptophyta and, with the loss of light-harvesting phyobilins, in cells of the Haptista to produce the Haptophyta, and in cells of the TSAR supergroup to produce the chromerids and dinoflagellates in the Alveolata, and the Ochrista in the Stramenopila. A very small minority of photolithotrophic eukaryotes (the genus Paulinella) arose by endosymbiosis of an α-cyanobacterium in a rhizarian (TSAR supergroup) (Nowack, 2014).

Eukaryotic algae: Archaeplastida (Glaucophyta, Rhodophyta, Chlorophyta, algal members of the Streptophyta)

The primary endosymbiosis of a β-cyanobacterium in an aerobic eukaryote that gave rise to chloroplasts of the Archaeplastida involved a cyanobacterium whose closest living relative is the freshwater Gloeomargarita lithophora (Brasier, 2013; Blank, 2013a; Lewis, 2017; Ponce-Toledo et al., 2017; Sánchez-Baracaldo et al., 2017). The basal extant Archaeplastida are the freshwater Glaucophyta; the basal Rhodophyta, the Cyanidiophyceae, are non-marine, but their habitat of acid hot springs cannot be described as freshwater (Dittami et al., 2017). There have been numerous freshwater–marine transitions in the Rhodophyta and, especially, in the Chlorophyta, but less so in the predominantly freshwater algal Streptophyta (Dittami et al., 2017). These habitat variations make it difficult to predict which primary active ion transporters occur in the plasmalemma of these organisms.

In the Rhodophyta, the non-marine (acid hot spring) Cyanidium caldarium, Cyanidioschyzon merolae and Galdieria sulphuraria (Cyanidiophyceae) have P-type H⁺-ATPases but not Na⁺-ATPases (Ohta et al., 1997; Lee et al., 2017). Two Na⁺-ATPases (PyKPA1 and PyKPA2) have been characterized in the marine bangiophycean Porphyra yezoensis (now Pyropia yezoensis) (Barrero-Gil et al., 2005; Uji et al., 2012a, 2012b; see also Chan et al., 2012; Kishimoto et al., 2013). This organism also has two plasmalemma Na⁺/H⁺ antiporters (Uji et al., 2012b). An Na⁺:H⁺ antiporter has also been described in Pyropia haitanensis by Chen et al. (2019), who assume, on the basis of vanadate inhibition, that there is a H⁺-ATPase at the plasmalemma of this alga. However, vanadate inhibition is the case for all P and F ATPases (Müller et al., 1999; Araki & González, 1998; Kuhlbrandt, 2004; Hong & Pedersen, 2008; Pedersen et al., 2012), but to a much smaller extent for V ATPases (Müller et al., 1996; Araki & González, 1998). Accordingly, vanadate is a general inhibitor of H⁺, Na⁺ and Ca²⁺ P-ATPases, and is not specific for H⁺ ATPases. Reed et al. (1981) and Reed & Collins (1981) examined the energetics of ion transport in Porphyra purpurea over a wide range of hypo- and hyper-saline (relative to seawater) media. Under all salinities (1/16 seawater to 3× seawater) there is active Na⁺ efflux (Reed et al., 1981). It should be noted that the measurement of Ψ _CO in the essentially non-vacuolate Porphyra purpurea cells by Reed and co-workers was determined using the distribution of the lipophilic cation TPMP⁺; there are reservations about the use of this method of measuring transplasmalemma electrical potential differences in eukaryotes (Ritchie, 1982, 1984). The floridiophycean Chondrus crispus has a P-type Na⁺-ATPase but no H⁺-ATPase (Lee et al., 2017). There seem to be no relevant data for freshwater red algae.

In the Chlorophyta, the microalgal Prasinophyceae occur in marine and also freshwater habitats (Tragin & Vaulot, 2018; Del Cortona et al., 2020). There is functional evidence for the presence in the plasmalemma of the marine Tetraselmis (=Platymonas) viridis of an ATP-driven Na⁺ pump (Balnokin & Popova, 1994; Balnokin et al., 1997, 1999, 2004; Gimmler, 2000; Pagis et al., 2001, 2003; Popova & Balnokin, 2013) and an ATP-driven H⁺ pump (Popova & Balnokin, 1992; Gimmler, 2000; Pagis et al., 2003). There is genomic evidence of a P-type Na⁺-ATPase in the marine Ostreococcus tauri (Rodríguez-Navarro & Benito, 2010). Gimmler (2000) provides an energetic background to infer the presence of active H⁺ and Na⁺ efflux in Tetraselmis viridis, although there are no data from that organism on the electrical potential difference across the plasmalemma, or Na⁺ and H⁺ concentrations in the cytoplasm.

The Chlorophyta: Chlorophyceae are mainly freshwater but with some marine representatives (Tragin & Vaulot, 2018; Del Cortona et al., 2020), the P-type ATPases of the genus Dunaliella has been the subject of considerable investigation (Wolf et al., 1995; Weiss & Pick, 1996; Popova et al., 2018). Sequences encoding P-type H⁺-ATPases have been found in the genomes of the halophilic Dunaliella bioculata (Smahel et al., 1990; Wolf et al., 1995), Dunaliella salina (Weiss & Pick, 1996; Katz et al., 2007), Dunaliella bioculata (Bertucci et al., 2010) and Dunaliella tertiolecta (Popova et al., 2018), as well as the acidophilic Dunaliella acidophila (Weiss & Pick, 1996; Bertucci et al., 2010). While Popova et al. (2018) could not find a P-type Na⁺-ATPase in the genome of Dunaliella tertiolecta, Popova et al. (2005; see also Shumkova et al., 2000) functionally identified an electrogenic Na⁺-translocating ATPase in Dunaliella maritima using inside-out plasmalemma vesicles (see also Popova & Balnokin, 2013). The possibility of a primary H⁺ pump with H⁺:Na⁺ antiport was ruled out by the observation of stimulation, rather than inhibition, of Na⁺ transport by the uncoupler CCCP (m-chlorophenyl carbonyl cyanide phenylhydrazone). Popova et al. (2005) did not attempt to identify whether the catalyst of Na⁺ transport was a P-type ATPase. Further investigation is needed into the possibility of direct redox energization of Na⁺ efflux from Dunaliella salina (Katz & Pick, 2001). The freshwater Chlamydomonas reinhardtii has a P-type Na⁺-ATPase (Barrero-Gil et al., 2005; Rodríguez-Navarro & Benito, 2010), as well as a P-type H⁺-ATPase (Campbell et al., 2001; Barrero-Gil et al., 2005; Bertucci et al., 2010).

An important aspect of the functioning of primary active transport of H⁺ and Na⁺ in driving symport, antiport and uniport of other solutes by Dunaliella is the free energy difference across the plasmalemma. There are several reports of intracellular Na⁺ concentrations (Katz & Avron, 1985; Bental et al., 1986; Pick et al., 1986; Wegmann, 1986; Gimmler, 2000), cytoplasmic pH (Burns & Beardall, 1987; Gimmler et al., 1988; Ginzburg et al., 1988; Gimmler, 2000) and Ψ _CO (Gimmler & Greenway, 1983; Oren-Shamir et al., 1990; see the critique by Ritchie, 1982, 1984) in a variety of halophilic Dunaliella species. Ψ _CO of Dunaliella acidophila has been measured using the preferable microelectrodes method (Remis et al., 1992), but not so far for halophilic Dunaliella species. The thermodynamic analysis by Gimmler (2000) dealt with Dunaliella salina and suggested active efflux of both H⁺ and Na⁺, and pre-dates the discovery of the electrogenic Na⁺ ATPase in Dunaliella maritima (Popova et al., 2005; see also Shumkova et al., 2000). Khramov et al. (2019) showed increased transcript abundance of a putative H⁺ P-ATPase under hypersaline conditions in Dunaliella maritima.

As with the Chlorophyceae, the Chlorophyta: Trebouxiophyceae are mainly freshwater but with some marine members (Tragin & Vaulot, 2018; Del Cortona et al., 2020). There is genomic evidence of both Na⁺-ATPase and H⁺-ATPases in the halotolerant Picochlorum sp. (Foflonker et al., 2014). The only contribution to a thermodynamic analysis of the need for active H⁺ and/or Na⁺ efflux is the work of Bock et al. (1996) who, using ³¹P NMR, found a cytoplasmic pH of 7.8 in the high intertidal–supralittoral macroalga Prasiola crispa. For a freshwater Chlorella sp. there is genomic evidence of a Na⁺ -ATPase (Uji et al., 2012a), and functional evidence consistent with a H⁺-ATPase (Komor et al., 1989).

The earliest Chlorophyta: Ulvophyceae fossils are from marine sediments, and most extant ulvophyceans are marine, exceptions being the freshwater Dichotomosiphon and some species of Cladophora and the terrestrial Trentepohliales (Del Cortona et al., 2020). Blount & Levedahl (1960) found active electrogenic Na⁺ efflux and Cl⁻ influx in the marine gametophyte Halicystis ovalis phase of the marine ulvophycean Derbesia marina. There is genomic evidence for a P-type Na⁺ ATPase of the marine ulvophycean Flabellia petiolata (formerly Udotea petiolata) (Rodríguez-Navarro & Benito, 2010), and for a number of P-type ATPases in Ulva (Zhang et al., 2012; De Clerck et al., 2018).

Blinks (1940, 1949) showed that Halicystis ovalis (the giant-celled coenocytic gametophyte phase of Derbesia marina) had a potential difference between the vacuole and seawater medium (Ψ _VO) of −75 to −80 mV; for Halicystis osterhoutii (the gametophyte phase of Derbesia osterhoutii) the value is −65 to −70 mV. Replacing external Cl⁻ with NO₃⁻ (or SO₄²⁻, or a number of organic anions) caused the potential difference to become negligible, or positive. This is consistent with the short-circuit current experiments of Blount & Levedahl (1960) showing the occurrence of an electrogenic Cl⁻ influx pump in Halicystis ovalis. Graves & Gutknecht (1976, 1977a, 1977b) examined ion concentrations and fluxes, and the effects of decreased external Cl⁻ and low temperatures and of clamped Ψ _VO, in the gametophyte Halicystis parvula phase of Derbesia tenuissima, and also concluded that there is an electrogenic Cl⁻ influx pump. Graves & Gutknecht (1976, 1977a, 1977b) infer that the Cl⁻ pump is at the plasmalemma, as is the case for Acetabularia, discussed below.

Saddler (1970a, 1970b) measured ion (K⁺, Na⁺, Cl⁻) fluxes between uninucleate giant-celled marine Acetabularia and the seawater medium, and electrical properties of the cell, concluding that there was an electrogenic Cl⁻ influx pump. The occurrence of active electrogenic Cl⁻ influx was confirmed by Mummert & Gradmann (1976) and by Gradmann et al. (1982). The Ψ _CO in the light is −170 mV (Saddler, 1970a) to −180 mV (Amtmann & Gradmann, 1994). Saddler (1970a, 1970b) suggested that cytoplasmic Cl⁻ in Acetabularia is the same (500 mol m⁻³) as that in seawater, although this is not consistent with observed inhibitory effects of Cl⁻ on metabolism (Raven, 2017). Using the value of 500 mol m⁻³ for cytoplasmic Cl⁻, the electrochemical potential gradient for Cl⁻ is 17–18 kJ mol⁻¹, driving Cl⁻ from the cytoplasm to the medium (Saddler, 1970a, 1970b). Mummert & Gradmann (1991a) showed the role of Cl⁻ in the action potential of Acetabularia, and also (Mummert & Gradmann, 1991a, 1991b) discovered the role of vesicular transport of ions across the cytosol to the vacuole. The Cl⁻ influx is driven by a Cl⁻-ATPase (Gradmann et al., 1982; Goldfarb & Gradmann, 1983; Ohhashi et al., 1992) inhibited by vanadate (Smahel et al., 1992; see discussion of vanadate inhibition of primary active ion-pumping ATPase under ‘Rhodophyta’ above). Goldfarb et al. (1984) reported that the Cl⁻ pump can be reversed, coupled to net ATP synthesis (as in the prediction of Mitchell, 1979). Ikeda et al. (1990a, 1990b) showed that the Cl⁻-ATPase was not identical with the (C)F ATPase of the F_OF₁/CF_OCF₁ ATP synthase, but Ikeda et al. (1997) demonstrated the interchangeability of the b subunit of the Acetabularia Cl⁻-ATPase and the β-subunit of the Escherichia coli F-ATPase. Finally, Moritani et al. (1997) determined the primary structure of the b subunit of the Acetabularia Cl⁻-ATPase. There seems to have been no further work on this Cl⁻-ATPase other than studies on action potentials (Raven, 2017).

For the major ions commonly subject to primary active transport, Na⁺ and H⁺, the cytoplasmic Na⁺ concentration in Acetabularia is 60 mol m⁻³ (Amtmann & Gradmann, 1994), and the cytoplasmic pH is estimated at 8.0–8.4 using pH indicators, and pH 7.6–7.7 from the pH at which isolated intact chloroplasts exhibit their highest rate of photosynthesis (Dodd & Bidwell, 1971). If external Na⁺ is 450 mol m⁻³, the internal:external Na⁺ concentration difference is 0.133, the electrochemical potential difference for Na⁺ is 22 kJ mol⁻¹ and will drive Na⁺ into the cell. For H⁺, the indicator dye-measured cytoplasmic pH of 8.0–8.2 (Dodd & Bidwell, 1971) is essentially identical to that of seawater, so the electrochemical potential difference for H⁺ is 17 kJ mol⁻¹, driving H⁺ into the cell. No evidence is available for the occurrence of primary active transport processes at the plasmalemma of Acetabularia other than the inward Cl⁻ pump and the inward H⁺ pump. If secondary active efflux of Na⁺ is driven by Cl⁻ symport, granted the electrochemical potentials for the two ions calculated above, the Cl⁻:Na⁺ ratio must be not less than 2. Active H⁺ efflux cannot be driven by the inwardly directed H⁺-pumping rhodopsin. Since the calculated electrochemical potential differences for H⁺ and Cl⁻ are equal and opposite, net H⁺ efflux driven by Cl⁻ efflux requires a ratio of Cl⁻:H⁺ > 1.0.

Other large-celled ulvophyceans include the Siphonocladales and Cladophorales, mainly marine and comprising one or more coenocytic cells. In the cases examined, the vacuole-positive electrical potential difference across the tonoplast is much greater than in other vacuolated organisms, in some cases making the inside-positive tonoplast–medium electrical potential difference greater than the inside-negative cytosol–medium electrical potential difference (Hope & Walker, 1975). There are data for the giant-celled marine Chaetomorpha darwinii (now Chaetomorpha coliforme) on the Ψ _CO of −72 mV, cytoplasmic Na⁺ concentration (25 mol m⁻³) and cytoplasmic pH (pH 8.0–8.3 in the light; pH 7.5−7.8 in the dark), with external Na⁺ 500 mol m⁻³ and external pH 8.0. The free energy difference across the plasmalemma, cytosol relative to medium, for H⁺ is −7.6 kJ mol⁻¹, driving H⁺ into the cell, and −12.6 kJ mol⁻¹ for Na⁺, also driving Na⁺ into the cell (Dodd et al., 1966; Findlay et al., 1971; Raven and Smith, 1980), in the light. No data are available for electrical potentials, or intracellular ion concentrations, in the dark. Less information is available for the closely related large-celled alga, i.e. species of Valonia, Valoniopsis and Ventricaria (Bisson et al., 2006), but the available information is consistent with a situation similar to that in Chaetomorpha darwinii.

Finally among the Ulvophyceae, there are important data on Ulva spp., now incorporating the genera Ulva and Enteromorpha (Hayden et al., 2003). Ulva is multicellular, with small cells of which half or less of the volume is taken up by a vacuole. Ritchie (1985) examined the energetics of ion transport in Enteromorpha (=Ulva) intestinalis. The cytoplasmic pH was not measured in that study so Ritchie (1985) used a value of pH 7.3 from a wide range of studies on (mostly non-marine) cyanobacteria, eukaryotic algae and plants, with an external pH of 8.0. With the measured Ψ _CO of −54 ± 5 mV in seawater in the light, and −30 ± 5 mV in the dark, the proton electrochemical potential difference across the plasmalemma is not significantly different from zero (Ritchie, 1985). For Na⁺, the ion most likely to be subject to primary active efflux in Ulva, the electrochemical potential difference (cytosol relative to medium) across the plasmalemma is −15.5 kJ mol⁻¹ in the light and −13.1 kJ mol⁻¹ in the dark (Ritchie, 1985).

There are also data for the closely related Ulva lactuca. Reed & Collins (1981) used the distribution of the lipid-soluble cation TPMP⁺ to measure Ψ _CO for Ulva lactuca cells in seawater; they found values of −54 ± 1.8 mV in the light and −44 ± 3.5 mV in the dark. Ritchie (1988), using microelectrodes, showed that Ψ _CO of U. lactuca in seawater was −39 ± 1.1 mV in the light and −25 ± 1.9 mV in the dark. In view of the comments by Ritchie (1982, 1984) on problems with the use of lipid-soluble cations to estimate the electrochemical potential difference between cells and the medium in eukaryotes, the following calculations use the electrical potential difference values of Ritchie (1988). Thus, using intracellular and extracellular Na⁺ concentrations, we calculate the electrochemical potential difference for Na⁺ (cytosol relative to medium) across the plasmalemma to be −13.8 kJ mol⁻¹ in the light and −12.2 kJ mol⁻¹ in the dark. Ritchie (1988) does not give values for the electrochemical potential differences for H⁺ across the plasmalemma, but with the assumption made by Ritchie (1985) for cytoplasmic pH (7.3), the H⁺ electrochemical potential difference (cytosol relative to medium) across the plasmalemma is +0.30 kJ mol⁻¹ in the light and +1.65 kJ mol⁻¹ in the dark. The choice of using the cytoplasmic pH value of Ritchie (1988) is supported by Lundberg et al. (1989) who, using ³¹P NMR, found a cytoplasmic pH of 7.2 in U. lactuca.

The algal members of the Streptophyta are the Charophyceae sensu lato (Del Cortona et al., 2020). The most morphologically complex of the Charophyceae are the Charales with most of the thallus volume occupied by giant cells (Beilby, 2015; Nishiyama et al., 2018). Most of the Charales are freshwater, although some species occur in brackish waters (e.g. in the Baltic) and Lamprothamnium spp. grows in coastal lagoons with very large changes in salinity, with the highest values twice that of seawater (Beilby, 2015). Energization of transport at the plasmalemma of Lamprothamnium, like that of freshwater Charales, involves a P-type H⁺ efflux ATPase, with active Na⁺ efflux driven by H⁺ antiport and active Cl⁻ influx driven by H⁺ symport (Beilby, 2015). The electrochemical potential difference across the plasmalemma for H⁺, with external pH of 8 and cytoplasmic pH of 7.7 and a Ψ_CO of −160 mV (Kirst & Bisson, 1982) at 25°C, is −13.7 kJ mol⁻¹. For the major ions the electrochemical potential differences, cytosol relative to medium, are −24.2 kJ mol⁻¹ (Na⁺), −3.14 kJ mol⁻¹ (K⁺) and +9.27 kJ mol⁻¹ (Cl⁻) (Kirst & Bisson, 1982). The primary active efflux of H⁺ energizes, directly or indirectly, the active efflux of Na⁺ and K⁺ and active influx of Cl⁻.

Eukaryotic algae: organisms with secondary and tertiary chloroplast endosymbiosis

The earliest fossils of the diatoms (Bacillariophyceae sensu lato; Ochrophyta) are from marine habitats, with later invasion of fresh waters (Falkowski et al., 2004; Siver et al., 2018). There is functional evidence of a plasmalemma Na⁺-ATPase in the colourless marine diatom Nitzschia alba (Bhattacharya & Volcani, 1980). The work of Flynn et al. (1987) on plasmalemma vesicles of Phaeodactylum tricornutum is also consistent with the occurrence of an Na⁺-ATPase. The available evidence on cytoplasmic pH is for pH 7.6 in a non-vacuolate marine pennate diatom Phaeodactylum tricornutum (Burns & Beardall, 1987), and pH 7.3 in the centric marine diatom Thalassiosira weissflogii at an external pH of 8 (Hervé et al., 2002). The Ψ _CO is −60 to −90 mV for the marine centric diatom Coscinodiscus wailesii (Gradmann & Boyd, 1995, 1999a, 1999b) and −84 mV for the marine centric diatom Odontella sinensis (Taylor et al., 2017). These values for a range of marine diatoms are consistent with the occurrence of active H⁺ efflux. For Na⁺ the only intracellular concentration values are for whole cells of the marine Coscinodiscus granii (46 mol m⁻³) and Coscinodiscus wailesii (125 mol m⁻³) (Kesseler, 1974). Boyd & Gradmann (1999) assume that these mean intracellular concentrations, dominated by the concentration in the largest cell compartment, the vacuole, also apply to the cytosol. With this assumption, and Ψ _CO of −75 mV across the plasmalemma in the marine C. wailesii (Gradmann & Boyd, 1999a, 1999b), the Na⁺ free energy difference across the plasmalemma is 12 kJ mol⁻¹, inside negative. Jones & Morel (1988) suggested that redox reactions in the plasmalemma of Thalassiosira weissflogii can act as an H⁺ efflux pump. Bertucci et al. (2010) cite genomic evidence for a P-type H⁺-ATPase in the diatom Phaeodactylum tricornutum. Diatoms also have vacuolar H⁺ pyrophosphatases and V-type H⁺-ATPases (Bussard & Lopez, 2014). While there is evidence from metazoans for the expression of V-type H⁺-ATPases in the plasmalemma (Beyenbach & Wieczorek, 2006), there is no evidence as to whether this occurs in diatoms (Wieczorek et al., 1999; Bussard & Lopez, 2014).

Almost all of the extant Ochrophyta: Phaeophyceae are marine. For the marine phaeophycean Ectocarpus siliculosus there is genomic evidence for a P Na⁺-ATPase (Uji et al., 2012a, 2012b). The only estimates of the electrochemical potential differences across the plasmalemma of the Phaeophyceae are for the development of the just-fertilized eggs of the fucoid Pelvetia fastigiata (Allen et al., 1972; Gibbon & Kropf, 1993) and the unfertilized eggs of the fucoid Fucus serratus (Taylor & Brownlee, 1993). Gibbon & Kropf (1993) used microelectrodes to measure the Ψ _CO of −60 mv, inside negative), and the pH difference (0.5 units, inside low) between the cytosol and seawater medium. Gibbon & Kropf (1993) calculated the proton motive force across the plasmalemma, equivalent to an electrochemical potential difference, cytosol relative to medium for H⁺ of −2.5 kJ mol⁻¹, i.e. close to electrochemical equilibrium. The data for ion content of Allen et al. (1972) are for the whole zygote, rather than the cytosol, so calculations of electrochemical potential difference across the plasmalemma for K⁺, Na⁺ and Cl⁻ are less accurate than the value for H⁺ (Gibbon & Kropf, 1993). The calculations of Gibbon & Kropf (1993) show that while K⁺ and Cl⁻ are near electrochemical equilibrium, the electrochemical potential difference, cytosol relative to medium, for Na⁺ is −14 kJ mol⁻¹. Gibbon & Kropf (1993) therefore suggest that the primary active transport at the plasmalemma is for Na⁺, with Na⁺:H⁺ antiport generating the much smaller H⁺ electrochemical potential difference. Taylor & Brownlee (1993) examined the electrical properties of the plasmalemma, and the ion content, of unfertilized eggs of Fucus serratus in seawater. The Ψ _CO using microelectrodes is −40 to −65 mV, with a mean of −50 mV. With the same cautions as for Pelvetia fastigiata zygotes, the driving force for H⁺ is 1 kJ mol⁻¹ directed inwards, for Na⁺ 12 kJ mol⁻¹, directed inwards, and for Cl⁻, zero kJ mol⁻¹, i.e. equilibrium. These values are consistent with the occurrence of a Na⁺ efflux, with very weak evidence of a H⁺ efflux pump, and for Cl⁻ at equilibrium. However, replacement of external Cl⁻ by isethionate⁻ makes the plasmalemma electrical potential difference 20 mV less negative, consistent with active electrogenic Cl⁻ influx, and a lower Cl⁻ concentration in the cytosol than in some other intracellular compartments. Further experiments are needed to examine this possibility.

Klenell et al. (2002, 2004) suggested that Laminaria digitata and Laminaria saccharina (now Saccharina latissima) have a P-type H⁺-ATPase that is involved in acidifying part of the thallus surface; however, the evidence (inhibition by vanadate) does not distinguish a P-type H⁺ ATPase from a P-type Na⁺-ATPase in parallel with a H⁺:Na⁺ antiporter (see discussion above for Rhodophyta). Klenell et al. (2004) also used erythrosin B as an inhibitor of the plasmalemma P-type H⁺-ATPase; however, erythrosin B also inhibits a range of other processes (Gimmler, 1988).

In the Ochrophyta: Raphidophyceae, inverted plasmalemma vesicles of the marine Heterosigma akashiwo show ATP-dependent Na⁺ accumulation. The Na⁺ accumulation was inhibited by vanadate, and was shown not to be an accumulation down an inside-negative electrical potential, or a result of Na⁺:H⁺ exchange following H⁺ accumulation by an H⁺-ATPase (Shono et al., 1995, 1996). These findings are supported by genomic evidence of a Na⁺-ATPase (Shono et al., 2001; Jo et al., 2010; Uji et al., 2012a).

For the Haptophyta the earliest known fossils of the calcified coccoliths of coccolithophores in the Class Prymnesiophyceae are from marine strata, and there is no evidence that the coccolithophores ever invaded fresh waters (Falkowski et al., 2004). The coccolithophore Emiliania huxleyi (Haptophyta: Prymnesiophyceae) has a putative P-type H⁺-ATPase in the plasmalemma (Lohbeck et al., 2014). The Ψ _CO of a calcifying strain of Emiliania huxleyi (Sikes & Wilbur, 1982) is −81 mV using K⁺-valinomycin and −145 mV using a fluorescent dye (cf. Ritchie, 1982, 1984). However, using the preferable microelectrode technique, Taylor et al. (2011) found a Ψ _CO of −46 mV. There are conflicting reports on the intracellular pH of Emiliania huxleyi (Dixon et al., 1989; Nimer et al., 1994; see also Suffrian et al., 2011). Using the fluorescent dye method the internal pH was determined as 7.28 (Dixon et al., 1989), 7.03 (Nimer et al., 1994) and 7.0 (Gibbin et al., 2014), at an external pH of 8 in the presence of 2 mol m⁻³ inorganic carbon. Lower values for internal pH are found with the DMO method and/or in the absence of inorganic C (Dixon et al., 1989; Nimer et al., 1994). Another coccolithophore, Pleurochrysis sp., has a P-type Ca²⁺-ATPase (Araki & González, 1998).

The earliest known fossil Dinophyta (Alveolata) are from marine sediments with subsequent invasion of fresh waters (Lenz et al., 2002; Falkowski et al., 2004). Symbiotic dinoflagellates of the Symbiodiniaceae may have a Na⁺-ATPase in the plasmalemma (Goiran et al., 1997), although a H⁺-ATPase and H⁺-Na⁺ antiport has not been ruled out as the mechanism of Na⁺ efflux. Bertucci et al. (2010) and Mies et al. (2017a, 2017b) showed that the gene for a plasmalemma-located P-type H⁺-ATPase in the Symbiodiniaceae shows increased expression during symbiosis with corals, possibly related to acidification of the perisymbiotic space (Bertucci et al., 2010). It is not clear which ion is pumped by the P-ATPase in the calcifying dinoflagellate Thoracosphaera helmii, but it is probably Ca²⁺ (Van de Waal et al., 2013).

Submerged marine flowering plants: seagrasses

Fernández et al. (1999), Garcia-Sánchez et al. (2000) and Rubio et al. (2011) provided electrophysiological evidence of a H⁺-ATPase of the leaf and root cell plasmalemmas of Zostera marina. The Ψ _CO is −150 to −160 mV, inside negative, and is hyperpolarized by fusicoccin, a compound known to stimulate non-halophytic flowering plant plasmalemma H⁺-ATPases; the cytosolic pH is 7.3 (Fernández et al., 1999; Garcia-Sánchez et al., 2000). With an external seawater pH of 8.0 and the cytosol −160 mV negative relative to the seawater, the H⁺ electrochemical potential difference, cytosol relative to medium, across the plasmalemma is thus −13.7 kJ mol⁻¹ kJ per mol, favouring H⁺ entry. Rubio et al. (2005) showed that the cytosol Na⁺ concentration (measured with Na⁺-selective microelectrodes) of 10.7 ± 3.3 mol m⁻³ in cells of Zostera marina in seawater has a Ψ _CO of −150 mV, so the electrochemical potential difference across the plasmalemma is 25 kJ mol⁻³, driving Na⁺ into the cytosol. Following Pak et al. (1995), Fukuhara et al. (1996) and Muramoto et al. (2002) characterized a salt-tolerant P-type H⁺-ATPase in the plasmalemma of Zostera marina. Generation of the −25 kJ mol⁻¹ gradient (cytosol relative to medium) for Na⁺ using a Na⁺:H⁺ antiporter and a −13.7 kJ mol⁻¹ gradient for H⁺ requires a Na⁺:H⁺ ratio of 0.5 or lower. Rubio et al. (2017, 2018) showed that the cytosol pH and the electrical potential difference across the plasmalemma of the seagrass Posidonia oceanica are very similar to those of Zostera marina.

Emergent marine flowering plants: tidal (=salt) marsh plants (herbaceous) and mangroves (trees)

Brügemann & Janiesch (1989) compared the plasma membrane ATPase from control specimens and those grown with added NaCl of the tidal marsh plant Plantago maritima. Wu & Seliskar (1998) investigated salinity adaptations in the H⁺-ATPase of the tidal marsh plant Spartina patens. The H⁺-ATPase also energizes NaCl secretion by salt glands in recretohalophytic tidal marsh plants and mangroves (Yuan et al., 2016; Dassanayake & Larkin, 2017). The suggestion that the Limonium salt gland has primary active transport involving a Cl⁻-ATPase has not been substantiated (Raven, 2017).

Evolutionary aspects of primary active ion pumps in marine photosynthetic organisms

The occurrence of primary active H⁺, Na⁺ and Cl⁻ at the plasmalemma of marine photosynthetic organisms is summarized in Table 3, encapsulating the outcomes of the analysis in the previous section. Among the Archaeplastida, the brackish Characeae and brackish and marine flowering plants only have P(II)-type H⁺-ATPases. However, the Streptophyta are not basal among the Archaeplastida, so it cannot be concluded that P(II)-type H⁺-ATPase is the ancestral energizer of the plasmalemma. No data seem to be available for Glaucophyta; among Rhodophyta, marine representatives have P(II)-type Na⁺-ATPases and acidophilic ‘freshwater’ Cyanidiophyceae have P(II)-type H⁺-ATPases. Marine Chlorophyta have both P(II)-type H⁺-ATPases and P(II)-type Na⁺-ATPases. In these two phyla of Archaeplastida it is likely that horizontal gene transfer has been involved, e.g. via virus-encoded P-ATPases: an example is a Ca²⁺-ATPase in a freshwater Chlorella virus (Bonza et al., 2010). A range of transporters are encoded by other viruses (Greiner et al., 2018) and horizontal gene transfer has been shown for ATPases in prokaryotes (Hilario & Gogarten, 1993) and other transporters in eukaryotic algae (Chan et al., 2011, 2012). Similar horizontal gene transfer is required to account for the distribution of P(II)-type H⁺-ATPases and P(II)-type Na⁺-ATPases among marine algae with plastids derived from secondary or tertiary endosymbiosis (Chan et al., 2011, 2012; Burki et al., 2020). The origin of the F-ATPase that pumps Cl⁻ in some ulvophycean Chlorophyta (Table 3) is unclear; metazoan Cl⁻-ATPases seem to be P-ATPases (Gerencser & Zhang, 2003).

Table 3. Phylogenetic distribution of plasmalemma ion transport ATPases in marine photosynthetic eukaryotes

For references see text.

Conclusions

In Archaea there are ion-pumping rhodopsins that actively transport H⁺ out of, and Cl⁻ into, the cells; in one archaean there is an inward H⁺-pumping rhodopsin. In Bacteria all three of the ions H⁺, Na⁺ and Cl⁻ are moved (H⁺ and Na⁺ out, Cl⁻ in) by ion-pumping rhodopsins. There is an H⁺influx rhodopsin in Acetabularia, and (probably) H⁺ efflux rhodopsins in diatoms. Photochemistry based on bacteriochlorophyll exports H⁺ from the cytosol in some marine anoxygenic photosynthetic bacteria, but chlorophyll-based redox reactions do not export H⁺ from cells of marine cyanobacteria. Exergonic redox reactions export H⁺ and Na⁺ in photosynthetic bacteria, H⁺ in cyanobacteria and possibly H⁺ in eukaryotic algae. H⁺-and/or Na⁺-ATPases occur in the plasmalemma of all photosynthetic marine organisms tested. P-type H⁺ efflux ATPases occur in the marine Streptophyta, i.e. marine charophycean algae and seagrasses and emergent marine flowering plants. P-type Na⁺-ATPases are the main primary active ion pumps in the plasmalemma of other marine green algae and non-green algae. However, there may be P-type H⁺-ATPases in some cases, and a F-type Cl⁻-ATPase occurs in the ulvophycean Acetabularia. Some assignments of P-type ATPases as H⁺ or as Na⁺ pumps using genomics are not conclusive. Despite the insights that genomics can provide, it seems that there are still large gaps in the availability of electrophysiological data from many of the ecologically (and economically) important marine (and freshwater) phototrophs. There is thus a need for more such data to improve our understanding of the functioning of primary active transport in these organisms.