4 | DISCUSSION
O. alismoides possesses three CCMs, including constitutive
abilities to (i) use HCO3- and (ii)
operate C4 photosynthesis, and a facultative ability to perform CAM when
acclimated to low CO2 concentrations (Zhang et al.,
2014; Shao et al., 2017; Huang et al., 2018). We confirm here that this
species has a constitutive ability to use
HCO3-, and this allows it to exploit a
large proportion of the Ci pool and drive CO2 to very
low concentrations.
In this study, multiple lines of evidence show that an external CA,
putative αCA-1, plays a major role in Ci uptake in O. alismoides :
(i) external CA activity was measured, (ii) AZ inhibited Ci uptake with
the slope of Ci uptake vs the concentration of
CO2 between 15 and 40 µM being about a quarter of the
control after treatment with 0.2 mM AZ, (iii) transcripts of putative
αCA-1 were detected. The CA was confirmed to be external since (i)
washing of leaves treated with AZ, restored CA activity and (ii) its
sequence bears a signal peptide consistent with a periplasmic location.
External CA is indeed widespread in photoautotrophs from marine and
freshwater environments (Moroney et al., 2001; Dimario et al., 2018).
The green microalga Chlamydomonas reinhardtii has three αCAs, of
which two (Cah1 and Cah2) are localized in the periplasmic space and one
(Cah3) in the thylakoid membrane (Fujiwara et al., 1990; Karlsson et
al., 1998; Moroney & Chen, 1998). While CAs have the same catalytic
activity, their sequence identity could be very low among different
classes (Jensen et al., 2019). The αCA-1 from O. alismoides has
around 30% sequence identity with the periplasmic Cah1 from C.
reinhardtii . Many CAs are regulated by the concentration of
CO2. The diatom Phaeodactylum tricornutum does
not possess external CA, but the internal CA (β-type CA) is
CO2 responsive and crucial for its CCM operation (Satoh
et al., 2001; Harada et al., 2005; Harada & Matsuda, 2005; Tsuji et
al., 2017). In the marine diatom, Thalassiosira pseudonana , the
two external CAs, δ-CA and ζ-CA, as well as a recently identified
chloroplastic ι-CA are induced by carbon limitation (Samukawa et al.,
2014; Clement et al., 2017; Jensen et al., 2019). In contrast, the
putative αCA-1 in O. alismoides is constitutive and its
expression was unaffected by the CO2 concentration. This
is also true for Cah3 in the thylakoid lumen of C. reinhardtii(Karlsson et al., 1998; Moroney & Chen, 1998), while the expression of
the periplasmic CA (Cah1) and the mitochondrial CAs (β-CA1 and β-CA2)
are highly CO2-sensitive (Moroney & Chen, 1998).
We show that the anion exchange proteins, one group of the SLC4 family
HCO3- transporters (Romero et al.,
2013), is involved in HCO3- uptake inO. alismoides . DIDS, a
commonly-used inhibitor of AE/SLC-type
HCO3- transporters (Romero et al.,
2013) significantly decreased the final pH of a drift, and increased the
final CO2 concentration to about 0.8 µM which is not
substantially less than that expected in the absence of a CCM: a
terrestrial C3 plant CO2 compensation
point of 36 µL L-1 (Bauer & Martha, 1981) is
equivalent to about 1.2 µM. Furthermore, transcripts of putative
HCO3- transporter family in O.
alismoides were found to contain Band 3 anion exchange proteins (SLC4
member 1), and the peptides shared 70.6-85.7% sequence identity with
HCO3- transporters from other
terrestrial plant species. Several genes which encode SLC4 family
transporters, has been found to be involved in the CCMs in the marine
microalgae Phaeodactylum tricornutum and Nannochloropsis oceanica(Nakajima et al., 2013; Poliner et al., 2015), as well as the marine
macroalga Ectocarpus siliculosus (Gravot et al., 2010). More
broad evidence from the physiological data have demonstrated that anion
exchange proteins play a role in HCO3-uptake in green, red and brown marine macroalgae (Drechsler et al.,
1993; Granbom & Pedersén, 1999; Larsson & Axelsson, 1999; Fernández et
al., 2014). Although HCO3- use by
seagrasses is known to involve an anion exchange protein, to our
knowledge, this is the first report that provides evidence of the
presence of a direct HCO3- uptake via
DIDS-sensitive SLC4 HCO3- transporters
in an aquatic angiosperm. Whatever, these transporters for direct
HCO3- acquisition, appears to be much
more restricted in distribution than the widespread external CA.
Three mechanisms of HCO3- use have
been proposed in aquatic plants: i) indirect use of
HCO3- based on dehydration of
HCO3-, facilitated by external CA, to
produce elevated CO2 concentrations outside the
plasmalemma; ii) direct uptake of
HCO3- by an anion exchange transporter
in the plasmalemma and iii) direct uptake of
HCO3- by a P-type
H+-ATPase (Giordano et al., 2005). In this study we
provide evidence for the first two mechanisms in O. alismoides .
Although we did not specifically check for a P-type
H+-ATPase, this process appears to be absent, or of
minor importance, in O. alismoides in contrast to Laminaria
digitata and L. saccharina (Klenell et al., 2004), because inO. alismoides, HCO3- use was
abolished by addition of either AZ or DIDS. An AE is mainly responsible
for HCO3- use in the brown marine
macroalga Macrocystis pyrifera (Fernández et al., 2014), while in
several other brown macroalgae such as Saccharina latissima(formerly Laminaria saccharina ) external CA plays the major role
in HCO3- use (Axelsson et al., 2000),
though in L. saccharina as in L. digitate , a P-type
H+-ATPase has been identified (Klenell et al., 2004).
In another brown macroalga, Endarachne binghamiae ,
HCO3- use was based on an external CA
and P-type H+-ATPase with no contribution from an AE
(Zhou & Gao, 2010). Another strategy to use
HCO3- has been shown in some species
of freshwater macrophytes that involves the possession of ‘polar leaves’
(Steemann-Nielsen, 1947). At the lower surface of these leaves, proton
extrusion generates low pH and at their upper surface, high pH often
generates calcite precipitation (Prins et al., 1980). Consequently, at
the lower surface with low pH, the conversion of
HCO3- to CO2 near the
plasmalemma facilitates the cells to take up Ci. Because of this, there
is some evidence for a lower reliance on external CA in macrophytes with
polar leaves. For example, in a species with polar leaves,Potamogeton lucens , external CA was absent (Staal et al., 1989)
and in the polar leaf species Elodea canadensis , external CA
activity was present but not influenced by the CO2concentration (Elzenga & Prins, 1988).
It was initially surprising that AZ completely inhibited
HCO3- use. However, Sterling et al.
(2001) also found that AZ inhibited AE1-mediated chloride-bicarbonate
exchange. This result could be explained by the binding of CA to the AE
resulting in the formation of a transport metabolon, where there was a
direct transfer of HCO3- from CA
active site to the HCO3- transporter
(Sowah & Casey, 2011; Thornell & Bevensee, 2015). Thus, when CA is
inhibited, then the transport of HCO3-is inhibited.
O. alismoides can perform C4 photosynthesis, however the final
CO2 concentration at the end of pH-drift, when
HCO3--use was abolished by the
inhibitors, was 0.8-1.6 µM, which could be supported by passive entry of
CO2 without the need to invoke a CCM. These are slightly
higher than the CO2 compensation point in the freshwater
C4 macrophyte Hydrilla verticillata at less than 10 ppm (Bowes,
2010), which is equivalent to a dissolved CO2~0.3 µM at 25 °C. If this difference between the species
is real and not methodological, it could suggest that in O.
alismoides C4 photosynthesis is more important to suppress
photorespiration than to uptake carbon.
A simple model of carbon acquisition (Figure 7a) was constructed to
quantify the contribution of the three pathways involved in Ci uptake inO. alismoides : passive diffusion of CO2,
HCO3--use involving αCA-1 and
HCO3--use involving SLC4
HCO3- transporters. Using the Ci
uptake rates at different CO2 concentrations in Figure
3, and assuming that 0.3 mM DIDS completely inhibited
HCO3- transporters and that 0.2 mM AZ
completely inhibited αCA-1 and HCO3-transporters, we calculated: i) passive diffusion of CO2as the rate in the 0.2 mM AZ treatment that inhibited both αCA-1 and
SLC4 HCO3- transporters; ii) diffusion
of HCO3- and conversion to
CO2 by αCA-1 at the plasmalemma as the difference
between the rate in the presence of 0.3 mM DIDS and that in the presence
of 0.2 mM AZ; and iii) diffusion of
HCO3- and transfer across the
plasmalemma by SLC4 HCO3- transporters
as the difference in the rate between the control and the 0.3 mM DIDS
treatment. At a CO2 concentration of about 50 µM,
passive diffusion of CO2 contributed 55.7% to total Ci
uptake, diffusion of HCO3- and
conversion to CO2 by αCA-1 contributed 42.7% and
transfer of HCO3- across the
plasmalemma by SLC4 HCO3- transporters
contributed 1.6% (Figure 7b). At ~9 µM (about 66% of
equilibrium with air at 400 ppm CO2) the contribution to
total Ci uptake of CO2-diffusion,
HCO3- diffusion and conversion to
CO2 by αCA-1 and transfer by SLC4
HCO3- transporters was 24.0%, 64.4%
and 11.5% respectively and at about 1 µM CO2 (close to
a typical C3 CO2 compensation point) diffusion was zero
and αCA-1 and SLC4 HCO3- transporters
contributed equally to carbon uptake. So, as CO2concentrations fall, passive CO2 diffusion can no longer
support Ci uptake and indirect and direct use of
HCO3- allows Ci uptake to continue.
The stimulation of absolute rates of SLC4
HCO3- transporters-dependent Ci uptake
is consistent with patterns seen for a number of freshwater macrophytes
during pH-drift experiments, where rates increase as CO2approaches zero before declining as Ci is strongly depleted (Maberly &
Spence, 1983). This could be caused by regulation or by direct effects
of pH on HCO3- transporters activity.
These results confirm the prevailing notion from seagrasses that
external CA plays an important role in contributing to Ci uptake.
External CA contributed 25% to Ci uptake in Posidonia australis(James & Larkum, 1996) and ~60% in Zostera
marina (approximately 2.2 mM Ci at pH 8.2, equivalent to a dissolved
CO2 ~23 µM at 25 °C; Beer & Rehnberg,
1997), albeit in the presence of Tricine buffer that might inhibit the
photosynthesis rate. The value reported here for O. alismoides at
a CO2 concentration of 23 µM, 56%, is similar toZ. marina .
In conclusion, O. alismoides has developed a jack of trades CCM,
the master of which, either external CA or SLC4
HCO3- transporters, depends on the
CO2 concentration. There are several future lines of
work that need to be pursued. The distribution of
HCO3-transporters in freshwater species should be determined. The apparent
relationship between polar leaves and low or absent external CA activity
could be tested using a range of species, especially within the genusOttelia where calcite precipitation differs among species (Cao et
al., 2019). The Ci acquisition mechanisms of more freshwater species
should be examined. The cause of the increasing rate of
HCO3- transporters-dependent
HCO3- uptake as Ci becomes depleted
needs to be understood. Finally, production and analysis of genome
sequences for freshwater macrophytes will be a powerful tool to answer
these and future questions concerning the strategies used by freshwater
macrophytes to optimize photosynthesis.