Journal of Experimental Botany, Vol. 68, No. 3 pp.

359–367, 2017
doi:10.1093/jxb/erw421  Advance Access publication 31 December 2016

OPINION PAPER

Chloride: essential micronutrient and multifunctional

beneficial ion
John A. Raven1,2,*
1 
Division of Plant Science, University of Dundee at the James Hutton Institute, Invergowrie, Dundee DD2 5DA, UK
2 
School of Plant Biology, University of Western Australia MO84, Stirling Highway. Crawley, WA 6009, Australia

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*  Correspondence: j.a.raven@dundee.ac.uk

Received 13 May 2016; Editorial decision 20 October 2016; Accepted 20 October 2016

Editor: Christine Raines, University of Essex

Abstract
Cl− is an essential micronutrient for oxygenic photolithotrophs. About half of global primary productivity is carried out
by oxygenic photolithotrophs exposed to saline waters with Cl− concentrations orders of magnitude higher than that
needed to satisfy the micronutrient requirement. The other half of primary productivity involves terrestrial and fresh-
water glycophytes sometimes in environments containing significantly more Cl− than is needed for the micronutrient
requirement, but less than the toxic Cl– concentration for glycophytes. Intracellular Cl− acts in regulation of cell turgor
and volume, including that of stomatal and pulvinar nastic movements, is a major ion in streptophyte and ulvophy-
cean action potentials, and is involved in ion currents flowing around apices of pollen tubes and Acetabularia cells.
More work is needed on the essentiality of Cl− in these processes, as well as the recent finding that Cl− at 1–5 mol m−3
increases water use efficiency of growth and leaf area in Nicotiana tabacum.

Keywords:  Action potential, active transport, beneficial nutrient, chloride, ion channels, micronutrient, pulvini, stomata, turgor
regulation, water use efficiency.

Introduction
Cl− (chloride) is rightly regarded as an essential micronu-
trient for oxygenic photosynthetic organisms, first noted
by Arnon and Whatley (1949) (see below), and is generally
present in the environment at concentrations well in excess
of what is needed to satisfy the micronutrient requirement.
Franco-Navarro et al. (2016) have added the suggestion that
Cl− acts as a beneficial nutrient when supplied to the glyco-
phyte Nicotiana tabacum at concentrations (1–5 mol m−3) in
excess of those needed to satisfy the micronutrient require-
ments, but insufficient to cause toxicity. Herein the evidence
relating to Cl− as an essential micronutrient in oxygenic pho-
tolithotrophs is analysed, and suggestions for further tests
are made.
Tolerance of high external Cl− (significantly above the ben-
eficial range) is clearly the case for the organisms contribut-
ing the almost 50% of global annual net primary production
that occurs in saline environments (Field et  al., 1998). This
is despite the evidence that not only halophytic embryo-
phytes (mainly flowering plants) are secondarily salt tolerant
(Flowers et al., 2010), but also the cyanobacteria (Blank and
Sanchez-Baracaldo, 2010) and the Archaeplastida; that is,
those eukaryotic algae with primary chloroplast endosym-
biosis (Blank, 2013). Also considered is the range of other
functions of Cl− in the plant. These processes include turgor
generation used in growth and nastic (stomata, pulvini) move-
ments, and information transfer over tens of micrometres to

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360  |  Raven
a metre by electric currents carried by Cl−. Also considered is
the role of Cl− in charge and acid–base regulation in relation
to the form in which N is supplied and the influence on water
use efficiency (WUE). Finally, the various functions attrib-
uted to Cl− in plants are considered in relation to the possibil-
ity that other solutes can substitute for Cl−.
Chloride as an essential micronutrient in
photosynthetic organisms
It is very difficult to investigate the essentiality of Cl− by
culturing algae and plants in the absence of Cl−. The high
solubility of almost all Cl− salts means they are difficult
to remove from even the most highly purified salts used in
making up hydroponic culture solutions (Broyer et al., 1954;
Johnson et  al., 1957; Brownell and Crosland, 1972, 1974).
Furtheremore, there is atmospheric NaCl input as aerosols
produced from the ocean–atmosphere interface by turbulent
winds interacting with wave action from the ocean, with 1.8
Pg Cl− entering the marine boundary layer—the lower 1000
m of the troposphere—each year (Winterton, 2000). Brownell
and Crossland (1972, 1974) maintained plant growth cham-
bers at above atmospheric pressure to minimize inputs of
atmospheric NaCl in their work on Na+ requirements of
plants with photosynthesis using C4 and Crassulacean acid
metabolism. Johnson et al. (1957) found Cl− concentrations
in 11 plant species that were 3.3–44.4 times the Cl− that could
be accounted for as contamination in the culture solution,
and the rest was assumed to result from atmospheric Cl−
input. Kirkby and Armstrong (1980) found 150–200  mmol
per kg dry matter in NO3−-grown Ricinus communis despite
no deliberate addition of Cl− to the culture medium. Eyster
(1958) used the critical Cl− concentration (the minimum
external concentration yielding the maximum growth rate) in
studies on the green microalga Chlorella, and the findings did
not permit conclusions on the role of Cl− as a micronutrient
in photolithotrophic cultures, although this can be shown for
chemo-organotrophically grown cells.
The considerations in the last paragraph show that it is
difficult to demonstrate the micronutrient role of Cl− from
whole-organism culture studies. Additional data on the sta-
tus of Cl− as a micronutrient come from work on subcellu-
lar systems, and especially a role in PSII of photosynthesis
(e.g. Arnon and Whatley, 1949; Winterton, 2000). The role
in PSII is in facilitating the proton flux from the water oxi-
dation complex to the thylakoid lumen; in the absence of
Cl−, H+ transfer is impeded by formation of a salt bridge
between lys317 of D2 and asp61 of D1 (Guskov et al., 2009;
Pokhrel et al., 2011; Rivalta et al., 2011; Boussac et al., 2012;
Kato et al., 2012; Wang et al., 2014; Zhang et al., 2014). The
photosynthetic role may be obscured (Terry, 1977) in less
resolved systems than were used by Rivalta et al. (2011),
and PSII is partially inhibited when growth-inhibiting exter-
nal Cl− concentrations (decoupled from external Na+) are
supplied to whole Phaseolus vulgaris L. plants (Tavakkoli
et al., 2010). Other roles for Cl− in plants and algae in acti-
vating catalysis by proteins involve asparagine synthetase
(Rognes, 1980) and tonoplast V-type H+ ATPase (Churchill
and Sze, 1984). More controversial is the role of Cl− in acti-
vating amylase activity. Eyster (1952) suggests that plant as
well as animal amylases are activated by Cl−; D’Amico et al.
(2000) point out that, although the known Cl−-binding motif
is confined to the amylases of metazoan and some Gram-
negative bacteria, an unrelated Cl−-binding site could be
involved in Cl− stimulation of other amylases.
Franco-Navarro et al. (2016) built on the role of Cl− as an
essential micronutrient and provided evidence that it also has
a role at higher external concentrations as a beneficial macro-
nutrient. Their work compared N. tabacum L. plants grown
with three anion treatments and constant cation concentra-
tions compared with the basal solution (BS) with no addi-
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tional salts; in all cases, 75 mmol m−3 Cl− was present to satisfy
the micronutrient Cl− requirement. The treatments were ‘CL’
with 5 mol m−3 added KCl, ‘N’ with 5 mol m−3 added KNO3,
and ‘SP’ with 1.25 mol m−3 KH2PO4, 0.625 mol m−3 MgSO4,
and 0.625 mol m−3 CaSO4. In all cases, the total cation con-
centrations were 4.62  mol m−3 K+, 2.63  mol m−3 Ca2+, and
1.63 mol m−3 Mg2+. The CL treatment increased leaf water
content, and decreased leaf osmotic potential (i.e. a higher
concentration of solutes in leaf cells), and increased turgor
of leaf cells, relative to the other treatments, and increased
(i.e. less negative) leaf water potential. The photosynthetic
rate and rate of fresh and dry matter production was greatest
for the N treatment; the stomatal conductance and transpira-
tion rate were lowest, and the instantaneous WUE (mol CO2
assimilated in photosynthesis per mol H2O lost in transpira-
tion) and the growth WUE (g FW gain per m3 water lost)
were highest, for the CL treatment. This higher WUE with
CL is typical of the higher WUE found in N-, K-, and (some-
times) P-replete conditions relative to deficiency of these
nutrients (Woodward, 1699; Raven et al., 2004; Raven, 2013).
Franco-Navarro et al. (2016) point out that there are several
data sets on the effects of Cl− addition, below salinization
concentrations, on plants (Xu et al., 2000) but that these gen-
erally have not adequately separated the effects of the cation
added with Cl− from the effects of Cl− itself (e.g. Braconnier
and d’Auzac, 1990; Braconnier and Bonneau, 1998). An
exception, cited in Xu et al. (1999), is Christensen et al. (1981)
who added (NH4)2SO4 in parallel with NH4Cl in the work on
Triticum aestivum L, and found increased leaf turgor pressure
and decreased leaf water and osmotic potentials when NH4Cl
rather than (NH4)2SO4 was added to the crop. The findings
on turgor pressure and osmotic potential are similar to those
of Franco-Navarro et al. (2016) on N. tabacum, but the effects
on water potential are different.
An important aspect of increased WUE by increased Cl−
(CL treatment) is that a decrease in nitrogen use efficiency
(NUE: rate of dry matter gain per unit plant N) is predicted
as a result of the decreased stomatal conductance resulting in
a lower steady-state intercellular CO2 concentration and cor-
responding decreased CO2 saturation of Rubisco carboxylase
and increased Rubisco oxgenase activity in C3 plants such as
N. tabacum. It may be asked why Cl− supply should regulate
the interaction between WUE and NUE when more obvious
signals are water availability relative to transpiratory water
 Chloride: essential micronutrient and multifunctional beneficial ion  |  361

demand, and N availability, respectively. However, Franco-
Navarro et  al. (2016) point out that there is no decrease in
the photosynthetic rate in the CL treatment relative to the
SP or BS treatments, and they suggest that this results from
an increased mesophyll conductance, compensating for the
decreased stomatal conductance. If this were the case, there
would be no decrease in NUE in the CL treatment relative
to the BS and SP treatments. The possibility of an effect of
Cl− on mesophyll conductance adds further complexity to
attempts to understand the mechanism of genotypic and phe-
notypic variability of mesophyll conductance (Barbour et al.,
2016; Raven and Beardall, 2016).
Franco-Navarro et al. (2016) propose that Cl− supplied at
concentrations well in excess of the critical concentration, but
to the ocean and, by evaporation of confined bodies of water,
forms halite (NaCl) that yields locally high concentrations
in sedimentary rocks (Berner and Berner, 1996; Winterton,
2000). The mean Cl− in rivers is 0.16 mol m−3 from soil and
groundwater derived from natural sources; anthropogenic Cl−
brings this to 0.23 mol m−3. The natural sources of Cl− soils in
river water are, in decreasing order of importance, weathering
of halite, sea salt from dry deposition or rain, and volcanic
rocks (Berner and Berner, 1996; Winterton, 2000). Cl− is the
dominant anion in seawater, and has the longest replacement
time (87 Ma) of any seawater component (Berner and Berner,
1996). The input of Cl− to the ocean today exceeds removal,
with anthropogenic Cl− input and an absence of major evap-
orative basins removing Cl− (and Na+) as halite (Berner and

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below the concentration at which the growth rate of glyco- Berner, 1996; Winterton, 2000).
phytes is decreased by increased salinity (i.e. 1–5 mol m−3) is Cl− is the main naturally occurring form of the element
best regarded as a ‘beneficial nutrient’, a term usually reserved Cl in the biosphere. However, other inorganic (perchlorate:
for non-essential elements. All of the functions of Cl− (fig. 9 ClO4−) and organic (mainly methyl chloride: CH3Cl) forms
of Franco-Navarro et al., 2016) as an essential micronutrient, occur naturally (see Supplementary Appendix S1 at JXB
or as a beneficial nutrient, in plants (and algae and almost online).
all cyanobacteria) require intracellular Cl−, generally involv- Cl− is usually present at ‘macronutrient’ concentrations
ing active transport (against the electrochemical gradient for in soils (Xu et  al., 1999; White and Broadley, 2001), but
Cl− and so requiring energy input). The Cl– beneficial nutri- there are chloride-deficient soils in terms of plant growth
ent concentration range is in the concentration range of the described for south Sumatra for coconut (Cocos nucifera;
low affinity Cl− influx mechanism: this has either a linear or Braconnier and D’Auzac, 1990; Braconnier and Bonneau,
a hyperbolic (K1/2 4–22  mol m−3) relationship to the exter- 1998), for Columbia for growth of oil palm (Elaeis guineen-
nal Cl− concentration (table 1 of White and Broadley, 2001; sis Jacq.; Ollagnier and Ochs, 1971; Dubos et  al., 2011), for
fig. 9 of Franco-Navarro et al., 2016). The high affinity influx the sandy pampas region of Argentina for the growth of
mechanism in terrestrial glycophytes, charalean freshwater
algae, and a freshwater cyanobacterium has a lower K1/2 of
7–57 mmol Cl− m−3 (Ritchie, 1992b; Beilby and Walker, 1981; Table 1.  Vacuolar (v) and whole vacuolated cell (wvc) Cl−
table 1 of White and Broadley, 2001; fig. 9 of Franco-Navarro concentrations in eukaryotic photosynthetic organisms
et al., 2016).
The exception to the requirement for intracellular Cl− for Organisms External Cl− Internal Cl−
the major role as a micronutrient is in the water-oxidizing Charophyceae aquatic (+wall) 0.1–10 87–179 (v)
and oxygen-producing reactions of PSII in the glycophytic Charophyceae aquatic (+wall) 10–500 174–520
rock-dwelling cyanobacterium Gloeobacter. This is because Chlorophyceae aquatic (+wall) 0.4 30 (v)
Gloeobacter has the two photosystems, with the other mem- Chlorophyceae aquatic (–wall) 400, 1500 283, 325 (v)
brane-associated redox processes of photosynthesis and of Embryophyta Not determined; 670 1–100 (wvc)
respiration, ATP synthetase, and H+-pumping proteorho- Spermatophytes terrestrial (+wall) species of wild collected
dopsin, in the cell membrane, and the Cl−-requiring water- glycophytes
Embryophyta 500 578 (wvc)
oxidizing machinery on the periplasmic (external medium)
Magnoliopsida
side of the membrane (Raven, 2009; Rexroth et  al., 2011).
Submerged halophyte=seagrass
Gloeobacter is the extant cyanobacterium phylogenetically (+wall)
closest to the ancestral cyanobacterium (Blank and Sánchez- Embryophyta 500 600–1620 (wvc)
Baracaldo, 2000). More derived cyanobacteria, with the pho- Magnoliopsida
tosystems in thylakoids, and no PSII in the plasmalemma, Terrestrial and emergent coastal
have active Cl− influx (Ritchie, 1992a, b), but it is not known halophytes(+wall)
if this is the case for Gloeobacter. Presumably such active Rhodophyta 425 74–635 (wvc)
influx is required for the other essential roles of Cl− (fig. 9 of Florideophyceae (+wall)
Franco-Navarro et al., 2016). Ochrophyta 380 44–619 (wvc)
Phaeophyceae (+wall)
Ochrophyta 492 78–445 (wvc)
Bacillariophyceae (+wall)
Availability of chloride Alveolata 492 530 (wvc)
Dinophyta (+wall)
The ultimate source of Cl− in the biosphere is igneous rocks,
with ~200 mg Cl kg–1 (Kuroda and Sandell, 1953). Weathering From Supplementary Appendix S2. 
releases the very soluble Cl− that cycles through water on land Concentrations of Cl− are in mol m−3.
362  |  Raven

Table 2.  External and “cytoplasmic” Cl− concentration (in mol m−3) in cyanobacterial and eukaryotic cells with a minimal fraction of
vacuoles in the cell volume, and the cytosol of vacuolate cells estimated in the specified way and in sieve tubes

Organism Compartment and method External Cl− Internal Cl−

Cyanobacteria Cytosol+thylakoids 0.51 17
Charophyceae Cytosol, Cl− microelectrode 2.1 10
Charales
Embryophyta Cytosol, Cl− microelectrode 1.2 7
Marchantiopsida
Embryophyta Cytosol, Cl− microelectrode 0.4 11
Magnolipiopsida
Chlorophyceae Cytosol, Cl− microelectode 0.6 2.2
Ulvophyceae Cytosol?, compartmental analysis 25.5 194
Ulvophyceae Cytosol?, compartmental analysis 547 231

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Enbryophyta, Cytosol?, compartmental analysis 10.2 10–11
Marchantiopsida
Embryophyta Cytosol?, compartmental analysis 0.1 5.7–12.1
Magnoliopsida
Embryophyta Cytosol?, compartmental analysis 1 17–21
Magnoliopsida
Embryophyta Sieve tube sap 0.6–4 0.1–52.1
Magnoliopsida
Embryophyta Sieve tube sap Freshwater 5.4
Halophytic Seawater 28.3
Magnoliopsida
Ochrista Phaeophyceae Sieve tube sap 530 230–310
Laminariales
Embryophyta Cytosol, X-ray microanalysis 0.02 0.5, 19.3
Magnoliopsida Chloroplast, X-ray microanalysis 0.02 0.4, 1.4
Embryophyta Cytosol, X-ray microanalysis 100 91, 176
Magnoliopsida Chloroplast, X-ray microanalysis 100 55, 91
Embryophyta Cytosol, X-ray microanalysis 200 238, 241
Magnoliopsida Chloroplast, X-ray microanalysis 200 87, 271
Chlorophyceae Cytosol, X-ray micoanalysis 400 26 (vacuole 283)
Chlorophyceae Cytosol, X-ray microanlysis 1500 30 (vacuole 325)
Embryophyta Chloroplast, aqueous extraction 0.02 13–20
Magnoliopsida
Embryophyta Chloroplast, aqueous extraction 20 24–36
Magnoliopsida
Embryophyta Chloroplast, aqueous extraction 400 42–64
Magnoliopsida
Chlorophyceae Whole non-vacuolate cell 1.0 1.34
Three freshwater Whole non-vacuolate cell ≤1.0 1.3–2.1
Microalgae
Three marine Whole non-vacuolate cell 100–600 9–35
Microalgae
Chlorophyceae Whole non-vacuolate cell 50–1000 295–497

From Supplementary Appendix S3.

wheat (Triticum aestivum; Diaz-Zorita et  al., 2004), and for
north-western NSW in Australia for growth of durum wheat
(Schwenke et  al., 2009). Cl− addition to wheat crops in the
Williamette Valley of western Oregon decreased the incidence
of Gaeumannomyces graminis var. tritici and increased yields
(Christensen et al.. 1981). Diaz-Zorita et al. (2004) included
a fungicide treatment so fungal disease was shown not to be
a factor in their Cl− stimulation of wheat productivity. Grain
yield of Zea mays in New Jersey in the USA under a ‘minimum
stress field environment’ was increased in some years by Cl−
addition as KCl, with a K+ effect ruled out by adding equimo-
lar K+ as K2SO4, and decreases in the incidence of corn stalk
rot (Heckman, 1995, 1998). Although not increasing yield,
Cl− fertilization of pastures (Pehrson et al., 1999; Goff et al.,
2007) showed that increased Cl− in plant dry matter decreased
the inorganic cation:inorganic anion ratio, and the charge-bal-
ancing organic anions decreased the pre-disposition to peri-
paturuient hypoglycaemia (milk fever) in cows.
 Chloride: essential micronutrient and multifunctional beneficial ion  |  363
Chloride transport
What is known of the transport of Cl− across membranes
of algae and plants (see Supplementary Appendix S2 at
JXB online) is based on physiological/biophysical data and
on molecular genetics of the expression of Cl− transporters
and channels at the cell and membrane level. Supplementary
Appendix S2 shows that the molecular data have been very
helpful in understanding, for example, the role of Cl− chan-
nels in the regulation of stomatal aperture. Less helpful in
understanding Cl− transport and its relationship to the ben-
eficial Cl− range in glycophytes is the absence of a molecu-
lar genetic explanation for the sound biophysical evidence of
a 2H+:1Cl− symporter at the plasmalemma of glycophytes,
and the physiological role of a cation–chloride co-trans-
porter (CCC) transporter identified by molecular genetics
(Supplementary Appendix S2).
Functions of Cl− other than as a
micronutrient
Phylogeny of the osmotic role of Cl−
This is considered in Supplementary Appendices S2 and S3,
showing that there is a significant phylogenetic and environ-
mental (aclimatory and adaptive) variation in the intracellu-
lar Cl− concentration, together with the range of functions
that turgor, in part maintained by Cl−, fulfils (Tables 1 and 2).
Influence of nitrogen source on the use of Cl− or
organic anions as an osmoticum
Franco-Navarro et  al. (2016) only used NO3− as the N
source for their work on Nicotiana, although globally
many photosynthetic organisms have NH4+ or organic N
as their main N source, and there are diazotrophs catalys-
ing N2 fixation in both natural and agro-ecosystems (see
Supplementary Appendix S4 at JXB online). Raven and
Smith (1976) and Raven (1986) examined published data,
and concepts, related to acid–base regulation in vascu-
lar plants as a function of N uptake and assimilation as
NH4− or NO3−, extended by Raven et  al. (1990) to diazo-
trophy. The N assimilation processes and other synthetic
processes produce ~1.3 excess H+ per NH4+-N, ~0.3 excess
H+ per N2- or NH3-N, and 0.7 excess OH− per NO3−-N
(Raven and Smith, 1976; Raven, 1986; Raven et  al., 1990)
(Supplementary Appendix S4).
There are energetic advantages of NO3− assimilation using
photosynthetic reductant in the shoot rather than by respiration
in the root; the resulting excess OH− is neutralized by organic
acid synthesis with accumulation of the organic anion with
the cation accompanying NO3− up the xylem, although there
are energetic costs of this synthesis that partly or wholly offset
the energetic advantages of photochemical NO3− assimilation
(Raven and Smith, 1976; Andrews et  al., 2013). The costs of
organic acid synthesis during NO3− assimilation in illuminated
shoots can be largely recouped by transport of the organic
anion salt to the root, where catabolism of the organic anions
generates NADPH and/or ATP with excretion of OH− (Raven
and Smith, 1976; Andrews et  al., 2013) (see Supplementary
Appendix S4 at JXB online). There is also accumulation of
organic anions in NH4+- or N2-grown plants, with H+ efflux and
influx of some other cation; this is clearly not part of acid–base
regulation in N assimilation, and is more energetically expensive
than accumulation of an external anion such as Cl− (Andrews
et al., 2013) (Supplementary Appendix S4).
The accumulation of additional organic anions in Ricinus
communis with NO3− as the N source involves a decrease in
Cl− per mol m−3 of plant water relative to growth on NH4+,
with corresponding greater H+ efflux on NH4+ than OH− on
NO3− (Allen and Raven, 1987; Tables 3, 4; Supplementary
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Appendix S4). These results are for plants growth on 4.1 mol
Cl− m−3 (i.e. within the ‘beneficial’ range of Franco-Navarro
et al., 2016), and Table 5 shows that WUE is higher on NO3−
than on NH4+. This is presumably superimposed on any
higher WUE resulting from Cl− concentration in the ‘benefi-
cial’ range. A less marked effect is found for Phaseolus vulgaris
grow on the two N sources when grown with 0.8 mol Cl− m−3
(i.e. just below that ‘beneficial’ range) (Allen et  al., 1988;
Table 5; Supplementary Appendix S4). Franco-Navarro et al.
(2016)) only used NO3− as the N source in showing increased
WUE, with ‘beneficial’ Cl− concentrations relative to basal,
micronutrient, Cl− concentrations.
Osmolytes driving pressure flow in the phloem
Cl− only contributes 1.5% of the total osmolarity of phloem
sap in the glycophyte R.  communis (Smith and Milburn,
1980). Cl− contributes a much larger fraction of osmo-
larity to sieve tube sap of the brown marine macroalga
Macrocystis pyrifera (see Supplementary Appendix S3 at
JXB online).
Nastic movements: stomata and pulvini
The opening phase in stomata and the energy-dependent
phase of pulvinar movements involve increased turgor mainly
driven by the accumulation of ions, with variable contribu-
tions from Cl− and organic anions such as malate, some-
times with contributions from NO3− (see Supplementary
Appendix S5 at JXB online). In some cases, at least for
stomata, Cl− is the only anion involved (Supplementary
Appendix S5). Aside from the work of Franco-Navarro
et al. (2016) there seem to be no direct studies of the effect
of Cl− in the beneficial range rather than at lower supplied
Cl− concentrations.
Action and variation potentials and circulating currents
in Ulvophyceae and Streptophyta
Action potentials are essential for the functioning of the
carnivorous flowering plant Dionaea, and are involved in
the the transmission of other stimuli with effects distant
from the stimulus (see Supplementary Appendix S5 at JXB
online). Circulating currents carried by Cl− within, and in
364  |  Raven

the aqueous medium around, a large cell or a multicellu-
lar structure, with internal and external currents linked
by transmembrane ion currents, are found in a number of
Ulvophyceae and Streoptophyta, where they occur in grow-
ing pollen tubes (Supplementary Appendix S5). The evi-
dence of a unique role for Cl− is often lacking, and even
where this is available it is not generally clear whether (in
glycophytes) a Cl− supply in the beneficial range is needed
(Supplementary Appendix S5).
Table 3.  Mol negative charge m−3 plant water for Ricinus
communis grown on NH4+ or NO3− as N source
Cl− and active water transport?
A further active transporter is the electroneutral CCC known
from genomic studies in some algae and plants (Teakle and
Tyerman, 2010; Raven and Doblin, 2014; Wegner, 2014;
Henderson et  al., 2015; see Boyd and Gradmann, 2002).
These transporters transport two Cl− with two singly charged
cations, typically one K+ and one Na+ with ~100 H2O, so they
have been suggested to be components of active water trans-
port mechanisms in xylem refilling (Wegner, 2014) and in
algae in buoyancy generation in marine diatoms and volume
regulation in effectively wall-less, flagellate, freshwater algal
cells (Raven and Doblin, 2014). However, the intracellular,
and among cell types, location of CCC in Arabidopsis thali-

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ana and Vitis vinifera makes functional assignment difficult
Anion NH4+ as N source NO3− as N source (Henderson et al., 2015).
Cl− 93 64
NO3− 0 37
SO42− 36 20 Influence on glycophytes of external Cl−
P− 24 35
-COO− 56 71
above the benefical range
Total 209 227 The effects of inhibitory NaCl concentrations on glycophytes
have been widely investigated on crop plants in the context of
Assuming all P (organic and inorganic) has one mol negative charge the effects of salinization of agricultural land (Flowers et al.,
per mol P.
Much of the P is in macromolecules (minimal contribution to mol 2015; Munns and Gillihan, 2015). However, separation of the
soluble negative charge) and phospholipids (no contribution to mol inhibitory effects of Cl− rather than Na+ has received much
soluble negative charge). less attention (Flowers et al., 2015); one of the few exceptions
Some of the -COO− is in macromolecules (proteins, cell wall
polysaccharides) with, respectively, little and no contribution to the mol is the experiments on Vicia faba and Hordeum vulgare with
soluble negative charge. Cl− using conterions other than Na+ (Tavakkoli et al., 2010,

Table 4.  Mol excess H+ generated in Ricinus communis growth on NH4+ and measured H+ efflux, and mol excess OH− generated in
growth on NO3− and measured OH− efflux, all in mol m−3 plant water

Reaction NH4+ as N source NO3− as N source

+ −3
Conversion of N source into neutral organic N 387 mol H m plant water
Production of macromolecular and low molecular mass carboxylic acids 56 mol H+ m−3 plant water
Conversion of SO42− into neutral organic S 24 mol OH− m−3 plant water
Total intracellular production of H+/OH− 419 mol H+ m−3 plant water
Measured H+/OH− efflux from roots to the bathing medium 381 mol H+ m−3 plant water
213 mol OH− m−3 plant water
71 mol H+ m−3 plant water
16 mol OH− m−3 plant water
158 mol H+ m−3 plant water
131 mol OH− m−3 plant water

From table 2 of Allen and Raven (1987).

Table 5.  Cl− concentration in the growth medium and in the plant, and water lost in transpiration, during growth on different N sources,
and water lost in transpiration during growth, for Ricinus communis (table 2 of Allen and Raven, 1987) and Phaseolus vulgaris (table 4
of Allen et al., 1988) g water lost in transpiration is calculated from g water uptake during growth on the specified N source minus the g
water in the plants and minus the water used in metabolism during photosynthetic growth (18 g water per mol C)

Plant, N source Mol Cl− m−3 water in growth medium Mol Cl− m−3 water in the plant mg dry matter gain g−1 water lost
Ricinus communis 4.1 93 1.67
NH4+
Ricinus communis 4.1 64 2.33
NO3−
Phaseolus vulgaris 0.8 73 2.27
NH4+
Phaseolus vulgaris 0.8 7 2.50
NO3−
Phaseolus vulgaris 0.8 37 1.79
N2
 Chloride: essential micronutrient and multifunctional beneficial ion  |  365
2011). Geilfus and Mühling (2013) showed for V.  faba that
Cl− (not Na+) at 70  mol m−3 in the culture medium causes
transient alkalinization of the leaf apoplast, modulated by
the plasmalemma H+ ATPase. Cl− at 70–100 mol m3 or more
inhibits a number of enzymes (Ritchie and Larkum, 1984),
and especially those with anionic substrates (Gimmler et al.,
1984), and Cl− is equally, or even more, inhibitory to in vitro
protein synthesis than Na+ (Flowers and Dalmond, 1992),
More work is needed in which the inhibitory effects of Cl−
are distinguished from those of Na+
Conclusions and recommendations for
further work
The role of Cl− as an essential micronutrient for oxygenic
photosynthetic organisms is widely accepted, largely on the
basis of structural and functional studies on isolated PSII
complexes. It would be useful to extend this detailed under-
standing to other known (V-type H+ ATPase, asparagine
synthetase) and possible (amylase) Cl−-stimulated enzymes,
as well as to extend attempts to demonstrate essentiality of
Cl− for the growth of oxygenic photolithotrophs.
The high concentrations of Cl− generally found in algal
and plant cells, and particularly in their vacuoles, when grown
with Cl− concentrations more than sufficient to satisfy micro-
nutrient concentrations have been known for many decades,
as has the major role it plays in turgor generation and in
cell expansion. It is also known that these roles of Cl− vary
phylogenetically and with the N source. Less attention has
been paid to how the organisms respond to manipulations of
the Cl− content. The work of Franco-Navarro et  al. (2016)
increases our knowledge of the outcome of such manipula-
tions, and proposes that Cl− at concentrations in excess of
the concentration needed to satisfy the micronutrient require-
ment but below those causing Cl− toxicity is a beneficial
nutrient, thus becoming the first essential micronutrient to
be suggested also to be a beneficial nutrient. The essential-
ity of Cl− for nastic (stomata, puvini) responses, circulating
currents, and action and variation potentials is difficult to
establish. This is because Cl− is a micronutrient that must
be present at low concentrations in the growth medium, and
there is a relatively higher affinity Cl− uptake system in many
organisms. However, progress is possible using experiments
maniputating internal Cl− concentrations with measurements
of the distribution of Cl− between and within cells, combined
with measurements of nastic (stomata, puvini) responses, cir-
culating currents, and action and variation potentials.
Finally, further data are needed on how the inhibitory
effects of high concentrations on Cl− are distinguished
from those of Na+ on plant growth and on in vitro enzyme
activities.
Supplementary data
Supplementary data are available at JXB online.
Appendix S1. Natural occurrence of Cl species other than Cl–.
Appendix S2. Phylogeny of Cl– transport and the osmotic
role of chloride from whole-organism and vacuolar analyses.
Appendix S3. Cl– concentration in the total cytoplasm and
compartments of the cytoplasm.
Appendix S4. Influence of nitrogen source on the use of
chloride rather than organic anions as an osmoticum in ter-
restrial embryophytes
Appendix S5. The roles of Cl– in nastic responses, action
and variation potentials, circulating ionic currents, response
to decreased external osmolarity, and active water transport.
Acknowledgements
Discussions with Susan Allen, Mitchell Andrews, Mary Beilby, Mary
Downloaded from https://academic.oup.com/jxb/article-abstract/68/3/359/2768889 by guest on 24 September 2019
Bisson, Tim Colmer, Martina Doblin, Linda Handley, Hans Lambers, Enid
MacRobbie, Janet Sprent, F.  Andrew Smith, N.  Alan Walker, and Philip
White are gratefully acknowledged. The University of Dundee is a registered
Scottish charity, No. 015096
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