Ions Channels/Transporters and Chloroplast Regulation

Cell Calcium 58 (2015) 86–97

Contents lists available at ScienceDirect

Cell Calcium

jou rnal homepage: www.elsevier.com/locate/ceca

Review

Ions channels/transporters and chloroplast regulation

a,b,c,d,∗ a,b,c,d a,b,c,d

Giovanni Finazzi , Dimitris Petroutsos , Martino Tomizioli ,

a,b,c,d a,b,c,d e a,b,c,d

Serena Flori , Emeline Sautron , Valeria Villanova , Norbert Rolland , a,b,c,d,∗∗

Daphné Seigneurin-Berny

CNRS, Laboratoire de Physiologie Cellulaire & Végétale, UMR 5168, 17 rue des Martyrs, F-38054 Grenoble, France

Univ. Grenoble Alpes, LPCV, F-38054 Grenoble, France

CEA, DSV, iRTSV, LPCV, F-38054 Grenoble, France

INRA, LPCV, USC1359, 17 rue des Martyrs, F-38054 Grenoble, France

Fermentalg SA, 4 bis rue Rivière, F-33500 Libourne, France

a r t i c l e i n f o a b s t r a c t

Article history: Ions play fundamental roles in all living cells and their gradients are often essential to fuel transports,

Received 15 July 2014

to regulate enzyme activities and to transduce energy within and between cells. Their homeostasis is

Received in revised form 1 October 2014

therefore an essential component of the cell metabolism. Ions must be imported from the extracellular

Accepted 4 October 2014

matrix to their ﬁnal subcellular compartments. Among them, the chloroplast is a particularly interesting

Available online 13 October 2014

example because there, ions not only modulate enzyme activities, but also mediate ATP synthesis and

actively participate in the building of the photosynthetic structures by promoting membrane-membrane

Keywords:

interaction. In this review, we ﬁrst provide a comprehensive view of the different machineries involved

Ions trafﬁcking

in ion trafﬁcking and homeostasis in the chloroplast, and then discuss peculiar functions exerted by ions

Chloroplast envelope

Thylakoids in the frame of photochemical conversion of absorbed light energy.

Proton motive force

1. Introduction named electrochemically-driven transporters) use the concentra-

tion gradient of co-transported molecules and therefore include

Ions play key roles in all living organisms being involved in antiporters and symporters.

all metabolic and cellular functions. Therefore, ions are found Transmembrane proteins have an essential role in the regula-

in all subcellular compartments and have to be imported from tion of ions homeostasis and in biological functions. In chloroplasts,

the extracellular matrix to their ﬁnal localization within cells. a large variety of ions are found, at micro to millimolar concen-

In Arabidopsis, acquisition and translocation of ions within plant trations. Since metal ions (zinc -Zn-, copper -Cu-, iron -Fe-, etc)

organs, cells and subcellular compartments involve large fam- can be toxic for the cell, they are not found as free ions, but

ilies of ionic transporters with various substrate speciﬁcities, are always chelated by proteins or biomolecules. Conversely, ions

expression patterns, and subcellular localization. These families like potassium (K), magnesium (Mg), can be free and reach mil-

were classiﬁed into three major categories: channels/porins, pri- limolar concentrations. In the last 40 years, the existence of ionic

mary transporters/pumps and secondary transporters (according ﬂuxes across the chloroplast envelope or the thylakoid membranes

to the Transport Classiﬁcation system [1]). Channels transport has been mostly deduced from physiological measurements or

solutes down their concentration gradient without consuming from knowledge of the chloroplast metabolism. However, most of

energy and display the fastest transport rates among transporters. the proteins responsible for these ﬂuxes have not been identiﬁed

Primary transporters (e.g. ATPases) directly use energy to trans- yet. Indeed, their low abundance, their localization in intracellular

port molecules across membranes. Secondary transporters (also membranes, their hydrophobicity and the difﬁculties encoun-

tered when trying to produce them in heterologous systems have

strongly limited their identiﬁcation and functional characterization

using classical approaches. Several chloroplast transporters have

∗

Corresponding authors at: LPCV, iRTSV, CEA Grenoble, 17 rue des Martyrs, F- been identiﬁed in the last years, thanks to proteomic approaches

38054 Grenoble, France. Tel.: +33 438784184.

∗∗ targeted to the chloroplast and its sub-compartments (e.g. [2–6]),

Corresponding authors at: LPCV, iRTSV, CEA Grenoble, 17 rue des Martyrs, F-

and to reverse genetic studies. Nonetheless, controversies still

38054 Grenoble, France. Tel.: +33 438782363.

exist about the sub-plastidial localization or function of some of

E-mail addresses: giovanni.ﬁ[email protected] (G. Finazzi), [email protected]

(D. Seigneurin-Berny). these transporters, and others are still missing. Again, proteomics

http://dx.doi.org/10.1016/j.ceca.2014.10.002

G. Finazzi et al. / Cell Calcium 58 (2015) 86–97 87

Fig. 1. Overview of Arabidopsis chloroplast ions transporters/channels. Metals transporters are represented in blue, anions transporters in grey and other ions in orange.

Transporters whose chloroplast localization is controversial are noted in italic (YSL4–YSL6 and ACA1). Substrates, protein or chloroplast localization that need to be further

validated are noted “?”. The regulation of TPK3 by Ca is highlighted.

data have provided a list of unknown proteins that, based on Fig. 2 [9]) plays a key role in the photosynthetic process and can

+ 2+ 2+

sequence similarities, could be involved in ion transport. However, exist under a reduced (Cu ) or oxidized form (Cu ). In its Cu form,

no functional characterization exist in most cases. Moreover, while it constitutes the redox cofactor of plastocyanin (PC), a protein

not identiﬁed through large-scale approaches, members of well- required for electron transport from the cytochrome b6f complex

characterized ion transporters families have also been predicted to (b6f) to photosystem I (PSI) [10]. Cu is also required for the activity

be localized in the chloroplast using bioinformatics tools (for recent of the Cu/Zn superoxide dismutase (Cu/Zn-SOD), a soluble enzyme

reviews on chloroplast ion transporters see [7,8]). Overall, when that scavenges reactive oxygen species produced by photosynthe-

considering the known, hypothetical and missing transporters, a sis under stress conditions, which is found in eucaryotes and some

scenario emerges where a plethora of transporters is involved in procaryotes. In Arabidopsis, Cu delivery to chloroplasts and thy-

ion ﬂuxes across the membranes to facilitate exchanges between lakoids requires three PIB-type ATPases: AtHMA1, AtHMA6 and

the cytosol, the stroma and the thylakoid lumen (Fig. 1). AtHMA8 (Fig. 1). AtHMA1 (At4g37270) and AtHMA6 (At4g33520)

In the ﬁrst part of this review, we will provide a comprehensive are localized in the chloroplast envelope [5,11,12] and AtHMA8

description of transporters identiﬁed in Arabidopsis chloroplast (At5g21930) in the stroma-lamellae, i.e. non-appressed fractions,

membranes, including those that are still only incompletely char- of the thylakoid membranes [6]. Genetic approaches have shown

acterized. In the second part, we will discuss the implication of ions that both AtHMA6 and AtHMA8 are Cu transporters, AtHMA6 being

concentration and ﬂuxes on the optimization of the photosynthetic the main route of Cu supply to the Cu/Zn SOD and to the thylakoid

process. transporter AtHMA8, as required for PC biosynthesis [11,13]. More

recent biochemical studies have demonstrated that AtHMA6 is a

high afﬁnity Cu transporter [14] while the biochemical properties

2. A global overview of chloroplast ion of AtHMA8 still remain unknown. AtHMA1, the second envelope

channels/transporters transporter, provides an additional way to import Cu into the

chloroplast, which would provide Cu to the Cu/Zn SOD, and which is

2.1. Transporters involved in metal homeostasis essential under light stress conditions [12,15]. AtHMA1 could also

transport other metal/divalent ions like Zn, cobalt (Co), calcium (Ca)

Metal ions are essential cofactors for numerous chloroplast pro- [16] and was also proposed to be involved in Zn or Cu/Zn export

teins involved in photosynthesis (Cu, Mg, manganese -Mn-, Fe), from Arabidopsis and Barley chloroplasts [17,18]. The ionic speci-

oxidative stress detoxiﬁcation (Cu, Zn, and Fe), nutrient assimi- ﬁcity of AtHMA1 is still controversial, strongly suggesting that this

lation (Fe), biosynthesis of aminoacids (e.g. Zn for cysteine and protein could transport a broad range of divalent cations, probably

methionine), etc. Cu (estimated chloroplast concentration ∼60 ␮M, depending on the physiological conditions. As for AtHMA8, there

88 G. Finazzi et al. / Cell Calcium 58 (2015) 86–97

Fe2+ ~ 150 µM Mn2+

ADP + Pi ATP Mg2+ ~ 30 µM

~ 5 mM Fd ATP ase PSII b6f PSI Lumen PC

Lumen CAS FTSH

Ca2+

~ 5- 10 mM Cu+/2+ ~ 60 µM Zn2+ Stroma ~ 130 µM

Cytosol

Fig. 2. Metals in thylakoids. Examples of major metal requirements for photosynthetic complexes, membranes and proteins linked to photosynthesis. Mn: oxygen evolving

complex; Fe: [4Fe–4S] and [2Fe–2S] clusters and non heme Fe of photosynthetic complexes (PSI, PSII, b6f); Cu: plastocyanin (PC); Mg: chlorophyll, thylakoid membrane

stacking; Zn: metalloproteases; Ca: calcium sensing, OEC. Estimated ion concentrations were determined from [9] for Cu, Zn, Fe, Mn; from [19] for Mg; and from [9,19] for

Ca.

is a strong need for direct biochemical characterization of AtHMA1 since NAP14 is a non-intrinsic protein. Recently, two members

to deﬁne its deﬁnite role in chloroplast metal homeostasis. of the Arabidopsis family of YSL transporters, YSL4 (At5g41000)

In chloroplast, Mg (estimated chloroplast concentration ∼5 mM and YSL6 (At3g27020), were proposed to participate to the con-

[19], Fig. 2) plays an essential role in photosynthesis, being the trol of Fe homeostasis in the chloroplast by mediating its release

coordinating ion in the chlorophyll molecule. Appropriate Mg con- from chloroplasts and preventing its accumulation into chloro-

centrations are also required for the function or activation of some plasts during de-differentiation [29]. However, their localization

chloroplast enzymes [20,21]. The AtMRS2-11 (At5g22830) protein, and ion speciﬁcity is controversial. Indeed, Conte and coworkers

belonging to the CorA super-family (transporters for both Mg and [30] found that both transporters are associated with the vacuolar

Co, for review see [22]), may transport Mg into the stroma (Fig. 1). membranes and ER, and propose therefore that these transporters

However, its function has been deduced from complementation should be involved in the supply of Mn and nickel to proteins

assays of yeast mutants and no phenotype is associated with its located in internal cellular compartments.

overexpression in planta [23]. Therefore, its role in Mg homeostasis Mn is a redox-active transition metal, which is required for plant

2+ 3+ 4+

in planta needs further experimental support. growth and can exist in several oxidized states, Mn , Mn , Mn ,

6+ 7+

Similar to Cu, Fe is a redox active metal ion that is able to Mn , Mn . In chloroplasts, Mn (estimated concentration ∼30 ␮M

2+ 3+

exist as Fe and Fe under physiological conditions. Around 80% [9], Fig. 2) is mainly required to form the Mn-cluster in photosystem

of the cellular Fe is found in the chloroplast (estimated chloro- II (PSII), an essential component of the oxygen evolving complex

plast concentration ∼130 ␮M [9], Fig. 2). Fe is used as a cofactor in (OEC) that catalyzes water oxidation and oxygen production in oxy-

three groups of Fe-containing proteins: proteins containing iron- genic photosynthesis. Until now, however, no Mn transporter has

sulfur (FeS) clusters, hemes, and other Fe-containing proteins. Fe been identiﬁed in the chloroplast membranes.

plays major functions in photosynthesis (being present in all the Zn (∼130 ␮M [9], Fig. 2) is a non-redox metal, playing important

complexes involved in photosynthetic electron ﬂow), chlorophyll role as a catalytic and structural element in several chloro-

biosynthesis, and other essential metabolic processes that occur plast enzymes including carbonic anhydrase, metalloproteases,

in chloroplasts [24]. Recent studies have identiﬁed several pro- methionine synthase and others. To date, while its role remains

teins, localized in the chloroplast envelope, which may play a role controversial in planta, several studies have demonstrated that

in Fe import into the chloroplast. PIC1 (At2g15290), a permease AtHMA1 could be involved in the control of Zn homeostasis in

ﬁrst identiﬁed as a component of the protein import machinery, chloroplast [12,16,17].

could be involved in Fe import into the chloroplast and Fe homeo-

stasis [25]. PIC was reported to interact with the NiCo protein 2.2. Anion transporters

2+ 2+

(At4g35080), a member of the Ni –Co transporters family. It was

suggested that PIC1 and NiCo might function together in plastid Fe Plants cells contain several main anions such as nitrate/nitrite,

−

transport and/or that this complex could interact with the protein sulfate/sulﬁte, chloride (Cl), bicarbonate (HCO3 ) and phosphate.

translocon machinery to deliver Fe for FeS-cluster biogenesis, and Nitrite, sulfate, and phosphate are metabolized in the chloroplast

therefore promote the assembly of FeS clusters into new proteins where they are involved in processes like ammonium assimila-

upon translocation [26]. MAR1 (At5g26820), a close homolog of the tion (nitrite), assimilation of sulfur for the biosynthesis of cysteine

−

IREG/Ferroportin efﬂux transporters, was also proposed to trans- and methionine (sulfate) and ATP synthesis (phosphate). HCO3

port Fe or Fe-chelating polyamine such as nicotianamine (Fe-NA) and Cl also have key role in carbon assimilation and photosynthe-

into chloroplasts [27]. Lastly, a nonintrinsic ABC protein, NAP14 sis.

(At5g14100) was found to be another candidate for the transport Cl concentration in chloroplasts was estimated around 1–50 mM

of Fe into the chloroplast or for the regulation of Fe or other metals [31,32]. A member of the Cl channel (CLCs) family, CLCe

homeostasis [28]. If NAP14 functions as a metal transport system, it (At4g35440) is found in thylakoid membranes [33]. CLCs genes

would require a transmembrane channel counterpart (“?” in Fig. 1) encode for Cl channel/transporter and also nitrate/H antiporter

G. Finazzi et al. / Cell Calcium 58 (2015) 86–97 89

[34]. CLCe could thus correspond to the Cl channel responsible for indicated around 100 genes responsive to low CO2 [50,54,55]. Deep

ion compensation during the generation of the proton motive force RNA sequencing approaches [56,57] have extended the list of CO2-

across the thylakoid membrane [35] (see Section 3.2). However, the related differentially expressed genes to several thousands and

ion selectivity of CLCe remains to be elucidated. In spinach chloro- have revealed possible undiscovered Ci transporters. Similar input

plast envelope, anion-selective channels have also been detected is expected from high-throughput genotyping approaches [58]. The

by physiological measurements without identiﬁcation of the cor- carbon concentrating system does not seem to exist in C3 plant

responding proteins [36]. chloroplasts, however the Arabidopsis thaliana genome contains

Nitrate is reduced in the cytosol into nitrite that is then trans- nineteen carbonic anhydrases localized in the plasma membrane,

ferred to the chloroplast (chloroplast concentration ∼10 mM [31]) cytosol, chloroplast and mitochondria [59]. It has been proposed

where it is reduced to ammonium and assimilated into glutamate. that mitochondrial CAs reduce leakage of CO2 from plant cells

The transporter CsNitr1-L (At1g68570), a member of the proton- and allow efﬁcient recycling of mitochondrial CO2 for carbon ﬁxa-

dependent oligopeptide transporter family (POT) was shown to be tion in chloroplasts [60]. In Nicotiana tabacum plants, an aquaporin

localized in the chloroplast envelope and to load cytosolic nitrite (NtAQP1), that functions as a water channel in the plasma mem-

into the chloroplast during nitrate assimilation [37]. brane and as a CO2 channel at the chloroplast envelope, suggests a

Sulfate is the major form of inorganic sulfur utilized by plants mechanism that plants may use to modify photosynthetic function

where chloroplasts are the main site of reduction of sulfate into [61]. In A. thaliana, carbonic anhydrases CA1 and CA4 have been

2−

sulfur before its assimilation (concentration ∼10 mM SO4 in proposed to function early in CO2 signalling, acting as upstream

chloroplast [31]). Mourioux and coworkers [38] have detected the regulators of CO2-controlled stomata movement [62].

2− − 2−

presence of an SO4 /HPO4 antiporter by physiological measure- Inorganic phosphate (Pi/HPO4 ) is essential for ATP synthe-

ments but the corresponding transporter remains unidentiﬁed. sis during the light phase of photosynthesis. In the chloroplast

Recently, the SULTR3;1 protein (At3g51895) was identiﬁed in the envelope, several transporters have been identiﬁed that mediate

chloroplast envelope and demonstrated to contribute to the sulfate the 1:1 counter-exchange of Pi with phosphorylated organic com-

uptake into chloroplast [39] (Fig. 1). This transport is pH dependent, pounds. However, these transporters do not allow a net import of Pi

thus conﬁrming previous studies that have measured the presence into the stroma (concentration ∼5–35 mM in chloroplast [63,64]).

of a proton/sulfate co-transporter for uptake into the chloro- This net import can be achieved by transporters of the PHT2 and

+ +

plast [40]. Data from Cao and coworkers [39] also suggest that PHT4 families that mediate H - and/or Na -dependent Pi transport.

three other members of the SULTR3 family (SULTR3;2/At4g02700, These latter transporters belong to the ubiquitous Major Facili-

SULTR3;3/At1g23090, and SULTR3;4/At3g15990) may also be tator Superfamily (MFS) of transporters. PHT2;1 (At3g26570) is

involved in sulfate uptake into the chloroplast. However, the a H -dependent phosphate importer localized in the chloroplast

chloroplast envelope localization of these three transporters envelope [65,66] (Fig. 1). This transporter inﬂuences the allocation

remains to be established. of Pi throughout the plant and affects the expression of Pi-

To overcome CO2 limitation, caused by a 10 slower rate of dif- starvation responses [65]. PHT4;4 (also named ANTR2/At4g00370)

fusion of CO2 in water relative to air, cyanobacteria and microalgae is mainly expressed in green tissues and associated with the

have developed a CO2 concentrating mechanism that leads to ele- chloroplast envelope [3,67,68]. Complementation of yeast mutants

vated intracellular inorganic carbon (Ci) increasing the apparent under limiting Pi condition and uptake assays using radiolabeled Pi

CO2/O2 speciﬁcity for Rubisco, enhancing photosynthetic perfor- demonstrated that all of the PHT4 proteins are capable of mediating

mance and decreasing the carbon ﬂux into the photorespiratory Pi transport [68] with a low afﬁnity for Pi. Guo and coworkers [68]

pathway [41]. Thirty years after the discovery and characterization suggested that protons could serve as a co-transported substrate for

of the carbonic anhydrase CAH1 [42], genome-wide transcriptomic PHT4;4. However, using complementation of yeast mutant strains

approaches using the model algae Chlamydomonas reinhardtii have and radiolabeled Pi import assays in Xenopus oocytes, we found

identiﬁed many genes that respond to CO2 limitation, including that Na could be the co-transported ion (S. Miras and N. Rol-

nine carbonic anhydrases and several candidate Ci transporters land, unpublished data). Similarly, the nature of the co-transported

(reviewed in [41,43,44]). Chloroplasts from C. reinhardtii have been ion is still unclear in the case of PHT4;1/ANTR1 (At2g29650).

− +

shown to transport both CO2 and HCO3 [45]. The product of the Indeed, this protein has a Na -dependent Pi transport activity when

chloroplast gene YCF10, was the ﬁrst identiﬁed Ci transport candi- expressed in Escherichia coli [69] and an H -dependent Pi trans-

date. Disruption of this gene resulted in diminished Ci uptake in the port activity when expressed in yeast [68]. Several studies have

chloroplasts [46]. The YCF10 protein was discovered in pea plants demonstrated, using different approaches (GFP fusions, western-

and originally named CemA [47], while its cyanobacterial ortholog blot and proteomic analyses), that this transporter is localized

pxcA (CotA) has a role in light-induced proton extrusion [48]. LCIA in the chloroplast envelope [3,5,68]. This localization is contro-

(NAR1;2) a putative chloroplast envelope protein with a suggested versial since Pavon and co-worker [69] suggested that PHT4;1

role in nitrite transport into the chloroplasts [49] has been pro- is a thylakoid protein. However, we have previously shown that

posed as a candidate Ci transporter because LCIA expression is overexpression of PHT4;1 in plant leads to its accumulation in

regulated by CO2 irrespective of the nitrate source [50]. CCP1 and envelope membranes [5]. More recently, we showed, by western

CCP2 are two chloroplast envelope proteins, members of a peroxi- blot analysis, that PHT4;1 cannot be detected in the stroma-

somal, mitochondrial and plastid metabolite carrier protein family. lamellae nor in the grana fraction of the thylakoids [6]. These

Both CCP1 and CCP2 are strongly up-regulated by growth in low complementary observations are thus consistent with a chloroplast

CO2 [51]. However RNAi knockdown lines of both CCP1 and CCP2 envelope localization of this transporter. Functional characteriza-

grew slower but showed no carbon concentrating defect [52]. In tion of plant affected in the expression of PHT4;1 have suggested

Chlamydomonas, the RHP1 gene encodes Rhesus proteins similar to that this transporter may be involved in Pi reallocation that could

those in the human red blood membrane. RHP1 protein is predicted generate a signal to regulated SA (salicylic acid)-mediated plant

to be localized into the chloroplast envelope and is up-regulated defense [70]. Transiently expressed GFP fusions have revealed

in high CO2. It functions as a bi-directional CO2 gas channel and that two other members of the PHT4 family, PHT4;2 (At2g38060)

therefore has been hypothesized to provide CO2 for photosynthe- and PHT4;5 (At5g20380), are also localized in the chloroplast

sis in the absence of a CCM (i.e. when algae are grown under high [68]. PHT4;2 was further shown to catalyze Na -dependent Pi

CO2) [53]. All above mentioned candidate transporters have been transport in root plastids and is not expressed in chloroplasts

identiﬁed using DNA microarray proﬁling approaches that have [71].

90 G. Finazzi et al. / Cell Calcium 58 (2015) 86–97

2.3. Other cation channels/transporters (At1g01790), KEA2 (At4g00630) and KEA3 (At4g04850) belong to

the K -efﬂux antiporters (KEA). KEA1 and KEA2 are targeted to

The chloroplast has an essential requirement for calcium (Ca) the chloroplast envelope and KEA3 to the thylakoid membrane

ions since Ca modulates metabolic reactions of the chloroplast (con- [5,81]. Proteomic analysis have shown that KEA3 is associated

centration ∼5–10 mM in chloroplasts [9,19], Fig. 2). For example, with the stroma-lamellae sub-compartment [6]. KEA1 and KEA2

+ +

elevated concentration of Ca inhibits enzymes of the Calvin- could release K from the chloroplast in exchange for H inﬂux to

Benson-Bassham cycle (CBB cycle; reductive pentose phosphate avoid osmotic swelling of the organelle, and KEA3 could import K

cycle) (see Section 4), Ca is an essential cofactor of the OEC, it into thylakoid lumen [81]. The two-pore potassium channel TPK3

seems to inﬂuence chloroplast division or protein import into (At4g18160) was shown to be localized in the thylakoid stroma-

the chloroplast, etc (for a recent review see [72]). Several trans- lamellae and involved in K export from the lumen [82]. The activity

porters/channels are hypothesized to be involved in Ca ﬂuxes of this channel is Ca dependent and increased upon acidiﬁcation

however none of them have been fully characterized and trans- suggesting that this channel may participate in the calcium medi-

porters involved in chloroplast Ca homeostasis will need further ated chloroplast signalling response (see Section 4). There are new

investigation. The Ca -ATPase ACA1 (At1g27770) was originally increasing evidences that these transporters are essential for pho-

associated to plastid envelope but its Ca -stimulated ATPase activ- tosynthetic activity (see Section 3.2). CHX23 (At1g05580) belongs

ity could not be detected in puriﬁed envelope fractions [73] (Fig. 1). to the cation/H exchanger (CHX) family and was ﬁrst thought to be

+ +

Furthermore, proteomic analyses revealed that ACA1 might be an a chloroplast envelope K /H antiporter [83]. However, this protein

ER or plasma membrane protein [74,75], and this ACA1 protein was then found to be targeted to the endoplasmic reticulum and

was never detected in chloroplast membranes using proteomic to be preferentially expressed in pollen [84,85]. This transporter

approaches [3–5]. Moreno and co-workers [16] suggested that the has never been detected in proteomic approaches targeted to the

PIB-ATPase, AtHMA1 could be involved in Ca uptake into plastids. chloroplast [3–6].

However their conclusions only rely on experiments carried out in The pH gradient established over the envelope membrane is

yeast expressing the precursor form of AtHMA1 (i.e. still contain- thought to be created by a still unidentiﬁed proton (H ) P-type

ing its chloroplast transit peptide), a form which was shown to be ATPase [86,87]. A scheme for concentrating dissolved inorganic

inactive in this system [12,17]. Recently, a member of the AtGLR carbon by unicellular green algae utilizes a vanadate-sensitive

subgroup 3, AtGLR3.4 (At1g05200) was shown to be present in the transporter at the chloroplast envelope for the CO2 pump. In this

Arabidopsis chloroplast in addition to the plasma membrane [76], model, CO2 would freely diffuse through the envelope membrane

−

and could play an important role in the Ca-ﬂuxes [77]. However and then be converted into HCO3 due to the more alcaline pH of

further characterization of this protein is needed to determine if the stroma (when cells are exposed to light) when compared to the

AtGLR3.4 catalyzes the ﬂux of Ca (and divalent cations) or if it can pH of the cytosol [88]. However, to date, such an ATPase was not

also participate in monovalent cations ﬂuxes. Finally, several men- detected at the chloroplast envelope using proteomics [5] and the

bers of the Ca -permeable mechano sensitive channels like (MSL) pH gradient established over the envelope membrane might only

family are found in the chloroplast envelope [5]. MSL2 (At5g10490) result from H pumping into the thylakoids during the light phase

and MSL3 (At1g58200) participate in maintaining the shape and of photosynthesis. In the thylakoid stroma-lamellae, the F-ATPase,

size of the plastid by altering ion ﬂux in response to changes in CF0F1, contains multiple subunits arranged in a hydrophilic struc-

membrane tension [78] and both channels are also required to pro- ture (F1) and a hydrophobic structure (F0) forming the machinery

tect plastids from hypo-osmotic stress during normal plant growth transducing H across the membrane. This latter process drives ATP

[79]. MSL2 and MSL3 could serve as organellar osmotic release synthesis from ADP and Pi in the F1 moiety.

valves, mediating the ﬂux of osmolytes out of the plastid stroma Overall, ions play essential roles in chloroplast metabolism and

in response to increased membrane tension under hypo osmotic development. However, due to the difﬁculties encountered when

swelling. trying to analyze these difﬁcult membrane proteins, it appears that

Potassium (K ) is the most abundant cation found in plant and the identiﬁcation and characterization of chloroplast ions chan-

plays key roles in osmoregulation, enzyme activation, setting of nels/transporters has been (and is still) a real challenge. While

the membrane potential along with the proton motive force, etc. ionic ﬂuxes have been physiologically measured across the chloro-

Although K is an essential nutrient (concentration ∼120–200 mM plast membranes for decades, most of the transporters allowing

in chloroplasts [31,32]), sodium (Na ) is toxic for plant growth these ﬂuxes have only been identiﬁed and characterized during

and development (concentration ∼7–20 mM in chloroplasts [31]), the last ten years (see a summary in Table 1), and many remain

+ +

and the [K ]/[Na ] ratio often determines plant growth rate. Sev- to be identiﬁed. Subcellular and suplastidial proteomic approaches

+ + +

eral physiological studies have reported the presence of K , K /H have provided new candidates (based on their primary sequence

ﬂuxes across chloroplast membranes, but the proteins involved in analysis) that will need now further functional characterizations

these transport activities remained largely unknown. Recently, a to validate their role in chloroplast ion homeostasis. Alternatively,

+ + + +

K channel and K –Na /H antiporters have been identiﬁed and gene co-expression analyses (for a recent review see [91]) appear to

characterized in the chloroplast membranes. NHD1 (At3g19490), a be a useful tool to identify and predict the function of new chloro-

member of the sodium hydrogen antiporter (NHAD)-type carriers, plast ions transporters.

is localized in the chloroplast envelope and was shown to function

+ +

as a Na /H antiport [80] (Fig. 1). It was proposed to play an impor-

tant role in protecting vital chloroplast reaction like photosynthesis 3. Ions and regulation of photosynthesis

from toxic Na levels. In a recent proteomic study, NDH1 was also

detected in the thylakoid stroma-lamellae fraction [6], suggesting 3.1. Ions and thylakoid membrane stacking

a possible dual localization. However, localization in thylakoid sub-

compartment must be conﬁrmed by Western-blot analysis and the The ion chloroplast content deeply affects fundamental

physiological function associated with this localization remains to properties of the chloroplast. Membrane stacking, chlorophyll

be determined. NDH1 was also found associated to the plasma ﬂuorescence yield and electron transport properties are highly sen-

membrane proteome [75]. Nevertheless the characterization of sitive to the ion composition and concentrations in the stroma and

plant nhd1 mutant suggests that this ﬁnding was probably due lumen compartments. As already shown in the 60s [92], isolated

to a contamination of the PM by chloroplast membranes. KEA1 thylakoids resuspended in a low salt medium lose their stacking

G. Finazzi et al. / Cell Calcium 58 (2015) 86–97 91

Table 1

Chloroplast channels/transporters from Arabidopsis.

Protein name/AGI Protein family Localization (approaches) Substrate speciﬁcity (approaches) Function

2+ +

HMA1/At4g37270 PIB-type ATPases Envelope (Fluo & WB [12], Prot. - Uptake Cu or Cu (yeast expression, Metal homeostasis

[3]) plant mutant) [12] Oxidative stress

2+ 2+ 2+

- Uptake Zn /Ca /Co (yeast expression) [16]

- Export Zn (yeast expression, plant

mutant) [17]

HMA6/At4g33520 PIB-type ATPases Envelope (Fluo [11], Prot. [5]) Uptake Cu (plant mutant [11], yeast Cu homeostasis

expression & biochemical characterization Photosynthesis [14])

HMA8/At5g21930 PIB-type ATPases Thylakoids/Stroma-lamellae Uptake Cu (plant mutant [13]) Cu homeostasis

(Fluo [13], Prot. [6]) Photosynthesis

MRS2-11/At5g22830 CorA Envelope (Fluo [23], Prot. [5]) Uptake Mg (yeast complementation, Mg homeostasis

plant overexpressor) [23] Photosynthesis

PIC1/At2g15290 PIC permease Envelope (Fluo &WB [25], Prot. Uptake Fe (yeast complementation, plant Fe homeostasis

[26]) mutant, transport uptake in yeast) [25] Photosynthesis

NiCo/At4g35080 Ni–Co transporters Envelope (Fluo [26]) Uptake Fe (prediction) FeS cluster biogenesis?

In complex with PIC1 [26]

MAR1/At5g26820 IREG/Ferroportin Envelope (Fluo [27]) Uptake Fe or Fe-NA (plant mutant) [27] Fe homeostasis

efﬂux transporter

NAP14/At5g14100 Non-intrinsic ABC Envelope (Fluo [28], Prot. [5]) Transport Fe and/or other metals (plant Fe/metal homeostasis

transporters mutant) [28]

YSL4/At5g41000 YSL transporters Envelope (Fluo, WB [29]) Export Fe (plant mutant) [29] Fe

2+ 2+ 2+

YSL6/At3g27020 Vacuole (RE?) (Fluo [30] Prot. Import Fe , Ni , Mn (plant mutant) [30] homeostasis-detoxiﬁcation

[89]) Metal stress response

− −

CLCe/AT4G35440 CLC channels Thylakoid (Fluo, WB [33]) Cl , NO3 ? (plant mutant [33]) Photosynthesis

−

CsNitr1-L/At1g68570 POT transporters Envelope (Fluo, WB [37]) NO3 uptake (plant mutant, yeast Ammonium assimilation

expression [37])

2−

SULTR3;1/At3g51895 SULTR3 Envelope (WB, import assay SO4 uptake (plant mutant, uptake assays Sulfate assimilation transporters [39]) [39])

+ −

PHT2;1/At3g26570 PHT2 transporters Envelope (Fluo [65], Prot. [5])H/Pi importer (yeast complementation, Pi (HPO4 )

plant mutant [65]) homeostasis/allocation

PHT4;4/At4g00370 PHT4 transporters Envelope (Fluo [3–68], WB H /Pi importer (yeast complementation n.d.

[67], Prot. [5]) [68]) or Na /Pi importer (yeast

complementation, import assay

[unpublished data])

+ −

PHT4;1/At2g29650 PHT4 transporters Envelope (Fluo [3,68], WB & H /Pi importer (yeast complementation Pi (HPO4 ) homeostasis/Pi

Prot. [5]) [68]) or Na /Pi importer (expression in E. reallocation

Thylakoid (WB [69]) coli, uptake assays [69])

PHT4;5/At5g20380 PHT4 transporters Envelope (Fluo [68]) H /Pi importer (yeast complementation n.d. [68])

ACA1/At1g27770 P2-Ca ATPases Envelope (WB [73]) n.d. n.d.

Plasma membrane (Prot. [74,75])

GLR3.4/At1g05200 GLR subgroup 3 Envelope and PM (Fluo, WB Ca ? Other cations? n.d. [76])

+ 2+ + −

MSL2/At5g10490 MSL Envelope (Fluo, WB [78], Prot. Efﬂux of osmolytes (Na , Ca , H , Cl ?) Response to osmotic stress

[5]) (plant mutant) [78,79]

+ 2+ + −

MSL3/At1g58200 MSL Envelope (Fluo, WB [78], Prot. Efﬂux of osmolytes (Na , Ca , H , Cl ?) Response to osmotic stress

[5]) (plant mutant) [78,79]

NHD1/At3g19490 NHAD-type Envelope (Fluo) [80] Export Na (plant mutant, E. coli Protecting vital chloroplast

carriers Thylakoids/Stroma-lamellae complementation) [80] function from toxic Na

(Prot.) [6] level

CF0 ATPase/AtCg00130 P3-ATPases Thylakoid/Stroma-lamellae H efﬂux from the lumen [90] ATP supply

(Prot. [6])

KEA1/At1g01790 KEA Envelope (Fluo, WB [81], Prot. K export (plant mutants) [81] Chloroplast

[5]) osmoregulation and pH regulation

KEA2/At4g00630 KEA Envelope (Fluo, WB [81], Prot. K export (plant mutants) [81] Chloroplast

[5]) osmoregulation and pH regulation

KEA3/At4g04850 KEA Thylakoid/Stroma-lamellae K import into lumen (plant mutant) [81] Regulation of the proton

(Fluo, WB [81], Prot. [6]) motrice force

TPK3/AT4G18160 TPK family Thylakoid/Stroma-lamellae K efﬂux from the lumen (plant mutant) Regulation of the proton

(Fluo, WB) [82] [82] motrice force

KEA: K -efﬂux antiporters; NHAD: sodium-hydrogen antiporter; GLR: glutamate receptor like; POT: proton-dependent oligopeptide transporter; CLC: chloride channel;

2+ 2+ 2+ 2+ 2+

YSL: Yellow Stripe Like; IREG: iron-regulated transporters; PIC: permease in chloroplasts; Ni-Co: Ni –Co transporters; NA: nicotianamine; CorA: Ni , Co , and Mg

+ + 2− + 2− + + 2− 2+

transporters; TPK: Tandem-Pore K Channel. SULTR3: H /SO4 co-transporters; PHT2: H /HPO4 co-transporters; PHT4: H –Na /HPO4 co-transporters; MSL: Ca -

permeable mechanosensitive channel like. Fluo: transient or stable expression of protein fused to ﬂuorescent protein or ﬂuorophores; WB: western blot; Prot: proteomic

analysis. n.d.: not determined.

and hardly hold together. Addition of salt restored their normal fea- of negative charges on the thylakoid surface. The screening mod-

tures. Analysis of the effects generated by cations having different ulates coulombic repulsion between surfaces possibly leading to

valences, has led Barber et al. [93] to propose that the mecha- conformational changes both in and between membranes [94]. In

nism controlling this phenomenon involves electrostatic screening principle, the nature of the cation could speciﬁcally affect thylakoid

92 G. Finazzi et al. / Cell Calcium 58 (2015) 86–97

stacking, by speciﬁcally modulating the surface charge density carriers. Thus, optimization of the linear and cyclic pathway

though binding or protonation of speciﬁc residues [95]. This effect requires them to be physically separated from each other and the

could be particularly evident under low salt conditions, when there presence of the grana and stroma lamellae likely provides a phys-

is a substantial negative surface potential, and the local concentra- ical platform to optimize their activity. The cyclic pathway would

tion of cations, including H , will be high and charge neutralization operate predominantly in stroma lamellae far away from the grana,

possible, as shown by theoretical studies [96]. On the other hand, with b6f located in the stroma lamellae. Ferredoxin-NADP reductase

experimental data suggest that the cation-induced phenomena (FNR) could play a key role in discriminating between the linear and

showed little or no speciﬁcity within the same valence group. cyclic routes, by binding to PSI for linear electron transport and to

Thus, the dominant mechanism controlling thylakoid stacking and b6f for cyclic electron transport. In the stacks, the binding of FNR to

chlorophyll ﬂuorescence changes under these experimental con- b6f is unlikely because of steric hindrance [106].

ditions is the electrostatic screening mechanism [94] between Overall, it appears that ion homeostasis in the stroma, achieved

negatively charged residues exposed to the stroma, and the PSII via the active ion ﬂow catalyzed by the transport systems described

antenna LHCII, generally considered as the main protein respon- above, plays a major role in the regulation of the so called ‘light

sible for the electrostatic interaction [97]. Consistent with this, phase’ of photosynthesis, by controlling light harvesting and elec-

Chlb-deﬁcient chloroplast mutants are largely unable to produce tron ﬂow through the segregation of the different photosynthetic

normal amounts of membrane stacks [98]. Note that while the role complexes. However, ions also affect the ‘dark phase’ of photosyn-

of van der Waals attractive forces [99] and the cation-mediated thesis. Indeed the proton pumping into the thylakoid lumen during

electrostatic interaction between proteins in opposing membranes photosynthesis (see Section 3.2) is accompanied by a release of

are recognized, the ﬁnal shape of the grana also depends on the Mg , the major counter-ion, from the thylakoid membrane into the

type and percentages of lipids [100] and on the effect of speciﬁc stroma (e.g. [107] for a review). This brings about a 1–3 mM increase

proteins [101]. of the stromal Mg concentration, which stimulates the activ-

One of the main consequences of the cation mediated stack- ity of several enzymes that depend on Mg for optimal function

ing of the thylakoid membranes is the physical separation of PSI [20,21]. Changes in the Mg-ion concentration represent therefore

and PSII. In photosynthetic eukaryotes belonging to the green lin- a paradigm example of how photosynthetic activity is regulated

eage (plants mosses and green algae), the photosynthetic thylakoid in a concerted manner by ions. Indeed, increased [Mg ] improves

membranes form a physically continuous three-dimensional net- the generation of ATP and NADPH (by controlling thylakoid stack-

work that encloses a single aqueous space, the thylakoid lumen. ing) and enhances the utilization of these molecules by speeding

Because of membrane stacking, the thylakoid membranes of vas- up enzymatic activities responsible for their consumption during

cular plants mainly consist of two main domains: the grana, CO2 assimilation.

which are stacks of thylakoids, and the stroma lamellae, which are

unstacked thylakoids and connect the grana stacks (Fig. 2). Steric 3.2. Protons and the generation of a transthylakoid proton

hindrance prevents the very bulky PSI and ATP synthase complexes motive force

to move into stacked PSII enriched membranes. Therefore a het-

erogeneous distribution of the photosynthetic membranes exists Oxidation of plastoquinol (PQH2) at the b6f is thought to be the

between the grana (enriched in PSII and LHCII) and the stroma limiting step of electron transfer, at least in isolated thylakoids

lamellae (enriched in PSI, LHCI and ATPase). This spatial segregation [108]. This process is pH-dependent, owing to the release of two

has profound consequences on both the light harvesting and the protons into the lumen per each PQH2 oxidized, and turnover rates

electron ﬂow capacity of the photosynthetic machinery. First, by are found to decrease by 10-fold as pH is lowered from 7.5 to 5.5 in

imposing physical separation of the antenna systems of PSI and PSII, isolated thylakoid membranes (see e.g. [109]). Similarly, this activ-

spatial segregation induced by thylakoid stacking allows avoiding ity is strongly pH dependent in vivo in microalgae, such as Chlorella

useless energy ﬂow from PSII to PSI (energy spillover). This phe- sorokiniana, though in this case, the pH-dependence of the turnover

nomenon would otherwise occur because in PSI the energy levels of the b6f was shifted to more acidic values [110]. This indicates that,

of the reaction centre is lower and the kinetics of the trapping of in vitro and in vivo, there is a lumen pH ‘set point’ for inhibition of

excitation energy is much faster than in PSII [102]. Without a phys- electron ﬂow. However, this point is likely to be different due to

ical barrier, energy absorbed by PSII would spontaneously ﬂow to a different ionic environment experienced in vivo and in vitro. In

PSI, and because CO2 assimilation requires the in-series activity isolated thylakoids, the bulk of available data indicates that lumen

of PSII and PSI, this phenomenon would signiﬁcantly decrease the pH controls photosynthetic electron transfer primarily by gover-

overall yield of photosynthesis. Second, by imposing the existence ning the rate of plastoquinol oxidation at the b6f [111]. In contrast

of different antenna systems for the two photosystems, lateral seg- to results in vitro, several authors have demonstrated that the half-

regation of PSII and PSI imposes the existence of other mechanisms time for b6f turnover (measured as the post illumination half-time

to ﬁne-regulate the light need of photosynthesis. This is typically for P700 reduction or directly by cytochrome f turnover) remains

exempliﬁed by the existence of state transitions, i.e. a reversible rapid in intact leaves, or even increases slightly, when illumina-

physical migration of LHCII between PSII and PSI (when PSII is tion is increased towards and above saturation [112–114]. This

over-excited), which is triggered by its phosphorylation by a redox suggests that the luminal pH is probably not reaching very low val-

sensitive kinase [103]. ues during steady state photosynthesis. According to Kramer et al.

Another major consequence of the physical separation of PSII [115], this value can be evinced based on the pH dependence of the

and PSI is the ﬁne-tuning of the balance between linear (LEF) and different components of the photosynthetic machinery. Enzymes

cyclic (CEF) electron transport (see, e.g. [104]). In LEF, electrons are are generally adapted to the pH range where they normally oper-

transferred from water to NADP via PSII, the b6f and PSI, generate. The OEC of PSII was inactivated at pH below about 6, where

ating a proton gradient which contributes to the formation of ATP Ca dissociates from the OEC [115,116]. In addition, lowering the

(see Section 3.2). In CEF, electrons generated at the acceptor side pH from 7 to 4.5 dramatically increased the sensitivity of PSII to

of PSI are recycled to its donor side in a process involving the b6f photo-damage, again by affecting the turnover of the OEC [117].

and a series of ancillary proteins and complexes (PGR5, PGRL1, This has led Kramer and colleagues [118] to propose that the lumen

NDH [105]). CEF generates ATP without accumulating reducing pH remains moderately acidic (perhaps above about 5.5) during

equivalents. Photosynthesis requires both activities, which act in normal photosynthesis (see however [110,119–121] for a differ-

competition because they share a certain number of electrons ent conclusion). To combine the requirement for a relatively high

G. Finazzi et al. / Cell Calcium 58 (2015) 86–97 93

Fig. 3. Photosynthesis, ions and the proton motive force. Electron transfer along the photosynthetic electron transport (blue lines) chain leads to the formation of NADPH, to

+ +

the generation of an electric ﬁeld () and of a proton gradient (pH = [H ]s − [H ]l) across the thylakoid membranes. Both contribute to the onset of a proton motive force

(pmf), which is used by the ATP synthase to produce ATP and ultimately to ﬁx CO2. While both components of the pmf contribute to ATP synthesis, the sole proton gradient

regulates electron ﬂow (red lines), and induces thermal dissipation in the PSII antenna (NPQ, green lines). This requires the activation of the xanthophyll cycle enzymes (VDE)

and possible conformational changes in the small subunit PsbS (S). NPQ prevents photodamage under high light or CO2 limitation. When too large, the pH inhibits the

activity of the b6f and of PSII, thereby limiting photosynthesis. Therefore the pmf must be properly partitioned between the pH and components. Recent data indicate

+ +

that TPK3, the two-pore potassium channel identiﬁed in the thylakoids, and the H /K exchanger KEA3 may help regulating the partitioning of the pmf between the and

pH, while keeping the pmf constant.

proton motive force for ATP synthesis with the requirement for a pmf across the thylakoid membrane (which equivalent to a pH

+ + +

moderate lumen pH, one has to consider that both components of 2.5 [110]) could drive K /H antiport mediating K uptake into

of the pmf (the proton gradient, pH, and the electric ﬁeld, ) the thylakoid lumen. Consistent with this, recent data suggest a

are equally necessary for ATP synthesis [108]. However, the pH possible role for this protein in modulating the pmf in the light.

speciﬁcally regulates the photosynthetic control and NPQ, by mod- Arabidopsis plants lacking KEA3 show a modiﬁed pmf in the light

ulating conformational changes of regulatory proteins (PsbS) in the [81] and, although their phenotype is different from the one seen

PSII antenna [122] and the rate of electron ﬂow in the b6f [110]. in tpk3 mutants, these results point towards a modiﬁed capacity

Thus, the relative size of the pH must be regulated in response to to build a pmf in the light upon removal of this exchanger. In par-

environmental stimuli to allow proper photoprotection, ATP syn- ticular, the was doubled in the tpk3 mutant [82], while it was

thesis and avoid photodamage to the electron ﬂow machinery. The decreased by 20% in the kea3 mutant [81].

mechanism for such regulation has remained elusive for rather long Besides the regulation of the luminal pH, transporters and/or

time, although early data have suggested that this could be done antiporters could also modulate the transthylakoid pmf by a differ-

through ion counterbalancing by either Cl or K channels [35,123]. ent mechanism. In the light, a pH gradient is established across the

These channels could modify the relative contribution of the pH chloroplast envelope membrane not only by H translocation into

and to the pmf, while maintaining its absolute value unchanged the lumen, but also by H -ATPases to ensure a pH value of ∼8 in

(Fig. 3). the stroma [125]. This stromal pH 8 ensures proper photosynthesis

Very recently, we were able to show that TPK3, a potassium by modulating the activity of the CBB cycle enzyme [126]. This pH

+ 2+

channel from A. thaliana [82] and its cynobacterial counterpart gradient is also used for H -coupled Fe uptake into chloroplasts

synK [124] actively modulate the composition of the chloroplast by an unknown transporter or Na release by the NHD1 transporter

pmf through ion counterbalancing. In plants, TPK3 is found in the [127,128]. The pH regulation in the stroma could involve the activ-

thylakoid stromal-lamellae and Arabidopsis plants silenced for the ity of other members of the KEA family, KEA1 and KEA2 [81], by a

TPK3 gene display reduced growth and altered thylakoid mem- similar mechanism as the one invoked for KEA3. Indeed, kea1kea2

brane organization. This phenotype reﬂects an impaired capacity to mutants caused downstream effects leading to a decreased pH

generate a normal pmf, resulting in reduced CO2 assimilation and across the thylakoid membrane.

deﬁcient non-photochemical dissipation of excess absorbed light. Overall it appears that activity of pumping H -ATPase and

Thus, the TPK3 channel manages the pmf necessary to convert pho- chloroplast channel in the envelope membrane and thylakoids is an

tochemical energy into physiological functions. The involvement of essential mechanism to control generation of the pmf and to alle-

a K channel in ion counterbalancing requires an additional mech- viate osmotic constraints during photosynthesis, as required for

anism for K equilibrate concentration, possibly in the light, but proper ATP synthesis, functioning of the electron ﬂow chain and

certainly in the dark period of photosynthesis, when the K gra- photoprotection. The idea that the activity of ion channels controls

dient has to recover to the situation preceding illumination. The the composition of the pmf in the chloroplast and mitochondria

chloroplast KEA3 exchanger is a likely candidate. Indeed, the steep was already conceived by the chemiosmotic theory (see e.g. review

94 G. Finazzi et al. / Cell Calcium 58 (2015) 86–97

[129]). However, experimental support for this notion has only be and showed diminished activity and recovery of PSII after pro-

achieved very recently, thereby opening the excited ﬁeld of the longed exposure to high light. Addition of the CaM antagonist W7

investigation of molecular mechanisms regulating ion homeostasis or the G-protein activator mastoparan in WT cells also impaired

and photosynthetic efﬁciency. the induction of LHCSR3 expression, strongly suggesting that over-

all CAS and Ca are critically involved in the regulation of the HL

response. [149]. The impact of the antagonist W7 on LHCSR3 expres-

4. A focus on Ca sion was recently conﬁrmed on the transcriptional level [150].

Down-regulation of CAS also decreased the capacity of the

Ca is a universal second messenger in all eukaryotic organisms chloroplast to use alternative electron sinks (e.g. CEF [151]) to

[130,131], and it functions in mediating a number of external and generate ATP for carbon assimilation. Interestingly, both CEF and

internal stimuli. In terms of signal recognition, Ca-based signal NPQ deﬁciencies can be rescued by elevated Ca concentrations in

transduction is known to be important for sensing environmental the growth medium [147,149]. Recently, Wang and co-workers

signals. In response to various stimuli, cellular free calcium concen- screened a library of 20,000 insertional Chlamydomonas mutants

trations are increased by means of extracellular and intracellular and discovered that a knock-out of CAS could not grow at ambient

stores, thereby allowing a temporary and spatially control of cel- CO2 indicating a new role of CAS in CO2 concentrating mechanism.

lular processes. Several studies have ﬁrst shown that this ion is Complementation of this mutant with hemagglutinin HA epitope-

essential for proper photosynthesis and later identiﬁed a wider role tagged CAS however did not rescue the high CO2 phenotype of this

of calcium in the regulation of this organelle [72,132]. Ca directly mutant [152].

affects H2O oxidation to O2 by PSII being a component of a cluster In higher plants, CAS was found to be phosphorylated in a

of three inorganic ions – Mn, Cl, and Ca – which directly performs light-dependent manner, strongly depending on the activity of the

this catalytic activity [133]. Although the presence of Ca in PSII has light-dependent STN8 kinase [153]. The phosphorylation site was

been conﬁrmed by structural studies [134], it is still not very well mapped to the stromal domain of CAS [153]. Interestingly, regula-

understood how Ca is delivered into the PSII complex. Inactivation tion of CEF in plants also requires the phosphorylation of speciﬁc

of oxygen evolution by acidiﬁcation of PSII appears to be due to a proteins (PGRL1) by STN8 [154], and impaired phosphorylation

reversible release of Ca and the recovery of that inactivation can be results in a transient decrease of the cyclic ﬂow activity in vivo.

suppressed by Ca channel blockers [116]. This suggests the existence of an interplay between Ca transi-

Recent proteomic studies have shown that the ATP-dependent ents, STN8 kinase-dependent phosphorylation and CEF in plants

peptidases VAR1/FTSH5 and VAR2/FTSH2 are targets of Ca- and algae. Together with the Ca mediated regulation of the pmf

dependent phosphorylation [135]. These thylakoid-localized pro- via TPK3 (see Section 2.3), these data strongly suggest that, in

teases play a role in the turnover of photodamaged subunits of plants, this ion is one of the master regulators of the photosynthetic

PSII [136], however neither the phosphorylation mechanism nor efﬁciency.

the speciﬁc function of Ca-dependent phosphorylation is known.

A further connection between redox and Ca regulation in chloro-

5. Conclusion

plasts is taking place at the level of PSI. Indeed the ultimate electron

acceptor of this complex, NADP, is synthesized from NAD by the

Chloroplasts contain several important membranes, vital for

NAD kinase (NADK) enzyme, the ﬁrst calmodulin (CaM) regulated

their function. Like mitochondria, chloroplasts have a double-

enzyme ever identiﬁed in plants [137]. Since then, more than 200

membrane envelope, but unlike mitochondria, chloroplasts also

putative CaM-binding partners were identiﬁed in the chloroplast

have internal membrane structures called thylakoids. While the

sub-compartments [138]. These proteins are involved in the main

outer envelope membrane is permeable to most ions and metabo-

chloroplast functions that could thus be regulated by Ca ﬂuxes or

lites, the inner membrane as well as the thylakoids are highly

oscillations.

specialized with transport proteins, to regulate ion homeosta-

The CBB cycle is the metabolic pathway that connects pho-

sis and ﬂuxes (for optimum photosynthetic function), as well as

tosynthetic energy production to the conversion of atmospheric

metabolic activities and storage/signalling. While the mains rules

CO2 into organic compounds. Two enzymes of this cycle are regu-

governing ion consequences on membrane stacking and ATP syn-

lated by Ca : the enzyme fructose-1,6-bisphosphatase (FBPase),

thesis have been established several years ago, it is only recently

is regulated at the level of enzyme activation and catalysis.

that, thanks to an integrated cell biology approach, we have started

Sedoheptulose-l,7-biphosphatase (SBPase) is also modulated by Ca,

discovering the molecular actors responsible for ion and metabo-

both in terms of activation as well as catalytic inhibition [139,140].

lite ﬂuxes, and the speciﬁc consequences of these processes on

Eventually, Ca plays a more general role via its effect on the

chloroplast behaviour. Coupling transcriptomic, proteomic, and

so-called calcium-sensing protein (CAS), a protein that binds cal-

metabolomic studies with a detailed analysis of the functional

cium with low afﬁnity but high capacity [141]. Initially, CAS was

properties of the photosynthetic apparatus represents one of the

reported to be localized in the plasma membrane, where it medi-

new frontiers in chloroplast research. These studies will set the

ates extracellular Ca sensing in guard cells [139]. It was later also

bases for a dynamic characterization of photosynthetic cells in an

identiﬁed as a thylakoid membrane protein and is now believed to

ever-changing environment, and highlight the extreme ﬂexibility

have an exclusive thylakoid localization [142–145]. It is well estab-

of this fascinating molecular machine.

lished that Ca inﬂuxes from extra- and intracellular stores lead to an

increase in free cytosolic Ca resulting in stomatal closure [146], and

Conﬂict of interest

CAS is believed to be a key regulator of this process [143,147]. CAS

has been suggested to regulate stomatal closure through hydrogen

The authors wish to conﬁrm that there are no known conﬂicts

peroxide and nitric oxide elevation in guard cells that trigger fur-

of interest.

ther Ca transients resulting ﬁnally in stomatal closure [148]. The

presence of CAS in unicellular algae strongly supports a function

beyond the regulation of stomatal opening. Indeed, photoacclima- Acknowledgements

tion and photosynthesis in C. reinhardtii are also regulated by CAS.

Knock-down lines of CAS could not properly induce the expression This study received ﬁnancial support from the French National

of LHCSR3 protein that is crucial for non-photochemical quenching Research Agency (ANR-10-LABEX-04 GRAL Labex, Grenoble

G. Finazzi et al. / Cell Calcium 58 (2015) 86–97 95

Alliance for Integrated Structural Cell Biology and ANR-2010- [28] E. Shimoni-Shor, M. Hassidim, N. Yuval-Naeh, N. Keren, Disruption of Nap14,

a plastid-localized non-intrinsic ABC protein in Arabidopsis thaliana results

GENOM-BTV-002-01 Chloro-Types), from an INRA BAP Department

in the over-accumulation of transition metals and in aberrant chloroplast

grant (project “Mixoalgues”) and the Region Rhone Alpes (Cible

structures, Plant Cell Environ. 33 (2010) 1029–1038.

project, “Elici-TAG-Screening”). Funds from the Marie Curie Initial [29] F. Divol, D. Couch, G. Conéjéro, H. Roschzttardtz, S. Mari, C. Curie, The Ara-

bidopsis Yellow Stripe Like4 and 6 transporters control iron release from the

Training Network Accliphot (FP7-PEOPLE-2012-ITN; 316427) are

chloroplast, Plant Cell 25 (2013) 1040–1055.

also acknowledged.

[30] S.S. Conte, H.H. Chu, D.C. Rodriguez, et al., Arabidopsis thaliana yellow stripe1-

Like4 and Yellow stripe1-like6 localize to internal cellular membranes and are

involved in metal ion homeostasis, Front. Plant Sci. 26 (2013) 283.

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