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Cell Calcium 58 (2015) 86–97

Contents lists available at ScienceDirect

Cell Calcium

jou rn al hom epage: www.elsev ier .com/ locate /ceca

eview

ons channels/transporters and chloroplast regulation

iovanni Finazzia,b,c,d,∗, Dimitris Petroutsosa,b,c,d, Martino Tomizioli a,b,c,d,erena Flori a,b,c,d, Emeline Sautrona,b,c,d, Valeria Villanovae, Norbert Rollanda,b,c,d,aphné Seigneurin-Bernya,b,c,d,∗∗

CNRS, Laboratoire de Physiologie Cellulaire & Végétale, UMR 5168, 17 rue des Martyrs, F-38054 Grenoble, FranceUniv. Grenoble Alpes, LPCV, F-38054 Grenoble, FranceCEA, DSV, iRTSV, LPCV, F-38054 Grenoble, FranceINRA, LPCV, USC1359, 17 rue des Martyrs, F-38054 Grenoble, FranceFermentalg SA, 4 bis rue Rivière, F-33500 Libourne, France

r t i c l e i n f o

rticle history:eceived 15 July 2014eceived in revised form 1 October 2014ccepted 4 October 2014vailable online 13 October 2014

a b s t r a c t

Ions play fundamental roles in all living cells and their gradients are often essential to fuel transports,to regulate enzyme activities and to transduce energy within and between cells. Their homeostasis istherefore an essential component of the cell metabolism. Ions must be imported from the extracellularmatrix to their final subcellular compartments. Among them, the chloroplast is a particularly interestingexample because there, ions not only modulate enzyme activities, but also mediate ATP synthesis and

eywords:ons traffickinghloroplast envelopehylakoids

actively participate in the building of the photosynthetic structures by promoting membrane-membraneinteraction. In this review, we first provide a comprehensive view of the different machineries involvedin ion trafficking and homeostasis in the chloroplast, and then discuss peculiar functions exerted by ionsin the frame of photochemical conversion of absorbed light energy.

hotosynthesisroton motive force

. Introduction

Ions play key roles in all living organisms being involved inll metabolic and cellular functions. Therefore, ions are foundn all subcellular compartments and have to be imported fromhe extracellular matrix to their final localization within cells.n Arabidopsis, acquisition and translocation of ions within plantrgans, cells and subcellular compartments involve large fam-lies of ionic transporters with various substrate specificities,xpression patterns, and subcellular localization. These familiesere classified into three major categories: channels/porins, pri-ary transporters/pumps and secondary transporters (according

o the Transport Classification system [1]). Channels transportolutes down their concentration gradient without consuming

nergy and display the fastest transport rates among transporters.rimary transporters (e.g. ATPases) directly use energy to trans-ort molecules across membranes. Secondary transporters (also

∗ Corresponding authors at: LPCV, iRTSV, CEA Grenoble, 17 rue des Martyrs, F-8054 Grenoble, France. Tel.: +33 438784184.∗∗ Corresponding authors at: LPCV, iRTSV, CEA Grenoble, 17 rue des Martyrs, F-8054 Grenoble, France. Tel.: +33 438782363.

E-mail addresses: giovanni.finazzi@cea.fr (G. Finazzi), daphne.berny@cea.frD. Seigneurin-Berny).

ttp://dx.doi.org/10.1016/j.ceca.2014.10.002143-4160/© 2014 Elsevier Ltd. All rights reserved.

© 2014 Elsevier Ltd. All rights reserved.

named electrochemically-driven transporters) use the concentra-tion gradient of co-transported molecules and therefore includeantiporters and symporters.

Transmembrane proteins have an essential role in the regula-tion of ions homeostasis and in biological functions. In chloroplasts,a large variety of ions are found, at micro to millimolar concen-trations. Since metal ions (zinc -Zn-, copper -Cu-, iron -Fe-, etc)can be toxic for the cell, they are not found as free ions, butare always chelated by proteins or biomolecules. Conversely, ionslike potassium (K), magnesium (Mg), can be free and reach mil-limolar concentrations. In the last 40 years, the existence of ionicfluxes across the chloroplast envelope or the thylakoid membraneshas been mostly deduced from physiological measurements orfrom knowledge of the chloroplast metabolism. However, most ofthe proteins responsible for these fluxes have not been identifiedyet. Indeed, their low abundance, their localization in intracellularmembranes, their hydrophobicity and the difficulties encoun-tered when trying to produce them in heterologous systems havestrongly limited their identification and functional characterizationusing classical approaches. Several chloroplast transporters havebeen identified in the last years, thanks to proteomic approaches

targeted to the chloroplast and its sub-compartments (e.g. [2–6]),and to reverse genetic studies. Nonetheless, controversies stillexist about the sub-plastidial localization or function of some ofthese transporters, and others are still missing. Again, proteomics

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.T 4–YSLv

dsnncbrcsit

dmacp

2c

2

tolm

ransporters whose chloroplast localization is controversial are noted in italic (YSLalidated are noted “?”. The regulation of TPK3 by Ca2+ is highlighted.

ata have provided a list of unknown proteins that, based onequence similarities, could be involved in ion transport. However,o functional characterization exist in most cases. Moreover, whileot identified through large-scale approaches, members of well-haracterized ion transporters families have also been predicted toe localized in the chloroplast using bioinformatics tools (for recenteviews on chloroplast ion transporters see [7,8]). Overall, whenonsidering the known, hypothetical and missing transporters, acenario emerges where a plethora of transporters is involved inon fluxes across the membranes to facilitate exchanges betweenhe cytosol, the stroma and the thylakoid lumen (Fig. 1).

In the first part of this review, we will provide a comprehensiveescription of transporters identified in Arabidopsis chloroplastembranes, including those that are still only incompletely char-

cterized. In the second part, we will discuss the implication of ionsoncentration and fluxes on the optimization of the photosyntheticrocess.

. A global overview of chloroplast ionhannels/transporters

.1. Transporters involved in metal homeostasis

Metal ions are essential cofactors for numerous chloroplast pro-

eins involved in photosynthesis (Cu, Mg, manganese -Mn-, Fe),xidative stress detoxification (Cu, Zn, and Fe), nutrient assimi-ation (Fe), biosynthesis of aminoacids (e.g. Zn for cysteine and

ethionine), etc. Cu (estimated chloroplast concentration ∼60 �M,

6 and ACA1). Substrates, protein or chloroplast localization that need to be further

Fig. 2 [9]) plays a key role in the photosynthetic process and canexist under a reduced (Cu+) or oxidized form (Cu2+). In its Cu2+ form,it constitutes the redox cofactor of plastocyanin (PC), a proteinrequired for electron transport from the cytochrome b6f complex(b6f) to photosystem I (PSI) [10]. Cu is also required for the activityof the Cu/Zn superoxide dismutase (Cu/Zn-SOD), a soluble enzymethat scavenges reactive oxygen species produced by photosynthe-sis under stress conditions, which is found in eucaryotes and someprocaryotes. In Arabidopsis, Cu delivery to chloroplasts and thy-lakoids requires three PIB-type ATPases: AtHMA1, AtHMA6 andAtHMA8 (Fig. 1). AtHMA1 (At4g37270) and AtHMA6 (At4g33520)are localized in the chloroplast envelope [5,11,12] and AtHMA8(At5g21930) in the stroma-lamellae, i.e. non-appressed fractions,of the thylakoid membranes [6]. Genetic approaches have shownthat both AtHMA6 and AtHMA8 are Cu transporters, AtHMA6 beingthe main route of Cu supply to the Cu/Zn SOD and to the thylakoidtransporter AtHMA8, as required for PC biosynthesis [11,13]. Morerecent biochemical studies have demonstrated that AtHMA6 is ahigh affinity Cu+ transporter [14] while the biochemical propertiesof AtHMA8 still remain unknown. AtHMA1, the second envelopetransporter, provides an additional way to import Cu into thechloroplast, which would provide Cu to the Cu/Zn SOD, and which isessential under light stress conditions [12,15]. AtHMA1 could alsotransport other metal/divalent ions like Zn, cobalt (Co), calcium (Ca)[16] and was also proposed to be involved in Zn or Cu/Zn export

from Arabidopsis and Barley chloroplasts [17,18]. The ionic speci-ficity of AtHMA1 is still controversial, strongly suggesting that thisprotein could transport a broad range of divalent cations, probablydepending on the physiological conditions. As for AtHMA8, there

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

Mg2+

~ 5 mM

Stroma

Cytosol

Ca2+

~ 5-10 mM

Fe2+

~ 150 µMMn2+

~ 30 µM

Cu+/2+

~ 60 µMZn2+

~ 130 µM

Lumen

Fd

PSIb6f

ATPas e

PC

ATPADP + Pi

CAS FTSH

PSII

Lumen

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

it

[cccbCHaoi

eoptspcbitifics(stttuIpi(ohw

omplex; Fe: [4Fe–4S] and [2Fe–2S] clusters and non heme Fe of photosynthetic

tacking; Zn: metalloproteases; Ca: calcium sensing, OEC. Estimated ion concentraa.

s a strong need for direct biochemical characterization of AtHMA1o define its definite role in chloroplast metal homeostasis.

In chloroplast, Mg (estimated chloroplast concentration ∼5 mM19], Fig. 2) plays an essential role in photosynthesis, being theoordinating ion in the chlorophyll molecule. Appropriate Mg con-entrations are also required for the function or activation of somehloroplast enzymes [20,21]. The AtMRS2-11 (At5g22830) protein,elonging to the CorA super-family (transporters for both Mg ando, for review see [22]), may transport Mg into the stroma (Fig. 1).owever, its function has been deduced from complementationssays of yeast mutants and no phenotype is associated with itsverexpression in planta [23]. Therefore, its role in Mg homeostasis

n planta needs further experimental support.Similar to Cu, Fe is a redox active metal ion that is able to

xist as Fe2+ and Fe3+ under physiological conditions. Around 80%f the cellular Fe is found in the chloroplast (estimated chloro-last concentration ∼130 �M [9], Fig. 2). Fe is used as a cofactor inhree groups of Fe-containing proteins: proteins containing iron-ulfur (FeS) clusters, hemes, and other Fe-containing proteins. Felays major functions in photosynthesis (being present in all theomplexes involved in photosynthetic electron flow), chlorophylliosynthesis, and other essential metabolic processes that occur

n chloroplasts [24]. Recent studies have identified several pro-eins, localized in the chloroplast envelope, which may play a rolen Fe import into the chloroplast. PIC1 (At2g15290), a permeaserst identified as a component of the protein import machinery,ould be involved in Fe import into the chloroplast and Fe homeo-tasis [25]. PIC was reported to interact with the NiCo proteinAt4g35080), a member of the Ni2+–Co2+ transporters family. It wasuggested that PIC1 and NiCo might function together in plastid Feransport and/or that this complex could interact with the proteinranslocon machinery to deliver Fe for FeS-cluster biogenesis, andherefore promote the assembly of FeS clusters into new proteinspon translocation [26]. MAR1 (At5g26820), a close homolog of the

REG/Ferroportin efflux transporters, was also proposed to trans-ort Fe or Fe-chelating polyamine such as nicotianamine (Fe-NA)

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

At5g14100) was found to be another candidate for the transportf Fe into the chloroplast or for the regulation of Fe or other metalsomeostasis [28]. If NAP14 functions as a metal transport system, itould require a transmembrane channel counterpart (“?” in Fig. 1)

exes (PSI, PSII, b6f); Cu: plastocyanin (PC); Mg: chlorophyll, thylakoid membraneere determined from [9] for Cu, Zn, Fe, Mn; from [19] for Mg; and from [9,19] for

since NAP14 is a non-intrinsic protein. Recently, two membersof the Arabidopsis family of YSL transporters, YSL4 (At5g41000)and YSL6 (At3g27020), were proposed to participate to the con-trol of Fe homeostasis in the chloroplast by mediating its releasefrom chloroplasts and preventing its accumulation into chloro-plasts during de-differentiation [29]. However, their localizationand ion specificity is controversial. Indeed, Conte and coworkers[30] found that both transporters are associated with the vacuolarmembranes and ER, and propose therefore that these transportersshould be involved in the supply of Mn and nickel to proteinslocated in internal cellular compartments.

Mn is a redox-active transition metal, which is required for plantgrowth and can exist in several oxidized states, Mn2+, Mn3+, Mn4+,Mn6+, Mn7+. In chloroplasts, Mn (estimated concentration ∼30 �M[9], Fig. 2) is mainly required to form the Mn-cluster in photosystemII (PSII), an essential component of the oxygen evolving complex(OEC) that catalyzes water oxidation and oxygen production in oxy-genic photosynthesis. Until now, however, no Mn transporter hasbeen identified in the chloroplast membranes.

Zn (∼130 �M [9], Fig. 2) is a non-redox metal, playing importantrole as a catalytic and structural element in several chloro-plast enzymes including carbonic anhydrase, metalloproteases,methionine synthase and others. To date, while its role remainscontroversial in planta, several studies have demonstrated thatAtHMA1 could be involved in the control of Zn homeostasis inchloroplast [12,16,17].

2.2. Anion transporters

Plants cells contain several main anions such as nitrate/nitrite,sulfate/sulfite, chloride (Cl), bicarbonate (HCO3

−) and phosphate.Nitrite, sulfate, and phosphate are metabolized in the chloroplastwhere they are involved in processes like ammonium assimila-tion (nitrite), assimilation of sulfur for the biosynthesis of cysteineand methionine (sulfate) and ATP synthesis (phosphate). HCO3

−

and Cl also have key role in carbon assimilation and photosynthe-sis.

Cl concentration in chloroplasts was estimated around 1–50 mM[31,32]. A member of the Cl channel (CLCs) family, CLCe(At4g35440) is found in thylakoid membranes [33]. CLCs genesencode for Cl channel/transporter and also nitrate/H+ antiporter

Calciu

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wscpmRcutoptSicr

fhvCmpoain(scdcap(rprCsBCgCttitsCi

G. Finazzi et al. / Cell

34]. CLCe could thus correspond to the Cl channel responsible foron compensation during the generation of the proton motive forcecross the thylakoid membrane [35] (see Section 3.2). However, theon selectivity of CLCe remains to be elucidated. In spinach chloro-last envelope, anion-selective channels have also been detectedy physiological measurements without identification of the cor-esponding proteins [36].

Nitrate is reduced in the cytosol into nitrite that is then trans-erred to the chloroplast (chloroplast concentration ∼10 mM [31])here it is reduced to ammonium and assimilated into glutamate.

he transporter CsNitr1-L (At1g68570), a member of the proton-ependent oligopeptide transporter family (POT) was shown to be

ocalized in the chloroplast envelope and to load cytosolic nitritento the chloroplast during nitrate assimilation [37].

Sulfate is the major form of inorganic sulfur utilized by plantshere chloroplasts are the main site of reduction of sulfate into

ulfur before its assimilation (concentration ∼10 mM SO42− in

hloroplast [31]). Mourioux and coworkers [38] have detected theresence of an SO4

2−/HPO4− antiporter by physiological measure-

ents but the corresponding transporter remains unidentified.ecently, the SULTR3;1 protein (At3g51895) was identified in thehloroplast envelope and demonstrated to contribute to the sulfateptake into chloroplast [39] (Fig. 1). This transport is pH dependent,hus confirming previous studies that have measured the presencef a proton/sulfate co-transporter for uptake into the chloro-last [40]. Data from Cao and coworkers [39] also suggest thathree other members of the SULTR3 family (SULTR3;2/At4g02700,ULTR3;3/At1g23090, and SULTR3;4/At3g15990) may also benvolved in sulfate uptake into the chloroplast. However, thehloroplast envelope localization of these three transportersemains to be established.

To overcome CO2 limitation, caused by a 104 slower rate of dif-usion of CO2 in water relative to air, cyanobacteria and microalgaeave developed a CO2 concentrating mechanism that leads to ele-ated intracellular inorganic carbon (Ci) increasing the apparentO2/O2 specificity for Rubisco, enhancing photosynthetic perfor-ance and decreasing the carbon flux into the photorespiratory

athway [41]. Thirty years after the discovery and characterizationf the carbonic anhydrase CAH1 [42], genome-wide transcriptomicpproaches using the model algae Chlamydomonas reinhardtii havedentified many genes that respond to CO2 limitation, includingine carbonic anhydrases and several candidate Ci transportersreviewed in [41,43,44]). Chloroplasts from C. reinhardtii have beenhown to transport both CO2 and HCO3

− [45]. The product of thehloroplast gene YCF10, was the first identified Ci transport candi-ate. Disruption of this gene resulted in diminished Ci uptake in thehloroplasts [46]. The YCF10 protein was discovered in pea plantsnd originally named CemA [47], while its cyanobacterial orthologxcA (CotA) has a role in light-induced proton extrusion [48]. LCIANAR1;2) a putative chloroplast envelope protein with a suggestedole in nitrite transport into the chloroplasts [49] has been pro-osed as a candidate Ci transporter because LCIA expression isegulated by CO2 irrespective of the nitrate source [50]. CCP1 andCP2 are two chloroplast envelope proteins, members of a peroxi-omal, mitochondrial and plastid metabolite carrier protein family.oth CCP1 and CCP2 are strongly up-regulated by growth in lowO2 [51]. However RNAi knockdown lines of both CCP1 and CCP2rew slower but showed no carbon concentrating defect [52]. Inhlamydomonas, the RHP1 gene encodes Rhesus proteins similar tohose in the human red blood membrane. RHP1 protein is predictedo be localized into the chloroplast envelope and is up-regulatedn high CO2. It functions as a bi-directional CO2 gas channel and

herefore has been hypothesized to provide CO2 for photosynthe-is in the absence of a CCM (i.e. when algae are grown under highO2) [53]. All above mentioned candidate transporters have been

dentified using DNA microarray profiling approaches that have

m 58 (2015) 86–97 89

indicated around 100 genes responsive to low CO2 [50,54,55]. DeepRNA sequencing approaches [56,57] have extended the list of CO2-related differentially expressed genes to several thousands andhave revealed possible undiscovered Ci transporters. Similar inputis expected from high-throughput genotyping approaches [58]. Thecarbon concentrating system does not seem to exist in C3 plantchloroplasts, however the Arabidopsis thaliana genome containsnineteen carbonic anhydrases localized in the plasma membrane,cytosol, chloroplast and mitochondria [59]. It has been proposedthat mitochondrial CAs reduce leakage of CO2 from plant cellsand allow efficient recycling of mitochondrial CO2 for carbon fixa-tion in chloroplasts [60]. In Nicotiana tabacum plants, an aquaporin(NtAQP1), that functions as a water channel in the plasma mem-brane and as a CO2 channel at the chloroplast envelope, suggests amechanism that plants may use to modify photosynthetic function[61]. In A. thaliana, carbonic anhydrases CA1 and CA4 have beenproposed to function early in CO2 signalling, acting as upstreamregulators of CO2-controlled stomata movement [62].

Inorganic phosphate (Pi/HPO42−) is essential for ATP synthe-

sis during the light phase of photosynthesis. In the chloroplastenvelope, several transporters have been identified that mediatethe 1:1 counter-exchange of Pi with phosphorylated organic com-pounds. However, these transporters do not allow a net import of Piinto the stroma (concentration ∼5–35 mM in chloroplast [63,64]).This net import can be achieved by transporters of the PHT2 andPHT4 families that mediate H+- and/or Na+-dependent Pi transport.These latter transporters belong to the ubiquitous Major Facili-tator Superfamily (MFS) of transporters. PHT2;1 (At3g26570) isa H+-dependent phosphate importer localized in the chloroplastenvelope [65,66] (Fig. 1). This transporter influences the allocationof Pi throughout the plant and affects the expression of Pi-starvation responses [65]. PHT4;4 (also named ANTR2/At4g00370)is mainly expressed in green tissues and associated with thechloroplast envelope [3,67,68]. Complementation of yeast mutantsunder limiting Pi condition and uptake assays using radiolabeled Pidemonstrated that all of the PHT4 proteins are capable of mediatingPi transport [68] with a low affinity for Pi. Guo and coworkers [68]suggested that protons could serve as a co-transported substrate forPHT4;4. However, using complementation of yeast mutant strainsand radiolabeled Pi import assays in Xenopus oocytes, we foundthat Na+ could be the co-transported ion (S. Miras and N. Rol-land, unpublished data). Similarly, the nature of the co-transportedion is still unclear in the case of PHT4;1/ANTR1 (At2g29650).Indeed, this protein has a Na+-dependent Pi transport activity whenexpressed in Escherichia coli [69] and an H+-dependent Pi trans-port activity when expressed in yeast [68]. Several studies havedemonstrated, using different approaches (GFP fusions, western-blot and proteomic analyses), that this transporter is localizedin the chloroplast envelope [3,5,68]. This localization is contro-versial since Pavon and co-worker [69] suggested that PHT4;1is a thylakoid protein. However, we have previously shown thatoverexpression of PHT4;1 in plant leads to its accumulation inenvelope membranes [5]. More recently, we showed, by westernblot analysis, that PHT4;1 cannot be detected in the stroma-lamellae nor in the grana fraction of the thylakoids [6]. Thesecomplementary observations are thus consistent with a chloroplastenvelope localization of this transporter. Functional characteriza-tion of plant affected in the expression of PHT4;1 have suggestedthat this transporter may be involved in Pi reallocation that couldgenerate a signal to regulated SA (salicylic acid)-mediated plantdefense [70]. Transiently expressed GFP fusions have revealedthat two other members of the PHT4 family, PHT4;2 (At2g38060)

and PHT4;5 (At5g20380), are also localized in the chloroplast[68]. PHT4;2 was further shown to catalyze Na+-dependent Pitransport in root plastids and is not expressed in chloroplasts[71].

9 Calciu

2

iceBcstphpiaiFEwaPHyiisAafAabfasmt[vis

ptAiaaefltKcmiatfdacpbmpt

0 G. Finazzi et al. / Cell

.3. Other cation channels/transporters

The chloroplast has an essential requirement for calcium (Ca)ons since Ca modulates metabolic reactions of the chloroplast (con-entration ∼5–10 mM in chloroplasts [9,19], Fig. 2). For example,levated concentration of Ca inhibits enzymes of the Calvin-enson-Bassham cycle (CBB cycle; reductive pentose phosphateycle) (see Section 4), Ca is an essential cofactor of the OEC, iteems to influence chloroplast division or protein import intohe chloroplast, etc (for a recent review see [72]). Several trans-orters/channels are hypothesized to be involved in Ca fluxesowever none of them have been fully characterized and trans-orters involved in chloroplast Ca homeostasis will need further

nvestigation. The Ca2+-ATPase ACA1 (At1g27770) was originallyssociated to plastid envelope but its Ca2+-stimulated ATPase activ-ty could not be detected in purified envelope fractions [73] (Fig. 1).urthermore, proteomic analyses revealed that ACA1 might be anR or plasma membrane protein [74,75], and this ACA1 proteinas never detected in chloroplast membranes using proteomic

pproaches [3–5]. Moreno and co-workers [16] suggested that theIB-ATPase, AtHMA1 could be involved in Ca uptake into plastids.owever their conclusions only rely on experiments carried out ineast expressing the precursor form of AtHMA1 (i.e. still contain-ng its chloroplast transit peptide), a form which was shown to benactive in this system [12,17]. Recently, a member of the AtGLRubgroup 3, AtGLR3.4 (At1g05200) was shown to be present in therabidopsis chloroplast in addition to the plasma membrane [76],nd could play an important role in the Ca-fluxes [77]. Howeverurther characterization of this protein is needed to determine iftGLR3.4 catalyzes the flux of Ca (and divalent cations) or if it canlso participate in monovalent cations fluxes. Finally, several men-ers of the Ca2+-permeable mechano sensitive channels like (MSL)amily are found in the chloroplast envelope [5]. MSL2 (At5g10490)nd MSL3 (At1g58200) participate in maintaining the shape andize of the plastid by altering ion flux in response to changes inembrane tension [78] and both channels are also required to pro-

ect plastids from hypo-osmotic stress during normal plant growth79]. MSL2 and MSL3 could serve as organellar osmotic releasealves, mediating the flux of osmolytes out of the plastid stroman response to increased membrane tension under hypo osmoticwelling.

Potassium (K+) is the most abundant cation found in plant andlays key roles in osmoregulation, enzyme activation, setting ofhe membrane potential along with the proton motive force, etc.lthough K+ is an essential nutrient (concentration ∼120–200 mM

n chloroplasts [31,32]), sodium (Na+) is toxic for plant growthnd development (concentration ∼7–20 mM in chloroplasts [31]),nd the [K+]/[Na+] ratio often determines plant growth rate. Sev-ral physiological studies have reported the presence of K+, K+/H+

uxes across chloroplast membranes, but the proteins involved inhese transport activities remained largely unknown. Recently, a+ channel and K+–Na+/H+ antiporters have been identified andharacterized in the chloroplast membranes. NHD1 (At3g19490), aember of the sodium hydrogen antiporter (NHAD)-type carriers,

s localized in the chloroplast envelope and was shown to functions a Na+/H+ antiport [80] (Fig. 1). It was proposed to play an impor-ant role in protecting vital chloroplast reaction like photosynthesisrom toxic Na+ levels. In a recent proteomic study, NDH1 was alsoetected in the thylakoid stroma-lamellae fraction [6], suggesting

possible dual localization. However, localization in thylakoid sub-ompartment must be confirmed by Western-blot analysis and thehysiological function associated with this localization remains to

e determined. NDH1 was also found associated to the plasmaembrane proteome [75]. Nevertheless the characterization of

lant nhd1 mutant suggests that this finding was probably dueo a contamination of the PM by chloroplast membranes. KEA1

m 58 (2015) 86–97

(At1g01790), KEA2 (At4g00630) and KEA3 (At4g04850) belong tothe K+-efflux antiporters (KEA). KEA1 and KEA2 are targeted tothe chloroplast envelope and KEA3 to the thylakoid membrane[5,81]. Proteomic analysis have shown that KEA3 is associatedwith the stroma-lamellae sub-compartment [6]. KEA1 and KEA2could release K+ from the chloroplast in exchange for H+ influx toavoid osmotic swelling of the organelle, and KEA3 could import K+

into thylakoid lumen [81]. The two-pore potassium channel TPK3(At4g18160) was shown to be localized in the thylakoid stroma-lamellae and involved in K+ export from the lumen [82]. The activityof this channel is Ca2+ dependent and increased upon acidificationsuggesting that this channel may participate in the calcium medi-ated chloroplast signalling response (see Section 4). There are newincreasing evidences that these transporters are essential for pho-tosynthetic activity (see Section 3.2). CHX23 (At1g05580) belongsto the cation/H+ exchanger (CHX) family and was first thought to bea chloroplast envelope K+/H+ antiporter [83]. However, this proteinwas then found to be targeted to the endoplasmic reticulum andto be preferentially expressed in pollen [84,85]. This transporterhas never been detected in proteomic approaches targeted to thechloroplast [3–6].

The pH gradient established over the envelope membrane isthought to be created by a still unidentified proton (H+) P-typeATPase [86,87]. A scheme for concentrating dissolved inorganiccarbon by unicellular green algae utilizes a vanadate-sensitivetransporter at the chloroplast envelope for the CO2 pump. In thismodel, CO2 would freely diffuse through the envelope membraneand then be converted into HCO3

− due to the more alcaline pH ofthe stroma (when cells are exposed to light) when compared to thepH of the cytosol [88]. However, to date, such an ATPase was notdetected at the chloroplast envelope using proteomics [5] and thepH gradient established over the envelope membrane might onlyresult from H+ pumping into the thylakoids during the light phaseof photosynthesis. In the thylakoid stroma-lamellae, the F-ATPase,CF0F1, contains multiple subunits arranged in a hydrophilic struc-ture (F1) and a hydrophobic structure (F0) forming the machinerytransducing H+ across the membrane. This latter process drives ATPsynthesis from ADP and Pi in the F1 moiety.

Overall, ions play essential roles in chloroplast metabolism anddevelopment. However, due to the difficulties encountered whentrying to analyze these difficult membrane proteins, it appears thatthe identification and characterization of chloroplast ions chan-nels/transporters has been (and is still) a real challenge. Whileionic fluxes have been physiologically measured across the chloro-plast membranes for decades, most of the transporters allowingthese fluxes have only been identified and characterized duringthe last ten years (see a summary in Table 1), and many remainto be identified. Subcellular and suplastidial proteomic approacheshave provided new candidates (based on their primary sequenceanalysis) that will need now further functional characterizationsto validate their role in chloroplast ion homeostasis. Alternatively,gene co-expression analyses (for a recent review see [91]) appear tobe a useful tool to identify and predict the function of new chloro-plast ions transporters.

3. Ions and regulation of photosynthesis

3.1. Ions and thylakoid membrane stacking

The ion chloroplast content deeply affects fundamentalproperties of the chloroplast. Membrane stacking, chlorophyll

fluorescence yield and electron transport properties are highly sen-sitive to the ion composition and concentrations in the stroma andlumen compartments. As already shown in the 60s [92], isolatedthylakoids resuspended in a low salt medium lose their stacking

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

Table 1Chloroplast channels/transporters from Arabidopsis.

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

HMA1/At4g37270 PIB-type ATPases Envelope (Fluo & WB [12], Prot.[3])

- Uptake Cu2+or Cu+ (yeast expression,plant mutant) [12]- Uptake Zn2+/Ca2+/Co2+ (yeast expression)[16]- Export Zn2+ (yeast expression, plantmutant) [17]

Metal homeostasisOxidative stress

HMA6/At4g33520 PIB-type ATPases Envelope (Fluo [11], Prot. [5]) Uptake Cu+ (plant mutant [11], yeastexpression & biochemical characterization[14])

Cu homeostasisPhotosynthesis

HMA8/At5g21930 PIB-type ATPases Thylakoids/Stroma-lamellae(Fluo [13], Prot. [6])

Uptake Cu+? (plant mutant [13]) Cu homeostasisPhotosynthesis

MRS2-11/At5g22830 CorA Envelope (Fluo [23], Prot. [5]) Uptake Mg2+ (yeast complementation,plant overexpressor) [23]

Mg homeostasisPhotosynthesis

PIC1/At2g15290 PIC permease Envelope (Fluo &WB [25], Prot.[26])

Uptake Fe2+ (yeast complementation, plantmutant, transport uptake in yeast) [25]

Fe homeostasisPhotosynthesis

NiCo/At4g35080 Ni–Co transporters Envelope (Fluo [26]) Uptake Fe2+ (prediction)In complex with PIC1 [26]

FeS cluster biogenesis?

MAR1/At5g26820 IREG/Ferroportinefflux transporter

Envelope (Fluo [27]) Uptake Fe2+ or Fe-NA (plant mutant) [27] Fe homeostasis

NAP14/At5g14100 Non-intrinsic ABCtransporters

Envelope (Fluo [28], Prot. [5]) Transport Fe2+ and/or other metals (plantmutant) [28]

Fe/metal homeostasis

YSL4/At5g41000YSL6/At3g27020

YSL transporters Envelope (Fluo, WB [29])Vacuole (RE?) (Fluo [30] Prot.[89])

Export Fe2+ (plant mutant) [29]Import Fe2+, Ni2+, Mn2+ (plant mutant) [30]

Fehomeostasis-detoxificationMetal 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

expression [37])Ammonium assimilation

SULTR3;1/At3g51895 SULTR3transporters

Envelope (WB, import assay[39])

SO42− uptake (plant mutant, uptake assays

[39])Sulfate assimilation

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

Pi (HPO4−)

homeostasis/allocationPHT4;4/At4g00370 PHT4 transporters Envelope (Fluo [3–68], WB

[67], Prot. [5])H+/Pi importer (yeast complementation[68]) or Na+/Pi importer (yeastcomplementation, import assay[unpublished data])

n.d.

PHT4;1/At2g29650 PHT4 transporters Envelope (Fluo [3,68], WB &Prot. [5])Thylakoid (WB [69])

H+/Pi importer (yeast complementation[68]) or Na+/Pi importer (expression in E.coli, uptake assays [69])

Pi (HPO4−) homeostasis/Pi

reallocation

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

n.d.

ACA1/At1g27770 P2-Ca2+ ATPases Envelope (WB [73])Plasma membrane (Prot.[74,75])

n.d. n.d.

GLR3.4/At1g05200 GLR subgroup 3 Envelope and PM (Fluo, WB[76])

Ca2+? Other cations? n.d.

MSL2/At5g10490 MSL Envelope (Fluo, WB [78], Prot.[5])

Efflux of osmolytes (Na+, Ca2+, H+, Cl−?)(plant mutant) [78,79]

Response to osmotic stress

MSL3/At1g58200 MSL Envelope (Fluo, WB [78], Prot.[5])

Efflux of osmolytes (Na+, Ca2+, H+, Cl−?)(plant mutant) [78,79]

Response to osmotic stress

NHD1/At3g19490 NHAD-typecarriers

Envelope (Fluo) [80]Thylakoids/Stroma-lamellae(Prot.) [6]

Export Na+ (plant mutant, E. colicomplementation) [80]

Protecting vital chloroplastfunction from toxic Na+

levelCF0 ATPase/AtCg00130 P3-ATPases Thylakoid/Stroma-lamellae

(Prot. [6])H+ efflux from the lumen [90] ATP supply

KEA1/At1g01790 KEA Envelope (Fluo, WB [81], Prot.[5])

K+ export (plant mutants) [81] Chloroplastosmoregulation and pHregulation

KEA2/At4g00630 KEA Envelope (Fluo, WB [81], Prot.[5])

K+ export (plant mutants) [81] Chloroplastosmoregulation and pHregulation

KEA3/At4g04850 KEA Thylakoid/Stroma-lamellae(Fluo, WB [81], Prot. [6])

K+ import into lumen (plant mutant) [81] Regulation of the protonmotrice force

TPK3/AT4G18160 TPK family Thylakoid/Stroma-lamellae(Fluo, WB) [82]

K+ efflux from the lumen (plant mutant)[82]

Regulation of the protonmotrice force

KEA: K+-efflux antiporters; NHAD: sodium-hydrogen antiporter; GLR: glutamate receptor like; POT: proton-dependent oligopeptide transporter; CLC: chloride channel;YSL: Yellow Stripe Like; IREG: iron-regulated transporters; PIC: permease in chloroplasts; Ni-Co: Ni2+–Co2+ transporters; NA: nicotianamine; CorA: Ni2+, Co2+, and Mg2+

t PHT2:p roteina

atvn

ransporters; TPK: Tandem-Pore K+ Channel. SULTR3: H+/SO42− co-transporters;

ermeable mechanosensitive channel like. Fluo: transient or stable expression of pnalysis. n.d.: not determined.

nd hardly hold together. Addition of salt restored their normal fea-ures. Analysis of the effects generated by cations having differentalences, has led Barber et al. [93] to propose that the mecha-ism controlling this phenomenon involves electrostatic screening

H+/HPO42− co-transporters; PHT4: H+–Na+/HPO4

2− co-transporters; MSL: Ca2+- fused to fluorescent protein or fluorophores; WB: western blot; Prot: proteomic

of negative charges on the thylakoid surface. The screening mod-ulates coulombic repulsion between surfaces possibly leading toconformational changes both in and between membranes [94]. Inprinciple, the nature of the cation could specifically affect thylakoid

9 Calciu

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2 G. Finazzi et al. / Cell

tacking, by specifically modulating the surface charge densityhough binding or protonation of specific residues [95]. This effectould be particularly evident under low salt conditions, when theres a substantial negative surface potential, and the local concentra-ion of cations, including H+, will be high and charge neutralizationossible, as shown by theoretical studies [96]. On the other hand,xperimental data suggest that the cation-induced phenomenahowed little or no specificity within the same valence group.hus, the dominant mechanism controlling thylakoid stacking andhlorophyll fluorescence changes under these experimental con-itions is the electrostatic screening mechanism [94] betweenegatively charged residues exposed to the stroma, and the PSIIntenna LHCII, generally considered as the main protein respon-ible for the electrostatic interaction [97]. Consistent with this,hlb-deficient chloroplast mutants are largely unable to produceormal amounts of membrane stacks [98]. Note that while the rolef van der Waals attractive forces [99] and the cation-mediatedlectrostatic interaction between proteins in opposing membranesre recognized, the final shape of the grana also depends on theype and percentages of lipids [100] and on the effect of specificroteins [101].

One of the main consequences of the cation mediated stack-ng of the thylakoid membranes is the physical separation of PSInd PSII. In photosynthetic eukaryotes belonging to the green lin-age (plants mosses and green algae), the photosynthetic thylakoidembranes form a physically continuous three-dimensional net-ork that encloses a single aqueous space, the thylakoid lumen.ecause of membrane stacking, the thylakoid membranes of vas-ular plants mainly consist of two main domains: the grana,hich are stacks of thylakoids, and the stroma lamellae, which arenstacked thylakoids and connect the grana stacks (Fig. 2). Stericindrance prevents the very bulky PSI and ATP synthase complexeso move into stacked PSII enriched membranes. Therefore a het-rogeneous distribution of the photosynthetic membranes existsetween the grana (enriched in PSII and LHCII) and the stroma

amellae (enriched in PSI, LHCI and ATPase). This spatial segregationas profound consequences on both the light harvesting and thelectron flow capacity of the photosynthetic machinery. First, bymposing physical separation of the antenna systems of PSI and PSII,patial segregation induced by thylakoid stacking allows avoidingseless energy flow from PSII to PSI (energy spillover). This phe-omenon would otherwise occur because in PSI the energy levelsf the reaction centre is lower and the kinetics of the trapping ofxcitation energy is much faster than in PSII [102]. Without a phys-cal barrier, energy absorbed by PSII would spontaneously flow toSI, and because CO2 assimilation requires the in-series activityf PSII and PSI, this phenomenon would significantly decrease theverall yield of photosynthesis. Second, by imposing the existencef different antenna systems for the two photosystems, lateral seg-egation of PSII and PSI imposes the existence of other mechanismso fine-regulate the light need of photosynthesis. This is typicallyxemplified by the existence of state transitions, i.e. a reversiblehysical migration of LHCII between PSII and PSI (when PSII isver-excited), which is triggered by its phosphorylation by a redoxensitive kinase [103].

Another major consequence of the physical separation of PSIInd PSI is the fine-tuning of the balance between linear (LEF) andyclic (CEF) electron transport (see, e.g. [104]). In LEF, electrons areransferred from water to NADP+ via PSII, the b6f and PSI, gener-ting a proton gradient which contributes to the formation of ATPsee Section 3.2). In CEF, electrons generated at the acceptor sidef PSI are recycled to its donor side in a process involving the b6f

nd a series of ancillary proteins and complexes (PGR5, PGRL1,DH [105]). CEF generates ATP without accumulating reducingquivalents. Photosynthesis requires both activities, which act inompetition because they share a certain number of electrons

m 58 (2015) 86–97

carriers. Thus, optimization of the linear and cyclic pathwayrequires them to be physically separated from each other and thepresence of the grana and stroma lamellae likely provides a phys-ical platform to optimize their activity. The cyclic pathway wouldoperate predominantly in stroma lamellae far away from the grana,with b6f located in the stroma lamellae. Ferredoxin-NADP reductase(FNR) could play a key role in discriminating between the linear andcyclic routes, by binding to PSI for linear electron transport and tob6f for cyclic electron transport. In the stacks, the binding of FNR tob6f is unlikely because of steric hindrance [106].

Overall, it appears that ion homeostasis in the stroma, achievedvia the active ion flow catalyzed by the transport systems describedabove, plays a major role in the regulation of the so called ‘lightphase’ of photosynthesis, by controlling light harvesting and elec-tron flow through the segregation of the different photosyntheticcomplexes. However, ions also affect the ‘dark phase’ of photosyn-thesis. Indeed the proton pumping into the thylakoid lumen duringphotosynthesis (see Section 3.2) is accompanied by a release ofMg2+, the major counter-ion, from the thylakoid membrane into thestroma (e.g. [107] for a review). This brings about a 1–3 mM increaseof the stromal Mg2+ concentration, which stimulates the activ-ity of several enzymes that depend on Mg2+ for optimal function[20,21]. Changes in the Mg-ion concentration represent thereforea paradigm example of how photosynthetic activity is regulatedin a concerted manner by ions. Indeed, increased [Mg2+] improvesthe generation of ATP and NADPH (by controlling thylakoid stack-ing) and enhances the utilization of these molecules by speedingup enzymatic activities responsible for their consumption duringCO2 assimilation.

3.2. Protons and the generation of a transthylakoid protonmotive force

Oxidation of plastoquinol (PQH2) at the b6f is thought to be thelimiting step of electron transfer, at least in isolated thylakoids[108]. This process is pH-dependent, owing to the release of twoprotons into the lumen per each PQH2 oxidized, and turnover ratesare found to decrease by 10-fold as pH is lowered from 7.5 to 5.5 inisolated thylakoid membranes (see e.g. [109]). Similarly, this activ-ity is strongly pH dependent in vivo in microalgae, such as Chlorellasorokiniana, though in this case, the pH-dependence of the turnoverof the b6f was shifted to more acidic values [110]. This indicates that,in vitro and in vivo, there is a lumen pH ‘set point’ for inhibition ofelectron flow. However, this point is likely to be different due toa different ionic environment experienced in vivo and in vitro. Inisolated thylakoids, the bulk of available data indicates that lumenpH controls photosynthetic electron transfer primarily by gover-ning the rate of plastoquinol oxidation at the b6f [111]. In contrastto results in vitro, several authors have demonstrated that the half-time for b6f turnover (measured as the post illumination half-timefor P700

+ reduction or directly by cytochrome f turnover) remainsrapid in intact leaves, or even increases slightly, when illumina-tion is increased towards and above saturation [112–114]. Thissuggests that the luminal pH is probably not reaching very low val-ues during steady state photosynthesis. According to Kramer et al.[115], this value can be evinced based on the pH dependence of thedifferent components of the photosynthetic machinery. Enzymesare generally adapted to the pH range where they normally oper-ate. The OEC of PSII was inactivated at pH below about 6, whereCa2+ dissociates from the OEC [115,116]. In addition, lowering thepH from 7 to 4.5 dramatically increased the sensitivity of PSII tophoto-damage, again by affecting the turnover of the OEC [117].

This has led Kramer and colleagues [118] to propose that the lumenpH remains moderately acidic (perhaps above about 5.5) duringnormal photosynthesis (see however [110,119–121] for a differ-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, tothe 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 gradientregulates 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 thea ust bet K+ exc�

pmoasuPTetmttTa(

csptTbgdTtaacdc

ctivity of the b6f and of PSII, thereby limiting photosynthesis. Therefore the pmf mhat TPK3, the two-pore potassium channel identified in the thylakoids, and the H+/

pH, while keeping the pmf constant.

roton motive force for ATP synthesis with the requirement for aoderate lumen pH, one has to consider that both components

f the pmf (the proton gradient, �pH, and the electric field, �� )re equally necessary for ATP synthesis [108]. However, the �pHpecifically regulates the photosynthetic control and NPQ, by mod-lating conformational changes of regulatory proteins (PsbS) in theSII antenna [122] and the rate of electron flow in the b6f [110].hus, the relative size of the �pH must be regulated in response tonvironmental stimuli to allow proper photoprotection, ATP syn-hesis and avoid photodamage to the electron flow machinery. The

echanism for such regulation has remained elusive for rather longime, although early data have suggested that this could be donehrough ion counterbalancing by either Cl or K channels [35,123].hese channels could modify the relative contribution of the �pHnd �� to the pmf, while maintaining its absolute value unchangedFig. 3).

Very recently, we were able to show that TPK3, a potassiumhannel from A. thaliana [82] and its cynobacterial counterpartynK [124] actively modulate the composition of the chloroplastmf through ion counterbalancing. In plants, TPK3 is found in thehylakoid stromal-lamellae and Arabidopsis plants silenced for thePK3 gene display reduced growth and altered thylakoid mem-rane organization. This phenotype reflects an impaired capacity toenerate a normal pmf, resulting in reduced CO2 assimilation andeficient non-photochemical dissipation of excess absorbed light.hus, the TPK3 channel manages the pmf necessary to convert pho-ochemical energy into physiological functions. The involvement of

K+ channel in ion counterbalancing requires an additional mech-

nism for K+ equilibrate concentration, possibly in the light, butertainly in the dark period of photosynthesis, when the K+ gra-ient has to recover to the situation preceding illumination. Thehloroplast KEA3 exchanger is a likely candidate. Indeed, the steep

properly partitioned between the �pH and �� components. Recent data indicatehanger KEA3 may help regulating the partitioning of the pmf between the �� and

pmf across the thylakoid membrane (which equivalent to a �pHof 2.5 [110]) could drive K+/H+ antiport mediating K+ uptake intothe thylakoid lumen. Consistent with this, recent data suggest apossible role for this protein in modulating the pmf in the light.Arabidopsis plants lacking KEA3 show a modified pmf in the light[81] and, although their phenotype is different from the one seenin tpk3 mutants, these results point towards a modified capacityto build a pmf in the light upon removal of this exchanger. In par-ticular, the �� was doubled in the tpk3 mutant [82], while it wasdecreased by 20% in the kea3 mutant [81].

Besides the regulation of the luminal pH, transporters and/orantiporters could also modulate the transthylakoid pmf by a differ-ent mechanism. In the light, a pH gradient is established across thechloroplast envelope membrane not only by H+ translocation intothe lumen, but also by H+-ATPases to ensure a pH value of ∼8 inthe stroma [125]. This stromal pH 8 ensures proper photosynthesisby modulating the activity of the CBB cycle enzyme [126]. This pHgradient is also used for H+-coupled Fe2+ uptake into chloroplastsby an unknown transporter or Na+ release by the NHD1 transporter[127,128]. The pH regulation in the stroma could involve the activ-ity of other members of the KEA family, KEA1 and KEA2 [81], by asimilar mechanism as the one invoked for KEA3. Indeed, kea1kea2mutants caused downstream effects leading to a decreased �pHacross the thylakoid membrane.

Overall it appears that activity of pumping H+-ATPase andchloroplast channel in the envelope membrane and thylakoids is anessential mechanism to control generation of the pmf and to alle-viate osmotic constraints during photosynthesis, as required for

proper ATP synthesis, functioning of the electron flow chain andphotoprotection. The idea that the activity of ion channels controlsthe composition of the pmf in the chloroplast and mitochondriawas already conceived by the chemiosmotic theory (see e.g. review

9 Calciu

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pdtPtApaNepsco

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4 G. Finazzi et al. / Cell

129]). However, experimental support for this notion has only bechieved very recently, thereby opening the excited field of thenvestigation of molecular mechanisms regulating ion homeostasisnd photosynthetic efficiency.

. A focus on Ca

Ca is a universal second messenger in all eukaryotic organisms130,131], and it functions in mediating a number of external andnternal stimuli. In terms of signal recognition, Ca-based signalransduction is known to be important for sensing environmentalignals. In response to various stimuli, cellular free calcium concen-rations are increased by means of extracellular and intracellulartores, thereby allowing a temporary and spatially control of cel-ular processes. Several studies have first shown that this ion isssential for proper photosynthesis and later identified a wider rolef calcium in the regulation of this organelle [72,132]. Ca2+ directlyffects H2O oxidation to O2 by PSII being a component of a clusterf three inorganic ions – Mn, Cl, and Ca – which directly performshis catalytic activity [133]. Although the presence of Ca in PSII haseen confirmed by structural studies [134], it is still not very wellnderstood how Ca is delivered into the PSII complex. Inactivationf oxygen evolution by acidification of PSII appears to be due to aeversible release of Ca and the recovery of that inactivation can beuppressed by Ca channel blockers [116].

Recent proteomic studies have shown that the ATP-dependenteptidases VAR1/FTSH5 and VAR2/FTSH2 are targets of Ca-ependent phosphorylation [135]. These thylakoid-localized pro-eases play a role in the turnover of photodamaged subunits ofSII [136], however neither the phosphorylation mechanism norhe specific function of Ca-dependent phosphorylation is known.

further connection between redox and Ca regulation in chloro-lasts is taking place at the level of PSI. Indeed the ultimate electroncceptor of this complex, NADP, is synthesized from NAD by theAD kinase (NADK) enzyme, the first calmodulin (CaM) regulatednzyme ever identified in plants [137]. Since then, more than 200utative CaM-binding partners were identified in the chloroplastub-compartments [138]. These proteins are involved in the mainhloroplast functions that could thus be regulated by Ca fluxes orscillations.

The CBB cycle is the metabolic pathway that connects pho-osynthetic energy production to the conversion of atmosphericO2 into organic compounds. Two enzymes of this cycle are regu-

ated by Ca2+: the enzyme fructose-1,6-bisphosphatase (FBPase),s regulated at the level of enzyme activation and catalysis.edoheptulose-l,7-biphosphatase (SBPase) is also modulated by Ca,oth in terms of activation as well as catalytic inhibition [139,140].

Eventually, Ca plays a more general role via its effect on theo-called calcium-sensing protein (CAS), a protein that binds cal-ium with low affinity but high capacity [141]. Initially, CAS waseported to be localized in the plasma membrane, where it medi-tes extracellular Ca sensing in guard cells [139]. It was later alsodentified as a thylakoid membrane protein and is now believed toave an exclusive thylakoid localization [142–145]. It is well estab-

ished that Ca influxes from extra- and intracellular stores lead to anncrease in free cytosolic Ca resulting in stomatal closure [146], andAS is believed to be a key regulator of this process [143,147]. CASas been suggested to regulate stomatal closure through hydrogeneroxide and nitric oxide elevation in guard cells that trigger fur-her Ca transients resulting finally in stomatal closure [148]. Theresence of CAS in unicellular algae strongly supports a function

eyond the regulation of stomatal opening. Indeed, photoacclima-ion and photosynthesis in C. reinhardtii are also regulated by CAS.nock-down lines of CAS could not properly induce the expressionf LHCSR3 protein that is crucial for non-photochemical quenching

m 58 (2015) 86–97

and showed diminished activity and recovery of PSII after pro-longed exposure to high light. Addition of the CaM antagonist W7or the G-protein activator mastoparan in WT cells also impairedthe induction of LHCSR3 expression, strongly suggesting that over-all CAS and Ca are critically involved in the regulation of the HLresponse. [149]. The impact of the antagonist W7 on LHCSR3 expres-sion was recently confirmed on the transcriptional level [150].

Down-regulation of CAS also decreased the capacity of thechloroplast to use alternative electron sinks (e.g. CEF [151]) togenerate ATP for carbon assimilation. Interestingly, both CEF andNPQ deficiencies can be rescued by elevated Ca concentrations inthe growth medium [147,149]. Recently, Wang and co-workersscreened a library of 20,000 insertional Chlamydomonas mutantsand discovered that a knock-out of CAS could not grow at ambientCO2 indicating a new role of CAS in CO2 concentrating mechanism.Complementation of this mutant with hemagglutinin HA epitope-tagged CAS however did not rescue the high CO2 phenotype of thismutant [152].

In higher plants, CAS was found to be phosphorylated in alight-dependent manner, strongly depending on the activity of thelight-dependent STN8 kinase [153]. The phosphorylation site wasmapped to the stromal domain of CAS [153]. Interestingly, regula-tion of CEF in plants also requires the phosphorylation of specificproteins (PGRL1) by STN8 [154], and impaired phosphorylationresults in a transient decrease of the cyclic flow activity in vivo.This suggests the existence of an interplay between Ca transi-ents, STN8 kinase-dependent phosphorylation and CEF in plantsand algae. Together with the Ca2+ mediated regulation of the pmfvia TPK3 (see Section 2.3), these data strongly suggest that, inplants, this ion is one of the master regulators of the photosyntheticefficiency.

5. Conclusion

Chloroplasts contain several important membranes, vital fortheir function. Like mitochondria, chloroplasts have a double-membrane envelope, but unlike mitochondria, chloroplasts alsohave internal membrane structures called thylakoids. While theouter envelope membrane is permeable to most ions and metabo-lites, the inner membrane as well as the thylakoids are highlyspecialized with transport proteins, to regulate ion homeosta-sis and fluxes (for optimum photosynthetic function), as well asmetabolic activities and storage/signalling. While the mains rulesgoverning ion consequences on membrane stacking and ATP syn-thesis have been established several years ago, it is only recentlythat, thanks to an integrated cell biology approach, we have starteddiscovering the molecular actors responsible for ion and metabo-lite fluxes, and the specific consequences of these processes onchloroplast behaviour. Coupling transcriptomic, proteomic, andmetabolomic studies with a detailed analysis of the functionalproperties of the photosynthetic apparatus represents one of thenew frontiers in chloroplast research. These studies will set thebases for a dynamic characterization of photosynthetic cells in anever-changing environment, and highlight the extreme flexibilityof this fascinating molecular machine.

Conflict of interest

The authors wish to confirm that there are no known conflictsof interest.

Acknowledgements

This study received financial support from the French NationalResearch Agency (ANR-10-LABEX-04 GRAL Labex, Grenoble

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lliance for Integrated Structural Cell Biology and ANR-2010-ENOM-BTV-002-01 Chloro-Types), from an INRA BAP Departmentrant (project “Mixoalgues”) and the Region Rhone Alpes (Cibleroject, “Elici-TAG-Screening”). Funds from the Marie Curie Initialraining Network Accliphot (FP7-PEOPLE-2012-ITN; 316427) arelso acknowledged.

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