Chlamydomonas reinhardtii cellular compartments and their contribution to intracellular calcium signalling.

Calcium (Ca2+)-dependent signalling plays a well-characterized role in the response to different environmental stimuli, in both plant and animal cells. In the model organism for green algae, Chlamydomonas reinhardtii, Ca2+ signals were reported to have a crucial role in different physiological processes, such as stress responses, photosynthesis, and flagella functions. Recent reports identified the underlying components of the Ca2+ signalling machinery at the level of specific subcellular compartments and reported in vivo imaging of cytosolic Ca2+ concentration in response to environmental stimuli. The characterization of these Ca2+-related mechanisms and proteins in C. reinhardtii is providing knowledge on how microalgae can perceive and respond to environmental stimuli, but also on how this Ca2+ signalling machinery has evolved. Here, we review current knowledge on the cellular mechanisms underlying the generation, shaping, and decoding of Ca2+ signals in C. reinhardtii, providing an overview of the known and possible molecular players involved in the Ca2+ signalling of its different subcellular compartments. The advanced toolkits recently developed to measure time-resolved Ca2+ signalling in living C. reinhardtii cells are also discussed, suggesting how they can improve the study of the role of Ca2+ signals in the cellular response of microalgae to environmental stimuli.

Introduction

From the early stages of life on Earth, unicellular organisms had to cope with the environment, and thus to control ion concentrations within the cell. Among them all, the regulation of cytosolic calcium ion concentration ([Ca2+]cyt) was an evolutionary necessity, forced by the cytotoxicity of this ion. A prolonged increase of calcium concentration in the cytoplasm ([Ca2+]cyt) above ~10–4 M causes aggregation of proteins and nucleic acids, and negatively influences the integrity of lipid membranes. Moreover, high [Ca2+]cyt leads to precipitation of phosphates, reducing the availability of this key precursor for nucleotide triphosphates, such as ATP, chosen early on by cells as the energy currency of life (Case ). Consequently, the first unicellular forms of life had developed effective transport systems to maintain Ca2+ homeostasis at far lower concentrations, ~100 nM, 10 000–20 000 times lower than the [Ca2+] in the extracellular space. Such a concentration gradient, initially designed for survival, together with the peculiar coordination chemistry of the Ca2+ ion, created a cellular environment for the evolution of a Ca2+ signalling system, which evolved through all the phylogenetic stages, being found ubiquitously from the most ancient prokaryote to the most specialized eukaryotic cell (Case ; Carafoli and Krebs, 2016). In the latter, the enormous electrochemical potential difference of Ca2+ across cellular membranes has been widely reported to allow rapid and transient increases of free [Ca2+]cyt, whose frequency (period), amplitude, and duration are determined by the acting calcium signalling toolkit (Bothwell and Ng, 2005; Verret ). Upon various stimulation events, the interplay between Ca2+ influx and efflux pathways rapidly changes, modulating [Ca2+]cyt and causing Ca2+ binding-induced conformational changes in a specific set of proteins, finally leading to a downstream activation of specific biological processes (McAinsh and Pittman, 2009; Edel ). In eukaryotes, Ca2+ signalling mechanisms have also evolved at the endomembrane level, exploiting intracellular organelles to allow better spatial and temporal regulation of free Ca2+ homeostasis in the cytosol. Many eukaryotic organelles have been shown to function as Ca2+ storage compartments, as in the case of the apoplast/cell wall, vacuole, and the endoplasmic reticulum (ER) of plant cells being important to ensure the transient nature of Ca2+ signals and fast response to environmental stimuli (Stael ). Chloroplasts, mitochondria, and the nucleus, in addition, can also generate intraorganellar Ca2+ signals, participating in a complex network of signal transduction pathways and influencing their own function (Stael ; Costa ). In plants, it was extensively demonstrated that most perceived stimuli can be associated with specific ‘Ca2+ signatures’, arising from variation of organellar and cytosolic [Ca2+], characterized by unique spatio-temporal patterns (Whalley and Knight, 2013).

Uptake and extrusion of Ca2+ across biological membranes is favoured by Ca2+ channels or Ca2+ transporters. The former allow ion diffusion down its electrochemical gradient, while the latter are primary or secondary Ca2+ transporters, respectively, also referred to as Ca2+-translocating ATPases and electrochemical potential-driven Ca2+ transporters, according to their mechanisms of Ca2+ transport. Ca2+-decoding ‘tools’ instead are represented by Ca2+-binding sensor proteins, such as Ca2+-dependent protein kinases (CDPKs), calmodulins (CaMs), calmodulin-like proteins (CMLs), and calcineurin B-like proteins (CBLs), which act either as primary effectors or as signal relays. These molecules are part of the Ca2+ signalling toolkits in both prokaryotes and eukaryotes (Verret ; Domínguez ), even though there are significant differences between different group of organisms. Unicellular microalgae, a large and diverse group of photosynthetic eukaryotes, show a surprising diversity in their Ca2+ signalling toolkits, differing significantly from both higher plants and animal counterparts, but also between microalgal lineages. Several excellent reviews have covered the evolution and the role of global Ca2+ signalling in the heterogeneous world of these organisms (Verret ; Edel ; Wheeler ). In the present review, therefore, we will focus on the Ca2+ signalling toolkits and Ca2+-based responses of the green model alga Chlamydomonas reinhardtii, widely exploited as a reference organism to study photosynthesis, chloroplast biology, organelle crosstalk, phototaxis, and for development of synthetic biology tools for the production of high-value products (Salomé and Merchant, 2019). In C. reinhardtii, Ca2+ signalling has been shown to play a central role in many motile responses (flagella beating, phototaxis, chemotaxis, mating, and de-flagellation), in stress responses to environmental abiotic stimuli, and also in the regulation of photosynthesis (Wheeler and Brownlee, 2008; Verret ; Bickerton ). Even though many individual components of these signalling pathways remain to be identified, the relatively reduced complexity of the Ca2+ signalling toolkit in this organism might represent an advantage to study the evolution of the Ca2+ signalling machinery. Indeed, C. reinhardtii is characterized by the presence of several ‘animal-like’ Ca2+ channels that are absent from plant genomes (Table 2), such as voltage-gated cation channels (VDCCs), transient receptor potential (TRP) channels, and inositol triphosphate receptors (IP3Rs) (Wheeler and Brownlee, 2008; Verret ). Conversely, specific plant components of the Ca2+ signalling responses are conserved in C. reinhardtii, such as OSCA (hyperosmolarity-gated calcium-permeable) channels, found to play a role in osmotic stress signalling in land plants (Edel and Kudla, 2015; Cronmiller ). Similarly, Ca2+/H+ antiporters, absent in mammalian cells, can be found in C. reinhardtii, even though they have an additional Na+/H+ exchange activity (Pittman ). Moreover, the CDPK family of Ca2+ sensor kinases, that play a central role in response to biotic and abiotic stimuli in land plants, have recently been found to be encoded by 15 different genes in C. reinhardtii, and to also be involved in nitrogen deficiency-induced oil accumulation (Y. Li ). Recently, 13 novel CBL-like proteins have also been identified through a genome-wide analysis in C. reinhardtii, displaying characteristic EF-hand Ca2+-binding domains like those present in Arabidopsis CBLs (Kumar ).

Table 2.

Summary of the Ca2+-related proteins identified in the genome of C. reinhardtii and their distribution in plant and animal genomes

Gene ID in C. reinhardtii	Alias	Protein family	Number of genes in family (C. reinhardtii)	Involved in	Plant homologues	Animal homologues	References
Ca 2+ sensors/binding proteins
Cre01.g038400	CRT2	Ca2+-binding protein/chaperone	1	Ca2+ buffer, molecular chaperone activity (?)	Present, classified into two groups of homologs, CRT1/2 and CRT3	Present (CALR in Homo sapiens)	Jia et al. (2009); Mariani et al. (2013)
Cre12.g497300	CAS	Ca2+ sensing receptor	1	Ca2+-mediated regulator of CCM functioning and photoprotection	Present, AtCAS	Not identified	Petroutsos et al. (2011); Edel et al. (2017)
Cre03.g202950	CRX	Ca2+-dependent sensor responder	1	Ca2+-mediated regulator of redox state and photosynthesis	Not identified in vascular plants	Not identified	Hochmal et al. (2016); Charoenwattanasatien et al. (2020
Cre01.g016300	EFh3, CAM3	Ca2+-binding protein	–	–	Not identified (43.1% sequence identity with calmodulin-like 38 (CML38) of Arabidopsis)	Not identified (up to 38% sequence identity to various mammalian calmodulin-like proteins)	Phytozome C. reinhardtii v5.5 proteome and BLAST searches with C. reinhardtii query sequence
Cre03.g197500	MOT6	Ca2+-binding protein	–	–	Not identified (25% sequence identity to an EF-hand Ca2+-binding protein in Arabidopsis)	Not identified (36% sequence identity to a human calcyphosin-like protein)	Phytozome C. reinhardtii v5.5 proteome and BLAST searches with C. reinhardtii query sequence
Cre17.g705000	CDPK1	Ca2+-dependent protein kinase (CDPK)	14	–	Present, 34 CDPK genes in Arabidopsis	Not identified	Pazour et al. (2005); Liang and Pan (2013); Motiwalla et al. (2014)
Cre01.g009500	CDPK3			Flagellar biogenesis
Cre13.g564500	CDPK11			–
Cre08.g363750	CBL-like1	Calcineurin B-like protein (CBL)	13	Ca2+ sensor, interacting with VDCC	Present, 10 CBLs genes in Arabidopsis	Not identified, closest similarity with the regulatory subunit (CNB) of yeast and animal calcineurin	Beckmann et al. (2016); Kumar et al. (2020)
Ca 2+ channels
Cre10.g452950	TRP1	Transient receptor potential (TRP) channel	21	Ca2+ release/exchange (?)	Not identified	Present, >100 genes identified in various animals	Arias-Darraz et al. (2015a); McGoldrick et al. (2019)
Cre16.g655950	TRP2			Ca2+ signalling in limiting CO2			Christensen et al. (2020)
Cre09.g398400	TRP5			Ca2+ release/exchange (?)			Fujiu et al. (2011)
Cre07.g341350	TRP11			Ca2+-mediated mechanoreception			Fujiu et al. (2011)
Cre09.g397142	ADF1, TRP15			Acid-activated entry route for Ca2+ in deflagellation			Hilton et al. (2016)
Cre17.g715300	PKD2, TRPP2			Ca2+-dependent regulation of the mating process and target and anchor of mastigonemes			Huang et al. (2007); Liu et al. (2020)
Cre14.g611300	ChR1	Microbial-type rhodopsin with light-gated ion conductance	2	Light-gated regulator of Ca2+-dependent phototaxis	Not identified	Not identified	Nagel et al. (2002); Berthold et al. (2008)
Cre02.g085257	ChR2						Nagel et al. (2003); Schmidt et al. (2006)
Cre11.g479350	POLLUX-related	CASTOR/POLLUX-related ion channel	2	–	Present, DMI1 in M. truncatula, and CASTOR and POLLUX in L. japonicus	Not identified	Phytozome C. reinhardtii v5.5 proteome
Cre14.g615800	POLLUX-related	CASTOR/POLLUX-related ion channel		–	Present, DMI1 in M. truncatula, and CASTOR and POLLUX in L. japonicus		Phytozome C. reinhardtii v5.5 proteome
Cre16.g665050	CAV2	Voltage-dependent cation channel (VDCC)	9	Ca2+ influx and regulation of flagellar waveform	Not identified	Present, 10 members in human with low sequence similarity	Fujiu et al. (2009); Verret et al. (2010)
Cre01.g013700	ASC1	Voltage-dependent anion channel (VDAC)	2	Entry and exit of ions and metabolites (?)	Present, five isoforms in Arabidopsis	Present, three isoforms in mammals	Tateda et al. (2011); Massoz et al. (2017)
Cre05.g241950	ASC2
Cre12.g553450	CNG1	Cyclic nucleotide-gated channels (CNGCs)	3	–	Present, 20 genes in Arabidopsis	Present, six genes in human	Wheeler and Brownlee (2008); Edel and Kudla (2015)
Cre12.g553900	CNG2			–
Cre17.g706900	CNG3			–
Cre16.g665450	RYR1, IP3R	Inositol 1,4,5-trisphosphate receptor (IP3R)	1	Ca2+ release/flux across flagella membrane or intracellular stores (?)	Not identified	Present, three genes in human	Pazour et al. (2005); Schönknecht (2013); Edel and Kudla (2015)
Cre01.g025000	Orai	Ca2+ release-activated Ca2+ (CRAC) channel	1	Modulating cytosolic [Ca2+] (?)	Not identified in angiosperms, but still present up to gymnosperms	Present, together with STIM in the SOCE	Edel et al. (2017)
Cre16.g685650	GLR1	GLR-type ligand gated cation channel	1	Ca2+ release/exchange (?)	Present, 20 genes in Arabidopsis	Present	Wheeler and Brownlee (2008); Verret et al. (2010); Wudick et al. (2018)
Ca 2+ transporters
Cre12.g505350	ACA2, FAP10	P-type IIB Ca2+-ATPases (ACAs)	3	Ca2+ active transport, against its concentration gradient (?)	Present, 10 ACAs in Arabidopsis	Present, as plasma membrane Ca2+-ATPase, PMCA	García Bossi et al. (2020)
Cre02.g145100	FAP39
Cre16.g681750	FAP381
Cre05.g242350	-	P-type IIA Ca2+-ATPases (ECAs)	2		Present, four ECAs in Arabidopsis	Present, as analogous sarcoplasmic-endoplasmic reticulum Ca2+-ATPases (SERCAs)
Cre11.g467795	-
Cre12.g519500	CAX1	H+/cation exchanger (CAX)	3	Ca2+ or Na+/H+ exchange, cation homeostasis	Present, six genes in Arabidopsis	Present, but absent in mammals	Pittman et al. (2009); Emery et al. (2012)
Cre02.g084350	CGLD1	PAM71-type Mn2+/Ca2+ cation transporter	1	Uptake/maintenance of Mn2+ or Ca2+	Present, AtPAM71	Present, human TMEM165 and yeast Gdt1p	Xing et al. (2017); Zhang et al. (2018)
Cre17.g729200	MCU	MCU-type Ca2+ uniporter	1	Ca2+ transport across IMM (?)	Present, six isoforms in Arabidopsis	Present	Teardo et al. (2017; 2019)
Cre15.g639150	LETM1	LETM-like H+/cation exchanger	1	Indirect influence on Ca2+ transport across IMM (?)	Present, two isoforms in Arabidopsis	Present, 2 isoforms in Homo sapiens	Wagner et al. (2016); Austin and Nowikovsky (2019)

The table includes Ca2+-binding proteins, Ca2+-permeable channels, and Ca2+ transporters predicted from the C. reinhardtii genome/proteome (Phytozome C. reinhardtii v5.5) or identified in previous studies. Articles reporting the original data are provided. The genomes were additionally searched for candidate Ca2+-related protein homologues, using Basic Local Alignment Search Tool (BLAST) searches with animal, plant, and algal proteins. When the proposed function is not yet supported by experimental evidence, a question mark (?) has been included in the corresponding column. ‘–’ denotes alias not yet assigned or molecular/physiological function not yet predicted.

These are all crucial components of C. reinhardtii Ca2+ signalling, putatively involved in the transport and sensing of the Ca2+ ion among and within the different intracellular compartments of the cell. In the following sections, all the different molecules involved in C. reinhardtii Ca2+ signalling will be addressed in the context of the different organelles or compartments. While Ca2+ signalling systems have been extensively studied in plants, reporting a crucial contribution of organelles in the shaping of the Ca2+ signature in response to a given stimulus (Costa ; Navazio ), the role of organelles in algal Ca2+ signalling still remains elusive. Numerous pieces of evidence depict plant Ca2+ signalling systems as complex networks of intersecting signal transduction pathways, requiring and being influenced by more than one source of Ca2+ (Dodd ; Costa ). Very few examples are reported of direct in vivo imaging of [Ca2+]cyt in green algae. Among these, a recent study in C. reinhardtii has shown an increase in cytosolic [Ca2+] in response to a bacterial cyclic lipopeptide, orfamide A, which appears to target a Ca2+ channel in the plasma membrane (Aiyar ). Furthermore, another work in C. reinhardtii demonstrated that different osmotic stimuli give rise to distinct [Ca2+]cyt elevations, also suggesting a crucial role for Ca2+ entry across the plasma membrane in the initiation of the signalling response (Bickerton ). Ca2+-based responses to osmotic stress, however, appear to be conserved between C. reinhardtii and plants, probably involving further release of Ca2+ from intracellular stores, for example during the propagation of the Ca2+ wave.

In this review, we highlight the recent advances in the understanding of the C. reinhardtii cellular mechanism underlying the generation, shaping, and decoding of Ca2+ signals, at the level of both the cytosol and intracellular compartments. In the following sections, an overview of the known and possible components of the Ca2+ signalling machinery is provided for each organelle, together with their associated role and function. Each Ca2+-binding protein, channel, and transporter putatively involved in Ca2+ homeostasis or fluxes among the different intracellular compartments of C. reinhardtii cells can be seen in Fig. 1. A brief survey of the recently developed and implemented toolkit to measure time-resolved cytosolic and organellar Ca2+ signalling in living C. reinhardtii cells is also provided.

Fig. 1.

Identified and/or predicted Ca2+ signalling toolkit in C. reinhardtii. Overview of the Ca2+-related molecular players possibly involved in Ca2+ fluxes across the different subcellular compartments of the C. reinhardtii cell. When the subcellular localization of the protein has not yet been experimentally determined a question mark (?) has been included. See text for further details.

The candidate algal calcium stores: contractive vacuoles, acidocalcisomes, and the endoplasmic reticulum

A finely regulated intracellular Ca2+ compartmentalization represents an essential requirement in all eukaryotic cells to avoid [Ca2+]cyt increases above the cytotoxicity limit, but especially to use Ca2+ as a versatile signalling molecule. The vacuole represents one of the major intracellular calcium stores in plants, as a single membrane-bounded compartment within the cytoplasm (Stael ). They are acidic organelles required for protein degradation, turgor control, temporary storage, or permanent accumulation of metabolites, and are fundamental in Ca2+ signalling, compartmentalization, and storage (Marty, 1999; Costa ). In algae and protozoa, including C. reinhardtii, small lytic acidic vacuoles are present, and can be distinguished from contractile vacuoles (CVs), which are instead osmoregulatory organelles. The vacuoles of yeasts and higher plants are thought to have evolved from lytic vacuoles of ancestral green algae, whereas CVs appear to have evolved when protists entered freshwater habitats (Becker, 2007; Pittman, 2011). Moreover, the structure of the vacuolar system in C. reinhardtii has been proven to be highly dynamic, being regulated during cell growth and autophagy, also forming autophagy-related vacuoles under specific stress conditions, and changing in number, volume, and composition of vacuolar compartments at high concentrations of heavy metals (Nishikawa ; Goodenough ).

Contractile vacuoles

CVs are membrane-bound cell compartments found in many unicellular freshwater protists, that cyclically accumulate and extrude water and excess ions—notably Ca2+—out of the cell, allowing survival under hypotonic conditions (Plattner, 2013; Komsic-Buchmann ). Chlamydomonas reinhardtii cells contain two CVs, whose structure and function have been investigated in detail. Despite some differences in morphology and behaviour, CVs of green algae and protists, such as Dictyostelum and Paramecium, possess a quite similar set of proteins and mechanism of function, including vacuolar-type H+-ATPases (V-ATPases), aquaporins, and vesicular transport (Komsic-Buchmann ). CVs have been shown to store Ca2+ in many protist species, and many specific Ca2+ transporters located at these organelles have already been characterized (Pittman, 2011). The CVs of the amoeba Dictyostelium discoideum, for example, display both a PMC1-like Ca2+-ATPase, called PAT1, and a Ca2+/H+ exchanger, and are believed to be involved in Ca2+ regulation, in addition to osmoregulation (Moniakis ; Malchow ).

It has not been proven yet whether in C. reinhardtii CVs can also function as intracellular Ca2+ stores; however, these organelles possess V-H+-ATPase and H+-pyrophosphatase (H+-PPase) pumps, that could generate a H+ gradient and energize Ca2+/H+ exchange through CAX transporters, sequestering Ca2+ from the cytoplasm (Pittman, 2011; Ruiz ). Indeed, three CAX genes have been identified in the genome of C. reinhardtii, of which CrCAX1 encodes a proven Ca2+/H+ exchanger that localizes to the vacuole when heterologously expressed in yeast (Pittman ; Emery ). CrCAX1, has been predicted to be vacuolar localized in C. reinhardtii, even though its localization remains to be experimentally confirmed (Table 1) (Hanikenne ). These transporters could in fact reside either in C. reinhardtii CVs or in the membrane of acidic lytic vacuoles, that can also act as calcium stores. Furthermore, besides its possible role in Ca2+ homeostasis, CrCAX1 exhibits Na+/H+ exchange activity, that could contribute to Na+ efflux from the cell during salt stress (Pittman ). CrCAX1 gene expression, however, was reported to be repressed by high concentrations of Ca2+, suggesting its important role in the generation/propagation of Ca2+ signals rather than for providing Ca2+ tolerance (Pittman ). Accordingly, CrCAX1 may be regulated in vivo to prevent excessive intracellular accumulation of ions, that could be cytotoxic for unicellular organisms with a limited capacity for intracellular ion storage. This could indicate a crucial role for C. reinhardtii vacuoles in Ca2+ homeostasis and signalling rather than in Ca2+ tolerance, as usually reported for plants (Hirschi, 1999). CrTRP5, identified as a member of the TRP channels family in C. reinhardtii (Tables 1, 2), was also localized in the anterior region within the cell body, where CVs are present (Fujiu ). TRP channels are known mediators for cellular sensing in many eukaryotic organisms, from protists to insects and animals, excluding plants. A proteome-wide search for putative TRP channel-coding sequences identified 21 protein candidates in C. reinhardtii, of which TRP1, TRP5, and TRP16 are suggested as the best candidates to serve as repolarization-associated channels (Arias-Darraz b). Among these, the putative flagellar CrTRP1 has recently been characterized as a functional channel, displaying voltage-dependent outward rectification, a temperature-dependent gating, and a cationic non-selective permeability, thus also being permeable to Ca2+ (Arias-Darraz a; McGoldrick ). TRP channels in C. reinhardtii thus might represent good candidates for the generation of input signals or the regulation of sensory input propagation. Furthermore, the presence of a TRP homologue in C. reinhardtii CVs, CrTRP5, might suggest a way for Ca2+ release from vacuoles, and their possible involvement in the propagation of Ca2+ signals.

Table 1.

Summary of the subcellular localization of the Ca2+-related proteins identified in the genome of C. reinhardtii

Gene ID	Alias	Subcellular localization	Localization evidence	Type	Function	References
Cre12.g519500	CAX1	Contractile vacuoles/acidocalcisomes	Predicted+FP fusion	Ion transporter	Cation/H+ exchange	Hanikenne et al. (2005); Pittman et al. (2009)
Cre09.g398400	TRP5	Contractile vacuoles (?)	Immunofluorescence	Ion channel	Ca2+ release/exchange (?)	Fujiu et al. (2011)
Cre16.g665450	RYR1, IP3R	Flagella	MS analysis	Ion channel	Ca2+ release (?)	Pazour et al. (2005)
		Acidocalcisomes (?)	–			–
		ER (?)	Predicted (DeepLoc-1.0)			–
Cre01.g038400	CRT2	ER (?)	Predicted	Ca2+-binding protein	Ca2+ buffer, molecular chaperone (?)	Zuppini et al. (1999); Mariani et al. (2013)
Cre16.g655950	TRP2	Chloroplast (?)	Predicted	Ion Channel	Ca2+ signalling in limiting CO2	Christensen et al. (2020)
Cre02.g084350	CGLD1	Chloroplast (?)	Predicted	Ion Transporter	Uptake/maintenance of Mn2+ or Ca2+	Xing et al. (2017)
Cre12.g497300	CAS	Chloroplast	Immunofluorescence+FP fusion	Ca2+-binding protein	Ca2+-mediated regulator of CCM functioning and photoprotection	Petroutsos et al. (2011); Wang et al. (2016); Yamano et al. (2018)
Cre03.g202950	CRX	Chloroplast	Predicted+immunoblot+FP fusion	Ca2+-binding protein	Ca2+-mediated regulator of redox state and photosynthesis	Hochmal et al. (2016); Charoenwattanasatien et al. (2020)
Cre14.g611300	ChR1	Eyespot (plasma membrane)	Immunofluorescence+MS analysis	Ion Channel	Light-gated regulator of Ca2+-dependent phototaxis	Nagel et al. (2002); Berthold et al. (2008); Schmidt et al. (2006
Cre02.g085257	ChR2	Eyespot (plasma membrane)	MS analysis	Ion channel	Light-gated regulator of Ca2+-dependent phototaxis	Nagel et al. (2003); Schmidt et al. (2006)
Cre01.g013700	ASC1	Mitochondria (?)	Predicted (DeepLoc-1.0)	Mitochondrial porin	Entry and exit of ions and metabolites (?)	Massoz et al. (2017)
Cre05.g241950	ASC2	Mitochondria (?)	Predicted (DeepLoc-1.0)	Mitochondrial porin	Entry and exit of ions and metabolites (?)	Massoz et al. (2017)
Cre17.g729200	MCU	Mitochondria (?)	–	Ion transporter	Ca2+ transport across IMM (?)	Teardo et al. (2017)
Cre15.g639150	LETM1	Mitochondria (?)	Predicted (PredAlgo+DeepLoc-1.0)	Ion transporter	Indirect influence on Ca2+ transport across IMM (?)	Phytozome C. reinhardtii v5.5 proteome
Cre01.g016300	EFh3	Mitochondria	MS analysis	Ca2+-binding protein	–	Atteia et al. (2009)
Cre03.g197500	MOT6	Mitochondria	MS analysis	Ca2+-binding protein	–	Atteia et al. (2009)
Cre11.g479350	POLLUX-related	Nucleus (?)	–	Ion channel	–	Phytozome C. reinhardtii v5.5 proteome
Cre14.g615800	POLLUX-related	Nucleus (?)	–	Ion channel	–	Phytozome C. reinhardtii v5.5 proteome
Cre16.g665050	CAV2	Flagella	Immunofluorescence+immunoblot on SF	Ion channel	Ca2+ influx and regulation of flagellar waveform	Fujiu et al. (2009)
Cre07.g341350	TRP11	Flagella	Immunofluorescence+immunoblot on SF	Ion channel	Ca2+-mediated mechanoreception	Fujiu et al. (2011)
Cre17.g715300	PKD2	Flagella	Immunofluorescence+immunoblot on SF +FP fusion	Ion channel	Ca2+-dependent regulation of the mating process	Huang et al. (2007); Liu et al. (2020)
Cre09.g397142	ADF1, TRP15	Flagella (?)	–	Ion channel	Acid-activated entry route for Ca2+ in deflagellation	Hilton et al. (2016)
Cre17.g705000	CDPK1	Flagella	MS analysis +immunofluorescence+immunoblot on SF	Ca2+-binding protein	–	Pazour et al. (2005); Motiwalla et al. (2014)
Cre01.g009500	CDPK3	Flagella	MS analysis+immunofluorescence+immunoblot on SF	Ca2+-binding protein	Involved in flagellar biogenesis	Pazour et al. (2005); Liang and Pan (2013)
Cre13.g564500	CDPK11	Flagella	MS analysis	Ca2+-binding protein	–	Pazour et al. (2005); Liang and Pan (2013)
Cre08.g363750	CBL-like1	Flagella	Immunofluorescence	Ca2+-binding protein	Ca2+ sensor, interacting with VDCC	Kumar et al. (2020)

The table includes Ca2+-permeable channels, Ca2+ transporters, Ca2+-binding proteins. and sensors predicted from the C. reinhardtii genome/proteome (Phytozome C. reinhardtii v5.5) or identified in previous studies. When the subcellular localization of the protein has not yet been experimentally confirmed, or its proposed function is not yet supported by experimental evidence, a question mark (?) has been included in the corresponding column. Experimental evidence or prediction of subcellular localization are reported (FP, fluorescent protein; SF, subcellular fractions), referring to the original articles. When specified, the subcellular localization has only been predicted here using PredAlgo and/or DeepLoc-1.0 software (Tardif ; Almagro Armenteros ).

Acidocalcisomes

Chlamydomonas reinhardtii cell also possess acidocalcisomes, typical electron-dense membrane-enclosed organelles with an acidic lumen, rich in polyphosphate and able to accumulate Ca2+ and other metals (Docampo and Huang, 2016). Acidocalcisomes have been proposed to represent the earliest form of an intracellular calcium pool, that can reach concentrations above millimolar levels (Patel and Docampo, 2010). They have been documented in bacteria, and in protist and mammalian cells, and proven to play a direct role in Ca2+ signalling in a range of protists and as a major Ca2+ internal store also in C. reinhardtii (Docampo and Huang, 2016). Recent evidence in C. reinhardtii has also highlighted their essential role in reshaping the cell during acclimation to changing environmental conditions, including nutrient deprivation (sulfate, nitrate, and phosphate) and metal deficiency conditions, accumulating manganese (Mn), zinc (Zn), and copper (Cu) (Aksoy ; Hong-Hermesdorf ; Tsednee ). In Chlamydomonas moewusii, acidocalcisomes have been described as electron-dense bodies (EDBs) rich in polyphosphate, in which the estimated total concentration of Ca2+ is ~2–4 M (Kuin ). Recent studies have reported, however, that most of the acidosomal calcium in C. reinhardtii is not in the form of soluble ions, but is precipitated by the polyphosphates into a distinct chemical phase (Gal ). However, data obtained by using 45Ca2+ and X-ray microanalysis (XRMA) indicate that a large fraction of the Ca2+ can be quickly mobilized from C. moewusii EDBs, suggesting the presence of a delicate and balanced equilibrium between soluble and insoluble forms of calcium and phosphate (Kuin ).

The same study showed that in C. moewusii calcium can be released from intracellular stores, including from EDBs, upon treatment with mastoparan analogues, which are known activators of G-proteins and phospholipase C (PLC), producing inositol-3-phosphate (InsP3). Vacuoles in the yeast Saccharomyces cerevisiae, similar in size, content, and electron density to EDBs, have been shown to release Ca2+ in an InsP3-dependent manner (Belde ). The genome of C. reinhardtii encodes a single InsP3 receptor (InsP3R, or IP3R) homologue (Tables 1, 2), detected as an abundant component of the flagellar proteome and involved in Ca2+-dependent de-flagellation, observed in pharmacological studies upon application of pH, osmotic, or temperature shock (Ruiz ; Pazour ; Schönknecht, 2013). The precise subcellular localization of C. reinhardtii InsP3R, however, has not been investigated yet, and its presence on the acidocalcisomal membrane cannot be excluded. An InsP3-dependent Ca2+ release mechanism from acidocalcisomes would indeed strongly support the role of these organelles as intracellular Ca2+ sources in C. reinhardtii, besides maintaining intracellular signalling or contributing to refill the excitable stores (Kuin ; Schönknecht, 2013). Acidocalcisomes in C. reinhardtii also carry transmembrane polyphosphate polymerase and, like CVs, two different classes of proton pumps: H+-PPases and V-type ATPases, that could drive a H+ concentration gradient and potentially energize Ca2+ sequestration through Ca2+/H+ exchangers (Ruiz ). As previously mentioned, however, it is not known whether any of the three CrCAX transporters localize to acidocalcisomes, to CVs, or to both.

In the protozoan Trypanosoma, acidocalcisomes are usually also observed in close contact with other organelles and intracellular structures, such as mitochondria, nuclei, lipid inclusions, and CVs (Docampo, 2016). Close contact sites could be relevant for the transmission of Ca2+ signals, creating microdomains of high Ca2+ concentration, as occurs between the ER and mitochondria in vertebrate cells (Rizzuto ). These types of interactions have not yet been investigated or documented in green algae, but could potentially represent an additional conserved role for acidocalcisomes in Ca2+ homeostasis and signal propagation.

Endoplasmic reticulum

The ER is the major Ca2+ store capable of its release in most eukaryotes, and is the largest and the major rapidly exchanging Ca2+ store in animal cells (Raffaello ). ER network 3D reconstruction in the green alga Botryococcus braunii has shown that most of the ER is present as sheets or fenestrated sheets, differing from the pattern in land plant cells, with a definite larger contact area with organelles (Suzuki ). Besides being in continuity with the double nuclear membrane, the ER has been shown to be in strict contact with chloroplasts, oil bodies, or plasma membrane with specific domains (Suzuki ). As proposed in land plants, such a unique architecture may facilitate the establishment of a communication network between cell compartments where, through specific contact sites, organelles could exchange metabolites and ions, such as Ca2+ (recently reviewed in plants by Wang and Dehesh, 2018). Even though not much is known about Ca2+ storage properties of the C. reinhardtii ER, it has been proposed as an intracellular Ca2+ source in other green algae, mediating Ca2+ release in response to extracellular signals (Kuin ; Gonzalez ). In the lumen of the ER of many eukaryotes, Ca2+ ions are also buffered efficiently by Ca2+-binding proteins, among which calreticulin (CR) is one of the most predominant forms. Chlamydomonas reinhardtii cells express a CR homologue (CRT2) that putatively localizes to the ER (Table 1), providing first evidence for the presence of an intraluminal Ca2+ storage machinery in the C. reinhardtii ER (Zuppini ). CR, as an effective Ca2+ buffer, may in fact allow the transient storage of the ion and its prompt mobilization when Ca2+ release is triggered, being characterized by high capacity and low affinity (Zuppini ). Interestingly, CR expression in C. reinhardtii is developmentally regulated during gamete formation, with a progressive increase of the level of the protein in both pre-gametes and gametes. This could suggest an involvement of CR in the two stages of differentiation, mainly as a molecular chaperone during the first, but as a Ca2+ buffer in the stage that prepares the cell to mate, when Ca2+ ions are rapidly sequestered in intracellular stores (Mariani ).

However, the information about a Ca2+ signalling toolkit in the ER of green algae is still limited. The C. reinhardtii genome contains several homologues of ER-localized channels and transporters that have crucial roles in animal or higher plant Ca2+ signalling and homeostasis, even if none of them has yet been associated with ER Ca2+ homeostasis in this green alga (Verret ). Among these, the IP3R Ca2+ channel is mainly located in the ER of animal cells and has a central role in different Ca2+ signalling mechanisms, mediating Ca2+ release from intracellular stores (Schwarz and Blower, 2016). In the marine chlorophyte alga Ulva compressa, a copper-induced calcium release mechanism has been identified that acts through ER-localized ryanodine-sensitive and IP3-sensitive calcium channels (González ). As discussed above, an IP3R homologue has also been identified in C. reinhardtii, as an abundant protein of the flagellar proteome, where it could mediate Ca2+ entry across the flagella membrane (Pazour ; Verret ). Nevertheless, using the DeepLoc-1.0 server for eukaryotic protein subcellular localization prediction (Almagro Armenteros ), the IP3R homologue is predicted to reside in the ER membrane, thus opening up a possible alternative localization of the protein. Furthermore, in the fission yeast Schizosaccharomyces pombe, two ER-localized mechanosensitive ion channels that belong to the MscS-like (MSL) protein family contribute to cytosolic Ca2+ elevations, especially Msy2, by Ca2+ release from the cortical ER after a hypo-osmotic shock (Nakayama ). In C. reinhardtii, three MSL proteins were identified, two of which have an unknown subcellular localization, that could act similarly to the yeast homologues if properly localized. The third one, MSC1, was instead demonstrated to be a chloroplast-localized Cl– channel (Nakayama ). A more recent review, however, has also expanded the number of identified gene sequences coding for MSL family members in C. reinhardtii to seven, annotated in the Phytozome C. reinhardtii v5.5 proteome as MSC1–MSC7 (Edel and Kudla, 2015). In human cells, a well-characterized store-operated Ca2+ entry (SOCE) also allows changes in luminal ER [Ca2+] to regulate the opening of a plasma membrane Ca2+ channel, modulating cytosolic [Ca2+] and influencing several biological processes. This system is composed of an ER Ca2+ sensor, STIM, and a plasma membrane Ca2+ channel regulated by STIM, called Orai. In the genome of C. reinhardtii, one Orai sequence has been identified, showing the preservation of many important sequence features (Collins and Meyer, 2011). However, the activating ER-localized STIMs appear to be missing, suggesting an evolutionary earlier appearance of Orai and the presence of alternative gating mechanisms for this Ca2+-permeable channel in this algal species (Collins and Meyer, 2011; Edel ).

In plants, as in animals, Ca2+-ATPases are also crucial in maintaining Ca2+ homeostasis within the different intracellular compartments, by controlling Ca2+ efflux from the cytosol to organelles and/or to the extracellular space. Plants contain P-type ATPases, grouped as P-IIA ER-type Ca2+-ATPases (ECAs), mainly localized in the ER and Golgi apparatus, and P-IIB autoinhibited Ca2+-ATPases (ACAs), found mainly in the plasma membrane but also in the ER, vacuoles, and chloroplast membranes (García Bossi ). Three homologues of Arabidopsis ACAs and two for ECAs were found in C. reinhardtii, having a possible role in the reuptake of cytosolic Ca2+ against its concentration gradient. ACA- and/or ECA-mediated Ca2+ active transport could have a signalling function, removing this secondary messenger from the cell or storing it in organelles, such as the ER, vacuoles, chloroplast, or mitochondria (García Bossi ). The subcellular localization and characterization of these Ca2+-ATPases in C. reinhardtii needs to be elucidated, possibly clarifying their role in organellar Ca2+ signalling and homeostasis.

Compared with the detailed information about intracellular Ca2+ stores in higher plants and protists, current knowledge on the role of C. reinhardtii vacuoles and ER in Ca2+ homeostasis and signalling still remains in its infancy. From the data obtained so far, it is clear that the Ca2+ toolkit of C. reinhardtii putative Ca2+ stores needs further investigation, in terms of both the identification of the specific molecular players involved and the investigation of the precise role of each compartment as a Ca2+ store and/or Ca2+ signal transducer.

The role of the main algal cellular compartments in Ca2+ signalling and homeostasis

Chloroplast Ca2+ signalling and dynamics in C. reinhardtii cells

Chloroplasts are heavily compartmented organelles, specific for eukaryotic photosynthetic organisms. The green alga C. reinhardtii houses a single cup-shaped chloroplast, that occupies almost half of the cell volume. It is surrounded by an outer and inner envelope, and contains a soluble stroma, thylakoid membranes, and the enclosed luminal space. The lobes of the organelle contain mature thylakoids that catalyse the light reaction of photosynthesis, whereas the large base of the chloroplast hosts the so-called pyrenoid, a specialized region enriched in the carbon-fixing enzyme, the Rubisco complex. Such organized membrane architecture evolved over a billion of years ago via primary endosymbiosis of photosynthetic cyanobacteria within an ancestral anaerobic eukaryote (Jensen and Leister, 2014). Already in the cyanobacterial ancestor, in fact, the light-dependent reactions of the photosynthetic process take place in thylakoid membranes, which are in direct contact with the cytosol (Mechela ).

Besides being the host of the fundamental photosynthetic process, however, the C. reinhardtii chloroplast is also the site for the biosynthesis of amino acids, fatty acids, carotenoids, and chlorophylls, and assimilation of sulfates and nitrogen (Rea ).

Several recent studies highlighted the role of plant chloroplasts in the shaping of cytoplasmic Ca2+ signals, influencing the cellular Ca2+ network and modulating cytosolic Ca2+ transients (reviewed by McAinsh and Pittman, 2009; Nomura and Shiina, 2014; Costa ). In addition, different biotic and abiotic stimuli were reported to trigger specific chloroplast Ca2+ signals, among which there are elicitors of plant defence responses, cold, oxidative, salt, and osmotic stresses (recently reviewed by Navazio ). Interestingly, increases in absolute temperature also stimulate free [Ca2+] elevation in plant chloroplasts but not in the cytosol (Lenzoni and Knight, 2019). The involvement of chloroplast Ca2+ in the regulation of the photosynthetic machinery was extensively studied in recent years, starting from light reactions and CO2 fixation, but also encompassing processes not directly related to photosynthesis, such as stomatal movements, chloroplast import of nuclear-encoded proteins, or photoacclimation in C. reinhardtii (reviewed by Rocha and Vothknecht, 2012; Hochmal ). Plant chloroplasts possess a high Ca2+ concentration, of which the predominant portion (~15 mM) is bound to the negatively charged thylakoid membranes or to Ca2+-binding proteins, keeping the resting free [Ca2+]stroma as low as 150 nM to avoid the precipitation of phosphates (Hochmal ). This concentration has been shown to be modulated in a light-dependent way, commonly observing a Ca2+ uptake into the chloroplast in the light, and a Ca2+ release into the cytosol upon dark transition (Stael ). In the characean alga Nitellopsis, a light-dependent depletion of cytosolic Ca2+ in the vicinity of chloroplasts was observed, thus suggesting the presence of an active Ca2+ uptake machinery in the envelope membranes and a conserved role for the chloroplast in the shaping of cytosolic Ca2+ signals, probably regulated by light/dark transition and photosynthesis (Sai and Johnson, 2002). A transient increase in the intracellular Ca2+ concentration was in fact also observed in cyanobacteria upon light to dark transition, from a resting level of 0.2 µM up to 4 µM of free Ca2+ (Torrecilla ). The precise mechanisms involved in these dark-induced chloroplast Ca2+ fluxes remain unknown to date, even though the circadian gating of dark-induced increases in chloroplast and cytosolic free Ca2+ was recently demonstrated in Arabidopsis (Martí Ruiz ). Moreover, two chloroplast-targeted Ca2+ transporters that could determine the amplitude of the dark-induced Ca2+ signal in the stroma have recently been identified, named bivalent cation transporter 1 (BICAT 1) and BICAT 2 (Frank ). The first has been proposed to mediate Ca2+ transport through the thylakoid membrane into the lumen, and BICAT2 to transport Ca2+ ions across the chloroplast inner envelope. However, previous work has reported a prevalent role for both these transporters (respectively also named PAM71 and CMT1) in Mn2+ homeostasis rather than in Ca2+ transport (Schneider ; Eisenhut ; Zhang ). Accordingly, a homologue of the PAM71 transporter of Arabidopsis can also be found in C. reinhardtii chloroplasts (CGLD1) (Table 1), where it has been proposed to be involved in Mn2+ uptake/homeostasis rather than Ca2+ transport (Xing ). Interestingly, loss of PAM71 in both Arabidopsis and C. reinhardtii can be restored by enhanced Mn additions, but remains unaffected by supplementation with Ca (Schneider ). In addition, Arabidopsis pam71 mutants even accumulate more Ca in the thylakoid lumen when compared with the wild type (Schneider ). Similarly, the cyanobacterial homologue, the thylakoid-located SynPAM71, has been suggested to be crucial in the maintenance of Mn2+ homeostasis through its export from the cytoplasm into the luminal compartments (Gandini ). Therefore, it cannot be excluded yet that Arabidopsis PAM71 and C. reinhardtii CGLD1 could also function as a Ca2+-permeable channel when needed, and further studies are required to elucidate their precise physiological role and characteristics.

Analysis of chloroplast Ca2+ signals and Ca2+-permeable channels/transporters have been extensively performed in higher plants whereas, in C. reinhardtii, the presence and identification of stimulus-specific chloroplast Ca2+ signals and transients, together with the Ca2+ channels and transporters responsible, remains unexplored. The most recent related studies in C. reinhardtii were addressed to study Ca2+-dependent regulation of photosynthesis and its associated photoprotective mechanisms, taking advantage of the presence of one single large chloroplast rather than many (reviewed by Hochmal ). In particular, the thylakoid-localized Ca2+-sensing receptor (CAS) of C. reinhardtii (Table 1) was shown to be crucial, as a Ca2+-binding protein, for the light-induced expression of Light-Harvesting Complex Stress-Related 3 (LHCSR3) which is an important actor for high light acclimation and photoprotection in this organism (Petroutsos ). In C. reinhardtii, LHCSR3 is required for the photoprotective energy-dependent thermal dissipation of energy absorbed in excess (Peers ). Besides a severe impact on LHCSR3 expression, CAS RNAi knockdown mutant strains exhibited a pronounced light sensitivity and a prominent defect in PSII recovery (Petroutsos ). Active down-regulation of LHCSR3 under high light conditions is deleterious for C. reinhardtii and can explain the high light phenotypes observed in these mutant strains. The addition of 10-fold higher Ca2+ concentration to the growth medium of CAS RNAi knockdown lines, however, rescues PSII recovery and the energy-dependent thermal dissipation of energy absorbed in excess, suggesting an involvement of CAS and Ca2+ in the regulation of the nuclear-encoded LHCSR3 gene expression, and therefore their importance in high light acclimation (Petroutsos ). Similarly, CAS has been reported to be involved in high light response in Arabidopsis thaliana (Cutolo ). In addition to these roles in acclimation of photosynthesis to high light, recently CAS has also been proposed to be involved in regulating the positive phototactic response at low light intensities (Trippens ). Accordingly, CAS has been detected in a purified fraction of the C. reinhardtii eyespot, where it might have a role in the processing of the light signal regulating phototaxis. A study of the C. reinhardtii CAS (CrCAS) knockout mutant, unable to grow at ambient CO2, has also proposed CAS as a Ca2+-mediated regulator of CO2-concentrating mechanism (CCM)-related genes (Wang ). Indeed, CrCAS is essential for the retrograde signalling from the pyrenoid to the nucleus, influencing the expression of 13 genes operating under limiting CO2 conditions (reviewed by Rea ). A recent study exploiting high-resolution fluorescence images of CAS fused with a fluorescence protein demonstrated that CAS could move in the chloroplast along the thylakoid membrane in response to lowering CO2, gathering inside the pyrenoid during the operation of the CCM (Yamano ). Two possible mechanisms have been hypothesized for the relocation of this protein: post-translational modification, such as the phosphorylation of specific and conserved Thr residues, as in the case of Arabidopsis CAS by state transition kinases (STNs) (Cutolo ), or by so far unknown structural dynamics of thylakoid membranes (Yamano ). Interestingly, the addition of a chelating agent (BAPTA), removing external Ca2+, prevents the accumulation of CCM-related genes, suggesting that CAS regulation might be mediated by Ca2+ signals resulting from influx of external Ca2+ (Wang ; Rea ). A recent study also proposed a role for a C. reinhardtii TRP family putative Ca2+-permeable channel (TRP2) in the chloroplast CCM (Tables 1, 2), specifically modulating Ca2+ signalling at the level of the pyrenoid (Christensen ). In particular, TRP2 is predicted to be localized in the chloroplast and it was suggested to function through Ca2+ signalling in the acclimation of algal cells to limiting CO2 (Christensen ). TRP2, being a putative Ca2+-permeable channel, probably influences Ca2+ dynamics inside the pyrenoid, having an indirect role also in CAS protein functioning. Indeed, CrCAS is down-regulated in the trp2 mutant, along with other CCM genes under the control of CrCAS itself (Christensen ). It might be important to note that C. reinhardtii pyrenoid is a dense protein complex, surrounded by a starch sheath and traversed by thylakoid membranes, which could carry transporters and allow the passage of Ca2+ ions through this region (Engel ). In summary, C. reinhardtii CCM functioning was reported to be highly dependent on chloroplast Ca2+ dynamics and CrCAS function, even though the underlying mechanisms of its regulation remains to be characterized.

Nevertheless, Ca2+ was shown also to be involved in cyclic photosynthetic electron flow (CEF) in C. reinhardtii, in both the PGRL1/PGR5-related and the NAD(P)H dehydrogenase (NDH)-dependent pathway (Hochmal ). CrCAS directly interacts with Proton Gradient Regulation Like 1 (PGRL1) and Anaerobic Response 1 (ANR1), in a multiprotein complex, thus being required with Ca2+ in the modulation of qE and CEF (Terashima ). Down-regulation of CAS protein by an RNAi approach, as well as ANR1 depletion by artificial miRNA expression, results in strong inhibition of CEF under anoxia, which was partially rescued by an increase in the extracellular Ca2+ concentration, whereas the activity of the pgrL1 mutant was only marginally restored (Terashima ). CAS and ANR1 were thus proposed as additional components of the CEF supercomplex and Ca2+-dependent key regulators of CEF activity. These components are in fact interconnected: CEF participates in the acidification of the thylakoid lumen, which is required for efficient qE induction. As regards the NDH-dependent pathway, C. reinhardtii possess a type II NADH dehydrogenase (NDA2), characterized by two EF-hands motifs and suggested to have a Ca2+ binding capability, that might modulate its activity (Hochmal ).

Recently a new Ca2+-dependent ‘sensor-responder’ protein, namely calredoxin (CRX), has been identified and characterized in C. reinhardtii (Tables 1, 2), and shown to be conserved in green algae but absent in vascular plants (Hochmal ; Charoenwattanasatien ). This signalling protein has been identified as a chloroplast stroma-resident thioredoxin (TRX), having Ca2+-dependent activity and harbouring four Ca2+-binding EF-hand domains, connected to a typical thioredoxin fold. CRX, being able to interact with a chloroplast 2-Cys peroxiredoxin (PRX1) upon Ca2+ binding, was suggested as a crucial point of interconnection of redox and Ca2+ signalling in the chloroplast, probably translating alteration of Ca2+ and the redox state into cellular signalling (Hochmal ). Having high Ca2+ binding affinity and a Kd value of ~122 nM of free Ca2+ (corresponding to resting stromal [Ca2+]), it could in fact respond to increases of stromal [Ca2+], accelerating the rate of PRX1 reduction and ultimately modulating stromal redox poise. Moreover, in vivo analysis revealed that CEF is induced on reducing or removing CRX activity, which could be caused by enhanced ROS production in light, thus suggesting a further indirect role of Ca2+ in the regulation of algal photosynthesis (Hochmal ).

Chloroplast Ca2+ fluxes are shaped by the activity of envelope- or thylakoid-localized Ca2+-permeable channels and transporters, the identity of which is almost unknown in C. reinhardtii. Nevertheless, the C. reinhardtii genome encodes several homologues of plant chloroplast-localized Ca2+ channels and transporters, whose function and localization, however, remain to be investigated. A single C. reinhardtii homologue of Arabidopsis glutamate receptor-like channels (GLRs) has been identified but not characterized yet, namely GLR1 (Wheeler and Brownlee, 2008). AtGLR3.4 and AtGLR3.5 are both Ca2+-permeable channels belonging to the GLR family, shown to also have a chloroplast localization and to play a role in abscisic acid (ABA) signalling under abiotic stress, thus opening up a potential plastidial localization also of C. reinhardtii GLR1 (Navazio ).

In addition to chloroplast Ca2+-permeable channels and transporters, other regulatory chloroplast cation fluxes have been shown to shape cytosolic Ca2+ signals in plants during stress. In particular, Arabidopsis chloroplast envelope-located K+/H+ antiporters (KEA1 and KEA2) have been shown to be intimately linked to cytosolic Ca2+ transients during hyperosmotic stress. In C. reinhardtii, three putative KEA transporters have been identified, each one of a different subtype, whose function and localization still remain to be defined (Chanroj ).

The current available data are not sufficient to clearly define the role of C. reinhardtii chloroplast in the algal intracellular Ca2+ signalling network, beside their capacity to transport Ca2+ across their membranes and store it by binding to specific proteins or structures. Several works have in fact reported the importance of CAS and Ca2+ in the most studied chloroplast physiological processes, but still a lot of information is lacking regarding the molecular players involved in the chloroplast Ca2+ signalling of this organelle. Future significant studies on the role and the localization of putative channels, transporters, and sensors, together with their linking with specific algal stress responses and physiological processes, would certainly be of great importance to determine the role of this organelle in algal Ca2+ signalling.

The eyespot: a specialized chloroplast/plasma membrane region

In green algae, the eyespot apparatus is a specialized region of the chloroplast and adjacent plasma membrane, primarily involved in phototactic responses by detecting the intensity and direction of light (Kreimer, 2009). Its structure, function, and physiological role have been widely investigated and reviewed in green algae, especially in C. reinhardtii, as a primordial visual system (Kreimer, 2009; Thompson ). It can be described as an ‘organelle’, asymmetrically located on the outer surface of the cell, where the plasma membrane and both chloroplast envelopes are tightly apposed. In particular, it is typically made up of two layers of carotenoid-rich lipid globules inside the chloroplast, each subtended by a single thylakoid membrane (Engel ). By sensing light through this specialized region, C. reinhardtii performs phototaxis in low light and initiates a photophobic response under strong illumination. Extracellular Ca2+ and Ca2+ fluxes are intricately involved at different levels in these photoresponses. Phototaxis signalling has in fact been shown to be mediated by the plasma membrane-located photoreceptors channelrhodopsin (ChR) 1 and 2, light-gated H+- and Ca2+-permeable channels able to initiate mainly Ca2+ fast inward-directed complex currents in the eyespot region (Table 1) (Nagel , 2003; Sineshchekov ). Changes of intracellular free [Ca2+] in the eyespot region were shown to affect the phosphorylation status of ChRs, potentially regulating their function (Wagner ). Accordingly, it was recently demonstrated that multiple ChR1 phosphorylations, regulated via a Ca2+-based feedback loop, act as important components in the adaptation of phototactic sensitivity in C. reinhardtii (Böhm ). Such an increase of free [Ca2+] in the eyespot region also elicits the light-activated flagella currents, thereby regulating the flagella beating balance and the subsequent phototactic response (Harz and Hegemann, 1991; Pazour ). Ca2+ inward fluxes coming from the extracellular space or the periplasm, at the level of the eyespot, thus seem to have a crucial role in the light-sensing and signal transduction processes, regulating several downstream physiological responses. The chloroplast could play an important role in these Ca2+-mediated signalling processes, being a source of Ca2+ and regulating the cytosolic or local [Ca2+]. Several Ca2+ transporters and binding proteins have been identified in the C. reinhardtii subcellular fraction of intact eyespots, including three P-type ATPases, a plasma membrane CaM-binding Ca2+-transporting ATPase, and the previously mentioned chloroplast-localized CrCAS (Eitzinger ; Trippens ). Ca2+ ATPase could be important for signalling and adaptation mechanisms, lowering the cytosolic free Ca2+ concentration in the eyespot region following the activation of the ChRs. CrCAS instead can be found associated with thylakoids, both in the stroma and in the eyespot region (Yamano ). Indeed, as previously mentioned, a recent study using CrCAS-overexpressing lines suggested its role also in the adaptation responses to low light at an intermediate time scale (Trippens ). Accordingly, besides the previously reported roles in the regulation of light acclimation and CEF, CrCAS could have a role in the regulation of the positive phototactic response under continuous illumination (Trippens ). These observations could further suggest an intimate connection between chloroplast-dependent Ca2+ signalling and the fine-tuning of the phototactic responses in C. reinhardtii. However, the mechanisms through which Ca2+ and this chloroplast-localized protein can achieve the regulation of these biological processes are still unknown.

In summary, the eyespot region of the chloroplast and plasma membrane displays a unique Ca2+ signalling toolkit, sensing specific external stimuli and eliciting corresponding intracellular responses, involving different organelles. Its role in the onset and the propagation of these intracellular Ca2+ signals remains to be clarified, together with the Ca2+-related molecular players and intracellular pathway involved.

The still unexplored mitochondrial role in C. reinhardtii Ca2+ dynamics

In contrast to the intensively studied chloroplast, the role of C. reinhardtii mitochondria in intracellular Ca2+ dynamics has been poorly investigated to date. Mitochondria, however, have been shown in animals and plants to accumulate Ca2+ rapidly and transiently, being crucial in regulating, shaping, relaying, and decoding Ca2+ signals, besides being influenced by Ca2+ in their own functions (Wagner ). Moreover, their role in the modulation of cytosolic Ca2+ signatures, participating in the intracellular Ca2+ homeostasis and regulating physiological and pathological events, has been widely demonstrated in animal cells, but is also rapidly emerging in plants (reviewed by Brini, 2003; Wagner ; Costa ). In contrast to animal and higher plant fields, however, relatively limited information is currently available on the role of algal mitochondria in cytosolic and organellar Ca2+ signalling.

Chlamydomonas reinhardtii mitochondria display the typical double membrane-bounded structure, with distinct interior cristae. Interestingly, they also show an elongate branching morphology, changing and possibly fragmenting during different cell cycle and/or life stages (Aoyama ). Such interconnected mitochondrial thread-like structures could facilitate the establishment of an interorganellar signalling and communication network, allowing localized exchanges of metabolites and ions, such as Ca2+. In plant leaves, in fact, mitochondria have often been observed in close physical associations with other organelles, such as chloroplasts and peroxisomes, providing major routes for metabolic exchange and interorganellar signalling (Bobik and Burch-Smith, 2015). Similarly, in diatoms, chloroplasts and mitochondria can be found closely juxtaposed: these tight physical associations have been observed regulating the ATP/NADPH ratio through the re-routing of plastidial reducing power to mitochondria and the import of mitochondrial ATP into the plastid (Bailleul ). In C. reinhardtii, a strong metabolic interconnection between mitochondria and chloroplasts was also recently evidenced, both showing that mitochondria have a crucial influence on photosynthetic reaction and also suggesting that the coupling between these two organelles allows safe dissipation of photosynthetically derived electrons (Larosa ; Kaye ). These studies strongly suggest the presence of mitochondria–chloroplast metabolic interactions that, together with mitochondria branched distribution and potential physical close associations, could permit the passage of Ca2+ ions and thus the transmission of Ca2+ transients/signals.

Ca2+ ions cross the outer mitochondrial membrane (OMM) presumably through voltage-dependent anion channels (VDACs, or porins), found to allow fluxes of metabolites and Ca2+ in both animals and plants (Wagner ). In C. reinhardtii, two VDAC proteins have been identified in the genome, namely ASC1 (Cre01.g013700) and ASC2 (Cre05.g241950) (Table 2), that could putatively mediate Ca2+ transport across the OMM toward the intermembrane space (Massoz ). However, their subcellular localization and molecular properties have not been investigated yet, and their role remains to be defined.

In mammals, mitochondrial electron transport chain (ETC) reactions, coupled with proton movement across the inner mitochondrial membrane (IMM), generate a well-defined proton motive force (pmf), composed of a proton gradient and an electric component, reaching values around –180/–220 mV (Szabo and Zoratti, 2014). Such an electric component of the negative matrix side is sufficient to drive the import of Ca2+ ions, which can passively flux across the IMM into the matrix through specific channels and/or transporters. Once in the mitochondrial matrix, Ca2+ can be bound by proteins and/or inorganic acids, allowing the accumulation of large amounts of total sequestered Ca2+, maintaining the resting [Ca2+] in the matrix at ~100–200 nM (Wagner ). Similarly, in plants, depending on the species and cell type, free [Ca2+] has been reported to reach concentrations in the mitochondrial matrix ranging from 100 nM to 600 nM (Costa ). In C. reinhardtii, [Ca2+] values in the mitochondrial matrix have not been reported yet, alongside the identity and characterization of the specific IMM Ca2+ channels or transporters. Nevertheless, some plant or animal homologues are present in the C. reinhardtii genome, suggesting the presence of a conserved Ca2+ handling mechanism also in green algae. A single homologue of the animal mitochondrial calcium uniporter (MCU) is present in C. reinhardtii (Table 2), whereas most studied higher plant species harbour three or more MCU homologues (Teardo ). In mammals, MCU is responsible for Ca2+ loading into mitochondria, helping the recovery of cytosolic resting [Ca2+], but in plants two MCU homologues have also been found to mediate Ca2+ transport across the mitochondria and chloroplast membranes, respectively named AtMCU1 and AtMCU6, later renamed AtcMCU (De Stefani ; Teardo , 2019). New evidence in particular supports cMCU involvement in the generation of the chloroplast stromal Ca2+ transient specific for the osmotic stress in plants; moreover, mutants lacking cMCU showed an improved drought tolerance (Teardo ). Nevertheless, whether the C. reinhardtii MCU homologue localizes to mitochondria or chloroplasts and has a role in Ca2+ fluxes, influencing organellar and cytosolic Ca2+ signalling, is still unknown. Similarly, at least one homologue of the mammalian LETM1, an EF-hand protein proposed to be involved in mitochondrial K+ and Ca2+ homeostasis as a H+/cation exchanger, can be found in the Phytozome C. reinhardtii v5.5 proteome (Cre15.g639150, Table 2), sharing up to 53% identity with the Arabidopsis mitochondrial homologues AtLETM1 and AtLETM2 (Zhang ; Austin ). The precise role of LETM1 in K+ and Ca2+ transport has been a matter of debate for a long time; however, most recent models and strong arguments support a K+/H+ exchange activity through LETM1, together with an indirect influence on Ca2+ fluxes by affecting the Ca2+ cycle (Wagner ; Austin and Nowikovsky, 2019). Further experiments are needed to validate this model, and also to study a possible similar and conserved function of the C. reinhardtii homologue. As regards the mitochondrial Ca2+-binding protein and Ca2+ sensors in C. reinhardtii, an extensive proteomic survey has revealed the presence of two EF-hand-containing proteins in the mitochondrial proteome, named EFh3 and MOT6 (Tables 1, 2) (Atteia ). Chlamydomonas reinhardtii EFh3 protein, also named CAM3 (Cre01.g016300), shows 43.1% sequence identity with the Ca2+-binding CaM-like 38 (CML38) protein of Arabidopsis, whereas MOT6 (Cre03.g197500) interestingly shows 36% sequence identity to a human calcyphosin-like protein, a Ca2+-binding protein that may play a role in the regulation of Ca2+ transport (Dong ; Atteia ). Even if these proteins have been found in the mitochondrial proteome, their function has not yet been clearly defined. To date, studies reporting the identification and/or functional characterization of these putative Ca2+ transport and sensor proteins in microalgae are still lacking, and the mechanisms involved in mitochondrial Ca2+ sensing and transport in C. reinhardtii remain to be defined. Besides their molecular and physiological characterization, a good strategy to possibly understand the role of EFh3 (CAM3) and MOT6 in Ca2+ signalling could be represented by the analysis of cytosolic and mitochondrial Ca2+ dynamics in C. reinhardtii mutant strains lacking one of these putative transporters or sensors. A further possible strategy to elucidate the role of mitochondria in algal Ca2+ signalling processes might be a forward genetic screening on C. reinhardtii mutants impaired in mitochondrial Ca2+ homeostasis in order to identify the molecular players involved in this process.

Nuclear Ca2+ signalling

When comparing the role of different organelles in intracellular Ca2+ signalling, the nucleus also has to be taken into consideration, given its proven importance in the spatial location and decoding of intracellular Ca2+ signalling, in both animals and plants. Characteristic nuclear Ca2+ dynamics and signals have been shown to be crucial in the regulation of gene expression, controlling root symbiosis in plants, but also being involved in perception of mechanical and temperature stimuli (reviewed in plants by Charpentier, 2018). The role of the nuclear compartment in intracellular Ca2+ signalling of algae, however, remains to be investigated, along with the possible nuclear molecular players involved.

The C. reinhardtii nucleus is a double membrane-bounded organelle, with the outer envelope membrane in continuation with the ER and usually flanked by one to four Golgi bodies (Harris, 2013). It shows a polarized architecture, with nuclear pore complexes (NPCs) spanning the nuclear envelope and mediating nucleocytoplasmic exchange (Colón-Ramos ). Chlamydomonas reinhardtii NPCs have been structurally characterized, showing substantial differences from their human counterparts, with an unexpectedly large diameter and an asymmetric oligomeric state that has not been observed or predicted previously (Mosalaganti ). In eukaryotic cells, ions and molecules of molecular masses <40–60 kDa can permeate through the NPCs, thus suggesting a possible route for the generation of Ca2+ signals and transients in the nucleoplasm (Charpentier, 2018). Alternatively, Ca2+ ions could be taken up or released through nuclear membranes by specific calcium channels and transporters, located either in the inner or in the outer membrane of the envelope, participating in the nuclear Ca2+-dependent signalling. In animal cells, the nuclear envelope was reported to accumulate Ca2+ through the Ca2+-ATPase pump and Na+/Ca2+ exchanger, located respectively in the outer and the inner membrane, and release Ca2+ through Ca2+-permeable channels, sensitive to InsP3, cADP-ribose (cADPR), and nicotinic acid adenine dinucleotide phosphate (NAADP), like Ins3PR and ryanodine receptors (Ryrs) (Oliveira ). In plants, over the past few years, nuclear-localized ion channels and ion pumps that are responsible for characteristic nuclear Ca2+ oscillations have also been identified. Among them, Does not Make Infection 1 (DMI1) from Medicago truncatula, and the homologues CASTOR and POLLUX from Lotus japonicus, have been identified as cation channels involved in the nuclear Ca2+ spiking typical of the early stage of the symbiotic signal transduction pathway, resulting in downstream symbiosis-related gene expression (Charpentier ; Charpentier, 2018). Moreover, interacting with DMI1, a cyclic nucleotide-gated channel (CNGC), CNGC15, together with the Ca2+-ATPase MCA8, both located on nuclear membranes, have been shown to participate in nuclear Ca2+ oscillation of this symbiotic signalling pathway of M. truncatula (Charpentier ). A current model considers these three components DMI1 (or CASTOR and POLLUX), CNGC15, and MCA8 sufficient to generate nuclear calcium oscillations, with DMI1, CASTOR, and POLLUX proposed as a K+-permeable channel, even with only a moderate preference for K+ over Na+ and Ca2+. In this model, a secondary messenger, hypothetically a cyclic nucleotide, binds to CNGC15, causing its activation and the release of Ca2+; DMI1 allows movement of K+ to balance the transmembrane charge induced by Ca2+ movement, and Ca2+ would be finally taken up again by the action of MCA8 (Charpentier ; Charpentier, 2018). Another recent work, however, showed that CASTOR exhibits high Ca2+ selectivity over K+ or Na+, and proposed that DMI1, POLLUX, and CASTOR can by themselves function as Ca2+ release channels in the nucleus of the respective organisms, behaving as a Ca2+ release channel in generating Ca2+ spiking (Kim ). Further experiments are thus needed to fully clarify the role and characteristics of these channels, but also to understand the mechanism by which Ca2+ spiking in the nucleus is activated upon ligand binding by receptors. Even though the DMI1 and CNGC15 gene seem to be conserved in all land plants, being involved in modulating their nuclear Ca2+ signatures, their presence and possible function in algae have not been investigated yet (Leitão ). Interestingly, referring to Phytozome C. reinhardtii proteome v5.5, two predicted proteins sharing respectively 30% and 25% sequence identity with M. truncatula DMI1 and annotated as POLLUX-related ion channels, are present (Cre11.g479350 and Cre14.g615800, Table 2). The C. reinhardtii genome also contains three CNGC genes and encodes an IP3R, that could have a role in nuclear Ca2+ dynamics as well as their plant or animal counterpart, respectively (Table 2); nevertheless, their functions and subcellular localization are still unknown (Verret ). In summary, it remains to be shown whether the nucleus could function as a Ca2+ signalling and/or decoding compartment in C. reinhardtii, along with the investigation of the possible nuclear Ca2+ toolkit and the mechanisms encoding Ca2+ signals into specific information.

Flagella as Ca2+ signalling compartments

As a motile organism, C. reinhardtii has two flagella emerging from one side of the cell that, together with basal bodies and flagellar root system, have been highly conserved throughout evolution (Pazour and Witman, 2009). Ca2+-dependent signalling processes have been shown to be crucial to many aspects of their function, including a direct control of beat frequency and waveform of flagella, but also to be involved in phototactic responses, chemotaxis, and mechanosensation (Smith, 2002; Sakato ; Fujiu ; Wakabayashi ). Moreover, Ca2+ signalling was reported to have a role in other flagella-related physiological functions of C. reinhardtii, such as in the mating process, in the regulation of gliding motility, and in the maintenance/excision of the flagella (Goodenough ; Huang ; Wheeler ; Collingridge ; Hilton ). A recent work has also provided new evidence that support the regulatory role of Ca2+ in the interactions between intrafragellar transport trains and flagella membrane adhesive glycoproteins, modulating flagella adhesion and gliding motility (Fort ). Chlamydomonas reinhardtii has been used as a model system to understand flagella structure and function, and many stimulus-specific flagellar Ca2+ signals have already been characterized and associated with the related physiological process, together with the participating Ca2+ signalling toolkit. Nevertheless, some underlying mechanisms remain unclear, and many individual components still have to be identified or compiled into the whole signalling pathway. Flagellar Ca2+ signalling in C. reinhardtii has recently been summarized by excellent and detailed reviews, which we advise readers to consult for more details on the above-mentioned flagellar and Ca2+-dependent processes and mechanisms (Wheeler, 2017; Wheeler ). Therefore, here we mainly focus on the possible roles of flagella as independent compartments in the intracellular Ca2+ signalling processes. Chlamydomonas reinhardtii flagella in fact display a characteristic Ca+ signalling toolkit, with several unique Ca2+-permeable channels, transporters, and Ca2+-binding sensors. Known examples include the Ca2+-permeable VDCC, CAV2, involved in Ca2+ influx and primarily localized toward the distal part of flagella, activated by the TRPV subfamily channel TRP11, located instead in the proximal region of the flagella; moreover, a homologue of a mammalian Ca2+-permeable TRP channel was also observed to be accumulated in the flagella, namely PKD2, together with a TRP channel that probably resides near the flagella and mediates Ca2+ entry, ADF1/TRP15 (Table 1, 2) (Huang ; Fujiu et al., 2009, 2011; Hilton ). Recent works have attributed the TRP15 protein name to the C. reinhardtii Cre09.g397142 gene (Arias-Darraz b; Hilton ), even though it has been previously associated with another gene (Cre10.g422750, accession number XP_001702589) (Fujiu ), thus a unified nomenclature would certainly be necessary to further classify and study this protein. Interestingly, PKD2 was recently reported to be attached to mastigonemes, extracellular polymers of the glycoprotein mastigoneme-like protein 1 (MST1), and the axoneme, suggesting its mechanosensory role in motile cilia (Liu ). In addition, three Ca2+ sensor kinases have been identified in the flagella proteome, namely CDPK1, CDPK3, and CDPK11, having a crucial role in the flagella length assembly and length control, whereas a Ca2+-binding CBL-like1 protein was recently also locali//////////zed in flagella, interacting with VGCCs (Table 1) (Pazour ; Liang and Pan, 2013; Kumar ). Such confined localization of these molecular players further supports the experimental evidence that a C. reinhardtii single flagellum can act as an independent Ca2+ signalling entity from the cell body. Ca2+ imaging of C. reinhardtii cells loaded with the Oregon green BAPTA dextran Ca2+ indicator, in fact showed highly compartmentalized Ca2+ elevations in individual flagella, differing from Ca2+ elevation in the cytosol or in the other flagellum (Collingridge ). Similarly, a recent study on C. reinhardtii Ca2+ responses further demonstrated compartmentalized Ca2+ elevations in flagella upon hypo-osmotic shock, suggesting that the flagella and cytosol can act as independent Ca2+ signalling compartments, enabling localized cellular responses (Bickerton ). However, even though these studies showed compartmentalized flagellar Ca2+ signals, other flagella signalling processes are closely associated with the cell body, including phototactic responses, in which light sensing by the eyespot leads to the activation of flagellar Ca2+ channels, controlling the beating balance (Harz and Hegemann, 1991). Mechanosensitive flagella that show compartmentalized Ca2+ signals, in fact, could also activate accessory pathways, generating mobile second messengers or activating alternative mechanisms, thus influencing cytosolic Ca2+ signalling and relaying the perceived information. Flagella in C. reinhardtii have thus been shown to represent a highly dynamic and excitable signalling compartment, able to act in Ca2+ signalling either independently or in combination with the cell body. Further analyses are required to elucidate the possible interaction between cytosolic and flagellar Ca2+ signalling, both in the perception and in the transmission of a stimulus, to aid in understanding the mechanosensitive properties of these organelles and their role in intracellular Ca2+ signalling.

Molecular tools to monitor and measure in vivo intracellular Ca2+ concentrations in microalgae

Ca2+ imaging, together with a suitable set of optical Ca2+ indicators, represents an extremely powerful technique to investigate how Ca2+ can exerts its action on the above-mentioned biological processes. Among the available Ca2+ indicators, synthetic Ca2+-sensitive dyes and genetically encoded Ca2+ indicators (GECIs) can be distinguished as efficient tools for in vivo measurements of both Ca2+ concentrations and fluxes. A study of Ca2+ dynamics in living cells was initially performed through fluorescent Ca2+-sensitive dyes (e.g. Fura-2, indo-1, and Calcium Green-1), which have been successfully loaded into animal, plant, and even algal cells (Malgaroli ; McAinsh ; Russ ; Braun and Hegemann, 1999). They are highly versatile tools for analysing cellular Ca2+ responses, and the use of their acetoxymethyl ester forms also conferred on them membrane-permeable properties. Acetoxymethyl ester derivatives, however, caused several issues in many plant and algal cells, due to unequal loading and dye compartmentalization (Braun and Hegemann, 1999; Hong-Hermesdorf ). Conversely, their dextran-conjugated form overcame these limitations, being membrane impermeable and allowing robust and reproducible cytoplasmic dye loading through biolistic delivery methods (Bothwell ). In C. reinhardtii, this technique enabled visualization of Ca2+ transients in both the cytosol and flagella of single cells, helping to elucidate the role of Ca2+ signalling in different algal biological processes. Dextran-conjugated Fluo-4, for example, was used in combination with a reference inert marker dye (Texas red) to study the role of cytosolic [Ca2+] elevation in the deflagellation process of C. reinhardtii cells (Wheeler ). Subsequently, two studies in C. reinhardtii exploited the versatility of Ca2+-responsive Oregon Green (OG)-BAPTA dextran to evidence the role of Ca2+ signalling in the gliding motility process and osmotic stress responses (Collingridge ; Bickerton ). OG, compared with the previously mentioned Fluo-4, has a much greater fluorescence in the Ca2+-unbound state, thus reducing technical issues related to chlorophyll autofluorescence, and aids imaging by total internal reflection fluorescence (TIRF) microscopy (Bickerton ). These are few examples of the Ca2+-sensitive dyes more recently used to investigate Ca2+ dynamics in C. reinhardtii, even though the commercially available list of their variants displays a broad range of Ca2+ affinities, brightness, and spectral properties. However, even though the use of these dyes has been proven to be reliable and allowed fundamental discoveries to be made, they clearly present some limitations and disadvantages. They in fact need a delivery method, with associated technical expertise and a low proportion of loaded cells, consequently suffering from low throughput, high variability, and being prone to artefacts.

More recently, the analysis of Ca2+ dynamics in living cells has been revolutionized by the introduction of GECIs, that enabled real-time, spatially and temporally resolved imaging of Ca2+ levels in different cell types and organisms, and even in different subcellular compartments by specific targeting of GECIs to organelles (Costa ). Readers are advised to consult the excellent recent reviews on this topic for more details on application and properties of GECIs in animal and plant cells, in addition to their use in Ca2+ imaging techniques (Koldenkova and Nagai, 2013; Costa and Kudla, 2015; Costa ). As stated in these works, one of the first GECIs to be developed was the aequorin-based Ca2+ probe, which allowed monitoring of Ca2+ dynamics by photon emission measurements in transformed cells after reconstitution of the aequorin holoenzyme with exogenously applied coelenterazine (Costa and Kudla, 2015). Such probes are an excellent tool to measure Ca2+ dynamics and signals triggered by different stimuli at the level of animal, plant, and algal single cells, but also in cell populations or entire tissues. Interestingly, as a bioluminescent reporter, it has an intrinsically high signal-to-noise ratio, and it requires no damaging excitation light; such properties have made it an excellent tool to measure Ca2+ levels in chlorophyll-containing cells (Martí ). Moreover, as a GECI, and being mostly insensitive to variation of pH and Mg2+, it was used to efficiently monitor [Ca2+] changes at the level of single organelles, or their specific subcompartments (Costa ). In plants, they were successfully targeted and applied to measure Ca2+ dynamics in the vacuole, nucleus, Golgi apparatus, mitochondria, plastids/chloroplasts, chloroplast stroma, the outer and inner membranes of its envelope, and the thylakoid lumen and membrane (Costa ). In microalgae, aequorin-based Ca2+ reporters so far have only been targeted to the cytosolic compartment (Falciatore ; Aiyar ). In particular, an aequorin-based Ca2+ reporter was expressed in the diatom Phaeodactylum tricornutum, which was able to reveal the crucial role of Ca2+ dynamics in sensing systems for detecting and responding to fluid motion, osmotic stress, and iron, as a key nutrient (Falciatore ). Thereafter, an aequorin Ca2+ reporter was also expressed and used to measure [Ca2+] transients in the cytosol of C. reinhardtii cells, showing that the cyclic lipopeptide orfamide A can trigger an increase in cytosolic [Ca2+] (Aiyar ). This study has also confirmed in C. reinhardtii the strong increase in cytosolic [Ca2+] in response to acidification and salt stress, previously reported by the use of the synthetic Ca2+ dye OG-BAPTA dextran (Bickerton ). These results further suggest that both the Ca2+ indicators could represent a reliable tool to measure these reported events. However, the possibility to target aequorin-based probes to specific subcellular compartments, together with the previously mentioned crucial advantages, strongly suggest these probes as a method of choice in order to advance the study of Ca2+ dynamics in microalgae, toward an organellar and localized point of view. Moreover, aequorin-based probes still remain one of the preferred methods when accurate quantifications of Ca2+ levels are needed, opening up the possibility to measure the as yet unknown algal organellar [Ca2+] (Ottolini ). Recently, a GECl (G-GECO) has been expressed in the flagella of C. reinhardtii, allowing direct visualization of localized or propagating flagellar [Ca2+] elevations but, to our knowledge, no other studies have reported the targeting of GECIs to different specific subcellular compartments of algal cells (Fort ). Similarly, dual-fluorescent protein (FP) ratiometric GECIs have not been applied so far to study Ca2+ signalling in microalgae. Ratiometric GECIs are typically based on combinations of green fluorescent protein (GFP)-related proteins (i.e. Cameleons) and display high spatio-temporal resolution and sensitivity, thus being extremely useful in Ca2+ signalling studies. Cameleons [e.g. yellow Cameleon 3.6 (YC3.6)] are Förster resonance energy transfer (FRET)-based indicator proteins, which harbour cyan and yellow fluorescent proteins (CFP and YFP, or other spectral variants) linked together by the Ca2+-binding protein CaM and the CaM-binding peptide M13 (Koldenkova and Nagai, 2013). Ca2+ recordings with these ratiometric indicators rely on ratio shifts, thus these measurements are not influenced by the expression level of the indicators. Moreover, as widely used GECIs, they are available for Ca2+ measurements at the level of different intracellular compartments and even for simultaneous measurement of Ca2+ dynamics in different subcellular localizations (Costa and Kudla, 2015). Other single-FP GFP-based Ca2+ biosensors, however, such as GCaMP3, GCaMP6, or the green and red variant of GECO1 (G-GECO1 and R-GECO1), were also localized to different compartments simultaneously, also exhibiting in some cases significantly higher signal change compared with YC3.6 in response to several stimuli (Kleist ; Kelner ). All these mentioned GECIs detect changes in [Ca2+] in the ranges that occur physiologically and have been widely used as the main Ca2+ indicators in both animal and plant cells. Hence, they could also be potentially applied and selectively targeted to subcellular compartments in algal cells. The recent development of efficient strategies to strongly enhance recombinant gene expression in C. reinhardtii, in particular, has substantially favoured the use of genetically encoded tools in this organism (Baier ). These strategies have in fact already allowed the expression of useful transgenes or the intracellular accumulation of heterologous proteins of interest (Baier ; Perozeni ; Pivato ). Moreover, the strategic design of a versatile vector toolkit for nuclear transgene expression paved the way for targeting a protein of interest to many different subcellular compartments in C. reinhardtii cells. A heterologous fluorescent protein has in fact been successfully targeted to the cytoplasm, the nucleus, cellular microbodies, the mitochondria, chloroplast stroma, and even the pyrenoid region of algal cells (Lauersen ). These novel tools thus could provide an efficient strategy to stably express and target GECIs to C. reinhardtii subcellular compartments. Accordingly, we recently obtained YC3.6 stable expression through this approach in C. reinhardtii cytosol and chloroplast (Fig. 2). GECI-expressing strains will allow routine imaging of Ca2+ signalling, potentially enabling the study of biotic and abiotic stress-induced cytosolic and intraorganellar Ca2+ transients in this green model alga. Besides C. reinhardtii, however, such Ca2+-related tools could certainly also be expressed in other algal model species, starting with those where genetic tools to stably express heterologous genes have been developed. The marine diatom P. tricornutum could be one of them, where Ca2+ was reported to have a central role in several aspects of cell biology, again including environmental perception of abiotic and biotic stimuli. As discussed in this section, an aequorin-based Ca2+ sensor has already been used in P. tricornutum; however, the development of transgenic lines, also stably expressing other novel GECIs in different subcellular compartments, could represent a useful tool to address the above-mentioned question in diatoms too. Examples relating to this are two recently published works in which the expression of the previously mentioned R-GECO1 in P. tricornutum and Thalassiosira pseudonana has allowed both the identification of a novel class of Na+- and Ca2+-permeable, voltage-gated channels, and the discovery of a Ca2+ signalling pathway in diatoms to sense and rapidly respond to increases in phosphorus availability (Helliwell , 2021).

Fig. 2.

Confocal microscopy images of C. reinhardtii cell lines expressing YC3.6. Different subcellular localizations of targeted YC3.6 are displayed: cytosol (A) and chloroplast (B). Channels and false colours: mVenus moiety of YC3.6 (YFP, yellow), chlorophyll autofluorescence (Chlorophyll, red), and the overlay of the previous channels (MERGE). Scale bars=5 µm.

Conclusions

Several pieces of evidence support a crucial role for Ca2+ signalling in many aspects of C. reinhardtii cell biology, including environmental perception of stimuli, stress responses, motility-related processes, and regulation of photosynthesis. The requirement for Ca2+ in these signalling processes has been shown to be conserved, with different algal lineages, plants, or even animal cells displaying common elements in the underlying cellular mechanisms used to generate and sense Ca2+ elevations. However, C. reinhardtii cells present a characteristic intracellular Ca2+ signalling toolkit, with several unique elements (i.e. ChR), accompanied by other Ca2+-related molecular players conserved among Viridiplantae and other ‘animal-like’ Ca2+ signalling components such as Ca2+ transporters and Ca2+-binding proteins (Wheeler and Brownlee, 2008; Verret ). It is clear, however, that our knowledge of the identity, roles, and functions of most of these different channels/transporters, possibly involved in Ca2+ fluxes among the different intracellular membranes, still remains limited. The study of C. reinhardtii knockout/knockdown mutants for the previously mentioned putative Ca2+-related proteins could greatly help the identification and characterization of many of these components. CRISPR/Cas9-induced knockout and knock-in mutant strains could indeed be exploited with this purpose, to study gene function and to elucidate the role of uncharacterized Ca2+-related proteins (Shin ). Furthermore, C. reinhardtii strains recently generated by the Chlamydomonas Library Project (CLiP), a genome-wide indexed library of mapped insertion mutants, could also represent a powerful resource to accelerate this process (X. Li ). Moreover, the development of high-throughput characterization systems of protein localization and protein–protein interactions could further enable the systematic placement of Ca2+-related genes into pathways and the discovery of new uncharacterized ones. Mechanisms of intracellular Ca2+ signal transduction were also not completely elucidated, including how influx and efflux through these molecular players are regulated in a concerted manner to translate specific information into unique Ca2+ transients. To some extent, many Ca2+-based signal transduction networks have been reported in land plant and animal cells, including the possible role of each subcellular compartment in the shaping of the global Ca2+ signal. Conversely, our understanding of intracellular and organellar Ca2+ signalling in algae, and in particular in the green model alga C. reinhardtii, remains in its infancy. A useful approach to understand how different organelles could act as a source of Ca2+, for example, could be represented by the study of C. reinhardtii mutant strains lacking specific Ca2+ transporters/channels, together with pharmacological approaches using Ca2+ chelators or inhibitors of differentially distributed Ca2+ channels (Costa ). Such methods, applied to C. reinhardtii mutant strains stably expressing selectively targeted GECIs, will allow the imaging of cytosolic and intraorganellar [Ca2+] transients, possibly leading to the characterization of the depleted channels/transporters and to the definition of their role in the generation of different cytosolic and organellar stimulus-induced Ca2+ transients. A clear knowledge of the molecular players involved in C. reinhardtii organellar Ca2+ signalling, together with a precise understanding of their role in the regulation of cytosolic and intraorganellar Ca2+ fluxes, could really help to clarify the role of each organelle in the shaping of intracellular Ca2+ dynamics and consequently in the regulation of each Ca2+-based biological process. Together these studies could elucidate many aspects of the functioning of Ca2+-dependent signalling pathways in C. reinhardtii, providing new important insights both into their evolution among eukaryotes and into the understanding of their role in the perception and response to environmental stimuli.

Microalgae currently represent a renewable and sustainable source of biomass and highly valuable metabolites, whose composition and production are highly affected by environmental factors, including biotic and abiotic stresses (Benedetti ). A further understanding of microalgal mechanisms of stress perception and response could thus greatly help the development of promising strategies for accumulation of biomass and high value compounds. Newly discovered approaches of metabolic improvement and genetic engineering could then be applied to other commercially relevant species, such as Chlorella vulgaris, Dunaliella salina, and Haematococcus lacustris, toward the optimization of biomolecules and biomass production in a microalgal biorefinery concept.