Calcium (Ca2+) signaling is involved in the regulation of diverse biological functions through association with several proteins that enable them to respond to abiotic and biotic stresses. Though Ca2+-dependent signaling has been implicated in the regulation of several physiological processes in Chlamydomonas reinhardtii, Ca2+ sensor proteins are not characterized completely. C. reinhardtii has diverged from land plants lineage, but shares many common genes with animals, particularly those encoding proteins of the eukaryotic flagellum (or cilium) along with the basal body. Calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, is an important effector of Ca2+ signaling in animals, while calcineurin B-like proteins (CBLs) play an important role in Ca2+ sensing and signaling in plants. The present study led to the identification of 13 novel CBL-like Ca2+ sensors in C. reinhardtii genome. One of the archetypical genes of the newly identified candidate, CrCBL-like1 was characterized. The ability of CrCBL-like1 protein to sense as well as bind Ca2+ were validated using two-step Ca2+-binding kinetics. The CrCBL-like1 protein localized around the plasma membrane, basal bodies and in flagella, and interacted with voltage-gated Ca2+ channel protein present abundantly in the flagella, indicating its involvement in the regulation of the Ca2+ concentration for flagellar movement. The CrCBL-like1 transcript and protein expression were also found to respond to abiotic stresses, suggesting its involvement in diverse physiological processes. Thus, the present study identifies novel Ca2+ sensors and sheds light on key players involved in Ca2+signaling in C. reinhardtii, which could further be extrapolated to understand the evolution of Ca2+ mediated signaling in other eukaryotes.

Introduction

Calcium ion (Ca2+) is a versatile second messenger, which is involved in the regulation of diverse signaling pathways. It is involved in the direct regulation of several molecular and physiological processes in diverse organisms [1–4]. In plants, Ca2+ signaling is reported to be involved in developmental and physiological processes including regulation of the abiotic and biotic stress responses [5–10]. A highly dynamic Ca2+ signaling mechanism also exists in the chlorophyte alga, Chlamydomonas, particularly, with the flagellar activities like motility, mating, flagellation/deflagellation and flagellar length regulation [11–20]. In addition to these activities, the role of Ca2+ signaling in the regulation of general stress responses as well as in processes pertaining to the cell body have not been thoroughly explored in Chlamydomonas. Some Ca2+ binding proteins have been characterized like Ca2+ dependent protein kinases (CDPKs), CDPK3 and CDPK4 and Ca2+ sensor, CAS in Chlamydomonas [11,21,22]. In plants, major proteins that are involved in regulating Ca2+ associated stress responses are CDPKs, calmodulin (CaMs) and calcineurin B-like proteins (CBLs) [23,5,24]. During Ca2+ signaling, CBLs and CaMs act as sensor relay proteins, which interact with their effector proteins to transmit Ca2+ signals [5]. However, CDPKs are sensor–effector proteins, which can directly phosphorylate and hence regulate their targets [25,26]. These Ca2+ sensors can target proteins such as protein kinases, metabolic enzymes, transcription factors, channels/transporters or cytoskeleton associated proteins. CBLs interact and regulate the activity of protein kinases, known as CBL-interacting protein kinases (CIPKs) and form the CBL-CIPK module, which regulates diverse stress signaling pathways, intracellular ion homeostasis, germination and seed development [27].

It has been reported that the CBL-CIPK signaling module originated during early eukaryotic evolution [28–30]. However, either component of this module was found to be missing in many green algae suggesting selective loss during evolution [28,29,31–33]. CBLs have been shown to work independent of CIPKs in plants [34]. Thus, owing to the importance of CBL-CIPK network in various signaling as well as in development and physiological processes in plants, we explored the existence and functional significance of this important Ca2+ signaling component in C. reinhardtii. CIPKs were not identified, however, 13 novel CBL-like proteins (CrCBL-likes) were identified in C. reinhardtii through genome-wide analysis. They have characteristic EF-hand like Ca2+ binding motifs similar to those present in Arabidopsis CBLs (AtCBLs). Furthermore, one of the members, CrCBL-like1, was characterized in detail for its Ca2+ binding property, expression pattern and interacting partners. This is the first report that demonstrates the presence of CBL-like Ca2+ sensors in C. reinhardtii and also provides functional relevance of one of the archetypical genes. In future, detailed functional characterization of the CrCBL-like proteins will pave the path for understanding their significance in different physiological as well as developmental processes of the green algae.

Materials and methods

In silico analysis for identification of putative homologs of CBL in Chlamydomonas

Multiple sequence alignment of AtCBLs was performed and used as an input to generate HMM profile using HMMER software [35]. The generated profile was used with default parameters in the HMM search program of the HMMER package against C. reinhardtii proteome (Uniprot id: UP000006906). All the hits with significant positive scores were selected and then examined individually for the accessory domains. These hits were individually confirmed by BLASTP. The sequence analysis and motif detection were carried out using Geneious Version 7 (https://www.geneious.com) software with default parameters. The structural modeling of the CrCBL-like1 protein was performed using web based SWISS-MODEL program [36] and the structure was visualized using PyMOL X11Hybrid (Ver 2) (https://pymol.org).

Chlamydomonas reinhardtii cell culture

Chlamydomonas reinhardtii wild type strain CC-124 was procured from Chlamydomonas resource center (www.chlamycollection.org). Culture was grown using TAP (tris-acetate-phosphate media, pH 7.4 and supplemented with Hutner trace elements) at 25°C in a shaker incubator (120 rpm) in a synchronized manner (14 h light and 10 h dark condition). Light exposure was provided using white fluorescent light (30–40 µmol m−2 s−1).

Cloning, expression and purification of the CrCBL-like1 protein

The ORF of Cre08.363750 (referred to as CrCBL-like1 in this study) was cloned into pET21a vector and the sequenced construct was transformed into Escherichia coli BL21λDE3 cells. The transformed cells were grown at 37°C until OD of 0.5–0.6 after which the temperature was reduced to 28°C and 0.1 mM IPTG (isopropyl thiogalactosidase) was used for induction. The cells were pelleted after 6–7 h by centrifuging at 5000 g for 10 min and stored at −80°C. The stored cells were thawed and re-suspended in 30 ml of resuspension buffer (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM Imidazole, 5 mM 2-mercaptoethanol and 5% Glycerol) for purification of the expressed protein. The cells were lysed by sonication for 10 min with 20 s on/10 s off cycle in presence of 1 mM PMSF (phenylmethylsufonyl fluoride) in the buffer. The sonicated cells were treated with 1% Triton X-100 for 30 min at 4°C and centrifuged at 20 000 g for 30 min. The supernatant was incubated with 1 ml of Ni-NTA beads (Qiagen) for 90 min. Further purification was performed according to Qiagen protocol with the following buffers — Wash buffer 1: resuspension buffer with additional 150 mM NaCl; Wash buffer 2: resuspension buffer + 20 mM Imidazole; Elution buffer: resuspension buffer + 350 mM Imidazole. One to two micrograms of protein was visualized on 12% SDS–PAGE and 1 µg of protein was used for Western blot analysis.

Antibody against CrCBL-like1 protein

Affinity purified CrCBL-like1 protein was used for raising antibody in rabbit using commercial facility provided by Genei Bangalore, India. The antibody was used for CrCBL-like1 detection.

Native PAGE, SDS–PAGE and Western blotting

For protein expression analysis, the frozen cells were re-suspended in 1× PBS supplemented with 1 mM PMSF and sonicated at 40% amplitude for 2 min. with 30 s on/off cycle. The prepared cell lysate was centrifuged at 10 000 rpm for 5 min. The clear supernatant containing the total cellular protein was dispensed and its concentration was estimated via nanodrop as well as Bradford assay. Thirty micrograms of protein was mixed with 6× Laemmli buffer and then resolved on 12% SDS–PAGE gel. Protein expression profile was visualized via Western blot using the antibodies as mentioned previously.

Anti-His antibodies and anti-mouse alkaline phosphatase (AP) conjugated secondary antibodies were used for detecting the recombinant proteins at a dilution of 1 : 1000 (Sigma H1029) and 1 : 10 000, respectively. For detecting endogenous CrCBL-like1 protein, rabbit anti-CrCBL-like1 antibody was used at a dilution of 1 : 3000 and anti-rabbit horseradish peroxidase (HRP) conjugated secondary antibody at a dilution of 1 : 5000, was used. Tubulin was visualized using mouse anti-tubulin antibody (Sigma) at a dilution of 1 : 5000 and anti-mouse HRP conjugated secondary antibody at a dilution of 1 : 5000. Western blot was performed according to the standard protocols. The blots were quantified using ImageJ tool (https://imagej.nih.gov/ij/).

Gel shift assay

For native PAGE, 6 µg of CrCBL-like1 was incubated with 2 mM CaCl2 and 5 mM EGTA for 30 min in 20 µl volume. Twenty microliters of 2× native PAGE dye was added and then separated on 12% polyacrylamide gel at 4°C for 3 h at 160 V. The same set of samples were analyzed on 15% SDS–PAGE.

Fluorescence spectroscopy

Ca2+ binding experiments were performed via steady state intrinsic fluorescence spectroscopy (Cary Eclipse fluorescence spectrophotometer, Agilent). CrCBL-like1 did not have any tryptophan residue, therefore, tyrosine (Tyr) fluorescence was used for Ca2+ binding studies. The 2 µM of CrCBL-like1 was used to measure the Tyr fluorescence. Change in fluorescence was detected upon adding Ca2+ and EGTA. The 2 mM CaCl2 and 5 mM EGTA was used in 50 mM Tris–HCl buffer pH 8 and 150 mM NaCl. Excitation wavelength — 280 nm, emission range — 285–400 nm, excitation bandwidth — 5 nm and emission bandwidth — 10 nm were used. For Ca2+ binding saturation curve, different concentrations of Ca2+starting from 0.01 µM to 3 mM were used. The saturation curve was plotted using fluorescence intensity measured at the wavelength 304.9 nm at Y-axis and free Ca2+ concentration in the X-axis. The data was fitted using non-linear regression in Graphpad Prism 5 and apparent Kd was calculated manually as midpoint of the sigmoidal curve assuming that 50% of CrCBL-like1 is bound to Ca2+. The experiment was performed thrice for statistical significance.

Gel exclusion chromatography

All the eluted fractions of proteins were pooled and concentrated to 20 mg/ml using 3 kDa Millipore filters. The protein was passed through Superdex 16/600 GL 75 Pg column at a 1 ml/min flow rate and the fractions containing the proteins were collected. Ca2+ binding was also observed via gel exclusion chromatography. Of CrCBL-like1, 1.2 mg was incubated with 2 mM CaCl2 and 5 mM EGTA for 30 min and run on Superdex 200 (10/300 GL) column. Gel filtration (GF) buffer: 50 mM Tris–HCl pH8 and 150 mM NaCl. During GF respective ligands (Ca2+/EGTA) were also added in the GF buffers.

RNA extraction, cDNA preparation and real-Time quantitative PCR

Chlamydomonas reinhardtii was grown in 50 ml medium until mid-log phase, i.e. OD of 0.5. Different stress conditions including cold (4°C), oxidative stresses i.e. methyl viologen (1 mM) and hydrogen peroxide (1 µM), heat (37°C) and salt (200 mM), were subjected by incubating the mid log phase culture from 30 min to 4 h. Samples were harvested at different time points and cells were frozen in liquid nitrogen and stored at −80°C. RNA was extracted via hot phenol method. The frozen cells were re-suspended into 300 µl of TES buffer (heated at 65°C) and then aliquoted into 1.5 ml micro-centrifuge tube. Magna lyzer beads (∼100 µl) were added to lyse the cells using hand held tissue grinder. Subsequently, 300 µl of hot phenol (1 : 1 ratio of acidic phenol pH 4.5 and chloroform) was added to the tube, mixed well by vortexing and incubated at 65°C for 30 min with constant shaking on dry bath heater. After 30 min, the tube was centrifuged at room temperature for 15 min at 12 000 rpm. The aqueous phase obtained after centrifugation was transferred to a new tube containing 240 µl of chilled isopropanol and 30 µl of sodium acetate (pH 5.2). The tubes were incubated at −80°C for 15–30 min. Subsequently, the tubes were centrifuged at 15 000 rpm for 30 min, supernatant was discarded and the pellet was washed with 75% ethanol for 5 min. The pellet was air dried for ∼30–40 min until residual ethanol evaporated and then, re-suspended in 50 µl of nuclease free water. The integrity of the RNA was visualized via agarose gel electrophoresis. One microgram of RNA was used for cDNA preparation (High-capacity cDNA Reverse Transcription kit, Thermo Fischer) and subsequently qPCR (USB HotStart-IT SYBR Green qPCR Master Mix, Affymetrix) was performed according to manufacturer's protocol. qPCR of CrCBL-like1 under salt and heat stress was performed as described above and data was evaluated to calculate the fold change in gene expression. The house keeping gene, Guanine nucleotide-binding protein subunit beta-like protein (Crgblp), was used as an internal control [37].

RNA-seq data analysis

The previously generated RNA-seq data were downloaded from chlamynet (http://viridiplantae.ibvf.csic.es/ChlamyNet/ChlamyNet.html) and used to analyze expression patterns of CrCBL-like orthologs from Chlamydomonas. The expression values of different genes were plotted as heat maps using complex heat map package of Bioconductor using R software.

CD spectroscopy

Purified recombinant CrCBL-like1 was dialyzed in 10 mM sodium phosphate buffer of pH 7, which was compatible for CD spectropolarimeter. The 2 mM CaCl2/EGTA was added to buffer solution containing 0.5 mg/ml of CrCBL-like1 and spectra were collected on a J-815 (Jasco, Japan) CD spectropolarimeter continuously purged by N2 and equipped with a temperature control system. Blank was set with 10 mM sodium phosphate buffer, pH 7. Spectral measurements were performed in far UV (188–260 nm) range using quartz cell of path length 1 nm in a thermostatic cell holder. CD spectra were recorded using average of three scans and data were collected and plotted using IGOR software version 3.14.

Immunofluorescence

Cells in early log phase were harvested and re-suspended in 1× PBS. Two hundred microliters aliquot of cell suspension was seeded on acid washed cover slips that were coated with poly-l-lysine. Cells were fixed with 3.7% paraformaldehyde in 1× PBS and permeabilized by submerging in cold 100% ethanol at −20°C for 10 min. Cells were then washed with 1× PBS containing 0.25 M NaCl at room temperature for 5 min. Brief washings were done with 1× PBST (PBS containing 0.5% triton X-100) followed by incubation with rabbit anti-CrCBL-like1 antibody (1 : 250 dilution) or pre-immune serum at 4°C overnight. Samples were then incubated with FITC conjugated anti-rabbit IgG antibody (Invitrogen), at 1 : 1000 dilutions. After three rounds of brief washings, cover slips were mounted on slides by applying antifade reagent (Slow Fade Gold, Molecular Probes). Slides were visualized by Leica TCS SP5 confocal microscope.

Statistical analysis

If not otherwise indicated, data were obtained from at least three independent experiments (n = 3). Mean values were calculated and used for the analysis of standard deviation (SD) or standard error (SEM). Data analysis was performed with Prism 6.0 software (GraphPad, La Jolla, California).

Results

Identification of putative CBL orthologs in Chlamydomonas reinhardtii

To identify calcineurin B-like Ca2+ sensor proteins in C. reinhardtii, a multiple sequence alignment of AtCBLs was performed and used as an input to generate HMM profile. This profile was used to search for CBL orthologs in Chlamydomonas proteome. A total of 35 proteins were identified, out of which 22 proteins were omitted because of high e-value or being the isoforms of other genes lacking EF-hand motif or having features of CDPKs. Thus, in total 13 proteins were identified as AtCBL orthologs and confirmed by BLASTP. Structural similarities and differences between these proteins were further analyzed. Multiple sequence alignments revealed sequence homology amongst these proteins especially at their EF-hand motifs, which is one of the major characteristics of the Ca2+ sensors (Figure 1A). Majority of AtCBLs studied so far, contained four EF-hand motifs and a similar trend was observed in C. reinhardtii. Figure 1A shows the presence of conserved EF-hand like motifs in all the identified proteins. Furthermore, structural modeling of Cre08.g363750 (CrCBL-like1) was performed using SWISS-MODEL. The PyMOL analysis revealed the presence of characteristic helix–loop–helix motif of EF-hands in the CrCBL-like1. EF-hand motif was more similar to calcineurin-B variant from Coccidioides immitis (PDB Id 5b8i) as the model showed one bound Ca2+ with the EF-hand 3, while no bound Ca2+ was detected using Arabidopsis thaliana CBL2 crystal structure (PDB id 2zfd.1) as template. The structural model of CrCBL-like1 showed the typical EF-hand fold with Ca2+ co-ordinated in a pentagonal-bipyramidal geometry with the side chain carboxylate of Asp-(X), Asp-(Z), Glu-(-Z), the main chain carbonyl of Asp-(Y), and that of Phe-(-Y) and serine with help of a water molecule (-X) (Figure 1B,C). CrCBL-like1 showed the presence of three canonical EF-hands (EF-hand 2, 3 and 4), while EF-hand 1 showed the EF-fold with some differences at 1st and 12th position, where Alanine and Threonine were present (Figure 1B,C). Thus, based on the presence of canonical and non-canonical EF-hands, the existence of CBL-like Ca2+ sensors in green algae C. reinhardtii was established.

Identification of CBL orthologs in Chlamydomonas reinhardtii.

Figure 1.
Identification of CBL orthologs in Chlamydomonas reinhardtii.

(A) Amino acid sequence alignment of the Chlamydomonas CBL orthologs showing conserved EF-hands. (B) Structural modeling of the Cre08.g363750 (CrCBL-like1), EF- hands 1, 2, 3 and 4 are shown in blue, green, red and magenta colors, respectively. (C) Ca2+-bound EF-hand 3 motif, Ca2+ is shown as an orange sphere. Amino acid residues involved in the Ca2+ coordination are shown in single letters.

Figure 1.
Identification of CBL orthologs in Chlamydomonas reinhardtii.

(A) Amino acid sequence alignment of the Chlamydomonas CBL orthologs showing conserved EF-hands. (B) Structural modeling of the Cre08.g363750 (CrCBL-like1), EF- hands 1, 2, 3 and 4 are shown in blue, green, red and magenta colors, respectively. (C) Ca2+-bound EF-hand 3 motif, Ca2+ is shown as an orange sphere. Amino acid residues involved in the Ca2+ coordination are shown in single letters.

Expression and purification analysis of CrCBL-like1 protein

CrCBL-like1 was expressed and purified (Materials and methods section) through nickel NTA chromatography and subsequently through gel exclusion chromatography. A single highly pure fraction was obtained at the size of ∼17 kDa. The identity of the protein was confirmed through Western blotting using anti-Histidine antibody (Figure 2A). The oligomerization of CrCBL-like1 was explored using gel exclusion chromatography. Protein eluted mostly as a dimer; however, higher-order oligomers were also visible but at lower concentrations (Figure 2B).

Heterologous expression, purification and Ca2+ binding studies of the CrCBL-like1.

Figure 2.
Heterologous expression, purification and Ca2+ binding studies of the CrCBL-like1.

(A) Purification of CrCBL-like1, P: Pellet after sonication and 20 000 g spin, S: Supernatant after 20 000 g spin, E1 and E2: Elutions (1 µg, 2 µg), M: Marker (precision plus protein dual color standards), WB: Western blot with penta-anti-Histidine monoclonal antibody. (B) Gel filtration profile of CrCBL-like1, 20 mg/ml protein was applied to Superdex 16/600 GL 75 Pg column at a 1 ml/min flow rate. The profile shows the presence of dimer and trace amounts of the higher-order oligomers. (C,D) Gel Shift assay upon Ca2+ binding via native PAGE and SDS–PAGE, 6 µg of CrCBL-like1 was incubated with 2 mM CaCl2 and 5 mM EGTA for 30 min in 20 µl volume. The 20 µl of 2× native PAGE dye was added and then run on 12% polyacrylamide gel at 4°C. The same sets of the samples were run on 15% SDS–PAGE at RT; (E,F) Detection of the Ca2+ binding through gel filtration (GF). The 1.2 mg of protein was incubated with 2 mM CaCl2 and 5 mM EGTA for 30 min and applied to Superdex 200 (10/300 GL) column. (E) GF profile of CrCBL-like1 without any treatment and with 5 mM EGTA. (F) GF profile of CrCBL-like1 with 2 mM Ca2+ and with 2 mM Ca2+ + 5 mM EGTA. GF buffer: 50 mM Tris–HCl pH8 and 150 mM NaCl. During GF, respective ligands (Ca2+ and EGTA) were also added in the GF buffers. (G) CD spectroscopy of CrCBL-like1 in presence of Ca2+ and EGTA. The 0.5 mg/ml of recombinant CrCBL-like1 protein was incubated with buffer containing indicated concentrations of Ca2+ and EGTA or both for 5 min on ice followed by CD analysis. (H) Fluorescence spectroscopy method for 2 µM of CrCBL-like1 was used to measure the tyrosine (intrinsic) fluorescence. Change in the fluorescence was detected upon adding Ca2+ and EGTA. The 2 mM CaCl2 and 5 mM EGTA were used in 50 mM Tris–HCl buffer pH8 and 150 mM NaCl. Excitation wavelength 280 nm, emission range 285–400 nm, excitation bandwidth 5 nm and emission bandwidth 10 nm. N = 4.

Figure 2.
Heterologous expression, purification and Ca2+ binding studies of the CrCBL-like1.

(A) Purification of CrCBL-like1, P: Pellet after sonication and 20 000 g spin, S: Supernatant after 20 000 g spin, E1 and E2: Elutions (1 µg, 2 µg), M: Marker (precision plus protein dual color standards), WB: Western blot with penta-anti-Histidine monoclonal antibody. (B) Gel filtration profile of CrCBL-like1, 20 mg/ml protein was applied to Superdex 16/600 GL 75 Pg column at a 1 ml/min flow rate. The profile shows the presence of dimer and trace amounts of the higher-order oligomers. (C,D) Gel Shift assay upon Ca2+ binding via native PAGE and SDS–PAGE, 6 µg of CrCBL-like1 was incubated with 2 mM CaCl2 and 5 mM EGTA for 30 min in 20 µl volume. The 20 µl of 2× native PAGE dye was added and then run on 12% polyacrylamide gel at 4°C. The same sets of the samples were run on 15% SDS–PAGE at RT; (E,F) Detection of the Ca2+ binding through gel filtration (GF). The 1.2 mg of protein was incubated with 2 mM CaCl2 and 5 mM EGTA for 30 min and applied to Superdex 200 (10/300 GL) column. (E) GF profile of CrCBL-like1 without any treatment and with 5 mM EGTA. (F) GF profile of CrCBL-like1 with 2 mM Ca2+ and with 2 mM Ca2+ + 5 mM EGTA. GF buffer: 50 mM Tris–HCl pH8 and 150 mM NaCl. During GF, respective ligands (Ca2+ and EGTA) were also added in the GF buffers. (G) CD spectroscopy of CrCBL-like1 in presence of Ca2+ and EGTA. The 0.5 mg/ml of recombinant CrCBL-like1 protein was incubated with buffer containing indicated concentrations of Ca2+ and EGTA or both for 5 min on ice followed by CD analysis. (H) Fluorescence spectroscopy method for 2 µM of CrCBL-like1 was used to measure the tyrosine (intrinsic) fluorescence. Change in the fluorescence was detected upon adding Ca2+ and EGTA. The 2 mM CaCl2 and 5 mM EGTA were used in 50 mM Tris–HCl buffer pH8 and 150 mM NaCl. Excitation wavelength 280 nm, emission range 285–400 nm, excitation bandwidth 5 nm and emission bandwidth 10 nm. N = 4.

CrCBL-like1 protein undergoes conformational change upon binding to calcium

Ca2+ binding property of CrCBL-like1 and the associated conformational change was explored through multiple methods. We initially, confirmed the Ca2+ binding through native PAGE and SDS–PAGE. Figure 2C demonstrates that CrCBL-like1 shows a shift in its position upon binding to Ca2+ and EGTA in native PAGE confirming the conformational transitions and hence, indicating Ca2+ binding. These conformational differences were also visible on SDS–PAGE (Figure 2D). Glutathione-S-transferase (GST) protein was used as a negative control, which did not show any Ca2+ and EGTA mediated mobility shift on both native PAGE and SDS–PAGE (Supplementary Figure S1). Additionally, distinct conformation adopted by Ca2+ bound and unbound states was confirmed through gel exclusion chromatography, which aligned well with native PAGE data. Ca2+bound and unbound CrCBL-like1 proteins eluted at different elution volumes, with bound protein eluting earlier than the unbound protein suggesting that it was in an extended conformation when bound to Ca2+ (Figure 2G,H). Effect of Ca2+ binding on the secondary structure of CrCBL1 was analyzed by far UV circular dichroism spectroscopy. CrCBL-like1 CD spectra showed intense negative ellipticity at 208 and 222 nm depicting its significant alpha helical content, which is characteristic of most Ca2+ binding proteins. In the presence of Ca2+ ions, a decrease in helical content was observed as the peaks at 208 and 222 nm shift to lower negative values as compared with the control (Figure 2E). The conformational change in CrCBL-like1 protein due to Ca2+ binding was also investigated using Tyr fluorescence assay. Figure 2F shows that the Tyr fluorescence is altered in the Ca2+ bound and unbound complexes, strengthening the conclusion that CrCBL-like1 protein binds Ca2+ leading to conformational changes. To estimate the Ca2+ binding affinity of CrCBL-like1 protein, a Ca2+ titration assay using fluorescence spectroscopy was performed. A two-step Ca2+ binding saturation curve of CrCBL-like1 protein was observed (Figure 3). At nano-molar Ca2+ ion concentrations, a Ca2+ dependent decrease in fluorescence maxima of Tyr was observed. However, upon increasing the Ca2+ concentration further to the micro-molar range, a Ca2+ dependent increase in fluorescence maxima of Tyr was observed. Before performing the Ca2+ titrations, the protein was dialyzed in a buffer containing 10 mM EGTA and Chelex to remove any bound Ca2+ from the protein as well as trace amounts of Ca2+present in buffer. The EGTA from the protein was removed by dialyzing it against the buffer treated with only Chelex. The apparent Kd estimated was 60 ± 2 nM and 30 ± 3 µM, respectively. The specificity of Ca2+ binding kinetics was confirmed by performing EGTA titration assay via fluorescence spectroscopy. A decrease in the Tyr fluorescence was observed thereby, confirming the specificity as well as reversibility of the Ca2+ binding reaction.

Ca2+ binding kinetics of the CrCBL-Like1.

Figure 3.
Ca2+ binding kinetics of the CrCBL-Like1.

The 2 µM of protein was used to measure the tyrosine fluorescence. Change in the fluorescence was detected upon adding the different concentrations of Ca2+ and EGTA. (A) The graph shows the dose-dependent change in tyrosine fluorescence upon Ca2+ titration. Ca2+ concentrations used were from 0.001 to 10 µM. The Kd calculated was 60 ± 2 nM. (B) Second dose–response curve was seen upon increasing the Ca2+concentration from 0.001 to 5 mM. The Kd calculated was 30 ± 3 µM. (C) Specificity of the Ca2+dependent change in tyrosine fluorescence was monitored by adding the different concentrations of EGTA as a chelator. EGTA concentration corresponds to (0.001–10 mM). The buffer used was 50 mM TRIS–HCl pH 8 and 150 mM NaCl. Excitation wavelength 280 nm, emission wavelength 305 nm, excitation bandwidth 5 nm and emission bandwidth 10 nm, N = 3.

Figure 3.
Ca2+ binding kinetics of the CrCBL-Like1.

The 2 µM of protein was used to measure the tyrosine fluorescence. Change in the fluorescence was detected upon adding the different concentrations of Ca2+ and EGTA. (A) The graph shows the dose-dependent change in tyrosine fluorescence upon Ca2+ titration. Ca2+ concentrations used were from 0.001 to 10 µM. The Kd calculated was 60 ± 2 nM. (B) Second dose–response curve was seen upon increasing the Ca2+concentration from 0.001 to 5 mM. The Kd calculated was 30 ± 3 µM. (C) Specificity of the Ca2+dependent change in tyrosine fluorescence was monitored by adding the different concentrations of EGTA as a chelator. EGTA concentration corresponds to (0.001–10 mM). The buffer used was 50 mM TRIS–HCl pH 8 and 150 mM NaCl. Excitation wavelength 280 nm, emission wavelength 305 nm, excitation bandwidth 5 nm and emission bandwidth 10 nm, N = 3.

In vivo expression of CrCBL-like1 and light induced change in expression pattern

CBLs have been extensively studied in plant systems with involvement in several physiological and developmental processes [38]. Most of the characterized CBLs are localized in plasma membrane and tonoplast where they regulate various proteins involved in sodium compartmentalization, potassium homeostasis and production of reactive oxygen species [38–42]. Since CrCBL-like1 protein was identified using A. thaliana CBLs as template; therefore, it may be speculated that CrCBL-like1 protein might also localize in plasma membrane and perform similar functions. Immunostaining with CrCBL-like1 specific primary antibodies followed by FITC labeled secondary antibodies revealed the expression of this protein around plasma membrane as well as near basal bodies (Figure 4).

Expression pattern of the CrCBL-like1 in Chlamydomonas and its interaction with voltage-gated Ca2+channel (VGCC).

Figure 4.
Expression pattern of the CrCBL-like1 in Chlamydomonas and its interaction with voltage-gated Ca2+channel (VGCC).

(A) CrCBL-like1 protein expression was observed near the plasma membrane and basal bodies of the Chlamydomonas cells. (B) CrCBL-L1 seen to be expressed in the flagella probed by CrCBL-like1 antibody. Cells showing the localization in flagella have been presented along with the focus on a single cell (inset). (C) CrCBL-like1 co-localized with VGCC in the flagella. Antibody used: CrCBL-like1 (anti-rabbit, red) and VGCC (anti-goat, green). (D) VGCC was present in immunoprecipitation (IP) elutions of CrCBL-like1. VGCC band detected at the expected molecular mass (300 kDa) corresponding to the input (total cell lysate; CrTCL). IP of pre-immune serum (PIS) serves as negative control where no bands corresponding to VGCC were detected. N = 3.

Figure 4.
Expression pattern of the CrCBL-like1 in Chlamydomonas and its interaction with voltage-gated Ca2+channel (VGCC).

(A) CrCBL-like1 protein expression was observed near the plasma membrane and basal bodies of the Chlamydomonas cells. (B) CrCBL-L1 seen to be expressed in the flagella probed by CrCBL-like1 antibody. Cells showing the localization in flagella have been presented along with the focus on a single cell (inset). (C) CrCBL-like1 co-localized with VGCC in the flagella. Antibody used: CrCBL-like1 (anti-rabbit, red) and VGCC (anti-goat, green). (D) VGCC was present in immunoprecipitation (IP) elutions of CrCBL-like1. VGCC band detected at the expected molecular mass (300 kDa) corresponding to the input (total cell lysate; CrTCL). IP of pre-immune serum (PIS) serves as negative control where no bands corresponding to VGCC were detected. N = 3.

The presence of any signal sequence in the protein might be responsible for trafficking of the protein near cell membrane. It has been reported that dual fatty acyl modification via N-myristoylation and S-acylation determines the plasma membrane associated targeting of CBLs and CIPKs in Arabidopsis [43]. Therefore, the protein sequences were analyzed for the presence of these motifs. Interestingly, a myristoylation site with very high confidence was detected; however, palmitolyation site was not detected using web based Expasy: Myristoylator palmitoylation tool (https://web.expasy.org/myristoylator/).

Furthermore, there are reports showing differential gene expression and localization pattern of CBLs inside the cells in plants under different stress conditions [44]. Since C. reinhardtii is a photosynthetic organism and needs light, therefore, any changes in CrCBL-like1 localization in response to changes in the light conditions were analyzed. As mentioned earlier, CrCBL-like1 predominantly localized near the plasma membrane and basal bodies (Figure 4A). However, in the middle of the light phase of 14 : 10 h light–dark cycle, CrCBL-like1 was found to be localized prominently in the flagella of C. reinhardtii cells (Figure 4B).

CrCBL-like1 interacts with voltage-gated Ca2+ channel (VGCC)

Ca2+ signaling is essential for flagellar movement and therefore, several VGCCs have been reported to be present along the flagellar membrane of C. reinhardtii [45], which regulate appropriate Ca2+ concentration in the flagella for their movement. Many Ca2+ binding EF-hand containing proteins have been reported to regulate these VGCCs through certain Ca2+ feedback mechanisms [46–48]. Since the light exposure led to migration of CrCBL-like1 protein to flagella, it might have a role in flagellar movement by regulating VGCC. Consequently, the interaction of CrCBL-like1 protein with VGCC was also analyzed. Indeed, the CrCBL-like1 protein co-localized with VGCC in the flagella during the mid-phase of light cycle (Figure 4C) as well as interacted with each other as established by co-immunoprecipitation analysis (Figure 4D).

CrCBL-like genes are involved in stress response

To understand the functional role of CrCBL-like genes in response to environmental stresses, the gene expression analysis of publically available RNA-Seq data of C. reinhardtii under multiple stress conditions was analyzed. The heatmap revealed varying expression patterns for most of the genes, except Cre03.g178150, which showed consistently higher expression in all condition. As noted, CrCBL-like1 as well as Cre15.g641250 expressed similar patterns, showing higher expression in various nutrient deprived conditions, suggesting that these genes respond transcriptionally to extrinsic cues (Supplementary Figure S2). The changes in CrCBL-like1 expression in response to additional stress conditions, such as heat, cold, salt, H2O2 and methyl viologen (not included in the RNA-seq data) were also analyzed. The CrCBL-like1 transcript as well as protein expression were tested by performing real-time PCR and Western blot analyses, respectively. In all stress conditions, a decrease in protein expression was observed when compared with untreated sample except in cold condition(s) (Figure 5A,B; Supplementary Figure S3). The gene expression analysis of CrCBL-like1 under the heat and salt stress conditions was performed and the results corroborated with the protein expression profiles (Figure 5C).

Expression analysis of the CrCBL-like1 in response to abiotic stress.

Figure 5.
Expression analysis of the CrCBL-like1 in response to abiotic stress.

(A) CrCBL-like1 protein expression upon cold, methyl viologen, hydrogen peroxide (H2O2) and heat treatments. (B) CrCBL-like1 protein expression upon salt stress. The expression of CrCBL-like1 protein changed differentially due to these treatments as detected by Western blot using CrCBL-like1 antibody. (C)CrCBL-like1 gene expression analysis upon heat and salt stress. The expression of CrCBL-like1 decreased after 2 h similar to the protein expression trend. N = 3.

Figure 5.
Expression analysis of the CrCBL-like1 in response to abiotic stress.

(A) CrCBL-like1 protein expression upon cold, methyl viologen, hydrogen peroxide (H2O2) and heat treatments. (B) CrCBL-like1 protein expression upon salt stress. The expression of CrCBL-like1 protein changed differentially due to these treatments as detected by Western blot using CrCBL-like1 antibody. (C)CrCBL-like1 gene expression analysis upon heat and salt stress. The expression of CrCBL-like1 decreased after 2 h similar to the protein expression trend. N = 3.

Discussion

Ca2+ is considered a second messenger in eukaryotes. The temporally and spatially defined ‘Ca2+ signatures’ are involved in signal transduction pathways regulating many biological processes. Ca2+-mediated signaling is widely employed in different physiological functions such as photobehavioural responses (phototaxis and photo-stop response), motility, chemotaxis and mating in C. reinhardtii [10–13]. A small variation in the intracellular Ca2+ concentration in the flagella of C. reinhardtii, leads to major changes in its flagellar beating patterns and hence, the photomovement [49]. Based on these variations in intraflagellar Ca2+, C. reinhardtii confronts flagellar biogenesis or deflagellation which involves various Ca2+ regulated proteins [14,15]. In spite of the prominent involvement of Ca2+ signaling in major biological functions, very few Ca2+ binding or Ca2+ signaling proteins have been reported in C. reinhardtii.

In recent years, Ca2+ sensor proteins have gained increasing attention due to their omnipresence in signaling pathways. The study presents the identification and molecular characterization of Ca2+ sensor proteins in C. reinhardtii using AtCBLs as baits through genome-wide analysis. In silico analysis revealed the presence of 13 orthologs of CBLs in C. reinhardtii (Figure 1A). Most of the characterized CBLs in other plant species contain typical Ca2+-binding motif known as EF-hands. They are present mostly in two pairs. The identified proteins contained 4 EF-hands like motifs as observed by multiple sequence alignment. Structural modeling of the potential EF-hand like motifs revealed similar pentagonal-bipyramidal geometry with conserved Ca2+ binding residues (Figure 1B). In plants, CBLs have been reported to bind Ca2+ and regulate several physiological and developmental processes by interacting with its cognate kinases, known as CIPKs [27]. CIPKs are specific kinases containing the NAF/FISL domains, along with kinase and phosphatase interaction motif. We did not find any CIPK orthologs in C. reinhardtii, which is in line with other reports [28,29]. Given that, we identified these putative Ca2+ sensor proteins using CBLs as input, it's intriguing to find the functioning of these identified proteins in the absence of CIPKs. However, CBL family members are reported to interact with other proteins as well, in addition to CIPKs. CBL3 interacts with AtMTAN, in addition to many CIPK family members through different regions, in Arabidopsis [50,51]. CBL10 also interacts directly with AKT1 that leads to inhibition of inward movement of K+ [52]. CBL10 was also shown to be involved in reproductive development without the involvement of any of the CIPKs in Arabidopsis [53]. These reports indicate that the interactions of the CBL or CBL-like proteins are not limited to CIPK members, suggesting that the identified proteins might have CIPK-independent novel physiological functions in C. reinhardtii.

The CrCBL-like1 protein was further characterized based on structural modeling. Its EF-hand motif showed similarity to that of calcineurin-B variant from Coccidioides immitis (PDB Id: 5b8i) as well as CreinCBL8 in C. reinhardtii [54]. CrCBL-like1 structural model showed the typical EF-hand fold with Ca2+ co-ordinated in a pentagonal-bipyramidal geometry as described in literature. The binding of Ca2+ to the EF-hand motifs is a functional feature of CBL proteins for relaying Ca2+ signals as shown in Figure 2. The binding of Ca2+ to CBLs using different methods like gel electrophoresis and mobility shift assays is well documented. The effect of Ca2+ binding on the mobility of the protein was assessed using native PAGE and SDS–PAGE. Under both denaturing and native conditions, the Ca2+ bound and unbound protein showed differences in their mobility suggesting a change in the conformation of the protein. In the native condition, the Ca2+-saturated protein had a larger apparent molecular mass compared with the Ca2+ depleted form, which suggests that Ca2+ binding leads to a more extended protein conformation (Figure 2C,D). This aligns well with the structural changes observed in most of the EF-hand containing proteins upon Ca2+ binding. It is well documented that the EF-hand motifs adopt an open conformation when bound to Ca2+, with both the alpha helices lying perpendicular to each other. In contrast, it adopts a closed conformation, with both the helices lying parallel to each other in Ca2+ free state.

The Ca2+-induced conformational changes in the protein observed using gel exclusion chromatography, were in agreement with the PAGE results. The data correlated well with the gel exclusion chromatography data wherein Ca2+ bound protein eluted earlier compared with the unbound protein. However, under the denaturing conditions, the Ca2+ bound protein migrated faster compared with EGTA bound protein suggesting vice versa (Figure 2E,F). Such faster movement has been observed in a few Ca2+-binding proteins when associated with Ca2+, in comparison with Ca2+ depleted proteins [55–58]. Since EGTA has a higher molecular mass compared with Ca2+, therefore, the net mass of the protein when bound to EGTA is higher, hence its mobility is retarded. Ca2+ binding often leads to change in the secondary structure of the protein. The distinct conformers of Ca2+ bound and unbound forms were confirmed through UV circular dichroism spectroscopy. Taken together, we established that Ca2+ associates with CrCBL-like1 protein.

In literature, the CBL proteins have been commonly referred to as Ca2+ sensors, however, there are very few reports about the estimation of Ca2+-binding affinity. Therefore, we determined Ca2+ binding kinetics of the protein by monitoring changes in the steady state intrinsic fluorescence of Tyr upon Ca2+ titrations. This method has been used previously to calculate the dissociation constant of Ca2+ binding proteins [59,60]. Since, CrCBL-like1 lacked tryptophan residues as also observed in most of the CBL proteins, we used the change in Tyr fluorescence upon Ca2+ titration to obtain a dose–response curve. CrCBL-like1 has 4 EF-hand motifs, of which, all except EF-hand 1, have Tyr residue(s) in their motif; therefore, change in the intrinsic fluorescence of Tyr could be easily used to study Ca2+ binding kinetics. CrCBL-like1 showed fluorescence maxima at 304.6 nm without any bound Ca2+, thus, to calculate the Ca2+-binding affinity, different concentrations of free Ca2+ were added and the change in the fluorescence at 304.6 nm was plotted. One high affinity Ca2+ binding site with apparent Kd of 60 ± 2 nM and a second site with Kd of 30 ± 3 µM were observed (Figure 3). Cells are known to maintain ∼20 000-fold gradient between their intracellular and extracellular Ca2+ concentrations, which range from nM to mM [4,61]. Our results indicate association around Kd of 60 nM, which is well within the physiological intracellular range of Ca2+, suggesting the intracellular role of CrCBL-like1. A higher Kd indicates a potential role of CrCBL-like1 under cellular conditions in which the Ca2+ levels are increased like a sudden change in the environment due to stress.

The CrCBL-like1 protein has a myristoylation motif at its extreme N-terminus similar to that of other well characterized CBLs [43]. This strengthens the localization pattern of the CBL-like1 protein near membrane as in our study. However, on exposure to light, specific redistribution and localization of CrCBL-like1 protein in flagella of C. reinhardtii was observed. This is an interesting result considering the fact that several VGCCs are reported to be present along the flagellar membrane of C. reinhardtii [45]. Many Ca2+ binding proteins containing EF-hands regulate VGCCs, directly or indirectly, through a Ca2+ feedback mechanism. Some EF-hand containing superfamilies like calcineurin, calmodulin or CaBPs (Calcium-Binding Protein) are involved in the regulation of the activity of VGCCs [46,62]. Thus, we observed an interaction between CrCBL-Like1 protein and VGCC in C. reinhardtii (Figure 4). VGCCs are involved in the regulation of Ca2+ levels in the flagella required for the movement. It is possible that CrCBL-like1 is involved in the regulation of VGCC and hence, maintenance of optimal concentration of Ca2+ in the flagella of C. reinhardtii. It has been reported that high Ca2+ concentration (>10−6 M Ca2+) is required along the flagella for its reverse movement and deflagellation occurs if Ca2+ ion concentration exceeds 10−6 M at basal bodies in C. reinhardtii [63]. This suggests the requirement of a Ca2+ ion sensor at such important location to regulate the Ca2+ concentration. Considering this, the presence of CrCBL-like1 in the basal bodies and its migration to flagella explains the requirement of CrCBL-like1 to potentially monitor the Ca2+ concentration. The photo-perception by the eyespot is followed by the activation of VGCCs localized along the flagella. Interaction between CrCBL-like1 and VGCC might also suggest Ca2+ dependent direct or indirect activation of VGCC. Furthermore, gene and protein expression analysis of CrCBL-like1 showed a decrease in overall expression pattern in response to various kinds of stresses (Figure 5), which is obvious, as Chlamydomonas tend to deflagellate under stress condition. Due to deflagellation, most of the CrCBL-like1 expression might be reduced as CrCBL-like1 is expressed in basal bodies and flagella.

This work opens a new avenue to study stress signaling in Chlamydomonas, considering that CrCBL-like1 could be used as a tool to study the role of Ca2+ in stress signaling as well as to explore the diversity of lesser-known CIPK-independent functions of CBL-like proteins.

Competing Interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was partly supported by the Department of Biotechnology (DBT), the Science and Engineering Research Board (SERB), the Department of Science and Technology (DST-PURSE grant), the Council for Scientific and Industrial Research (CSIR) and the University Grant Commission (UGC- SAP, DRSIII grant), India to G.K.P. and S.K.

Author Contribution

G.K.P. and S.K. conceptualized and designed the research plan. M.K., K.S., A.K.Y., K.K. and M.B. conducted the experiments. M.K., K.K., S.K. and G.K.P. interpreted and analyzed the data. M.K., K.K. and K.S. prepared the figures. M.K., K.S., K.K., S.K. and G.K.P. wrote the manuscripts with active inputs from others. All authors have reviewed the manuscript.

Acknowledgements

We are thankful to CIF-UDSC for the confocal microscopy facility. We are thankful to Prof. Dr. Peter Hegemann, Institute for Biology, Humboldt University, Germany for providing Chlamydomonas strains. We are also thankful to Dr. Malathi Bheri, Department of Plant Molecular Biology, University of Delhi South Campus for copy-editing the manuscript. M.K. acknowledges funding support from DST-SERB. K.K. acknowledges funding support from DBT. K.S. thanks ICMR for junior and senior research fellowship.

Abbreviations

     
  • Ca2+

    calcium

  •  
  • CaBP

    calcium-binding protein

  •  
  • CaM

    calmodulin

  •  
  • CAS

    calcium sensor

  •  
  • CBL

    calcineurin B-like protein

  •  
  • CDPK

    Ca2+-dependent protein kinases

  •  
  • CIPK

    CBL-interacting protein kinase

  •  
  • VGCC

    voltage-gated Ca2+ channel

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Author notes

*

These authors contributed equally.

Supplementary data