Research
Spatial and temporal specificity of Ca2+ signalling in Chlamydomonas reinhardtii in response to osmotic stress
Peter Bickerton1,2*, Simone Sello1,3*, Colin Brownlee1,4, Jon K. Pittman2 and Glen L. Wheeler1 1Marine Biological Association, Citadel Hill, Plymouth, PL1 2PB, UK; 2Faculty of Life Sciences, University of Manchester, Oxford Road, Manchester, M13 9PT, UK; 3Department of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy; 4School of Ocean and Earth Science, University of Southampton, Southampton, SO14 3ZH, UK
Summary Author for correspondence: Ca2+-dependent signalling processes enable plants to perceive and respond to diverse envi- Glen Wheeler ronmental stressors, such as osmotic stress. A clear understanding of the role of spatiotempo- Tel: +1 01752 633275 ral Ca2+ signalling in green algal lineages is necessary in order to understand how the Ca2+ Email: [email protected] signalling machinery has evolved in land plants. Received: 16 March 2016 We used single-cell imaging of Ca2+-responsive fluorescent dyes in the unicellular green Accepted: 25 June 2016 alga Chlamydomonas reinhardtii to examine the specificity of spatial and temporal dynamics of Ca2+ elevations in the cytosol and flagella in response to salinity and osmotic stress. New Phytologist (2016) 212: 920–933 We found that salt stress induced a single Ca2+ elevation that was modulated by the doi: 10.1111/nph.14128 strength of the stimulus and originated in the apex of the cell, spreading as a fast Ca2+ wave. By contrast, hypo-osmotic stress induced a series of repetitive Ca2+ elevations in the cytosol 2+ Key words: calcium, Chlamydomonas that were spatially uniform. Hypo-osmotic stimuli also induced Ca elevations in the flagella reinhardtii, flagella, green algae, osmotic that occurred independently from those in the cytosol. stress, signalling. Our results indicate that the requirement for Ca2+ signalling in response to osmotic stress is conserved between land plants and green algae, but the distinct spatial and temporal dynam- ics of osmotic Ca2+ elevations in C. reinhardtii suggest important mechanistic differences between the two lineages.
(CBL) calcium sensor proteins and their CBL-interacting pro- Introduction tein kinases (CIPKs)) (Hetherington & Brownlee, 2004; Edel 2+ The ability of an organism to sense and respond to its environ- & Kudla, 2015). As [Ca ]cyt elevations are generated by a ment is fundamental for its survival. In plants, calcium (Ca2+)- wide variety of stimuli, the spatial and temporal dynamics of 2+ dependent signalling processes play a central role in the response each [Ca ]cyt elevation are important in conveying specificity to many environmental stimuli, including mechanical stimula- (McAinsh & Pittman, 2009; Whalley et al., 2011). For exam- 2+ tion, osmotic stress, oxidative stress and temperature shock ple, plant [Ca ]cyt elevations can take the form of brief (Knight et al., 1992, 1997; Takahashi et al., 1997; Kiegle et al., spikes, extended elevations or a series of oscillations. In com- 2000; Pei et al., 2000). It is likely that the development of these bination with the broad range of downstream responders, the 2+ signalling pathways helped plants to colonize land and become distinct spatiotemporal dynamics of [Ca ]cyt elevations enable the dominant primary producers in terrestrial ecosystems. How- the cell to use Ca2+ signalling in response to many different ever, our ability to understand how and why these important sig- stimuli. nalling processes have evolved in plants is limited, as the role of Vascular plants lack clear homologues of several important Ca2+ signalling in response to environmental stimuli in the green classes of Ca2+ channel found in animal cells, such as the four- algae (Chlorophyta), which represent the other major lineage in domain voltage-dependent Ca2+ channels (VDCCs), the tran- the Viridiplantae, is not well characterized. sient receptor potential (TRP) channels and the inositol triphos- Many of the diverse environmental stimuli encountered by phate receptor (IP3R) (Wheeler & Brownlee, 2008; Verret et al., plants give rise to activation of Ca2+-permeable channels in 2010; Edel & Kudla, 2015). However, these channels are present the plasma membrane or internal membranes, leading to tran- in green algal genomes (Merchant et al., 2007; Wheeler & 2+ 2+ sient elevations in cytosolic Ca ([Ca ]cyt) which are sensed Brownlee, 2008), suggesting that there may be important differ- by a range of downstream effector proteins (such as calmod- ences in the signalling mechanisms between plants and green 2+ ulin (CaM), calmodulin-like proteins (CMLs), the calcium- algae. While [Ca ]cyt elevations in plants have been extensively dependent protein kinases (CDPKs) or the calcineurin B-like characterized (McAinsh & Pittman, 2009), direct observations of 2+ 2+ [Ca ]cyt elevations in green algae remain limited. [Ca ]cyt eleva- *These authors contributed equally to this work. tions have been observed in response to ‘light-off’ stimuli in the
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2+ freshwater alga Eremosphaera viridis (Bauer et al., 1997) and dur- the dynamics of [Ca ]cyt elevations generated by C. reinhardtii ing the settlement of Ulva linza zoospores (Thompson et al., cells in response to osmotic stress. We found that salinity stress 2+ 2+ 2007). [Ca ]cyt elevations relating to the deflagellation process and hypo-osmotic stimuli induced [Ca ]cyt elevations with dis- have been extensively characterized in the freshwater alga tinct spatial and temporal characteristics. Hypo-osmotic stress 2+ Chlamydomonas reinhardtii (Wheeler et al.,2008).Chlamydomonas also induced repetitive [Ca ]fla elevations that were independent 2+ 2+ reinhardtii can excise its flagella via a Ca -dependent signalling of [Ca ]cyt, indicating that the flagella, although continuous pathway in response to various stressors, including osmotic stress with the cytosol, can act as distinct Ca2+ signalling compartments (Quarmby, 1996; Meijer et al., 2002). The addition of 20 mM in response to environmental stimuli. 2+ 2+ external Ca to C. reinhardtii led to a series of repetitive [Ca ]cyt elevations that were linked to the process of flagellar excision Materials and Methods (Wheeler et al., 2008). The rapid and dynamic nature of these 2+ [Ca ]cyt elevations in C. reinhardtii was considerably different 2+ Algal strains and growth conditions from those observed in vascular plants, where [Ca ]cyt transients 2+ + commonly last for many seconds. Repetitive [Ca ]cyt spiking in Chlamydomonas reinhardtii Dangeard strains CC1021 mt (wild vascular plants also generally occurs over timescales of minutes type) and cw15 (CCAP 11/32 CW15+, cell wall deficient) were rather than seconds (e.g. Ca2+ spiking in root hairs induced by obtained from the Chlamydomonas Resource Center (University nodulation factors) (Wais et al.,2000). of Minnesota, St Paul, MN, USA) and the Culture Collection of Chlamydomonas reinhardtii is a motile organism, which may Algae and Protozoa (Scottish Association for Marine Science, account for the dynamic nature of some of the signalling pro- Oban, UK), respectively. The ift20 IFT20-mCherry strain was a cesses within the cell. The motile responses of swimming C. gift from Karl Lechtreck (Lechtreck et al., 2009). Cultures were reinhardtii cells to light have been well documented and are grown in standard Tris-acetate-phosphate (TAP) liquid medium 2+ 2+ ° mediated by changes in flagellar Ca ([Ca ]fla). Voltage-gated at 23 C with a 16 h : 8 h, light : dark cycle and a light intensity of Ca2+ channels in the flagella are activated by a whole-cell depolar- 100 lmol m 2 s 1. ization mediated by channelrhodopsin, a light-gated ion channel situated in the plasma membrane adjacent to the eyespot (Harz Biolistic loading of dextran-conjugated fluorescent dyes & Hegemann, 1991; Fujiu et al., 2009). In addition to swim- ming, C. reinhardtii cells can move by adhering to a substrate via Cells were simultaneously loaded with two fluorescent dyes, the their flagella and gliding along that surface (Bloodgood, 1981). Ca2+-responsive dye Oregon Green-BAPTA Dextran (OG) Gliding motility is mediated by the movement of adherent glyco- (10 000 MW) and the reference dye Texas Red Dextran (TR) proteins in the flagellar membrane, which are driven along the (10 000 MW) (Invitrogen Ltd, Paisley, UK). Dextran- length of the flagellar axoneme by the microtubule motors conjugated dyes were used to avoid the problems with dye com- responsible for intraflagellar transport (IFT) (Collingridge et al., partmentalization commonly found with plant and algal cells 2013; Shih et al., 2013). We have previously demonstrated that (Bothwell et al., 2006). OG was used for this study rather than the movement of IFT proteins during gliding motility is regu- Fluo-4 dextran used previously (Wheeler et al., 2008) as OG lated by flagellar Ca2+ signalling (Collingridge et al., 2013). The has a much greater fluorescence than Fluo-4 when in the Ca2+- flagella of gliding C. reinhardtii cells are arranged at 180° to each unbound state, which reduces technical issues associated with other and gliding motility initiates when the pulling force in one chlorophyll autofluorescence and also aids imaging of the flagellum overcomes the resistance of the other (Bloodgood, flagella by Total Internal Reflection Fluorescence (TIRF) 2+ 2+ 1981). Direct observation of flagellar Ca ([Ca ]fla) in gliding microscopy. Chlamydomonas reinhardtii cells were concentrated C. reinhardtii cells indicates the potential for complex and highly by centrifugation (400 g for 5 min) and washed with biolistic dynamic signalling, with each individual flagellum capable of loading buffer (BLB) (10 mM HEPES, pH 7.4, 20 lMK+ glu- 2+ generating very rapid, repetitive [Ca ]fla elevations indepen- tamate and 50 mM sorbitol), and then biolistic loading was per- dently of the cytosol and the other flagellum (Collingridge et al., formed using a Bio-Rad PDS-1000 delivery system with 1100- 2013). psi rupture discs, as described previously (Wheeler et al., 2008). While the role of Ca2+ signalling in flagellar-related processes Samples were washed and resuspended in TAP medium and left in C. reinhardtii is well established, its role in regulating processes for 2 h to recover under normal growth conditions. After this in the cell body has been less thoroughly explored. Recent pro- period, loaded cells were chosen that were able to swim and gress indicates a role for Ca2+ in the cellular responses to nutrient glide normally, indicating that they were fully recovered from starvation (Motiwalla et al., 2014) and in regulating photoaccli- the loading process. We confirmed that the biolistically loaded mation, through the activity of the chloroplast-localized calcium fluorescent dyes were correctly localized to the cytosol of C. sensor protein (CAS) (Petroutsos et al., 2011). However, the reinhardtii, using confocal laser microscopy (Zeiss LSM510; 2+ – – dynamics of [Ca ]cyt elevations in response to these and other excitation 488 nm; emission 500 530 and 650 710 nm). The environmental stimuli in C. reinhardtii have not been character- dyes demonstrated a homogenous cytosolic localization, with no ized. evidence of compartmentalization. Chlorophyll autofluorescence In order to gain a better understanding of the role of Ca2+ sig- was negligible relative to OG, allowing us to subsequently use 2+ nalling in green algae, we performed a detailed examination of epifluorescent microscopy to image [Ca ]cyt.
Ó 2016 The Authors New Phytologist (2016) 212: 920–933 New Phytologist Ó 2016 New Phytologist Trust www.newphytologist.com New 922 Research Phytologist
Corporation, Northampton, MA, USA) (Supporting Informa- Ca2+ imaging tion Fig. S1). The smoothed OG/TR traces were divided by the Before imaging, cells were resuspended in Chlamydomonas Assay baseline trace to determine the relative change in fluorescence. 2+ Buffer (CAB) containing 5 mM HEPES, 1 mM HCl, 1 mM [Ca ]cyt elevations were defined as any increase in OG/TR fluo- KCl, 0.2 mM Ethylene glycol-bis(2-aminoethylether)-N,N,N0,N0- rescence above a threshold value (3%) that persisted for more ≥ tetraacetic acid and 0.5 mM CaCl2 with pH adjusted to 7.4 using than one frame. We calculated that an OG/TR 3% required a 2+ N-methyl-D-glucamine (NMDG). Free Ca was calculated to be minimum signal to noise ratio (SNR) of 10, with SNR defined as 301 lM using MAXCHELATOR (http://maxchelator.stanford. signal intensity/SD of noise. Contractile vacuole activity was edu/). Cells were placed in 35-mm glass-bottomed dishes (In detected by measuring changes in the fluorescence intensity of Vitro Scientific, Sunnyvale, CA, USA) coated with 0.01% poly- TR in a region of interest encompassing each contractile vacuole. 2+ L-lysine (Sigma-Aldrich, St Louis, MO, USA) to encourage For visualization of [Ca ]cyt dynamics in cells, IMAGEJ software adherence of the cells. Cells were perfused with CAB buffer at a was used to divide each OG image by a rolling median image flow rate of 3 ml min 1 during imaging. from 20 frames to produce a pseudocoloured ratio image. Previ- Cells were imaged by epifluorescence microscopy at room tem- ous researchers have demonstrated that standard Ca2+ ionophores perature using a Nikon Eclipse Ti with a 9100, 1.49 NA oil do not work well in C. reinhardtii (Braun & Hegemann, 1999), immersion objective and detection with a Photometrics Evolve which precludes an in vivo calibration of OG. We therefore per- EM-CCD camera (Photometrics, Tucson, AZ, USA). Simultane- formed an in vitro calibration of this dye using Calcium Calibra- ous excitation of OG and TR was performed using 488- and tion Buffer Kit no. 1 (Life Technologies Ltd, Paisley, UK), 2+ 561-nm lasers (Coherent, Santa Clara, CA, USA). A Dual-View allowing the increase in [Ca ]cyt for a given change in fluores- 2+ beam splitter device (Photometrics, Tucson, AZ, USA) was used cence to be estimated, assuming a resting [Ca ]cyt of 100 nM to detect emission at 500–550 and 575–625 nm, respectively. (Fig. S2). Data obtained from all treatments are shown as Images were captured using NIS-ELEMENTS v.3.1 software (Nikon, mean SE. Japan) with a 200–300-ms exposure (frame rate of 3.33– 5 frames s 1). The same microscopy equipment was used in Results TIRF mode for flagellar imaging. IFT20-mCherry was visualized in TIRF mode using 561-nm laser excitation and 575–625-nm Salt stress induces a single [Ca2+] elevation in emission. cyt Chlamydomonas reinhardtii NaCl shocks were delivered by rapidly switching the perfusion from CAB to CAB + 50–400 mM NaCl. Hypo-osmotic treat- In order to examine whether Ca2+ signalling processes are con- ments were delivered by switching the perfusion to deionized served between vascular plants and green algae, we applied four l 2+ water containing 50 M CaCl2. To investigate the response to external stimuli that routinely induce [Ca ]cyt elevations in hypo-osmotic shock in the absence of contractile vacuole activity, plants to C. reinhardtii strain CC1201. We found that hydrogen cells were equilibrated to CAB containing 100 mM sucrose for at peroxide (up to 20 mM) and mannitol (up to 400 mM) did not 2+ 2+ least 1 h. In order to assess whether extracellular Ca is involved induce [Ca ]cyt elevations in C. reinhardtii (Fig. 1a,b). However, 2+ 2+ in [Ca ]cyt elevations in response to osmotic shock, cells were NaCl and hypo-osmotic shock resulted in [Ca ]cyt elevations initially perfused with Ca2+-free CAB (containing 200 lM and these stimuli were therefore examined in greater detail. We 2+ EGTA without added CaCl2) and a hypo-osmotic shock was examined Ca signalling in response to salt stress in two C. administered using deionized water containing 50 lM EGTA reinhardtii strains, the CC1021 wild-type strain and cw15, a cell (adjusted to pH 7). In order to assess the involvement of wall deficient mutant. Application of 300 mM NaCl to CC1021 2+ 2+ mechanosensitive ion channels in the generation of [Ca ]cyt ele- resulted in [Ca ]cyt elevations in nine out of 61 cells. These cells 2+ vations, cells were pretreated for at least 10 min with a final con- exhibited a single rapid [Ca ]cyt elevation lasting for centration of either 5 lM GsMTx4 (Smartox Biotechnology, 2.04 0.33 s that occurred 7.04 0.73 s after the stimulus was Saint-Martin-d’Heres, France) or 10 lM Ruthenium red (RuR) applied. The mean maximal amplitude of the OG/TR increase 2+ (Sigma-Aldrich) before the perfusion with distilled water or was 8.93 0.97%, which equates to a mean maximal [Ca ]cyt l 2+ distilled water containing 10 M RuR, respectively. RuR did of 184 nM, assuming a resting [Ca ]cyt of 100 nM. The nature 2+ 2+ not exhibit any fluorescent properties that interfered with Ca of the [Ca ]cyt elevations was highly consistent, although it is imaging. clear that only a small proportion of the cells exhibit this response (14.8%), suggesting that a threshold stimulus is required to trig- ger a [Ca2+] elevation. Very few [Ca2+] elevations were Data processing cyt cyt observed at lower concentrations of NaCl (no cells responded at 2+ = Changes in [Ca ]cyt were identified by calculating the fluores- 150 mM (n 11) and only one out of 12 cells at 200 mM NaCl). cence intensity ratio between OG and TR for a defined region of Higher concentrations of NaCl (400 mM) shrank the cells exces- 2+ interest within each C. reinhardtii cell. Ratio traces were sively, preventing accurate determination of [Ca ]cyt. Previous smoothed using a Savitsky–Golay filter (window 5, order 2) and researchers have demonstrated that Ca2+-dependent signalling a baseline trace was created using an asymmetric least squares pathways in C. reinhardtii can be influenced by the concentration 2+ smoothing baseline algorithm (ORIGINPRO 2016; OriginLab of external Ca in the surrounding media (Quarmby & Hartzell,
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(a) Chlorophyll OG BAPTA Texas Red Merge
CV
(b) 1.20 1.20 400 mM 300 mM D-mannitol NaCl 1.15 1.15
1.10 1.10
1.05 1.05 F ratio (OG : TR) 2+ Fig. 1 Osmotic shock induces [Ca ]cyt 1.00 1.00 elevations in Chlamydomonas reinhardtii. (a) Confocal laser microscopy image of a CC1021 C. reinhardtii cell demonstrating 0.95 0.95 –20 020406080 –20 020406080 that the biolistically loaded Ca2+-responsive fluorescent dye Oregon Green BAPTA dextran (OG) is correctly localized to the 1.20 20 mM 1.20 cytosol. The localization of the reference dye Hypo-osmotic Texas Red dextran (TR) is also shown, along H2O2 shock with chlorophyll autofluorescence. The 1.15 1.15 position of a contractile vacuole (CV) is marked. Bar, 5 lm. (b) Ca2+ signalling 1.10 1.10 viewed by epifluorescent microscopy in response to four different stimuli applied to 2+ cells for 30 s. No [Ca ]cyt elevations were 1.05 1.05 observed following the addition of 400 mM = mannitol (n 18) or 20 mM hydrogen F ratio (OG : TR) peroxide (H2O2)(n = 18), but there were 1.00 1.00 2+ distinct [Ca ]cyt elevations in response to 300 mM NaCl (n = 9 out of 61 cells) or to hypo-osmotic shock (induced by deionized 0.95 0.95 –20 020406080 –20 020406080 water) (n = 22 out of 31 cells). F ratio denotes fluorescence ratio of OG/TR. Time (s) Time (s)