Inhibition of protein or glutamine biosynthesis affect the light-induced dephosphorylation of the SBiP1 chaperone in Symbiodiniaceae Open Access

The marine photosynthetic dinoflagellate Symbiodinium microadriaticum CassKB8 (henceforth CassKB8) was originally isolated from the jellyfish Cassiopea xamachana in its endosymbiotic stage and has been widely used as a model of study in its in vitro cultured stage [1-6]. These microorganisms can live either freely in the water column or as endosymbionts of a variety of cnidarian hosts. Since they respond to diurnal/nocturnal photoperiod cycles, they require the necessary signal-transduction machinery to adapt their responses depending on the environmental conditions.

Protein kinases and phosphatases are responsible for the exquisite regulation of key target molecules through phosphorylation and dephosphorylation within cellular signal-transduction cascades and a significant representation of kinase sequences has been reported in Symbiodininaceae [2,7]. Among the target molecules reported to be modulated by phosphorylation are the endoplasmic reticulum (ER)-resident BiP chaperones [5,6,8-11]. In this compartment, they assist in the folding and assembly of newly synthesized proteins as they undergo translocation into the ER, and they can also associate with misfolded and/or underglycosylated proteins [8,12].

Post-translational phosphorylation of BiP results in profound intracellular functional changes; for example, phosphorylated (inactive) BiP is believed to assemble into oligomers, whereas only the dephosphorylated monomeric (active) form can bind to clients [8,13]. Thus, phosphorylation/dephosphorylation of BiP correlates with inactivation/activation, respectively, of the chaperone activity [5,6,8,10,11,13]. This inactivation was observed as an increase in CrBiP phosphorylation in the presence of the inhibitor of protein synthesis cycloheximide (CHX) in Chlamydomonas reinhardtii [10]. The conclusion was that the CrBiP phosphorylation state was indirectly regulated by target of rapamycin (TOR) through the control of protein synthesis after a screen for targets of TOR in this alga [10].

Recently, we reported the light-modulated phosphorylation/dephosphorylation of the BiP-like protein, SBiP1 from CassKB8 [5,6]. The SBiP1 molecule is highly conserved [6,11], and SBiP1 sequences have been observed in all Symbiodiniaceae species reported so far [6,11], but we have focused on determining its properties from cultured CassKB8. SBiP1 was shown to be highly phosphorylated on Thr during the 12-h dark phase of the CassKB8 growth cycle and but upon shifting to light, Thr phosphorylation decreased within 30 min [6]. Dephosphorylation occurred independent of photosynthesis and the wavelength of the visible light spectra but was highly sensitive to light intensity as it occurred with as low as a 1 µmole photon m⁻² s⁻¹ stimulus [6]. Furthermore, in highly phosphorylated SBiP1 in the absence of light, the stressful temperature of 32°C for this species also induced its Thr dephosphorylation [5]. These data suggested an activation/inactivation of the SBiP1 chaperone by regulation of its Thr phosphorylation levels [10,13] under homeostasis and/or stress conditions in CassKB8.

Nutritional status is a key regulator of protein synthesis and chaperone activity in various organisms. In eukaryotic cells, amino acid availability directly affects the activation of the mTOR pathway which, in turn, regulates translation initiation and protein folding through BiP chaperones [14,15]. For instance, studies in yeast and mammalian cells have demonstrated that nutrient depletion leads to reduced BiP expression and an increased unfolded protein response (UPR) due to an imbalance between protein load and chaperone capacity [16,17]. Additionally, in photosynthetic organisms such as C. reinhardtii, nitrogen starvation has been shown to significantly reduce protein synthesis [18], while UV-C light stress also modulates chaperone function [19]. Given that Symbiodiniaceae are highly dependent on nutrient exchange with their hosts [20], it is likely that fluctuations in nutrient availability influence BiP function in a similar manner, either through direct transcriptional regulation or via post-translational modifications such as phosphorylation.

To obtain further knowledge on the relationship of the SBiP1 modulation by phosphorylation with cellular events related to nutritional status and confirm our hypothesis that there was a correlation of the chaperone phosphorylation/dephosphorylation with those events, we evaluated the effect of protein synthesis and glutamine synthetase inhibition on the phosphorylation status of the protein. We found that light-induced dephosphorylation of SBiP1 was inhibited upon treatment with CHX but not with chloramphenicol (CPL); however, this phenomenon was only delayed by the glutamine synthetase inhibitor, glufosinate (GLUF). Finally, short-term heat shock restored the dephosphorylation of the chaperone thus overriding the CHX-induced effect. These results suggest a strong interrelationship between SBiP1 chaperone phosphorylation/dephosphorylation and cellular processes in a concerted regulation by nutritional and stress cues in addition to light.

Since it has been previously documented that BiP chaperones from C. reinhardtii are activated by dephosphorylation at the onset of de novo protein synthesis, which occurs downstream of the TOR pathway, and the effect is reversed by the protein synthesis inhibitor CHX [10], we tested the effect of this inhibitor on the phosphorylation behavior of SBiP1 under maximum dephosphorylation conditions, starting 3 h after the onset of the light cycle. We observed that the Thr phosphorylation level of SBiP1 (Figure 1A, SBiP1-p, upper panels) was indeed minimum 3 h after the onset of light in the control treated for 30 additional min under light with vehicle alone (Figure 1A, lane EtOH, upper panel, 30 min; Figure 1B, white bar, 30 min) and remained unchanged for the next 1, 4, and 7 h after the addition of vehicle (Figure 1A, lanes EtOH, upper panel, 1, 4, and 7 h, respectively; Figure 1B, white bars, 1, 4, and 7 h, respectively). In contrast, after 3 h of light and treatment with CHX for further 30 min under light, a clear increase in the Thr phosphorylation level was observed (Figure 1A, lane CHX, upper panel, 30 min; Figure 1B, gray bar, 30 min). The increase in phosphorylation was sustained for the next 1 and 4 h after CHX was added (Figure 1A, lanes CHX, upper panel, 1 and 4 h, respectively; Figure 1B, gray bars, 1 and 4 h, respectively). This increase in Thr phosphorylation was statistically significant after quantitation by densitometry of the bands and normalization against the total SBiP1 protein detected by anti-SBiP1 antibodies (Figure 1A, lower panels, SBiP1) from three biological replicates (Figure 1B, asterisks on gray bars). At the same time, the SBiP1 detection was used as an internal loading control for each respective lane (Figure 1A, lower panels, SBiP1). After 7 h of treatment with CHX to the cells, the SBiP1 Thr phosphorylation level decreased to statistically non-significant values (Figure 1B, gray bar, 7 h), although the SBiP1-p band corresponding to phosphorylated SBiP1 (Figure 1A, lane CHX, upper panel, 7 h) was still observed more intense than in the control (Figure 1A, lane EtOH, upper panel, 7 h). In parallel, we tested the effect of the chloroplast-specific protein synthesis inhibitor CPL on the level of SBiP1 phosphorylation. We observed no significant effect on the Thr phosphorylation levels of SBiP1 when CPL was added 3 h after the onset of light and the cells further incubated for 30 min, 1, 4, and 7 h (Figure 1A, lanes CPL, upper panel, 30 min, 1, 4 and 7 h, respectively; Figure 1B, dotted bars, 30 min, 1, 4, and 7 h, respectively). As a positive control for the CPL inhibitory effect on chloroplast protein synthesis, we followed the presence of the photosystem II, D1 protein, throughout the treatment. We observed that, indeed, the protein level diminished as a function of the time of CPL treatment compared with the control (Supplementary Figure S1). These results indicated that global protein synthesis inhibition exerts an effect on light-induced SBiP1 dephosphorylation whereas specific chloroplast protein synthesis inhibition does not.

We wanted to assess whether CHX reverted or rather prevented the dephosphorylation of SBiP1 after the transition from darkness to the light phase of growth. Thus, we added CHX under darkness 2 h prior to the onset of the light phase of the growth cycle and monitored the SBiP1 Thr phosphorylation after the switch to light for 30 and 60 min. As previously observed, the high level of Thr phosphorylation of SBiP1 typical of cells under darkness was present in cells treated with vehicle for 2 h prior to the onset of light (Figure 2A, left upper panel [SBiP1-p], dark, 12 h; Figure 2B, black bar, control). A similar level of Thr phosphorylation was observed in cells treated with CHX for 2 h prior to the onset of light (Figure 2A, right upper panel [SBiP1-p], dark, 12 h; Figure 2B, black bar, CHX). As expected, in vehicle-treated cells, the typical dephosphorylation of SBiP1 was observed 30 and 60 min after the onset of light (Figure 2A, left upper panel [SBiP1-p], lanes 30 and 60 min, respectively; Figure 2B, left white bars, control, respectively). Conversely, the SBiP1 Thr phosphorylation level remained with little change in CHX-treated cells after light exposure for 30 or 60 min (Figure 2A, right upper panel [SBiP1-p], lanes 30 and 60 min, respectively; Figure 2B, light gray bars, CHX, respectively). These results indicated that the presence of CHX in CassKB8 cells under darkness is sufficient to prevent the light-induced Thr dephosphorylation of SBiP1 for at least the time tested of 1 h after the onset of light and suggest that light triggers processes that impinge on protein biosynthesis.

One set of the CHX-treated cells after 60 min of light stimulation in the presence of CHX were subjected to treatment with 32°C for 30 additional min and then analyzed by western blot with anti-pThr antibodies. We observed that, even though CHX prevented the Thr dephosphorylation of SBiP1 after the light stimulus, the heat shock overrode this effect and promoted the dephosphorylation of the chaperone (Figure 2A, right upper panel [SBiP1-p], 90 min; Figure 2B, dark gray bar, CHX). The results were statistically significant from three biological replicates (asterisks on bars). This indicated that a short heat shock treatment was sufficient to induce dephosphorylation of the SBiP1 chaperone.

Since the inhibition of protein biosynthesis appeared to prevent dephosphorylation (thus maintaining the chaperone inactive state) of SBiP1, we explored the effect of inhibition of another biosynthetic process. We tested the competitive inhibition of glutamine synthetase (which aminates glutamate to produce glutamine) by 1 mg/ml GLUF, on the light-stimulated SBiP1 dephosphorylation. GLUF was added to the cell cultures at the onset of the dark cycle and incubated for 12 h in the dark to first determine whether this treatment would affect the viability of the cells at the times assayed. When we checked viability of the GLUF treated cultures by Evans blue [21], we observed that after 12 h in the presence of GLUF, the cells did not show any blue staining ( Figure 3A, middle panel, GLUF) and quantitation showed 100% viability (Figure 3C, diagonally hatched bar), similar to the untreated cells (control in both Figure 3A, upper panel, and Figure 3C, white bar, respectively). This indicated that the cells were undisturbed by the treatment. On the other hand, heat-killed cells were both stained blue (Figure 3A, lower panel, boiled) and less than 50% viable (Figure 3C, dotted bar). Cell concentration was ~30% lower in the treated cells (Figure 3B, diagonally hatched bar), compared with the untreated cells (Figure 3B, white bar), indicating a possible delay in cell division induced by GLUF.

The effect of GLUF on SBiP1 phosphorylation was then assessed just prior to the onset of the light phase (dark 12 h), as well as 30 and 120 min of light. The cells under darkness both treated with vehicle (control) or GLUF displayed the typical behavior of highly Thr phosphorylated SBiP1 (Figure 4A , Dark 12 h, left upper panels labeled SBiP1-p, respectively; and Figure 4B, left black bar, respectively). In the control group, upon light exposure for 30 and 120 min, the expected prompt SBiP1 dephosphorylation occurred (Figure 4A, left upper panel [SBiP1-p], 30 and 120 min, respectively; Figure 4B, white bars, 30 and 120 min, respectively). Conversely, in the presence of GLUF, SBiP1 was not significantly dephosphorylated after 30 min of light (Figure 4A, right upper panel [SBiP1-p], 30 min; Figure 4B, diagonally hatched bar, GLUF, 30 min). The SBiP1 phosphorylation signal diminished significantly only after 2 h of light (Figure 4A, right upper panel [SBiP1-p], 120 min; Figure 4B, diagonally hatched bar, GLUF, 120 min). These data suggested that inhibition of processes involved in nitrogen assimilation profoundly affected the SBiP1 chaperone phosphorylation status.

HSP70 family chaperones have been reported to be closely linked to cellular metabolism [10,22]. As previously mentioned, in C. reinhardtii, the specific inhibition of CrTOR by rapamycin promoted phosphorylation (inactivation) of the ER resident BiP chaperone (CrBiP) and assigned a role to TOR on the control of BiP modification [10]. The CrBiP chaperone inactivation effect was also promoted by treatment with CHX, implicating that CrTOR regulates CrBiP phosphorylation through controlling protein biosynthesis [10]. These data suggested that a close link between the chaperone activation switch and the metabolic status of the cell exists, although the external stimulus that triggers the process remains unknown. We have found the use of alkaline phosphatase-conjugated secondary antibodies on western blots followed by densitometric analysis a powerful tool to analyze changes in the level of phosphorylation of Symbiodiniaceae proteins [4-6]. Even though this technique provides a narrow dynamic range [23], we have succeeded in obtaining reproducibility and reliability to assign statistically significant changes in Thr phosphorylation in CassKB8 SBiP1 [4-6]. Using this technique, we previously found that light profoundly affects such SBiP1 Thr phosphorylation in these cells [5,6], and here, we demonstrate that a protein synthesis-SBiP1 Thr phosphorylation status correlation occurs similar to CrBiP [10]. We observed that under light conditions, the inhibition of protein synthesis with CHX increased the level of SBiP1 phosphorylation (Figure 1). Additionally, the dark-adapted CassKB8 cells in the presence of CHX did not show an observable change in the Thr phosphorylation status of SBiP1 after 60 min of light stimulation (Figure 2, CHX), contrary to the untreated control (Figure 2, EtOH). These results indicate that inhibition of protein synthesis maintains the phosphorylated SBiP1 status which, in turn, would render the chaperone inactive [10,13] and suggests that protein biosynthesis is the process through which light induces dephosphorylation of SBiP1 which represents its active state. At present, it is unknown whether the TOR pathway is involved in regulation of the chaperone activity as reported for C. reinhardtii [10]. Upon BLAST analysis to search for TOR in Symbiodiniaceae databases, we could not find any significant homologous sequence, suggesting that a canonical TOR homolog is not present in these organisms and alternate pathways are at play. On the other hand, as expected for a chaperone, the SBiP1 hypothetically inactive status which resulted from the CHX treatment was reversed upon heat shock; that is, the CHX-treated cells after 60 min of light stimulation further treated for 30 min with high temperature prompted a hypothetical re-activation of the chaperone, observed as a significant dephosphorylation (Figure 2, CHX, Heat Shock). A previous similar result was found when highly phosphorylated SBiP1 under dark conditions was observed to dephosphorylate with the same heat shock treatment [5]. These results indicate that beyond the dephosphorylation of SBiP1 by light, heat shock also induces SBiP1 dephosphorylation by an unknown alternate mechanism, possibly to assist heat-induced unfolded proteins at the ER. It is important to note that inhibition of cytosolic protein synthesis by CHX was concomitant to a sustained phosphorylation of the chaperone whereas inhibition of chloroplastic protein synthesis by CPL had no effect as light-induced dephosphorylation of SBiP1 occurred analogous to control (Figure 1). These data suggest that global protein synthesis reflecting the intracellular nutritional status [24,25] is closely linked to a chaperone activity switch as it is proposed to occur in C. reinhardtii [10]. Protein synthesis is an anabolic process, and similar converging pathways are likely to have impact on regulation of the chaperone activity. Thus, inhibition of another metabolic pathway should result in a similar effect on the chaperone. Glutamine synthetase (GS) is a fundamental enzyme for nitrogen assimilation and glutamine synthesis in photosynthetic organisms [26,27]. In Symbiodiniaceae, as well as in plants, inhibition of GS by GLUF inhibits growth (Figure 3) and the cells eventually perish [28,29]; however, cell death does not occur immediately, and cells are still viable during the first 12 h of GLUF treatment (Figure 3). Therefore, we could still observe short-term effects, if any, of inhibition of GS on SBiP1. Interestingly and consistent with the effect of CHX on the cells, GLUF treatment for 12 h in dark-adapted cells subsequently stimulated with light for 30 min, also appeared to prevent the dephosphorylation of SBiP1 (Figure 4A and B, GLUF, 30 min). The effect of GLUF inhibition of SBiP1 Thr dephosphorylation appeared to be transient, as dephosphorylation was observed after 2 h of light stimulation (Figure 4, GLUF, 120 min), compared to the untreated control (Figure 4A and B, control, 120 min).

Although an indirect effect of GLUF on global protein synthesis by its growth inhibiting activity cannot be ruled out as the cause of the transient inhibition of SBiP1 dephosphorylation, the data suggest a scenario where a GLUF-induced delay on SBiP1 dephosphorylation through GS inhibition occurs given that a) the GS/GOGAT pathway is the most expedite for ammonium assimilation into amino acids, and this is the main pathway in dinoflagellates [30,31]; b) light improves ammonium and soluble aminoacid assimilation [32-34]; and c) SBiP1 participates in de novo protein synthesis. This could be due to a downshift in ammonium assimilation which translates into a temporal deficit in nitrogen uptake for biosynthetic processes [35] until an alternative nitrogen arrival happens through a catabolic pathway such as the pathway through glutamine dehydrogenase which also exists in Symbiodiniaceae [36]. Altogether, the data further supported the findings that inhibition of metabolic processes such as protein and glutamine synthesis have an observable effect on the Thr phosphorylation levels of the SBiP1 chaperone. These results are also consistent with the notion that chaperones of the HSP70 family are influenced by the nutritional status of the cell [10,37], and this seems to be the case also in CassKB8 cells and probably in Symbiodiniaceae since SBiP1 appears to be a ubiquitous protein in this family [6].

The findings on cultured Symbiodiniaceae cannot be extrapolated to symbionts in hospite; however, it has become more evident that nitrogen metabolism is critical for symbiosis and the endosymbiont itself. For example, it was recently reported that endosymbionts under optimal light conditions for photosynthesis make more carbon available and stimulate nitrogen assimilation in the host. This in turn, limits nitrogen available to algal endosymbionts resulting in low densities within the holobiont [38]. In fact, it has been demonstrated that cnidarian hosts use a carbon-negative feedback loop to control symbiont proliferation [20] where supplementing additional carbohydrates significantly reduced the symbiont density through limiting their nitrogen availability. Thus, it is quite likely that the natural diel cycles and light-regulated pathways leading to protein synthesis and nitrogen metabolism in the endosymbiont are coupled to diverse signaling cascades that control the activity of chaperones such as SBiP1 to maintain symbiont-host homeostasis. In addition, it is highly likely that heat stress would profoundly affect chaperone activity in hospite.

A proposed hypothetical model incorporating our previous data [5,6] and current findings is shown in Figure 5. Initially, a kinase has rendered SBiP1 fully Thr phosphorylated (thus inactive; light green croissant) under darkness at the normal 26°C growth temperature (right purple arrow). At the onset of the light phase, phototransduction activates light-stimulated pathways (left upper dashed green arrows); among these, a highly sensitive photoreceptor (PR) with broad light spectrum sensitivity [6] upstream of SBiP1 triggers the activation of a currently unknown pathway (lower left dashed green arrow) that is related to the activation of metabolic processes such as protein synthesis (right lower black arrow); the signaling cascade eventually results in the activation of a phosphatase (middle dashed and solid green arrows) leading to the dephosphorylation of Thr phosphorylated SBiP1 (light green croissant) to hypothetically activate its chaperone activity (green croissant). The active chaperone is now able to assist client proteins being newly synthesized in the ER (SBiP1-protein X-ribosome complex). The PR highly sensitive to the broad visible light spectrum would be a requirement for photoreception and phototransduction in cells such as Symbiodiniaceae living in the water column or in symbiosis deep within the tissue of other underwater hosts. This dephosphorylation occurs independent of photosynthesis since CPL (this work) or DCMU [6] do not affect SBiP1 dephosphorylation (upper left red blunt arrow and upper dashed green arrow). This also suggests that alternative nutrient sensing pathways independent of photosynthesis including an orthologous TOR pathway may come into play (left thick green arrow); thus, the hypothetical chaperone activity switch would also be connected to cues reflecting the nutritional status of the cell (i.e., active protein and glutamine biosynthesis) which would result in activation of the chaperone (middle green dashed and solid arrows). On the other hand, inhibition of global protein synthesis by CHX (middle horizontal blunt arrow at the left) results in a decrease in the protein biosynthetic process (right upper black arrow), which somehow promotes (or is the result of) chaperone inactivation (upper middle purple arrow). Alternatively, inhibition of glutamine biosynthesis delays but does not block the activation of the pathways that lead to the activation of the phosphatase (left dashed blunt arrows) which eventually ensue (left pink arrow following the pink blunt arrows and middle dashed and solid green arrows) stimulating the phosphatase activity that dephosphorylates SBiP1 (green croissant). It is currently unknown whether protein synthesis is up- or downstream the chaperone activation switch. Stress responses also affect the chaperone status; under heat stress (32°C) either under light (lower vertical solid green arrow) or darkness (right long solid green arrow), the chaperone activity is required prompting the phosphatase to dephosphorylate SBiP1 [6]. Heat shock would also prompt activation of the chaperone even when protein synthesis is inhibited (horizontal left red blunt arrow and vertical solid green arrow). The UPR and other stress responses resulting in reactive oxygen species production trigger mechanisms critical for cell survival and, in some cases, they could also override the light-stimulated dephosphorylation of the chaperone through various alternative mechanisms including inhibition of protein synthesis (lower vertical red blunt arrow). We have shed light on possible links between the modulation of the SBiP1 chaperone phosphorylation levels by nutritional cues and phototransduction processes that hypothetically control its activity in Symbiodiniaceae and are currently searching the molecular participants of the pathways. This will lead us to eventually dissect all the components of this light-stimulated signaling mechanism in these photosynthetic potentially endosymbiotic microorganisms.

CassKB8 culture (also known as MAC-CassKB8 and previously classified as clade A Symbiodinium) was a kind gift of Dr. Mary Alice Coffroth (State University of New York at Buffalo). Cultures have been routinely maintained in our laboratory for over twenty years, grown in ASP-8A medium under photoperiod cycles of 12 h light/dark at 26°C at a light intensity of 80–120 μmole photon m⁻² sec⁻¹ under aerated conditions.

Polyclonal anti-phosphothreonine (anti-pThr) antibodies were from Cell Signaling Technology ™, Inc. (Danvers, MA; cat. 9381S) and Abcam (Cambridge, UK; cat. Ab9337). Anti-PsbA to the C-terminal of the PsbA protein (AS05 084) was from Agrisera (Vannas, Sweden). Rabbit anti-SBiP1 polyclonal antibodies were used as the purified IgG fraction (~1 mg/ml IgG as stock) as previously described [5,6]. Alkaline-phosphatase (AP)-conjugated polyclonal anti-rabbit IgG antibodies raised in goat were from Zymed^™-Life Technologies (Grand Island, NY). Reagents 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitro blue tetrazolium (NBT) were from Promega (Madison, WI). The inhibitors of protein synthesis CXH and CLP were from Sigma. The inhibitor of glutamine synthetase (GS) ammonium GLUF was from SantaCruz Biothecnology. All other reagents were from Sigma.

The protein extracts (see below) were separated in discontinuous denaturing gels [39] of 10% polyacrylamide in the separation zone (375 mM Tris-HCl, pH 8.8; 10% acrylamide/bis-acrylamide [29:1]; 0.1% SDS; 0.1% ammonium persulfate [APS]; 0.106 % N, N, N, N'-tetramethylethylenediamine [TEMED]) and 4% polyacrylamide in the stacking zone (125 mM Tris-HCl, pH 6.8; 4% acrylamide/bisacrylamide [29:1]; 0.1% SDS; 0.1% APS; 0.066% TEMED), in a Mini-PROTEAN^™3 System (Bio-Rad, Hercules, CA). After electrophoresis, the proteins were transferred to PVDF membranes in ‘friendly buffer’ [40] (25 mM Tris-HCl, 192 mM glycine, 10% isopropanol) at a constant current of 300 mA for 1 h. The membranes were blocked in a solution of 3% bovine serum albumin (BSA) in PBS (2.79 mM NaH₂PO₄, 7.197 mM Na₂HPO₄, 136.9 mM NaCl, pH 7.5) added with 0.01 % TX-100 (PBS-T) for 1 h at 50°C, with gentle agitation. After blocking, the primary antibodies, anti-pThr (Cell Signaling, 1:2,500), anti-PsbA (1:2,500), or anti-SBiP1 (0.404 µg/ml IgG fraction) in PBS-T, were added to the membranes and incubated overnight with gentle rocking at 25°C. Alternatively, when Abcam anti-pThr antibodies were used, TBS-T (20 mM Tris-Base, 150 mM NaCl, 0.01 % TX-100, pH 7.5) was the buffer for the BSA blocking solution (5% BSA) and primary antibody dilution (1:500). Blocking for at least 2 h and all other incubations were carried out at 4°C. After overnight incubation, the membranes were washed five times, 5 min each, in PBS-T or TBS-T, and incubated with secondary antibody (alkaline-phosphatase-conjugated goat anti-rabbit IgG) at 1:2500 dilution in PBS-T or TBS-T for 2 h at 25 or 4°C, buffer and temperature depending on the primary antibody used. Subsequently, the membranes were washed again five times, 5 min, and the final wash was followed by a brief rinse with alkaline developing solution (100 mM Tris-HCl, 150 mM NaCl, 1 mM MgCl₂, pH 9) at 25°C. Finally, the membranes were developed with a commercial solution of BCIP and NBT according to the manufacturer (Promega, Madison, WI), in alkaline developing solution. In the case of anti-pThr from Cell Signaling, development was carried out in PBS-T to ensure more astringency and less background. It is important to note that we have previously determined that incubations in PBS-T or TBS-T yielded identical results when anti-pThr antibodies from Cell Signaling were used [4].

Three to four different cultures inoculated and collected on different days were used as experimental replicates for the analyses. Densitometric values from samples were subjected to the Shapiro–Wilk test for normal distribution and then to ANOVA with post-hoc Tukey Honesty Significant Difference (HSD) analysis for statistical significance. In the one case when the data did not present normal distribution, the Kruskal–Wallis with post-hoc Dunn test was applied. After the corresponding statistical analyses all data were plotted and significance indicated as corresponded. In some cases, Student’s t-test without correction was also applied but only for confirmation of the increasing or decreasing trends in phosphorylation levels and not used for statistical significance determination.

After each time point of treatments, the Erlenmeyer flask was shaken briefly to suspend the cells and equal 10 ml aliquots were placed into 15 ml Falcon tubes. Then, the cells from each tube were sedimented by centrifugation at 2,600×g for 1 min at 26°C, and immediately suspended in 300 μl Laemmli buffer [39], supplemented with 0.2 mM NaVO₃, 10 mM NaPPi and a cocktail of protease inhibitors (Complete™; Roche, Basel, Switzerland), mixed with~250 μl total volume of glass beads (425–600 μm diameter). Subsequently, the cells were lysed with a MINI-BEAD BEATER-1™ (Biospec Products) at maximum speed for 3 min. The lysate was heated at 95°C for 5 min centrifuged at 12,000×g for 10 min, and the supernatant used for western blot analysis.

Six-day-old CassKB8 cultures were sedimented by centrifugation at 2,600×g for 3 min and suspended in fresh ASP-8A medium to reach a concentration of 4–6 × 10⁵ cells/ml. Two equal portions of 40 ml in Erlenmeyer flasks wrapped with aluminum foil were incubated for 12 h at 26°C under darkness (nocturnal phase). The cultures were then treated 2 h (after 10 h of darkness) before the change to light with either: 0.1 mM CHX (16 µl of 0.25 M CHX stock) in ethanol (EtOH) or with vehicle (16 µl EtOH) as negative control. Protein extraction was carried out at either 12 h darkness, 30, or 60 min of light for each treatment, as described above. For the CHX treatment, a further short-term heat shock was applied after 60 min of light stimulation (see below). The proteins were analyzed by 10% SDS-PAGE gels, and western blot with either anti-pThr (Abcam), or anti-SBiP1 antibodies. The bands on the blots from triplicate samples were captured with a ChemiDoc-It™2 Imager (UVP-Analytik Jena, Up- land, CA, USA), analyzed by densitometry with the system’s VisionWorks LS software, and normalized as previously described [4,5].

Six-day-old CassKB8 cultures were sedimented by centrifugation at 2,600×gg for 3 min and suspended in fresh ASP-8A medium to reach a concentration of 4–6 × 10⁵ cells/ml. Then, three 40 ml portions of cell suspension were separated in Erlenmeyer flasks wrapped with aluminum foil and incubated at 26°C for the 12 h of their nocturnal phase. After this phase, light ensued (80–120 µmol photon m⁻² s⁻¹ and 26°C) and after 3 h, each of the flasks were treated with either 0.1 mM CPL (16 µl of 0.25 M CPL stock in EtOH), 0.1 mM CHX (16 µl of 0.25 M CHX stock in EtOH), or vehicle (16 µl EtOH), as negative control. Prior to protein extraction, the flasks were shaken after each treatment in order to take 10 ml homogeneus aliquots into 15 ml Falcon tubes after 30 min, 1, 4, or 7 h, and the cells were then processed as above. The proteins were analyzed by SDS-PAGE gels, and western blot with either anti-pThr (Cell Signaling), anti-SBiP1, or anti-PsbA antibodies. The bands on the blots were analyzed by densitometry as above. Normalization was carried out with the corresponding immunostained SBiP1 band density.

A parallel sample (10 ml suspension) of CassKB8 cells from the CHX treatment (CHX applied 2 h before the dark to light transition) was taken after 60 min of light stimulation at 26°C, centrifuged at 2,600×g for 1 min, resuspended in 10 ml ASP-8A medium with 0.1 mM CHX and incubated for additional 30 min at 32°C under the same light intensity. After this time, the sample was processed for protein extraction and analyzed as above by SDS-PAGE gels, western blot with either anti-pThr (Abcam) or anti-SBiP1 antibodies, and densitometry as described above.

A six-day-old culture of CassKB8 was concentrated by centrifugation at 2,600×g for 3 min and suspended in 60 ml of ASP-8A medium to reach a cell concentration of 4–6 × 10⁵ cells/ml. The suspension was then divided in 30 ml portions in two separate Erlenmeyer flasks. To one flask, GLUF (ChemCruz®, Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to a final concentration of 1 mg/ml, and the equivalent amount of vehicle (sterile milliQ water) was added to the other one as control. The flasks were covered with aluminum foil and allowed to incubate for 12 h of their dark photoperiod. In parallel, protein extractions were performed from 10 ml homogeneous aliquots, prior to treatment with light (12 h dark), or after 30- and 120 min light exposure. The samples were analyzed with anti-pThr (Abcam) and anti-SBiP1 antibodies. The assay was performed to four biological replicates. Band intensities of the phosphorylated SBiP1 were normalized to the corresponding total SBiP1 protein band, and the obtained values averaged averaged, checked for normal distribution, subjected to Kruskal–Wallis with post-hoc Dunn test analysis, and plotted. Cell viability in the presence and absence of GLUF was determined by Evans blue staining as previously described [21] under a Leica DM500 microscope.

All data generated or analyzed during this study are included in this article. Raw data showing the original western blot membranes and densitometric measurements are provided as Online Supplementary Figure 1 and Uncited Online Supplementary Material 1, respectively.

The authors declare no competing interest.

We are thankful for the support by the Secretary of Science, Humanities, Technology, and Innovation (SECIHTI-Mexico) for a) research grant 285802 to MAV; b) postdoctoral fellowship to E.M.-R.; and c) Ph.D. fellowship No. 255464 to R.C.-M.

R.C.-M.: Methodology, Writing—original draft, Writing—review and editing. T.I.-F. and E.M.-R.: Data curation, Formal analysis, Methodology, Writing—original draft, Writing—review and editing. M.A.V.: Conceptualization, Methodology, Writing—original draft, Writing—review and editing.

No ethical approval was required for the live materials used in this work since the Symbiodiniaceae cell cultures are publicly available.

We are grateful for the technical help of M.O. Edgar Escalante-Mancera, M.I. Miguel Ángel Gómez-Reali, Dr. Edén Magaña-Gallegos, M.S. Fernando Negtete-Soto, and M.T.I.A. Gustavo Villarreal-Brito.

CHX

cycloheximide

CPL

chloramphenicol

CassKB8

Symbiodinium microadriaticum CassKB8

ER

endoplasmic reticulum

GLUF

glufosinate

TOR

target of rapamycin