Broad scale proteomic analysis of heat-destabilised symbiosis in the hard coral Acropora millepora

Coral physiology in response to elevated temperature

Sustained declines in photosynthetic health and symbiont density are well-defined characteristics of coral bleaching⁴¹. Consistent with previous studies^10,11, the photosynthetic health of the coral symbionts, measured as dark-adapted quantum yield of PSII (F_V/F_M), decreased towards the end of the temperature ramping period (from day 4), declining further over the following three days (rmANOVA; F_6,39 = 129.9, P < 0.001; Fig. 1a). Similarly, midday measurements of effective quantum yield of PSII (ΔF/F_M)′ showed continuous decline under elevated temperature from day 5 (rmANOVA; F_6,39 = 21.85, P < 0.001; Fig. 1b). After two days at target temperature (mean 31.9 °C), mean symbiont cell density had declined significantly (t = 3.95, df = 3, P = 0.028) to less than 50% of control corals (Fig. 1c), indicating bleaching via symbiont loss, but with no change in chlorophyll a or c2 per cell (Fig. 1d). Congruent with these data, single-celled measurements of F_V/F_M showed a significant shift in population distribution (KS; D = 0.797, P < 0.001), from a tight cluster of healthy endosymbiotic (in-hospite) cells (median = 0.709) to a broad spread in F_V/F_M (median = 0.567) of the in-hospite cells under elevated temperature (Fig. 1e).

Biomolecular profile of the symbiont using FTIR microspectroscopy

There were significant differences in biomolecular content of coral symbionts with elevated temperature (Fig. 2) with increases in the relative concentrations of saturated fatty acids (t = − 15.65, df = 3, P < 0.001), saturated lipids (t = − 10.53, df = 3, P = 0.002), ester carbonyl (t = − 4.19, df = 3, P = 0.025), carboxylates (t = − 3.59, df = 3, P = 0.037), and free amino acids I (t = − 4.31, df = 3, P = 0.023) and II (t = − 4.7013, df = 3, P = 0.018), consistent with the findings of Petrou et al.¹⁰. In contrast to this previous study however, there were no declines in protein-related biomolecules (Amide II, CH-stretch II) and an increase in Carbohydrate II (t = − 11.50, df = 3, P = 0.001). The overall accumulation of energy stores (lipid and carbohydrate), despite the downregulation of energy production (photosynthesis), could be indicative of reduced translocation of metabolites to the host.

Overview of proteomic analysis

A total of 1,230 unique proteins were identified (774 host and 456 symbiont) at a false discovery rate ≤ 0.01. Of those, 107 were significantly increased in abundance and 125 decreased in abundance (see Supplementary Tables 2 [host] and 3 [symbiont] for all differentially abundant proteins), while the remainder (998) displayed no significant change in abundance between control and treatment (see supplementary data file). Of the 113 proteins that were identified to be differentially expressed between the control and treatment in the host (Fig. 3a), 37 proteins had log2 fold changes ≥ 1 (increased abundance), while 17 proteins yielded log2 fold changes ≤ − 1 (decreased abundance). In the symbiont, of the 119 proteins that were significantly increased or decreased in abundance, 24 were identified to have a fold change of ≥ 1 and 48 proteins had a fold change of ≤ − 1 (Fig. 3a). Given that the total number of proteins within a given metabolic pathway were often low, we assessed the overall reorganisation of the host and symbiont proteomes in response to thermal stress at the level of biological and molecular function, based on groupings of GO-term classifications (Fig. 3b; Supplementary Tables 2 and 3).

Functional analysis of the host proteome response to elevated temperature

Host: antioxidant function, protein modification and degradation

The greatest number of differentially abundant proteins in the host were associated with protein modification and degradation (Fig. 4). Of the 66 linked to modification processes, 14 were differentially expressed. Most of the proteins that showed an increase in abundance in this groups were heat shock proteins (HSPs), other chaperones and proteins involved in folding, quality control, and proteolysis. Specifically, we detected SGT1 homolog A, possibly involved in the ubiquitination of HSP90 client proteins and an Activator of 90 kDa HSP ATPase homolog 1, a co-chaperone of HSP90AA1. As expected, there were significant increases in HSP90 kDa (0.65 FC) and HSP90-alpha (0.48 FC), consistent with temperature shocked anemones and corals²⁹. Also, we detected increases in two endoplasmic reticulum (ER) disulfide-isomerases (A4, 0.45 FC; A3, 0.21 FC) and the chaperone protein BiP (0.94 FC), which are important for the identification and translocation of misfolded proteins⁴². BiP is an HSP70 molecular chaperone located in the lumen of the ER and, while abundant under all growth conditions, it is strongly induced under conditions that lead to the accumulation of unfolded polypeptides in the ER⁴³.

Of the 66 proteins detected that were associated with proteolytic function (protein degradation), seven were increased in abundance, including the metalloprotease component of the 26S proteasome (1.55 FC), a peptidase involved in the regulation of intracellular protein levels and selective removal of damaged or incorrectly folded proteins^44,45. Previous studies have found this proteasome to be responsive to oxidative stress, becoming elevated in both anemones²⁹ and foraminifera⁴⁶ under thermal stress. Counter to this, we detected a decrease in the Proteasome subunit alpha type-6 (-0.71 FC), a component of the 20S core proteasome complex involved in the proteolytic degradation of most intracellular proteins. This complex plays numerous essential roles within the cell by associating with different regulatory particles including participating in ATP-dependent degradation of ubiquitinated proteins. These differential changes to proteolytic regulation indicate a change in the hosts ability to maintain proteostasis via efficient protein quality control.

We detected seven ‘response to stress’ proteins that were regulated in the host. Among them, Heme-binding protein 2 (− 1.55 FC), a protein that can promote mitochondrial permeability and facilitate necrotic cell death under different types of stress conditions, decreased in abundance⁴⁷. We also detected a Transmembrane emp24 domain-containing protein 4, a protein involved in ER stress response that may play a role in the regulation of heat-shock response and apoptosis⁴⁸. Taken together, the increase in chaperones, changes to proteolytic function and decrease in proteins able to control necrotic cell death suggests a loss in regulatory processes essential to general proteostasis.

Reactive oxygen species (ROS) are a natural by-product of aerobic metabolism, but their over-production has been implicated in the bleaching response to thermal stress^2,49,50. We detected 22 proteins related to antioxidant function in the host, of which only five were identified to have significantly changed abundances relative to the control (Fig. 4). Well studied antioxidants, like Superoxide dismutase, Catalase and Thioredoxins, were detected, but were not differentially abundant (see Supplementary data file). Instead, temperature stress resulted in the unique detection of Glutathione S-transferase (undetected in control samples), which catalyses the binding of the reduced form of glutathione (GSH) to xenobiotic substrates for the purpose of detoxification. The host also increased synthesis of Methionine adenosyltransferase 1 (MAT 1), a protein in the methionine cycle responsible for the synthesis of S-adenosyl methionine (SAMe). SAMe is a primary methyl donor in eukaryotic cells and an important precursor for GSH. This increase in MAT in the cnidarian host is consistent with changes observed in heat shocked Aiptasia²⁹, and in Acropora spp. under a variety of stressors^51,52 and given the intermediary link between MAT and GSH, it is possible that an increase in MAT, and thus SAMe levels, may be complicit in supporting the antioxidant system.

For a range of marine invertebrates, the thioredoxin-peroxiredoxin system has been implicated in scavenging hydrogen peroxides, but its activity is not often measured⁵³. Here we detected an increase in peroxiredoxin-4 (0.48 FC), a thiol-specific peroxidase that catalyses the reduction of hydrogen peroxide (H₂O₂) and organic hydroperoxides to water and alcohols, respectively. Surprisingly however, both peroxiredoxin-5 (− 0.23 FC) and peroxiredoxin-6 (− 0.28 FC), which also function in cell protection against oxidative stress by detoxifying peroxides, decreased in abundance following heat stress. Together, these data indicate that although the response of the coral host to thermal stress involves upregulation of several components of the antioxidant system, it may also simultaneously have reduced levels of other proteins required for detoxification.

Elevated levels of three proteins annotated in the cell death category (of a total of 27), including a caspase-7-related protein, again suggest that the host may have suffered extensive oxidative damage. Caspase-7 (0.23 FC) is an executioner caspase, relatives of which have been implicated in apoptosis in corals^54,55. Executioner caspases are constitutively synthesised but normally activated (by cleavage) only after cells have sustained irreversible damage. The heterogeneity in the responses of tissues or cell-types to temperature-induced oxidative stress could potentially explain the apparent contradictions observed in these data. Symbionts reside exclusively in the gastrodermal (= endodermal) cells of the coral, and it is these, rather than the ectodermal cells, which are likely to be the immediate victims of oxidative stress caused by the symbionts.

Host: central metabolism and calcification

Cell homeostasis relies on balancing energy requirements (e.g. ATP and NADPH) with the production and utilisation of metabolites, which means that shifts in metabolic function are needed to acclimate to environmental change. Of the 149 host proteins identified that were classified into the functional group ‘central metabolism’, 23 were present at different levels in heat-treated and control samples (Fig. 4).

Nitrogen metabolism in the host Amongst the differentially abundant proteins associated with nitrogen metabolism under heat stress were Glutamate dehydrogenase (GDH; 0.54 FC), which increased in abundance, and Glutamine synthetase (GS; − 1.55 FC), which decreased in abundance. Increased GS activity has been reported as a characteristic of symbiotic cnidarians^28,56,57 and hypothesised to serve as a mechanism for imposing nitrogen limitation on the endosymbiont, restricting its growth³⁰. As ammonium assimilation via the high-affinity GS system is energetically costly, decreased levels of this protein reflect metabolic expediency during the collapse of the symbiotic state. To compensate for the decline in GS activity, increased levels of the low affinity, but energetically inexpensive, GDH protein are presumably required to maintain cellular homeostasis during symbiosis breakdown. Note that the dramatic decline in the level of Phosophoserine aminotransferase (FT − 3.63; the most reduced of all host proteins) is also consistent with the host transitioning away from reliance on ammonium assimilation via the GS pathway⁵⁸. These results are supported by a recent study on the heat stress response in the coral Stylophora pistillata²², in which gene expression of GS decreased and the catabolic version of GDH increased, driving amino acid degradation likely to meet the coral’s increased energy requirements (increased respiration). In line with these findings, we measured increased proteolysis and detected increases in proteins associated with respiration (see Supplementary Results and Discussion). If, as proposed by Rädecker et al. (2021), translocation of carbon from the symbiont is insufficient to meet host demand under elevated temperatures, then the host would be forced to degrade its own energy stores. Our study detected an increase in proteins involved in the degradation of lipids, proteins and fatty acids, all of which could be attributed to a direct response to energy limitation in the host.

Carbohydrate and lipid metabolism in the host Although few changes in abundance were observed in proteins associated with carbohydrate or lipid metabolism (5/46 and 4/32, respectively; see Fig. 4), most of these reflect a shift from reliance on translocated photosynthate to storage lipid breakdown. Cnidarians are amongst the few animal groups in which the glyoxalate pathway is present; by enabling the product of beta-oxidation of lipids (acetyl CoA) to support gluconeogenesis, the combined actions of isocitrate lyase and malate synthase in the glyoxylate cycle facilitate breakdown of storage lipids, enabling their use for energy production. In heat stressed Porites asteroides⁵⁹, gene expression of isocitrate lyase increased in abundance but expression of malate synthase did not, and the same scenario was seen at the proteomic level in our Acropora millepora data. The observed decline in the level of Fumarylacetoacetate hydrolase (FAH) domain-containing protein 2 can also be rationalised in terms of operation of the glyoxalate pathway, as many members of the FAH domain-containing protein family have oxaloacetate hydrolase activity⁶⁰, which would interfere with operation of the glyoxalate pathway. Also consistent with a metabolic switch from reliance on carbohydrate metabolism was an observed reduction in the levels of UTP–glucose-1-phosphate uridylyltransferase (− 0.91 FC) after heat stress. This enzyme is involved in glycogen biosynthesis and is upregulated during the establishment of symbiosis in cnidarians^{<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 61" title="Lin, M.-F., Takahashi, S., Forêt, S., Davy, S. K. & Miller, D. J. Transcriptomic analyses highlight the likely metabolic consequences of colonization of a cnidarian host by native or non-native Symbiodinium species. Biol. Open 8, bio038281.
https://doi.org/10.1242/bio.038281}

(2019).” href=”https://www.nature.com/articles/s41598-021-98548-x#ref-CR61″ id=”ref-link-section-d89473859e1135″>61. The increased abundance of a Phospholipase B-like 1 (0.54 FC; Fig. 4), which is capable of releasing fatty acids from phospholipids, and the electron transfer Flavoprotein subunit alpha (1.29 FC), a protein required for beta-oxidation of fatty acids and normal amino acid metabolism⁶², together with decreased abundance (twofold) of an Acetyl-CoA synthetase 2-like protein, the roles of which include fatty acid biosynthesis from carbohydrates, provides further support for the hypothesis that the host was mobilising lipid stores to compensate for decreased availability of algal photosynthate.

Calcification and symbiosis Two of the three carbonic anhydrases identified significantly decreased in abundance in the heat-treated corals (-1.2 FC), a response consistent with previous work⁶³. In reef building corals, carbonic anhydrases (CAs) play two key roles in calcification of the skeleton: supplying dissolved inorganic carbon (DIC) for calcium carbonate precipitation, and the removal of carbonic acid from the precipitation site⁶⁴. Thus, a reduction in this enzyme suggests significant disruption to calcification processes under thermal stress^63,65. Also consistent with suppression of calcification by heat stress, lower levels (− 2.30) of an acidic protein that is a component of the skeletal organic matrix^25,66 were detected after heat stress.

Of the total 111 proteins classified as being involved in intracellular, transmembrane and nitrogen transport, 19 differed in abundance following heat treatment (Fig. 4). Of these, the decreased abundance of a V-ATPase subunit A (-0.60 FC) protein may be directly relevant to the collapse of the symbiosis. V-ATPases are responsible for acidifying intracellular compartments and in corals, by acidifying the symbiosome, a V-ATPase has been shown to both promote symbiont photosynthesis, essentially acting as a component of a carbon-concentrating mechanism (CCM), and to facilitate translocation of photosynthetic products⁶⁷. Therefore, decreased levels of this protein will result in reduced photosynthetic exchange between partners, as both algal photosynthesis and translocation of photosynthate will decline.

Host: cell structure and organisation

More than 80 proteins associated with cell structure and organisation were detected in the coral host proteome, 10 of which were differentially expressed (Fig. 4). Actin, a principal constituent of the microfilament network and a key component of muscle fibres, is one of the most abundant proteins in eukaryotic cytoskeletons⁶⁸. Several proteins that have known roles in the binding and assembly of actin filaments decreased in abundance in this study, including coactosin-like protein (− 0.79 FC), filamin-A (− 0.14 FC), radixin (− 0.08 FC) and alpha-adducin (− 0.15 FC). There were also decreases in proteins contributing to actin organisation, cell shape and microtubule microstructural dynamics in the cytoskeleton, namely amplaxin (− 0.45 FC) and EBF3 (− 0.54 FC). Actin filaments are known to be sensitive to oxidative damage⁶⁸, and similar negative effects of elevated temperature have been observed in both anemones²⁹ and foraminifera⁴⁶. Notably, concomitant with a decline in actin-related proteins, we detected an increase in the cell membrane-cytoskeletal-associated F-actin-uncapping protein LRRC16A (0.93 FC), which plays a role in the disassembly of actin filaments (Fig. 4), corroborating increased actin breakdown. The decline in the structural and cytoskeletal proteins and increase in protein disassembly in this study are consistent with previous work that has shown loss in host tissue integrity with thermal stress^23,24 and provides further evidence of oxidative damage in the host, suggesting significant cell reorganisation and possibly impaired control over cell structure mechanisms.

Of the 29 proteins associated with the host extracellular matrix, Matrilin-2, a protein which may play a role in adhesion or anchoring of filaments increased in abundance (1.08 FC). Proteins strongly decreased in abundance included integrin alpha-6 (VLA-6), which plays a role in cell adhesion processes (− 1.06 FC). The changes in adhesion proteins and matrix assembly processes suggest that coral tissue integrity may be compromised by heat stress, and together with the overall degradation of adhesion proteins, may be the host’s way of encouraging symbiont expulsion.

Functional analysis of the symbiont proteome to elevated temperature

Symbiont: antioxidant function, protein modification and degradation

Thermal stress in algae can lead to over-production of ROS via photosystem malfunction, resulting in denaturation of cellular proteins and their subsequent degradation⁴⁹. Of the 50 proteins detected likely to be involved in protein modification, fourteen were differentially expressed under elevated temperature (Fig. 4). As found in the host, the largest group of proteins to increase in abundance were heat shock proteins (HSPs). Heat shock like 85 kDa protein, which is linked to ATPase activity, increased 0.9-fold, while the fold increases in other HSPs (HSP90, HSP70-14, HSP70, HSP70 (DnaK) and HSP60-2) ranged between 0.43 and 0.82. A small number (three of eight;) of proteins involved in detoxification and stress responses in the symbiont changed in abundance with heat treatment (Fig. 4). Peroxiredoxin-2 and -5 increased in abundance 0.80 and 0.84 FC, respectively, whereas phosphoglucan water dikinase, a protein important for starch degradation in the chloroplast, was significantly depleted (− 3.85 FC) after heat treatment. Several proteins associated with death and senescence (Dipeptidyl peptidase 8, cysteine proteinase SAG39 [− 0.72 FC], Serine/threonine-protein phosphatase 5 [− 3.02 FC] and DAP kinase 3 [− 0.22 FC]) also decreased in abundance.

While the strong expression of chaperones is consistent with previous studies on coral symbionts under thermal stress^29,32,33,69, the limited antioxidant response and lack of any evidence for cell death or protein degradation were unexpected, and consistent with the idea that symbionts mount a damage control response but remain viable under the type of heat-stress imposed here. Furthermore, we found no change in relative protein content in the biomolecular data (Fig. 2), consistent with the absence of increased protease levels. In line with previous studies^11,12,13,70, the relatively slow, yet prolonged, thermal stress treatment reduced photosynthetic performance but may not have induced heavy oxidative stress and subsequent deterioration of symbiont cells.

Symbiont: central metabolism

Nitrogen metabolism Tight regulation of nitrogen cycling between the host and symbiont is often considered central to the evolutionary success of the coral symbiosis^56,71,72. Of the 29 symbiont proteins detected that were nominally associated with nitrogen metabolism, ten were affected by elevated temperature and the majority of these decreased in abundance (Fig. 4). Both methionine synthase (MS), which methylates homocysteine to regenerate methionine, and type-3 glutamine synthetase (GS), an enzyme essential in ammonium assimilation and glutamine biosynthesis⁷³ were undetectable following heat-treatment. While these changes may indicate nitrogen limitation, it must be noted that two other proteins also classified as GS and with higher spectral counts did not change significantly following the treatment (see Supplementary Data File), suggesting glutamine biosynthesis may not have been severely affected in the symbiont; a finding consistent with symbionts of Acropora aspera³² and Stylophora pistillata²² under thermal stress.

Carbohydrate metabolism A total of 17 symbiont proteins classified as associated with carbohydrate metabolism changed during the heat stress experiment. Proteins that increased in abundance included Glycerol-3-phosphate dehydrogenase (GPDH) (0.61 FC), an important link between carbohydrate and lipid metabolism. GPDH catalyses the reduction of NADH and dihydroxyacetone phosphate (DHAP) to form NAD⁺ and Glycerol-3-phosphate (G3P), an intermediary metabolite that connects multiple metabolic pathways such as glycerolipid synthesis, glycolysis and gluconeogenesis. Overexpression of GPDH in the marine diatom Phaeodactylum tricornutum was shown to result in significant increases (6.8-fold) of glycerol and a 60% increase in neutral lipid content⁷⁴. Thus, the increase in GPDH here could equate to increase in G3P and therefore more glycerol and lipid, a response commonly observed in the coral symbionts under temperature stress^10,35,37,75.

The heat-treated proteome also showed evidence of increased carbohydrate production, consistent with the FTIR biomolecular data (Fig. 2). UDP-sugar pyrophosphorylase (0.71 FC), which converts sugar-1-phosphate into UDP-glucose, and UDP-glucose 6-dehydrogenase (2.81 FC), responsible for the interconversion of UDP-glucose and UDP-glucuronate, an intermediate in polysaccharide biosynthesis including that of hemicellulose and pectin in plants⁷⁶, were both significantly more abundant. UDP-sugars are the main precursors of biomass production and primary metabolites in plants (e.g. sucrose, cellulose, hemicellulose and pectin), as well as glycoproteins and glycolipids^{<a data-track="click" data-track-action="reference anchor" data-track-label="link" data-test="citation-ref" aria-label="Reference 77" title="Kleczkowski, L. A., Decker, D. & Wilczynska, M. UDP-Sugar Pyrophosphorylase: A new old mechanism for sugar activation77, and starch synthesis in dinoflagellates is thought to involve the incorporation of UDP-glucose by granule-bound starch synthase78.}

We also detected increases in phosphoenolpyruvate carboxykinase (PEPCK; 0.79 FC), which is part of the gluconeogenesis pathway (the production of glucose from non-carbohydrate compounds). In addition to the loss of two pyruvate kinases (PKLR, PK), co-associated with the glycolysis pathway (see Supplementary Results and Discussion), there was a decrease in the abundance of proteins associated with CoA transferase processes in carbohydrate metabolism. CaiB/b (− 3.82 FC) and pentafunctional AROM polypeptide (− 0.59 FC) both decreased and there was decreased abundance of a glycogen branching enzyme (1,4 alpha; − 2.1 FC), a protein involved in glycogen biosynthesis and accumulation⁷⁹. In direct contrast with the animal host, the decreased abundance of Isocitrate lyase 1 (− 0.28 FC) detected in the symbiont presumably limits the breakdown of storage lipids, thereby eliminating a potential nutrient source via storage lipid mobilisation^80,81. The loss of glycolytic enzymes combined with declines in proteins involved in glycogen biosynthesis, suggest a slowing in the symbiont’s energy production. Both lipid accumulation and higher carbohydrate content that was observed in the FTIR data (Fig. 2) are congruent with the increase in UDP sugar production for sucrose, cellulose or glycolipid synthesis observed in the proteome.

Fatty acid and lipid metabolism All six of the 23 detected proteins associated with fatty acid and lipid metabolism that were differentially regulated, decreased in the symbiont proteome (Fig. 4). The symbiont decreased soluble inorganic pyrophosphatase 1 to below detection level; a protein which catalyses the conversion of pyrophosphate into phosphate ions, releasing energy that can be used to catalyse otherwise unfavourable chemical reactions. This mechanism is particularly important for the activation of fatty acids for beta-oxidation⁸². There was also a strong decline in the abundance of acetyl-CoA carboxylases (− 1.58 to − 1.61 FC), which provide the malonyl-CoA substrate for fatty acid biosynthesis⁸³. Congruent with the above, there was a decrease in glycerol-3-phosphate acyltransferase (GPAT; -1.5 FC), the rate-limiting enzyme in the de novo pathway of glycerolipid synthesis, which plays a pivotal role in the regulation of triglyceride (TAG) and phospholipid synthesis⁸⁴. Together with the downregulation of ATP-citrate synthase subunit 1 (− 0.89 FC), a protein that catalyzes the formation of cytosolic acetyl-CoA that is mainly used for the biosynthesis of fatty acids and sterols, these changes to the symbiont proteome signal a decline in fatty acid and lipid biosynthesis. Despite this apparent downregulation in lipid production, we observed an accumulation of lipid in the cell (Fig. 2), an inconsistency that may be explained by reduced fatty acid translocation to the host and increased glycerol production within the cell.

Symbiont: photosynthesis and carbon fixation

When in hospite, the symbiont delivers a major proportion of the reduced energy derived from photosynthesis to the host. Any alteration in this activity and/or in nutrient exchange may be indicative of imminent breakdown of the host-symbiont relationship. In this study, the photosynthetic responses as measured by fluorometry (Fig. 1) were consistent with published studies on bleaching, showing downregulation of photosynthetic efficiency and electron transport in the symbiont under elevated temperatures^7,9,14. The proteomic data reported here show that declines in photosynthetic efficiency and electron transport coincide with the decreased abundance of proteins associated with photosynthesis, chlorophyll biosynthesis and carbon fixation (Figs. 4 and 5). Congruent with the measured decline in photosynthetic efficiency, we detected a decrease in the abundance of photosynthetic electron transport chain proteins including the cytochrome b6-f complex (-0.68 FC), and the PSII-reaction centre protein subunits, D2 (− 0.56 FC) and D1 (− 0.35 FC). The decline in these PSII subunits are indicative of photoinhibition^14,15,17, whereby protein repair mechanisms are unable to keep pace with degradation⁸⁵, leading to increased ROS production and subsequent fatty acid oxidation. However, the absence of a strong oxidative stress response in the symbiont suggests a possible controlled downregulation of activity, either to conserve nitrogen use and/or limit ROS production.

Decreases in abundance of several proteins related to photoprotection and photosynthetic pigment biosynthesis were also observed. We detected a strong decrease in Zeaxanthin epoxidase (− 2.75 FC), a protein responsible for converting zeaxanthin into antheraxanthin and PSII stability/assembly factor HCF136 (− 2.43 FC), essential for PSII biogenesis (including psbD and cytochrome b559). Major decreases in abundance were observed in the cases of magnesium protoporphyrin IX methyltransferase (− 3.82 FC) and Mg-chelatase subunit ChlD (− 3.11 FC), proteins involved in chlorophyll biosynthesis, as well as in Glutamate-1-semialdehyde aminotransferase 1, which is involved in the biosynthesis of tetrapyrroles (− 0.55 FC). This predominant decrease in photosynthetic protein complexes and pigment biosynthesis proteins of PSII indicate a possible reduction in PSII units either due to dissociation of light harvesting complexes from the thylakoid membrane because of thermal damage^86,87 or a decreased dependency on photosynthesis by the symbiont. The increase in peridinin-chlorophyll a binding proteins (0.34 FC) however, lends support to the idea that the symbiont is simply altering pigment composition to better suit life outside the symbiosis. Indeed, the significant changes to the symbiont proteome—the decline in photosynthetic performance, absence of extensive oxidative damage, combined with increased accumulation of lipids—might be reflective of the symbiont adjusting to a non-symbiotic lifestyle.

The decrease in light reaction processes was paralleled with a decline in the carbon fixation protein RuBisCO, which decreased -1.46-fold under heat stress. Dinoflagellates contain the bacterial-type (Form II) RuBisCo, which is more thermally stable than the typical plant (Form I) enzyme⁸⁸. Thus, the decreased abundance of RuBisCO may reflect broad changes in symbiont metabolism or DIC limitation from the decreased abundance of V-ATPase (which acts as a CCM).

The results from this study (Fig. 5) are largely consistent with a model recently proposed²² in which host energy limitation from reduced carbon translocation results in the collapse of the coral–symbiont relationship. In our study, the increase in proteins involved with proteolysis and lipid catabolism are indicative of energy limitation in the host, and changes in key nitrogen cycling proteins, like those found by Rädecker et al. (2021), provide support for possible amino acid breakdown. Additionally, the reduction in symbiont photosynthesis and increased carbon storage provides further evidence for a reduction in carbon translocation to the host, supporting a model of coral symbiosis breakdown based on host energy limitation originating from increased demand and/or reduced carbon flow from the symbionts. Importantly, our study uncovered a decrease in the proton-pump V-ATPase that is known to maintain a low pH in the symbiosome helping concentrate DIC at the host/symbiont interface. This change in symbiosome condition could explain a loss in carbon translocation to the host, as DIC limitation in the symbiont would result in a decline in photosynthesis while also reducing the efficiency of photosynthate translocation through the symbiosome⁶⁷. While the model presented by Rädecker et al. 2021, does not attempt to explain symbiont expulsion from the host, our data showed considerable changes to proteins involved in maintaining the structural integrity of the host tissue, and as such, symbionts may be lost through general deterioration of host tissue stability. Finally, our data revealed significant ROS-driven physiological deterioration in the host, which was not as evident in the symbiont. Instead, the symbiont displayed proteomic changes consistent with a general reduction in metabolic activity. While previous studies have established a connection between elevated temperature and ROS production in coral symbionts⁷⁰, the data presented here suggests that, under the environmental conditions employed in this study, the symbionts are able to cope with increased ROS or at least manage it by reducing activity. As such, it is likely that damage in the host is primarily a result of host-produced ROS as opposed to ROS generated by the symbiont, as is frequently proposed⁸⁹.

Source: Ecology - nature.com