Quantitative proteomics insights into Chlamydomonas reinhardtii thermal tolerance enhancement by a mutualistic interaction with Sinorhizobium meliloti

ABSTRACT Interactions between photosynthetic microalgae and bacteria impact the physiology of both partners, which influence the fitness and ecological trajectories of each partner in an environmental context-dependent manner. Thermal tolerance of Chlamydomonas reinhardtii can be enhanced through a mutualistic interaction with vitamin B12 (cobalamin)-producing Sinorhizobium meliloti. Here, we used label-free quantitative proteomics to reveal the metabolic networks altered by the interaction under normal and high temperatures. We created a scenario where the growth of Sinorhizobium requires carbon provided by Chlamydomonas for growth in co-cultures, and survival of Chlamydomonas under high temperatures relies on cobalamin and possibly other metabolites produced by Sinorhizobium. Differential abundance analysis identified proteins produced by each partner in co-cultures compared to mono-cultures at each temperature. Proteins involved in cobalamin production by Sinorhizobium increased in the presence of Chlamydomonas under elevated temperatures, whereas in Chlamydomonas, there was an increase in cobalamin-dependent methionine synthase and certain proteins associated with methylation reactions. Co-cultivation and heat stress strongly modulated the central metabolism of both partners as well as various transporters that could facilitate nutrient cross-utilization. Co-cultivation modulated expression of various components of two- or one-component signal transduction systems, transcriptional activators/regulators, or sigma factors, suggesting complex regulatory networks modulate the interaction in a temperature-dependent manner. Notably, heat and general stress-response and antioxidant proteins were upregulated in co-cultures, suggesting that the interaction is inherently stressful to each partner despite the benefits of mutualism. Our results shed insight into the metabolic tradeoffs required for mutualism and how metabolic networks are modulated by elevated temperature. IMPORTANCE Photosynthetic microalgae are key primary producers in aquatic ecosystems, playing an important role in the global carbon cycle. Nearly every alga lives in association with a diverse community of microorganisms that influence each other and their metabolic activities or survival. One chemical produced by bacteria that influence algae is vitamin B12, an enzyme cofactor used for a variety of metabolic functions. The alga Chlamydomonas reinhardtii benefits from vitamin B12 produced by Sinorhizobium meliloti by producing the amino acid methionine under high temperatures which are required for Chlamydomonas thermotolerance. Yet, our understanding of this interaction under normal and stressful temperatures is poor. Here, we used quantitative proteomics to identify differentially expressed proteins to reveal metabolic adjustments made by Chlamydomonas and Sinorhizobium that could facilitate this mutualism. These findings will enhance our understanding of how photosynthetic algae and their associated microbiomes will respond as global temperatures increase.

bacterium Sinorhizobium meliloti 1021 (Sm 1021) in mono-and co-cultures under normal and thermal stress conditions.Here, we created a scenario where the growth of Sinorhizobium relies on carbon provided by Chlamydomonas for growth in co-cultures and the survival of Chlamydomonas under high temperatures relies on B 12 and possibly other metabolites produced by Sinorhizobium.The differentially expressed proteins (DEPs) and the related pathways in the co-cultures under different temperatures were analyzed to predict the metabolic adjustments, including stress responses, made by Chlamydomonas and Sinorhizobium to facilitate this mutualism.These data shed new insights into the molecular bases of the mutualistic interactions between a photosyn thetic microalga and B 12 -producing bacterial partner and how those cellular responses may facilitate algal thermal tolerance enhancement.

RESULTS
Mono-cultures of Chlamydomonas and Sinorhizobium and their co-cultures were grown at 25°C (normal temperature) for 3 days prior to exposing them to a temperature upshift to 42°C (high temperature).Consistent with our previous study (22) on solid media, liquid cultures grew well at 25°C and Chlamydomonas mono-cultures exhibited visible chlorosis within 3 days after a 42°C temperature upshift (Fig. 1A).Following the temperature upshift, the Chlamydomonas population size neither increases nor decreases in the co-cultures, although it begins to decrease within 2 days after the temperature upshift in the Chlamydomonas monocultures (Fig. 1C).In contrast, Sinorhizobium survival was little influenced by co-cultivation with Chlamydomonas or by the temperature upshift (Fig. 1B).To reveal insight into the adaptive responses of each member of the co-cul ture to a temperature upshift and how the interactions between Chlamydomonas and Sinorhizobium (mono-vs co-culture) influences protein expression, we chose to examine the Chlamydomonas and Sinorhizobium proteomes 2 days after the temperature upshift since it preceded the dieoff that occurred in Chlamydomonas monocultures.

Overview of C. reinhardtii and S. meliloti proteome
The proteomes of Chlamydomonas and Sinorhizobium were obtained and analyzed as outlined in the "Materials and methods." After LC-MS/MS ( liquid chromatography−tan dem mass spectrometry) analysis, a total of 2,447 C. reinhardtii (about 13% of the total predicted) and 2,396 S. meliloti (about 34% of the total predicted) measurable proteins were identified.There was high congruence between experimental replicates, with correlations (R 2 ) ranging from 0.90 to 0.97 for C. reinhardtii samples and 0.97 to 0.99 for S. meliloti samples.
We used distributed normalized spectral abundance factor (dNSAF), a label-free quantitative measure of protein abundance (25) based on spectral counts corrected for peptides shared by multiple proteins, for comparative proteomic analyses.Based on pair-wise comparisons, we identified DEPs in both Chlamydomonas and Sinorhi zobium under each growth condition (Fig. 2; Tables S1 to S8).When comparing high vs normal temperatures, we detected slightly more differentially expressed Chlamy domonas proteins in the mono-than co-cultures (Fig. 2A; Tables S1 and S2), while for Sinorhizobium, there were no such differences (Fig. 2B; Tables S5 and S6).Interest ingly, when comparing co-with mono-cultures, we detected twofold more differen tially expressed Chlamydomonas proteins under normal compared to high-temperature upshifts (Fig. 2C; Tables S3 and S4).Yet, for Sinorhizobium, there were slightly more DEPs under high compared to normal temperature treatments (Fig. 2D; Tables S7 and S8).We also identified differentially expressed proteins that responded to both treatments, as illustrated by the overlapping circles in the Venn diagrams (Fig. 2).Significantly, we identified uniquely differentially expressed Chlamydomonas and Sinorhizobium proteins in response to the temperature upshift in mono-and in co-cultures, suggesting that responses to a temperature upshift can be modulated by the presence of a mutualistic partner.

Comparative proteomics reveal changes in Chlamydomonas and Sinorhi zobium metabolism at high growth temperature
The broader gene ontology (GO) analyses revealed the magnitude and nature of how a temperature upshift in co-and mono-cultures influenced proteomes.Changes in the Chlamydomonas proteome when co-cultured with Sinorhizobium were substantial and distinct from the proteome in the absence of Sinorhizobium, particularly proteins associated with protein folding, structural constituents of ribosomes, and organelle subcompartments (Fig. 3A and B; Tables S1 and S2).Following heat stress, those 572 DEPs (Fig. 2A) of the Chlamydomonas proteome were unaffected by co-cultivation with Sinorhizobium, particularly small molecule binding, protein binding, organic cyclic compound binding, and heterocyclic compound binding proteins (Fig. 3C).Yet, several heat shock proteins and chaperones (e.g., CLPB4 and HSP70C) are enriched in both Chlamydomonas mono-and co-cultures at elevated temperatures, indicating thermal tolerance relies on the same cellular responses to heat stress in the absence or presence of Sinorhizobium.
The temperature-upshift had a more dramatic effect on the metabolism of Sinorhi zobium than Chlamydomonas (compare Fig. 2A with 2B), and the metabolic processes most affected were those involved in organic cyclic and heterocyclic compound binding and organic substance (Fig. 3D through F).Many of the metabolic processes up-regula ted in Sinorhizobium by a temperature upshift included those involved in small molecule metabolic process and intracellular anatomical structures.In comparison, there were more unique downregulated proteins in Sinorhizobium when exposed to heat stress as a mono-than as a co-culture with Chlamydomonas (402 compared to 311 as shown in Fig. 2B, compare Fig. 3D with 3E).When co-cultured with Chlamydomonas under heat stress, Sinorhizobium metabolism changes included, for example, the upregulation of ion binding, small molecule binding, organic substance metabolic process, and biosynthetic processes.

Comparative proteomics reveals changes in Chlamydomonas and Sinorhi zobium metabolism when grown in coculture with each other
The GO analyses also revealed that Chlamydomonas and Sinorhizobium proteomes were strongly influenced by interactions with each other, although the extent of the response was modulated by heat stress (Fig. 4).Many processes, such as organic substance metabolic process, primary metabolic process, and intracellular anatomical structure, were downregulated in Chlamydomonas co-cultures under heat stress (Fig. 4B).Following the temperature upshift, however, the magnitude of metabolic changes was vastly greater in Sinorhizobium than in Chlamydomonas co-cultures, with an expression of the majority of proteins upregulated (28 compared to 2, compare Fig. 4D and B).The processes most affected by heat-stress in Sinorhizobium co-cultures were involved in organic cyclic and heterocyclic compound binding and various metabolic (organic substance, nitrogen, biosynthetic, cellular, and primary) processes (Fig. 4D).Importantly, there were few proteins in Chlamydomonas (such as DNJ8 and DAD1) and Sinorhizobium (such as Sma1231 and AatB) co-cultures whose expression was not influenced by growth temperature (Fig. 2C and D), which may be essential or contribute to maintaining the mutualistic association.

Heat stress and co-culturing with S. meliloti alter C. reinhardtii methionine biosynthesis
Consistent with a previous report (22), Chlamydomonas under normal temperature treatments relies on the cobalamin-independent METE (Cre03.g180750.t1.2) rather than the cobalamin-dependent METH (Cre06.g250902.t1.1) methionine synthase (Fig. 5A).Moreover, in co-culture following a temperature upshift, there was nearly a 2.5-fold increase in cobalamin-dependent methionine synthase METH expression (Fig. 5A) compared to Chlamydomonas monocultures.Given that methionine contributes to cellular SAM production, it is not surprising that we observed increased expression of SAM synthesis and SAM-dependent methylation reactions in Chlamydomonas following a temperature up-shift (Fig. 5B).

Co-cultivation with C. reinhardtii and high temperature increases expression of S. meliloti proteins for B 12 biosynthesis
De novo cobalamin biosynthesis is complex and includes the production of the tetrapyrrole uroporphyrinogenIII which is converted to precorrin-2, an intermediate in the aerobic pathway of cobalamin synthesis by Sinorhizobium (26).In Sinorhizobium co-cultures, the expression of several cobalamin and uroporphyrinogenIII biosynthesisrelated proteins was upregulated (Fig. 6A; Table S9).Based on summing the dNSAF values of these proteins involved in cobalamin biosynthesis, there is a significant (P = 0.003) increase in B 12 biosynthesis pathway expression by Sinorhizobium in co-cultures under high-temperature condition (Fig. 6B).Interestingly, we observed significantly increased expression of components of a highaffinity B 12 uptake system (btuBCDF) salvage pathway in Sinorhizobium during co-culture with Chlamydomonas (Table 1) under normal and high temperatures, although at levels that were slightly below our 1.5-fold differential expression threshold.

Co-cultivation and heat stress modulate Chlamydomonas and Sinorhizobium central metabolism
To evaluate how environmental temperature and co-culturing may influence the other metabolic pathways, we analyzed the changes in the expression of central metabolic pathways of Chlamydomonas and Sinorhizobium based on KEGG (Kyoto Encyclopedia  of Genes and Genomes) categories.The results revealed that there are overall 12 pathways in Chlamydomonas at high temperature (Fig. 7), for example, tryptophan metabolism [Fold change (FC) = 0.39, P < 0.001] and glycolysis/gluconeogenesis (FC = 0.87, P < 0.001), which were significantly downregulated, while only fatty acid degradation (FC = 2.59, P < 0.001) and other types of O-glycan biosynthesis (FC = 2.17, P = 0.019) were upregulated (Fig. 7, blue and red bars under "21gr-H").In con trast, Chlamydomonas co-culture at normal temperature (Fig. 7, bars under "21gr-N") exhibited increased expression of a variety of amino acid (e.g., lysine, FC = 1.46,P = 0.011), citrate (TCA) cycle (FC = 1.24,P < 0.001), and co-factor (e.g., nicotinate, FC = 1.24,P = 0.04) biosynthesis pathways.However, in Sinorhizobium co-cultures at high temperature, the number of upregulated pathways was greater than co-cultures at normal temperature (24 vs 7), primarily pathways associated with amino acid, carbo hydrate, and energy functions (Fig. 7, bars under "1021-H" and "1021-N").Increased expression of various amino acid metabolism, carbohydrate, and fatty acid degradation likely reflects access to carbon and energy resources derived from Chlamydomonas (actively or passively produced) that as a consequence alters Sinorhizobium's metabolic networks.The altered Sinorhizobium metabolic network is also reflected in the enrich ment of several putative adenylate/guanylate cyclases (e.g., CyaF1 is enriched >11-fold; Table S8) that produce cAMP/cGMP (cyclic adenosine monophosphate/cyclic guanosine monophosphate) which are involved in catabolite control/regulation.Interestingly, we detected several pathways whose expression was opposing in Chlamydomonas compared to Sinorhizobium in high-temperature co-cultures.For example, tryptophan metabolism, valine, leucine, and isoleucine degradation, glycoly sis/gluconeogenesis, and methane metabolism were upregulated in Sinorhizobium but downregulated in Chlamydomonas (Fig. 7).However, we did not observe a similar opposing expression pattern in the Chlamydomonas and Sinorhizobium co-cultures grown at normal temperature, except for valine, leucine, and isoleucine degrada tion.Exchanges of metabolites between Sinorhizobium and Chlamydomonas could be facilitated with increased expression of specific transporters during mutualistic interactions, based on different expression levels in the co-cultures compared to mono-cultures (Table 1).

Co-cultivation and heat stress modulate regulatory networks
Co-cultivation and heat stress alter the abundance of various regulatory proteins, many of which are components of two or one-component systems, or transcriptional activators/regulators (Table 2; Tables S1 to S8).In Chlamydomonas, the abundance of several well-characterized signal transduction systems (e.g., MAPK6 and RAPTOR) is among the most highly upregulated proteins in mono-culture, but their expression is greatly modulated by Sinorhizobium (Table 2).Interestingly, the downregulation of glycogen synthase kinase 3 (GSK3) (27) was detected in Chlamydomonas co-cultures at both normal and high temperatures, suggesting a complex regulation between growth and stress response in the co-cultures.
In Sinorhizobium, specific regulators are also required to address nutritional and physicochemical conditions during the mutualism.For example, the expression of the phosphate regulon transcriptional regulatory proteins PhoR and PhoB is highly increased in co-culture, particularly under high temperature (Table 2); this is consis tent with increased expression of various phosphate transport system components, including PhoD and PhoU (Table S8) and PstB (Table 1).A reduction of Glk (Gluco kinase) in Sinorhizobium co-cultures may facilitate the utilization of various carbon sources provided by Chlamydomonas.Interestingly, several regulatory proteins involved in nitrogen metabolism or oxygen sensing (e.g., FixJ, NtrC, and NtrX) or activated by blue light (Q92W49) were significantly upregulated in Sinorhizobium co-cultures exposed to high temperatures.

DISCUSSION
Our findings confirm and expand on the biology of mutualistic interactions between algae and bacteria, highlighting the role of vitamin B 12 in these interactions (11,12,34) and in algal thermotolerance (22,35).Chlamydomonas METE repression occurs either when co-cultivated with Sinorhizobium or during temperature upshifts.Since methio nine is a direct precursor for S-adenosylmethionine, the major cellular methyl donor, methionine may influence downstream methylation reactions (36,37).As reported previously, when exposed to high temperature, increased methylations of DNA [such as 5-methylcytosine (5mC) and N 6 -methyladenine (6mA)] and histone may occur, which triggers expression of heat-responsive genes critical for plant basal and acquired thermal tolerance (23,38,39).Here, the upregulation of S-adenosylmethionine synthase (SAS1) and certain methyltransferases by heat stress suggests the possible role of B 12 -medi ated methionine biosynthesis for supplying sufficient methyl groups for downstream methylation reactions required for the heat stress response.In addition, since methionine is a structural amino acid and the first amino acid in the initiation of most proteins, its availability can be critical for overall protein synthesis and hence cellular physiology.Collectively, this suggests that methylation reactions and protein translation are central factors involved in C. reinhardtii thermal tolerance when co-existing with B 12 -producing bacteria (Fig. 8).It also provides a foundation on which to develop new hypotheses for testing how organisms can adapt to a changing environment, and how the environment modulates microbial interspecies interactions (40).
As a key vitamin, B 12 can only be synthesized by certain prokaryotic organisms, and its direct or indirect release into the environment can be a key metabolite that facilitates recruitment of B 12 producers to the alga phycosphere (14,17,41).Consistent with prior observations (22), we observed upregulation of the B 12 biosynthesis pathway in S. meliloti in co-cultures, particularly under high-temperature treatments (Fig. 5).Several proteins, such as HemB, HemD, and CobA, are enriched in the co-cultures under both normal and high temperatures (Fig. 5), which are consistent with the hypothesis that B 12 production is regulated during algal-bacterial interactions (42).Why there is increased B 12 expression is still unclear, whether it is a consequence of a specific signal produced by Chlamydomonas that up-regulates Sinorhizobium B 12 expression and/or as a consequence of the nutrients provided by Chlamydomonas that alters expression levels.It is important to emphasize that the growth of S. meliloti in co-culture relies on carbon released (actively or passively) by Chlamydomonas since no other carbon source is provided in the medium.Whether increased expression of the cobalamin biosynthesis pathway is solely intended to meet Sinorhizobium's needs or a direct consequence of stimulation by Chlamydomonas for overproduction, the net result is cross-feeding.
In addition to B 12 , other metabolites may also play roles in the thermal tolerance of this Chlamydomonas-Sinorhizobium co-culture.Our data showed several opposing expression patterns of metabolic pathways present in high but not normal temperatures of co-cultures (Fig. 7).These results imply that extensive interactions may be occurring based on a division of labor, whereby Chlamydomonas provides photosynthate as a Sinorhizobium carbon source, and Sinorhizobium provides specific energy-expensive metabolites in addition to vitamin B 12 .Of particular interest are differences in trypto phan metabolism since it is considered the most energetically expensive amino acid to produce (43) and one precursor of indole-3-acetic acid (IAA), a key signaling molecule in algal-bacterial interactions (44).It is conceivable that the elevated tryptophan metabo lism in Sinorhizobium may increase the production of IAA, which can promote the growth of Chlamydomonas under high temperature, in a manner that may be similar to the role of tryptophan and IAA in stimulating diatom Pseudo-nitzschia multiseries cell division when co-cultured with bacterium Sulfitobacter (44).
Another important question is how B 12 and the other metabolites are transported and sensed.In bacteria, exogenous corrinoids are transported into the cell via an ABC transport system composed of Ton-B-dependent outermembrane permease, a periplasmic-binding protein, a cytoplasmic membrane permease, and an ATPase (BtuB, F, C, and D) (45).The increased expression of BtuC and BtuF in Sinorhizobium co-cul tures (Table 1) suggests that during co-cultivation, leakage of cobalamin out of the cell is potentially compensated for by re-importing cobalamin or cobinamide back into Sinorhizobium for salvaging purposes (8,46,47).However, whether B 12 secretion is passive or, if active, how it is regulated in co-cultures still remains unclear.In diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana, cobalamin acquisition protein 1 (CBA1) has been implicated in cobalamin uptake (48).Recently, CBA1 homolog protein (Cre02.g081050.t1.2) was identified in Chlamydomonas (49).Our data showed that CBA1 expression is slightly, albeit not significantly, increased in the mono-cultures after a temperature upshift (FC of Mo-H:Mo-N = 1.08,P = 0.53) and the co-cultures under normal temperature (FC of Co-N:Mo-N = 1.077,P = 0.69).However, its expression is decreased slightly in the co-cultures when comparing high vs normal temperature (FC of Co-H:Co-N = 0.619, P = 0.086) or when comparing co-cultures vs mono-cultures under high temperature (FC of Co-H:Mo-H = 0.617, P = 0.12).These patterns imply that the Chlamydomonas-Sinorhizobium microenvironment can be rich in B 12 under high temperature since CBA1 expression can be downregulated when B 12 is abundant as reported recently (49), or Chlamydomonas does not solely rely on CBA1 to obtain B 12 from Sinorhizobium during heat stress.Additionally, the other DEPs associated with signal transduction and transportation (Tables 1 and 2) imply that there are comprehensive interspecies recognitions and other metabolic communications within this coculture.
Prolonged heat stress causes enhanced rates of protein unfolding as reflected by increased expression of various molecular chaperones and proteases designed to correct folding and prevent protein aggregation (50,51).In Chlamydomonas, heat stress increased their abundance dramatically, but co-cultivation with Sinorhizobium had no apparent ameliorative effect.Interestingly, in co-cultures at normal temperatures, there was a small (~1.62-fold) increase in the expression of a variety of heat shock proteins (e.g., peptidylprolyl isomerases, HSP-like chaperone superfamily proteins; Table S3).Similarly, in co-cultures at normal temperature (Table S3), there was a slight increase in expression by Chlamydomonas of proteins for glutathione S-transferase, thioredox ins, and a manganese superoxide dismutase involved in the oxidative stress response.Collectively, these findings indicate that Chlamydomonas interactions with Sinorhizobium are modestly stressful, which has also been observed in other alga-bacterial interactions (16), although why is currently unclear.Protective strategies, such as expression of chaperone or universal stress proteins, to ameliorate the stress or to increase expression of proteins for exopolysaccharide/biofilm formation to minimize the effects of stress indicate that there is a fine balance between beneficial and detrimental consequences of the interaction.
Compared with their mono-cultures, the regulation of growth and stress responses by Chlamydomonas and Sinorhizobium in co-culture is complex.This is reflected in changes in the expression of some key, multi-function regulators.For example, glycogen synthase kinase 3 (GSK3) proteins, the highly conserved serine/threonine kinases in eukaryotes, are required for growth, development, and biotic and abiotic stress responses in plants (27).In Chlamydomonas-Sinorhizobium co-cultures, GSK3 expression levels are downre gulated (Table 2).This suggests that one or more Sinorhizobium-derived signals can greatly reduce the expression of GSK3 independent of environmental temperature.This reduction may result in either the common (e.g., the shared DEPs in Fig. 2C) or specific downstream responses (e.g., the unique DEPs in Fig. 2C) by Chlamydomonas in the presence of Sinorhizobium.Future functional studies of these proteins will contribute to our understanding of the molecular mechanism of algal-bacterial interactions.
The mutualistic exchange of nutrients between algae or diatoms and microbes (in particular, algae and B 12 -producing bacteria) has become an area of increasing interest given the impact that these interactions could have on global nutrient cycling (12,16,22,44).Thus far, a common molecular interaction mechanism is still not well known, suggesting that there is a certain degree of specificity in the interaction, likely in a context-dependent manner.Here, our label-free quantitative proteomics approach has provided new insights into the molecular basis of algal-bacterial interactions and highlights the balance between the beneficial properties required for maintaining co-existence (metabolite exchange) and the potentially detrimental challenges each partner must overcome to survive.This information provides additional insight into the processes governing community interactions and function in the phycosphere.

Growth of Chlamydomonas and Sinorhizobium
C. reinhardtii 21gr (CC1690, Chlamydomonas Resource Center) and S. meliloti 1021 (52) were maintained on Tris-acetone-phosphate and Tryptone-Yeast Extract media, respectively (53,54).Co-inoculation of Chlamydomonas and Sinorhizobium and thermal tolerance assays were performed as described previously (22), with the exception that the thermal tolerance assays were performed in liquid cultures rather than on solid media.Briefly in the thermal tolerance assays, Chlamydomonas and Sinorhizobium were inoculated in minimal salts (MM) liquid medium (55) at approximately a 1:10 (Chlamydo monas: Sinorhizobium) cell density ratio.For the Sinorhizobium mono-culture controls in MM medium, 0.1% sucrose was supplemented as the carbon source.These cultures were grown under continuous illumination (120 µmol photons m −2 s −1 ) at 25°C.For inducing a temperature upshift, mono-and co-cultures were grown at 25°C for 3 days prior to transferring cultures to 42°C.

Protein extraction and digestion
Two days after the temperature upshift, the mono-and co-cultures of Chlamydomonas and Sinorhizobium were collected, and protein samples were prepared as described previously (56).Briefly, cells were collected and resuspended in a lysis buffer consisting of 20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% DDM (N-dodecyl-β-D-maltoside), 50 mM DTT (dithiothreitol), and 1 × Roche protease inhibitor cocktail.Cells were lysed with a sonication probe for 10 min on ice (135 W output, 5 s on, and 5 s off), and the whole cell lysate was centrifuged (12,000 g at 4°C for 15 min) to remove cell debris.The supernatant-containing proteins were precipitated with −20°C cold acetone for 2 h, and the precipitate was dissolved in 50 mM ammonium bicarbonate digestion buffer.Protein concentration was determined using the BCA Protein Assay Kit (Beyotime, Beijing, China).Protein aliquots (100 µg) were reduced with 10 mM dithiothreitol for 0.5 h at 37°C before they were alkylated by 15 mM iodoacetamide in the dark for 30 min.Then, the proteins were digested with trypsin (Promega, UK) at a ratio of 1:45 (wt/wt) for 18 h.Desalting was performed using ZipTip C18 columns solid phase extraction (Millipore) and 0.1% formic acid (FA).The peptides were then eluted with 75% acetonitrile and 0.1% FA.

LC-MS/MS analysis
Proteomics samples were analyzed using an Exactive Plus Orbitrap Mass Spectrometer coupled with an EASY-nLC 1200 System (Thermo Fisher Scientific, Rockford, USA).Briefly, about 1 µg of protein sample was separated using a C18 column (150 Å pores, 15 cm length, and 100 µm diameter) at a flow rate of 300 nL/min for 120 min.The segmented linear gradient of mobile phase A (5% acetonitrile/0.1% FA) and 10%-80% of mobile phase B (90% acetonitrile/0.1% FA) was used to elute peptides prior to nanoelectros pray ionization at 2.0 kV.Mass spectrometry was performed using a data-dependent acquisition mode, and full precursor scans were collected with a mass range ranging from 350 to 2,000 m/z at 70 k resolution.The 20 most abundant precursor ions per scan were chosen for higher-energy collisional dissociation fragmentation.For MS-MS analysis, a resolution of 17,500 and an isolation window of 1.8 m/z were used in the Orbitrap Mass Spectrometer.The dynamic exclusion duration was 40 s, and the automatic gain control (AGC) target value was 5e 4 .
Protein sequences of Chlamydomonas and Sinorhizobium were searched using Proteome Discoverer 2.1 against Chlamydomonas protein database (v5.6, www.phyto zome.net)appended with NCBI chloroplast and mitochondrial databases (BK000554.2and NC_001638.1)and S. meliloti Uniprot proteome database, respectively.Only proteins detected in at least three replicate samples of treatment and included at least one unique peptide [false discovery rate (FDR ≤ 0.01)] met our criterion to be considered as a measurable protein.

Data analysis
For protein expression level comparisons, dNSAF was calculated for each protein as previously described (24).The data of each treatment were derived from four inde pendent replications, except for Chlamydomonas mono-cultures which were from three independent replications.Null spectral count values were replaced with a small spectral fraction before performing the calculations (57).Natural log-transformed dNSAF data were compared using Student's t test for two groups or one-way ANOVA followed by Duncan post-hoc tests for multiple groups with SPSS 19 software (IBM, USA).DEPs with FC ≥ ±1.5 and P < 0.05 were considered as upregulated and downregu lated proteins, respectively.GO enrichment analyses of DEPs were performed using TBtools (58).Pathway analyses were performed according to KEGG category annotations (WWW.KEGG.JP) and the approaches described previously (24).and editing | Bo Xie, Conceptualization, Funding acquisition, Investigation, Supervision, Visualization, Writing -original draft, Writing -review and editing

FIG 1
FIG 1 Enhancement of C. reinhardtii thermal tolerance by co-cultivation with S. meliloti.(A) Photographs illustrate how co-cultivation with S. meliloti (Sm) reduces the extent of C. reinhardtii (Cr) chlorosis 2 days after an upshift from normal (25°C) to high (42°C; H) temperatures.The controls are cultures at a normal temperature of 25°C (N).For the high-temperature treatment, cultures were grown at 25°C for 3 days prior to the 42°C temperature upshift.(B and C) Survival of Chlamydomonas (B) and Sinorhizobium (C) in different treatments.Co-H, co-culture at high temperature; Co-N, co-culture at normal temperature; Mo-H, mono-culture at high temperature; Mo-N, mono-culture at normal temperature; DAI, days after inoculation.The bottom arrows indicate the growth periods with normal temperature (25°C; N) or with a 42°C temperature upshift (H).Values are the mean ± SEM of three to four independent experiments.

FIG 2
FIG 2 Influence of co-culture and heat stress on the number of DEPs in C. reinhardtii 21gr (A and C) and S. meliloti 1021 (B and D).(A and B) Comparisons between temperature upshift and normal temperature in mono-and co-culture.(C and D) Comparisons between co-cultures and mono-cultures under normal and temperature upshift conditions.Co-H, co-cultures following a high (42°C) temperature upshift; Co-N, co-cultures at normal temperature (25°C); Mo-H, mono-cultures at high temperature; Mo-N, mono-cultures at normal temperature.Black values are number of proteins whose expression is upregulated, and blue values are number of proteins whose expression is downregulated.White and yellow bold values are total number of DEPs.

FIG 3
FIG 3 GO classifications of the upregulated (red) and downregulated (blue) DEPs in C. reinhardtii (A-C) and S. meliloti (D-F) when comparing high and normal temperature treatments.The DEPs are categorized by whether the proteins are defined to be involved in metabolic functions (MFs), biological processes (BPs), or as a cellular component (CC), as indicated on the right y-axis.(A and D) Unique DEPs in Cr 21gr or Sm 1021 mono-cultures.(B and E) Unique DEPs in Cr 21gr or Sm 1021 co-cultures.(C and F) DEPs that respond to a temperature upshift in mono-and co-cultures.

FIG 5
FIG 5 Expression levels of select proteins involved in C. reinhardtii methionine synthesis and methylation.(A) Abundance of cobalamin-independent methionine synthase METE and cobalamin-dependent methionine synthase METH.(B) Abundance of selected proteins involved S-adenosylmethionine synthesis and methylation reaction.SAS1, S-adenosylmethionine synthetase; TEF11, S-isoprenylcysteine O-methyltransferase related protein; UMM6, UbiE/COQ5 methyltrans ferase family protein.NOP1, NOP58, and NOP56, rRNA methyltransferase family proteins.Values represent the mean ± SEM of 3-4 independent experiments, and for each protein, bars marked with the same letter are not significantly different based on a one-way ANOVA with a Duncan post-hoc test (P < 0.05).Mo-N, mono-cultures at normal temperature (25°C); Co-N, co-cultures at normal temperature; Mo-H, mono-cultures at high temperature (42°C) upshift; Co-H, co-cultures following a high-temperature upshift.

FIG 6
FIG 6 Expression levels of proteins involved in S. meliloti B 12 synthesis.(A) Representive proteins involved in B 12 synthesis.(B) Sum of dNSAF values of B 12 synthesis pathway proteins.Values represent the mean ± SEM of four independent experiments, and for each protein, the bars marked with the same letter are not significantly different based on a one-way ANOVA with a Duncan post-hoc test (P < 0.05).Mo-N, mono-cultures at normal temperature (25°C); Co-N, co-cultures at normal temperature; Mo-H, mono-cultures at high temperature (42°C) upshift; Co-H, co-cultures following a high-temperature upshift.

FIG 7
FIG 7 Comparison of metabolism-associated differentially expressed KEGG categories of the C. reinhardtii (Cr 21gr) and S. meliloti (Sm 1021) proteomes.The heatmaps show the zero-to-one row-scaled (using TBtools) expression of KEGG metabolic categories, and the bars show ratios of each KEGG category in co-cultures vs mono-cultures (Co:Mo) at normal (25°C; 21gr-N and 1021-N) or high (42°C; 21gr-H and 1021-H) temperatures.Bars in red and blue indicate significantly upregulated (ratio > 1.0, P < 0.05, t test) and downregulated categories (ratio < 1.0, P < 0.05, t test), respectively, and the others are in gray.

FIG 8
FIG 8 Schematic diagram showing the predicted molecular interactions between C. reinhardtii and S. meliloti in the coculture vs monoculture during heat stress conditions.Proteins or pathways that are found in higher amounts or are upregulated in cocultures compared with monoculture following a temperature upshift are represented in red, and those that are less abundant or downregulated are blue.The diamond and circular shapes represent the possible exchanged metabolites from C. reinhardtii and S. meliloti, respectively.

TABLE 1
Expression of representative transporters a a Values are the fold changes of dNSAF values of the proteins.The bolded values are significantly different according to a t test (P < 0.05).b H: high (42°C) temperature stress; N: normal (25°C) temperature.

TABLE 2
Expression of representative proteins with signal transduction functions a a Fold change of dNSAF values of the proteins.The bolded values are significant changed accord to t test (P < 0.05).b H: high (42°C) temperature stress; N: normal (25°C) temperature.