Chlamydomonas sp. UWO241 exhibits constitutively high cyclic electron flow and rewired metabolism under high salinity

The Antarctic green alga Chlamydomonas sp. UWO241 (UWO241) was isolated from the deep photic zone of a permanently Antarctic ice-covered lake. Adaptation to permanent low temperatures, hypersalinity, and extreme shade has resulted in survival strategies in this halotolerant psychrophile. One of the most striking phenotypes of UWO241 is an altered photosystem I (PSI) organization and constitutive PSI cyclic electron flow (CEF). To date, little attention has been paid to CEF during long-term stress acclimation and the consequences of sustained CEF in UWO241 are not known. In this study, we combined photobiology, proteomics, and metabolomics to understand the underlying role of sustained CEF in high salinity stress acclimation. High salt-grown UWO241 exhibited increased thylakoid proton motive flux and an increased capacity for NPQ. A Bestrophin-like Cl- channel was identified in the whole cell proteomes and transcriptome of UWO241 which likely supports ion homeostasis during high transthylakoid pH. Under high salt, a significant proportion of the upregulated enzymes were associated with the Calvin Benson Bassham Cycle (CBB), secondary metabolite biosynthesis, and protein translation. Two key enzymes of the Shikimate pathway, DAHP synthase and chorismate synthase, were also upregulated, as well as indole-3-glycerol phosphate synthase, an enzyme involved in biosynthesis of L-tryptophan and indole acetic acid. In addition, several compatible solutes (glycerol, proline and sucrose) accumulated to high levels in high salt-grown UWO241 cultures. We suggest that UWO241 maintains constitutively high CEF with associated PSI-cytb6f supercomplex to support robust growth and strong photosynthetic capacity under a constant growth regime of low temperatures and high salinity.


52
During photosynthesis light is transduced into stored energy through two major pathways, linear 53 electron flow (LEF) and cyclic electron flow (CEF). LEF involves the flow of electrons from 54 Photosystem II (PSII) to Photosystem I (PSI) resulting in the production of both ATP and 55 NADPH, which are consumed during carbon fixation in the Calvin Benson Bassham cycle 56 (CBB). CBB requires 3 molecules of ATP and 2 molecules NADPH to fix 1 molecule of CO2; 57 however, LEF produces an ATP:NADPH ratio of only 2.57:2 (Kramer and Evans, 2011). CEF 58 constitutes electron transfer from PSI to soluble mobile carriers and back to PSI via cytochrome 59 b6f (cyt b6f) and plastocyanin (Cardol et al., 2011;Nawrocki et al., 2019). As electrons are 60 shuttled between PSI and cyt b6f, a proton gradient is produced that leads to ATP production 61 only. In addition to satisfying the ATP shortage for efficient carbon fixation, CEF-generated 62 ATP may be used for other energy-requiring processes, such as the CO2-concentrating 63 mechanism of C4 photosynthesis (Takabayashi et al., 2005;Ishikawa et al., 2016), N2 fixation in 64 cyanobacteria heterocysts (Magnuson et al., 2011;Magnuson and Cardona, 2016), and survival 65 under environmental stress (Suorsa, 2015). 66 When the light harvesting antennae absorb light energy in excess of what is required for 67 growth and metabolism, energy homeostasis is disrupted, increasing the risk of formation of 68 reactive oxygen species (ROS) (Hüner et al., 1998). While this phenomenon is associated with 69 excess light, numerous environmental stresses can lead to imbalances in energy demands, 70 including high and low temperatures, high salinity, and nutrient deficiency. Moreover, the 71 duration of the environmental stress can vary over broad time scales, from a few minutes to days 72 or years. Survival of plants and algae requires coordination of short-and long-term acclimatory 73 strategies to maintain energy homeostasis. These acclimation responses are often triggered via 74 UWO241 was isolated from the deep photic zone (17 m sampling depth) of the hypersaline, 143 perennially ice-covered lake (Lake Bonney, McMurdo Dry Valleys, Victoria Land) (Neale and 144 Priscu, 1990;Neale and Priscu, 1995). As a consequence of more than two decades of study, this 145 photopsychrophile has emerged as a model for photosynthetic adaptation to permanent low 146 temperatures (Morgan-Kiss et al., 2006;Dolhi et al., 2013;Cvetkovska et al., 2017). In addition 147 to psychrophily, UWO241 exhibits robust growth and photosynthetic performance under high 148 salt (0.7 M NaCl, Supplemental Fig. S1; Morgan et al., 1998;Pocock et al., 2011). 149 Low temperature fluorescence spectra of mid-log phase cultures of C. reinhardtii and 150 UWO241 grown in control, low salt (LS) growth medium (standard BBM medium, 0.43 mM 151 NaCl) under optimal growth temperatures (20 o C and 8 o C, respectively) and light conditions (100  152 μmol m -2 s -1 for both algae) confirmed that C. reinhardtii possesses a typical 77K fluorescence 153 emission spectrum with prominent peaks at 685 nm (F685) and 715 nm (F715), representing 154 LHCII-PSII and PSI, respectively. In agreement with past reports (Morgan et al., 1998;Szyszka 155 et al., 2007), PSI fluorescence was significantly reduced (1.60-fold) in UWO241 relative to C. 156 reinhardtii grown under optimal temperature/light conditions in the LS growth medium (Fig.  157 1A). Moreover, PSI fluorescence was reduced by an additional 1.59-fold in cultures of UWO241 158 grown in high salinity (HS) growth medium (0.7 M NaCl), relative to LS-grown cells (Fig. 1A). 159 PSI activity was monitored by far red (FR) light inducible P700 photooxidation (Fig. 1B). 160 Following a rise in absorbance at 820 (A820), reflecting FR-induced P700 oxidation, we 161 compared rates of P700 re-reduction in the dark in LS cultures of C. reinhardtii as well as  and HS-grown cells of UWO241 (Fig. 1B). Since FR preferentially excites PSI and not PSII, 163 reduction of P700 following FR exposure is mainly due to alternative electron donors (Ivanov et 164 al., 1998). In agreement with other reports (Morgan-Kiss et al., 2002b;Cook et al., 2019), 165 UWO241 grown in standard LS growth medium exhibited a significantly shorter re-reduction 166 time for P700+ (t½ red ) compared with LS-grown C. reinhardtii (Fig. 1B). Moreover, HS-grown 167 UWO241 exhibited a 4.4-fold faster t½ red compared with LS-grown cultures (43±42 vs. 188±52 168 ms respectively; Fig. 1B). These data indicate that relative to the model C. reinhardtii, UWO241 169 exhibits a high capacity for PSI-driven CEF, which is further enhanced during acclimation to 170 long-term high salinity stress. 171 Higher rates of CEF in HS-grown UWO241 were also confirmed by electrochromic shift 172 (ECS) kinetics which estimates transthylakoid proton flux driven by light-dependent 173 photosynthesis (Fig. 2, Supplemental Fig. S2). The ECS signal was measured by the change in 174 absorbance of thylakoid pigments at 520 nm during application of light dark interval (Baker et 175 al., 2007). The total amplitude of ECS signal (ECSt), , was used to estimate the total proton 176 motive force (pmf) across thylakoid membranes (Kramer et al., 2003). UWO241 grown in HS 177 exhibited 6 to 7.5 fold higher ECSt than that of LS-grown cells under all light intensities ( Fig.  178 2A), suggesting HS-grown cells generate higher pmf than LS-grown cells at the same light 179 intensity. High pmf can be caused by either increased proton flux from LEF or CEF, reduced 180 proton efflux, or decreased ATP synthase activity (Kanazawa and Kramer, 2002;Livingston et 181 al., 2010;Carrillo et al., 2016). To verify which process(es) were contributing to high pmf in HS-182 grown cells, proton conductance (ɡH+) and fluxes through ATP synthase activity (νH+) were 183 analyzed. The inverse of the lifetime of the rapid decay of ECS (ɡH+) represents proton 184 permeability or conductivity of the thylakoid membrane and is largely dependent on the activity 185 of ATP synthesis (Supplemental Fig. S2D;Baker et al., 2007). The ɡH+ of HS-grown cells was 186 ~50 to 60% of that of LS-grown cells ( Figure 2B); however, the proton flux rate (νH+) showed 187 that the amount of ATP produced was still higher in HS-grown cells (Fig. 2C). The relationship 188 between νH+ and LEF can be used to estimate proton contribution from CEF (Baker et al., 2007). 189 In the linear plots of νH+ versus LEF, the slope of the HS-grown cells was higher than that of  grown cells (Fig. 2D), indicating that CEF contributes significantly to the total proton exffluxes 191 (νH+) in HS-grown cells. In close agreement with our P700 findings, UWO241-HS exhibited 192 higher rates of CEF compared to  HS-UWO241 based on P700 and ECS measurements, respectively). Last, HS-grown cells 194 exhibited downregulation of PSII and increased capacity for NPQ (Supplemental Fig. S2A (DalCorso et al., 2008;Iwai et al., 2010). An earlier report showed that high 201 salinity-acclimated cultures of UWO241 form a PSI supercomplex (UWO241-SC); however, the 202 yield of the UWO241-SC from fractionated thylakoids was relatively low and only a few 203 proteins were identified (Szyszka-Mroz et al., 2015). In agreement with this report, the sucrose 204 gradient from thylakoids isolated from LS-UWO241 had 3 distinct bands corresponding to major 205 LHCII (Band 1), PSII core complex (Band 2) and PSI-LHCI (Fig. 3A). In contrast, UWO241-HS 206 thylakoids lacked a distinct PSI-LHCI band, but exhibited several heavier bands, including the 207 UWO241-SC band (Band 4; Fig. 3B). We significantly improved recovery of the UWO241-SC 208 by solubilizing thylakoids with the detergent -DDM rather than β-DDM, which was used by 209 other groups (Fig. 3B). Formation of band 4 of C. reinhardtii thylakoids isolated from State 2 210 conditions, was more diffuse compared to the UWO241-SC (Fig. 3D). 211 Low-temperature fluorescence spectra were analyzed for the four bands extracted from 212 the sucrose density gradients shown in Fig. 3B and D (i.e. HS-UWO241 and State 2 C. 213 reinhardtii, respectively). In C. reinhardtii, Band 1 exhibited a major emission peak at 680 nm 214 ( Fig. 4C), corresponding to fluorescence from LHCII (Krause and Weis, 1991). Band 2 exhibited 215 emission peak at 685 nm, consistent with PSII core (Fig. 4A). Band 3 exhibited a peak at 685 nm 216 and a strong peak at 715 nm; the latter consistent with PSI-LHCI (Fig. 4A). However, Band 4 217 exhibited a strong fluorescence peak at 680 nm and a minor peak at 715 nm (Fig. 4A). 218 Fractionated thylakoids from HS-grown UWO241 exhibited emission spectra for Band 1 219 and Band 2 which were comparable with that from C. reinhardtii (Fig. 4B). In contrast, both 220 Band 3 and Band 4 (PSI and SC bands, respectively) exhibited highly reduced or a lack 221 fluorescence associated with PSI. However, we confirmed the presence of the PSI reaction center 222 protein, PsaA, in the UWO241-SC by immunoblotting (Fig. 4C). 223 224

Protein composition of the supercomplex. 225
Protein components of the UWO241-SC were analyzed using LC-MS/MS. We identified a total 226 of 39 proteins in the isolated band 4, significantly more proteins than the previously reported 227 supercomplex of UWO241 isolated using β-DDM (Szyszka-Mroz et al., 2015). The most 228 abundant proteins in the supercomplex were proteins of the PSI reaction center and cytochrome 229 b6f. In total we identified seven out of 13 subunits of PSI reaction center (Table 1; Supplemental  230   Table S1). Only two LHCI subunits, Lhca3 and Lhca5, and one LHCII minor subunit, CP29 231 were associated with the UWO241-SC. The Calcium sensing receptor (CAS) was identified as 232 the third most abundant protein in the UWO241-SC. We also identified four subunits of ATP 233 synthase in the UWO241-SC (α, β, γ, δ). In agreement with an earlier report, we found FtsH and 234 PsbP in the UWO241-SC band (Szyszka-Mroz et al., 2015). 235 Bands 3 and 4 contained many PSI core proteins but lacked almost all Lhca proteins 236 (Supplemental Table S1). These results agreed with an earlier report that failed to detect most 237 Lhca proteins in UWO241 thylakoids (Morgan et al., 1998). To determine whether the absence 238 of Lhcas in the UWO241 proteome was due to a loss of Lhca genes, we searched a UWO241 239 transcriptome generated from a culture grown under low temperature/high salinity (Raymond et 240 al., 2009). Surprisingly, we identified 9 Lhca homologues which were transcribed under high 241 salinity, suggesting that all or most of the LHCI genes are expressed in UWO241 (Supplemental 242 cleavage site, suggesting that it is localized to the chloroplast. Modeling of the UWO241 BEST1 251 protein using KpBest as a template, indicated that the UWO241 BEST channel may form a 252 pentamer, with the Clentryway and exit is located on the stromal and luminal sides, respectively 253 of the thylakoid membrane (Fig. 5D), similar to a recent study on AtBEST1 (Duan et al., 2016).  Table S2). One protein of 269 chloroplastic ATP synthase, the epsilon subunit, was upregulated (3.8 fold) in UWO241-HS. 270 Last, both FtsH proteins that were detected in the UWO241-SC, FtsH1 and FtsH2, were 271 upregulated in UWO241-HS (Table 1; Supplemental Table S2).  Table S2). The RubisCO large subunit was upregulated in 274 UWO241-HS, along with two chaperone proteins involved in RubisCO assembly, RuBA and 275 RuBB. A class 2 fructose-1,6 -bisphosphatase (FBPase) was the third highest upregulated 276 protein (5-fold), and fructose bisphosphate aldolase and transketolase were also upregulated in 277 UWO241-HS (Fig. 7A). 278 279 Metabolism: The TCA cycle protein aconitate hydratase was upregulated in UWO241-HS, while 280 pyruvate carboxylase, which is involved in anaplerotic reactions, was downregulated ( Fig. 7B; 281 Supplemental Tables S2 and S3). In addition, malate dehydrogenase (MDH) was downregulated 282 in UWO241-HS. MDH participates in the malate shunt and helps shuttle excess reducing power 283 from the chloroplast to the mitochondria by converting oxaloacetate to malate. In this process 284 excess NADPH are used and NADP pool is regenerated (Scheibe, 2004).  Table S2). In addition, the 289 enzyme glycerol-3-phosphate dehydrogenase (G3PDH), involved in glycerol biosynthesis, was 290 the highest upregulated enzyme in UWO241-HS cultures (6-fold) ( Fig. 7B; Supplemental Table  291 S2). G3PDH is involved in conversion of DHAP to sn-glycerol-3-phosphate that leads to 292 glycerol production through glycerol kinase (GK) or G3P phosphatase (GPP) (Driver et al., 293 2017). The G3P produced in this reaction is also a precursor for TAG synthesis and can also lead 294 to increased lipid production under salinity stress (Herrera-Valencia et al., 2012). On the other 295 hand, Alcohol-aldehyde dehydrogenase (AADH) was significantly downregulated in UWO241-296 HS (5-fold; Fig. 7B; Supplemental Table S3).  Table S2). Chorismate, the product of the Shikimate pathway, is a substrate for 303 both aromatic amino acids and many phenylpropanoid secondary metabolites. We found that 304 indole-3-glycerol phosphate synthase (IGP synthase) was upregulated significantly in the HS 305 conditions. IGP synthase is a branching enzyme that can either enter tryptophan pathway or lead 306 to de novo biosynthesis of the plant phytohormone indole acetic acid (IAA) (Ouyang et al., 307 2000). 308 309

Primary metabolome analysis. 310
Comparing the whole cell proteome of LS and HS grown cultures suggested that salinity has a 311 strong effect on primary and secondary metabolism in UWO241. To further explore this, 312 UWO241 metabolic extracts from LS and HS cultures were analyzed using GC-MS. We detected abundances indicated clustering and a discrete population of metabolites that accumulate at high 322 levels in HS-grown cultures when compared to LS-grown cells (Fig. 8). To better understand the 323 effect of high salinity on the metabolic profile of UWO241, we performed a detailed analysis on 324 the subset of primary metabolites that were positively identified. Overall, 59 metabolites (32%) 325 from different chemical categories were significantly different (p<0.01), out of which 9 were 326 present in higher abundance (Supplemental Table S4) and 50 were present in lower abundance 327 (Supplemental Table S5) Table S4). We 332 also observed a high accumulation of phytol (12.6 FC), suggesting chlorophyll degradation. 333 Tocopherol was also detected in our HS experiment in high amounts (9.5 FC), however its 334 accumulation was variable and thus not statistically different between samples (p>0.05). 335

336
Metabolites that accumulate in lower amounts in HS grown cultures: UWO241 cultures grown in 337 high salinity exhibited decreased amounts of 17 amino acids and compounds associated with 338 amino acid metabolism (Supplemental Table S5). Most notable metabolites from this class were 339 lysine (29.4 FC) and ornithine (14.0 FC), which could signify a shift in amino acid metabolism 340 to proline during exposure to high salinities. We also observed lower levels of the amino acid 341 tryptophan (2.8 FC) in HS grown cultures. Metabolites involved in purine and pyrimidine 342 metabolism were present in lower amounts in UWO241 exposed to high salinity, suggesting that 343 these cells have shifted their metabolism from maintenance of the cell cycle and nucleic acid 344 synthesis to producing osmoprotectants and compatible solutes. We also observed a reduction of Our study shows that UWO241 maintains robust growth and photosynthesis under the combined 349 stress of low temperature and high salt. This ability differs markedly from other model plants and 350 algae that typically display downregulation of photosynthesis and growth when exposed to 351 environmental stress, mainly as a consequence of bottlenecks in carbon fixation capacity (Hüner 352 et al., 1998;Hüner et al., 2003;Ensminger et al., 2006;Hüner et al., 2016). Previous research has 353 thoroughly described adaptive strategies for survival under permanent low temperatures, while 354 survival under hypersalinity has received less consideration (Morgan et al., 1998;Morgan-Kiss 355 et al., 2002a;Szyszka et al., 2007;Possmayer et al., 2011). 356 One of the more distinct photosynthetic characteristics of UWO241 is the presence of a 357 strong capacity for PSI-driven CEF (Morgan-Kiss et al., 2002b;Szyszka-Mroz et al., 2015;Cook 358 et al., 2019). We validated that CEF rates are high in HS-grown cultures using the ECS signal, 359 which was purported to mitigate problems with using P700 absorbance changes for CEF 360 estimates (Lucker and Kramer, 2013). While CEF appears to be essential in plants and algae for 361 balancing the ATP/NADPH ratio and protecting both PSI and PSII from photo-oxidative damage 362 supercomplex (Szyszka-Mroz et al., 2015). In this current study, following optimization of 369 thylakoid protein complex solubilization by substituting β-DDM with -DDM, the vast majority 370 of PSI shifts from free PSI in the LS-grown cultures to association with the UWO241-SC in the 371 HS-grown cultures. PSI supercomplexes have been described in several plant and algal species 372 (Iwai et al., 2010;Li et al., 2018;Steinbeck et al., 2018). The UWO241-SC is distinct from that 373 of C. reinhardtii because: i) its assembly is independent of short-term exposure to dark anaerobic 374 conditions or other state transition-inducing treatments (Fig. 3), ii) the vast majority of PSI in 375 UWO241 is associated with the UWO241-SC (Fig. 3), and iii) isolated UWO241-SC and PSI 376 bands as well as whole cells lack typical PSI fluorescence emission at 77K, despite the presence 377 of several PSI core proteins ( Fig. 4 and functional changes to PSI are major targets for long-term stress acclimation in UWO241 380 ( Fig. 9A). 381 While the UWO241-SC contains most of the PSI core proteins, both the UWO241-SC 382 and PSI bands, as well as whole cell proteomes isolated from LS and HS conditions lacked 383 homologues for most LHCI proteins (Table 1; Supplemental Table S1). This agrees with an 384 earlier study which was unable to detect most of the LHCI polypeptides by immunoblotting in 385 Morgan- Kiss et al., 2002b;2005;Szyszka et al., 2007;Cook et al., 2019). It also explains the 394 differences in the 77K emission spectra of the UWO241-SC and PSI bands between UWO241 395 and C. reinhardtii (Fig. 4). Last, a recent study reported that UWO241 transfers light energy 396 from PSII to PSI via constitutive energy spillover through an undescribed mechanism (Szyska-397 Mroz et al., 2019). Thus, UWO241 favors downregulated LHCI and constitutive energy spill-398 over in response to its extreme habitat, most likely the natural light environment of extreme 399 shade enriched in blue wavelengths (Neale and Priscu, 1995). preventing ROS-induced PSI damage (Munekage et al., 2008;Shimakawa et al., 2016;Chaux et 411 al., 2017;Huang et al., 2017). A strong constitutive CEF mechanism in UWO241 could be 412 beneficial for one or most of the above purposes. First, HS-grown cultures possess a higher 413 capacity for NPQ (Supplemental Fig. S2), supporting a role for CEF in constitutive 414 photoprotection ability. Expression of a thylakoid BEST ion channel also suggests CEF may be 415 used for NPQ. High CEF rates also correlate with a higher Y(PSI) and a lower PSI acceptor side 416 limitation in HS-grown cultures (Supplemental Fig. S3), suggesting enhanced PSI 417 photoprotection in UWO241-HS cells. 418 ATP synthase subunits were associated with the UWO241-SC (Table 1), suggesting CEF 419 contributes extra ATP in UWO241. HS-grown cultures exhibited significantly higher ECSt and 420 νH+ compared to LS-grown cultures, suggesting a high flux of protons through the chloroplastic 421 ATP synthase in spite of slow activity of ATP synthase (compare Fig. 2A and C with Fig. 2B). 422 Slower activity of ATP synthase could be overcome by higher ATP synthase subunits in the HS-423 grown UWO241 which is reported here (Figs. 6 and 7) and in an earlier report (Morgan et al., 424 1998). Recently it was shown that in a salt-tolerant soybean, increased CEF contributes to excess 425 ATP that is used to drive import of Na + in the vacuole (He et al., 2015). Taken together, 426 constitutively high rates of CEF in UWO241 are likely to provide dual benefits, that of 427 constitutive photoprotection of both PSI and PSII and extra ATP to support downstream 428 processes important for low temperature and/or high salinity adaptation (Fig. 9A). also higher in HS-grown cells (Fig. 9B). Overexpression of key bottleneck CBB enzymes such as 438 FBPase and SBPase enhances carbon fixation and RuBP regeneration (Lefebvre et al., 2005;439 Tamoi et al., 2006), while also supporting improved photosynthesis during stress (Driever et al., 440 2017). Low levels of 3-PGA, the direct biproduct of RubisCO activity also suggests strong 441 carbon sinks for fixed CO2 in HS-grown cultures (Supplemental Table S5). Last, overproduction 442 of these key CBB enzymes is supported by a robust protein translation ability, as several 443 ribosomal proteins are also overexpressed in HS-grown cells (Supplemental Table S2). 444 Enhanced CBB pathway activity would support robust photosynthetic activity and growth 445 in UWO241. However, proteomic evidence revealed other potential carbon sinks, including 446 carbon storage in the form of starch (Supplemental Table S2 act as a strong carbon sink to support high rates of carbon fixation (Fig. 9B). Starch stored in the 458 chloroplast is also transitory, and is often rapidly turned over (Thalmann et al., 2016). Starch 459 content was comparable between LS-and HS-grown UWO241 cultures (Supplemental Fig. S6). 460 Thus, transiently stored starch could be an additional adaptive strategy in UWO241, acting as an 461 energy and carbon buffer which can be rapidly mobilized when needed. This theory is supported 462 by other publications that reported accumulation of starch under cold or salinity stress (Siaut et 463 al., 2011;Wang et al., 2013), suggesting that transitory starch synthesis and mobilization may be 464 important during stress acclimation. 465 Glycerol is a compatible solute that accumulates at molar levels in the salt-tolerant alga 466 Dunaliella (Avron, 1986;Brown, 1990;Goyal, 2007a,b). Glycerol is synthesized through two 467 independent pathways localized in the chloroplast and the cytosol. In the presence of light, the 468 chloroplast pathway dominates at the expense of starch synthesis (Gimmler and Möller, 1981), 469 while in the dark, stored starch is degraded to provide substrates for the cytosolic pathway (Ben-470 Amotz and Avron, 1973). Under high salinity stress, D. tertiolecta utilizes both the chloroplast 471 and cytosolic pathways for glycerol synthesis (Goyal, 2007a, b). Overexpression of D. bardawil 472

SBPase in C. reinhardtii led to increased accumulation of glycerol and improved photosynthesis 473
under salinity stress (Fang et al., 2012). This current study showed that UWO241 also 474 accumulates glycerol in response to increased salinity (Supplemental Table S4). Synthesis of 475 glycerol could occur through either the chloroplast or cytosolic pathway, since isoforms of both 476 the cytosolic and chloroplast glycerol-3 phosphate dehydrogenases (GPDH, EC 1.1.1.8) were 477 upregulated under high salinity (Supplemental Table S2). GPDH is responsible for the first step 478 in glycerol synthesis, conversion of dihydroxyacetone phosphate (DHAP) to glycerol 3-479 phosphate (G3P), and also supplies G3P for chloroplastic glycerolipid synthesis (Chandra-480 Shekara et al., 2007). The cytosolic GPDH is highly overexpressed in UWO241, suggesting that 481 glycerol production via starch breakdown may be the dominant pathway in this organism. 482 The shikimate pathway is an essential link between primary and secondary metabolism 483 for producing precursors for aromatic amino acids (tryptophan, phenylalanine, tyrosine) as well 484 as many other aromatic metabolites such as, indole compounds, alkaloids, lignin and flavonoids 485  Table  499 S2). Thus, the CBB cycle and glycolysis are likely to be coordinated in order to provide 500 substrates to support high flux through the shikimate pathway. Last, there is evidence linking 501 CEF with the Shikimate pathway. One recent study involving a CEF mutant of Arabidopsis 502 thaliana (lacking pgr5 protein) showed that the levels of Shikimate metabolites were 503 significantly reduced in the CEF mutant as compared to wild type, suggesting a link between 504 CEF and chorismate synthesis (Florez-Sarasa et al., 2016). 505 Acclimation to a variety of stresses in plants and algae often involves upregulation of 506 heat shock proteins, stress metabolites, as well as signaling molecules such as plant hormones 507 and signal transduction pathways (eg: Ca 2+ ) (Montero- Barrientos et al., 2010;Suzuki et al., 508 2016). Several stress metabolites utilize chorismate as a substrate; although, many of these are 509 typically associated with plant hormone production. We provide evidence in the proteome of 510 UWO241 that a biosynthetic enzyme of the tryptophan pathway, indole-3-glycerol phosphate 511 synthase (IGPS, EC 4.1.1.48) is highly expressed under high salt (Supplemental Table S2). This 512 enzyme is the fourth step in the biosynthesis pathway of L-tryptophan (L-Trp) from chorismite 513 (Fig. 9B). Therefore, it is possible that a product of the shikimate pathway in HS grown 514 UWO241 is the aromatic amino acid L-Trp. However, the metabolome data showed that L-Trp 515 levels were reduced in the HS-grown cultures (Supplemental Table S5). L-Trp is also a major 516 substrate for production of the phytohormone, indole-3 acetic acid (IAA), and the product of 517

IGPS, indol-3-glycerol phosphate, is a branch point between L-Trp synthesis and a L-Trp 518
independent IAA synthesis pathway (Ouyang et al., 2000). IAA and several other 519 phytohomormones have been detected in a few cyanobacteria and algal species; however, their 520 putative function is largely based on exogenously added plant phytohormones to algal cultures 521 (Lu and Xu, 2015). Exogenously added IAA stimulates carbon fixation and growth and enhances 522 stress tolerance in algae (Lu and Xu, 2015). Last, IAA production increases Ca 2+ levels in plants 523 during acclimation to abiotic stress (Vanneste and Friml, 2013). Ca 2+ signaling has been linked 524 to both CEF and assembly of PSI supercomplexes (Terashima et al., 2012). Indeed, the Calcium 525 sensing receptor (CAS) was an abundant protein associated with the UWO241-SC (Table 1). 526 More work will be needed to ascertain whether IAA and Ca 2+ play roles in CEF and assembly of 527 the UWO241-SC. 528 Despite more than 2 decades of study, the enigmatic UWO241 still has secrets to share. hypersalinity by proposing a model for sustained PSI-CEF that supports a robust CBB pathway 534 and a regular growth rate (Fig. 9). Under permanent environmental stress, CEF supplies 535 constitutive photoprotection of PSI and PSII while also producing extra ATP for downstream 536 metabolism (Fig. 9A). The restructured photosynthetic apparatus is accompanied by major 537 rewiring of central metabolism to provide a strong carbon fixation potential which is used in part 538 to produce stored carbon and secondary metabolites (Fig. 9B). Algae adapted to multiple 539 stressors such as low temperatures combined with high salinity are robust fixers of CO2, Far red light induced photooxidation of P700 was used to determine rates of CEF as described by 576 Morgan- Kiss et al. (2002b). A volume of exponential phase cultures representing 25 μg Chl a 577 was dark adapted for 10 min and then filtered onto 25 mm GF/C filters (Whatman). Filters were 578 measured on the Dual-PAM 100 instrument using the leaf attachment. The proportion of 579 photooxidizable P700 was determined by monitoring absorbance changes at 820 nm and 580 expressed as the parameter (∆A820/A820). The signal was balanced and the measuring light 581 switched on. Far red (FR) light (λmax=715 nm, 10 Wm −2 , Scott filter RG 715) was then 582 switched on to oxidize P700. After steady-state oxidation levels were reached, the FR light was 583 switched off to re-reduce P700. The half time for the reduction of P700 + to P700 (t½ red ) was 584 calculated after the FR light was turned off as an estimate of relative rates of PSI-driven CEF 585 (Ivanov et al., 1998). The re-reduction time for P700 was calculated using Microcal TM Origin TM 586 software (Microcal Software Inc., Northampton, MA, USA). 587 588

In vivo spectroscopy measurements. 589
Saturation-pulse chlorophyll fluorescence yield changes and dark interval relaxation kinetics 590 (DIRK) of ECS were measured at 8 °C with the IDEA spectrophotometer as described 591 previously with some modifications (Sacksteder and Kramer, 2000;Zhang et al., 2009). A 2.5 592 mL of cell supplemented with 25 μL of 0.5 M NaHCO3 was pre-incubated in the dark for 10 min 593 and followed by 10 min illumination of far-red light. The chlorophyll fluorescence and ECS 594 were measured with the cells acclimated for 5 min in various actinic light intensities provided by 595 red LEDs. The PSII operating efficiency (ΦPSII) was calculated as Fq'/Fm', NPQ as (Fm-Fm')/Fm'. 596 The linear electron transport (LEF) was calculated from following equation: LEF = A × 597 (fractionPSII) × I × ΦPSII, where I is the light intensity, A is the absorptivity of the sample, which 598 is generally assumed to be 0.84 and fractionPSII is the fraction of absorbed light stimulating PSII 599 (Baker, 2008). The fractionPSII of UWO241 grown in low-salt and high salt, measured by 77K 600 fluorescence spectra, were 0.709 and 0.746, respectively. The total amplitude of the ECS signal 601 (ECSt) was used to estimate the proton motive force (pmf). The aggregate conductivity of the 602 thylakoid membrane to protons (gH+) was estimated from the inverse of lifetime of the rapid 603 decay of ECS (τECS) (Baker et al., 2007). All ECS signals were normalized to the rapid rise in 604 ECS induced by a single turnover flash to account for changes in pigmentation (Livingston et al. Thylakoids were isolated according to Morgan-Kiss et al. (1998). Mid-log phase cultures were 609 collected by centrifugation at 2500g for 5 min at 4°C. All buffers were kept ice-cold and 610 contained 1 mM Pefabloc Sc (Sigma, USA) and 20 mM NaF. The pellet was resuspended in 611 grinding buffer (0.3 M sorbitol, 10 mM NaCl, 5 mM MgCl2, 5 mM MgCl2, 1 mM benzamidine, 612 1mM amino-caproic acid). The cells were disrupted using chilled French press at 10,000 lb/in 2 613 twice, and then and centrifuged at 23,700g for 30 min. The thylakoid pellet was resuspended in 614 wash buffer (50 mM Tricine-NaOH [pH 7.8], 10 mM NaCl, 5 mM MgCl2) and centrifuged at 615 13,300xg for 20 min. The pellet was resuspended in storage buffer (0.3 M sorbitol, 10% glycerol, 616 50 mM Tricine-NaOH [pH 7.8], 10 mM NaCl and 5 mM MgCl2) and stored at -80°C until 617 analysis. 618 619 SDS-PAGE and Immunoblotting. 620 SDS-PAGE was performed using Bio-Rad Mini-Protean system and 12% Urea-SDS gel 621 (Laemmli, 1970). Thylakoid membranes were denatured using 50 mM DTT and incubated at 622 70°C for 5 min. Samples were loaded on equal protein basis (10 μg total protein). Proteins were 623 transferred to nitrocellulose membrane using cold-wet transfer at 100 V for 2.5 hours. The 624 membrane was blocked with TBST (Tris Buffer Saline Tween) buffer with 5% milk (Carnation). 625 A primary antibody against PsaA (Cat No. AS06-172; Agrisera, Sweden) was used at 1:1000 626 dilution to probe for major reaction center protein of PSI. Membranes were then exposed to 627 Protein A conjugated to horseradish peroxidase and blots were detected with ECL Select TM 628 Western Blotting Detection Reagent (Amersham). was ultra-centrifuged at 288,000g for 1 hour at 4°C using Sw40Ti rotor (Beckman coulter, 644 USA). Purified thylakoids were collected and diluted (3-fold) in Buffer 6 (5 mM Hepes-KOH 645 [pH 7.5], 10 mM EDTA) and centrifuged at 50,000xg to pellet the membrane. Linear sucrose 646 gradients were made using freeze thaw method with Buffer 7a (1.3 M Sucrose, 5 mM Hepes-647 KOH [pH 7.5], 0.05% α-DDM) and Buffer 7b (0.1 M Sucrose, 5 mM Hepes-KOH [pH 7.5], 648 0.05% α-DDM). Briefly, two dilutions of Buffers 7a and 7b were made, Buffer 7-1 (2x Buffer 7a 649 + 1x Buffer 7b) and Buffer 7-2 (1x Buffer 7 a + 2x Buffer 7b). To make the gradient, first 3 ml 650 of Buffer 7a was poured into 12 ml ultra clear tubes followed by flash freezing in liquid nitrogen. 651 Next, Buffer 7-1 was poured on top, followed by flash freezing. This was repeated for Buffer 7-2 652 and Buffer 7b respectively. The frozen gradients were kept at 4°C overnight to thaw. For 653 supercomplex isolation, thylakoid membranes (0.4 mg Chl) were resuspended in 1% n-dodecyl- Whole cell proteins were extracted as described previously (Valledor and Weckwerth, 2014). 663 Mid-log phase cells were collected by centrifugation at 2500g for 5 min (50 mg wet weight). The 664 cell pellets were resuspended in an extraction buffer containing 100 mM Tris-HCl (pH 8.0), 10% 665 (v/v) glycerol, 2 mM Pefabloc Sc, 10 mM DTT, and 1.2 % (v/v) plant protease inhibitor cocktail 666 (Cat. No. P9599,Sigma). Samples were transferred to 2 mL screw cap tubes containing 25 mg of 667 zirconia beads (Cat. No. A6758,Biorad) and homogenized 3 times for 45 seconds in a 668 BeadBeater (BioSpec). 20% SDS solution was added to the tubes and samples were incubated 669 for 5 min at 95°C. The denatured proteins were centrifuged at 12,000g to pellet any insoluble 670 material. Protein pellets were resuspended in 1.5 ml tris buffer (50mM Tris-HCl, pH 8.0) 671 containing 0.02% n-dodecyl-beta-maltoside (Glycon Biochemicals, Germany) and supplemented 672 with 1X Halt™ protease and phosphatase inhibitor cocktail (Thermo-Scientific, Rockford, IL). 673 After the protein extraction, the sample preparation for proteomics were conducted following our MS/MS raw data was analyzed by first converting into MS2 files, followed by database search 690 using ProLuCID (Xu et al., 2006). The UWO241 protein database was generated based on our 691 transcriptomics data supplemented with 37 common contaminants, and their reversed sequences 692 as quality control system to restrain false positive discovery to 0.05. Differentially expressed 693 proteins were analyzed using PatternLab for Proteomics (Carvalho et al., 2008). The proteomics 694 results have been deposited to the MassIVE repository with the identifier MSV000084382. 695 For identifying protein components in the supercomplex, the complex was harvested and 696 30 μg of total protein was processed similarly as described above to get the digested peptides. 697 Different from the whole cell proteomics, the processed the peptides were directly loaded onto a 698 capillary C18 column without fractionation, and further analyzed in a Thermo LTQ Orbitrap XL 699 mass spectrometer. The full mass spectra were recorded in the range of 350-1800 m/z with the 700 resolution of 30,000. The top 12 peaks of each scan were selected for MS/MS analysis. The data 701 analysis was conducted similarly as described above. For determination of the primary metabolome UWO241 were grown in four biological replicate 714 cultures as described above. Algal cells were harvested by centrifugation (6,000g, 5 min, 4°C) 715 and washed once with fresh media. The supernatant was decanted, and the algal cells were flash 716 frozen in liquid nitrogen and stored at -80°C. The metabolite extraction protocol was adapted 717 from (Fiehn et al., 2008). In brief, metabolites were extracted from 20 mg of frozen tissue in 1 ml 718 cold extraction buffer (methanol: chlroform: dH2O; 5:2:2). The samples were homogenized using 719 glass beads (500 μm i.d.) in a Geno/Grinder 2010 instrument (SpexSamplePrep, Metuchen, NJ, 720 USA), followed by centrifugation (14,000g, 2 min, 4°C). Samples were further processed and 721 derivatized for GC-TOF mass spectrometry as described (Lee and Fiehn, 2008). GC-MS 722 measurements were carried out on an Agilent 6890 gas chromatograph (Agilent, Santa Clara, 723 CA, USA), controlled by a Leco ChromaTOF software v 2.32 (Leco, St. Joseph, MI, USA). 724 Separation was performed on a Rtx-5Sil MS column (30m x 0.25mm x 0.25μm) with an 725 additional 10 m empty guard column (Restek, Bellefonte, PA, USA) using helium as a carrier (1 726 ml/min flow rate). The oven temperature was held constant at 50°C for 1 min, the ramped at 727 20°/min to 330°C at which it was held constant for 5 min. A Leco Pegasus IV mass spectrometer 728 (Leco, St. Joseph, MI, USA) was operated in electron impact (EI) mode at -70 eV ionization 729 energy with unit mass resolution at 17 spectra/s with a scan range of 80-500 Da. The transfer line 730 temperature between gas chromatograph and mass spectrometer was set to 280°C. Ionization 731 Electron impact ionization at 70V was employed with an ion source temperature of 250°C. 732 Mass spectra were processed using BinBase, an application system for deconvoluting and 733 annotating mass spectral data, and analyzed as described in (Fiehn et al., 2005). Metabolites were 734 identified based on their mass spectral characteristics and GC retention times, by comparison 735 with compounds in a plant and algae reference library (West Coast Metabolomics Center, UC 736 Davis, CA, USA). Peak heights for the quantification ion at the specific retention index 737 corresponding to each metabolite were normalized by the sum of peak heights in the sample. 738