Yasuyo Yamaoka
email: yamaoka1982@postech.ac.kr

Bae Seungjun Shin

Young Choi

Hanul Kim
email: hanulkim@postech.ac.kr

Sunghoon Jang

Masataka Kajikawa
email: kajikawa@lif.kyoto-u.ac.jp

Takashi Yamano
email: tyamano@lif.kyoto-u.ac.jp

Fantao Kong
email: kongfantaohit@163.com

Bertrand Legeret
email: bertrand.legeret@cea.fr

Hideya Fukuzawa
email: fukuzawa@lif.kyoto-u.ac.jp

Bae Young Choi

Bertrand Légeret

Yonghua Li-Beisson
email: yonghua.li@cea.fr

Youngsook Lee
email: ylee@postech.ac.kr

Mishiba, K Y Nagashima

The bZIP1 Transcription Factor Regulates Lipid Remodeling and Contributes to ER Stress Management in Chlamydomonas reinhardtii

Keywords: bZIP, Chlamydomonas, DGTS, ER stress, pinolenic acid

INTRODUCTION

Chlamydomonas reinhardtii is a model microalga widely used for studies of photosynthesis, flagella biogenesis, and lipid metabolism (Harris, 2001). Similar to many other microalgae, Chlamydomonas produces a large amount of triacylglycerol (TAG) under stress conditions (Merchant et al., 2012), and this prompted intensive studies of the mechanisms underlying stress responses in this organism. The endoplasmic reticulum (ER) stress response is an important stress response that is common to many eukaryotes. ER stress can be induced by biotic and abiotic factors, such as pathogen infection, temperature, high salinity, wounding, and ER stress inducing agents (Howell, 2013), and results in the accumulation of unfolded proteins in the ER. In mammals, the unfolded proteins accumulated in the ER are sensed by proteins in the ER membrane that transmit this information to the nucleus, inducing the expression of genes and restoring normal protein metabolism. This process is called the unfolded protein response (UPR). UPR genes encode molecular chaperones, folding enzymes, lipid biosynthesis enzymes, and components of the ER-associated protein degradation pathway [START_REF] Ron | Signal integration in the endoplasmic reticulum unfolded protein response[END_REF].

Recently, lipid metabolism was also found to be altered in many types of cells exposed to ER stress. In animal cells, ER stress increases de novo lipogenesis and TAG accumulation (reviewed in Basseri and Austin 2012; Mandl et al 2013; [START_REF] Zhou | ER stress and hepatic lipid metabolism[END_REF]Han and Kaufman 2016). In animal and yeast cells, exposure to ER stress promotes the biosynthesis of phosphatidylcholine (PC), a major ER membrane lipid, expanding the ER membrane area [START_REF] Sriburi | Coordinate regulation of phospholipid biosynthesis and secretory pathway gene expression in XBP-1(S)-induced endoplasmic reticulum biogenesis[END_REF][START_REF] Schuck | Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response[END_REF] and thereby attenuating the damage caused by the stress. The biosynthesis and storage of TAG in lipid droplets might also ameliorate the damage caused by various stress conditions, since stress often leads to the degradation of membrane lipids and the release of free fatty acids to toxic levels (Listenberger et al., 2003). However, little is known about the changes in other lipid molecules that occur during ER stress. Thus, it would be useful to determine the entire lipid profile of a cell, to monitor how it changes in response to ER stress, and to establish how it differs in mutants that do not have a normal ER stress response.

The C. reinhardtii genome encodes only one ER stress sensor, i.e., inositol-requiring enzyme 1 (CrIRE1). IRE1 is the best conserved ER stress sensor among metazoans, yeast, and plants [START_REF] Yamaoka | Identification and functional study of the endoplasmic reticulum stress sensor IRE1 in Chlamydomonas reinhardtii[END_REF]. IRE1 is a dual-function protein that has both kinase and ribonuclease (RNase) activity. Under ER stress, IRE1 dimerizes and then auto-phosphorylates, triggering its RNase activity. Active IRE1 splices mRNA encoding a transcription factor named Hac1 in budding yeast, XBP1 (X-box binding protein-1) in metazoans, and bZIP60 in Arabidopsis thaliana [START_REF] Yanagitani | Translational pausing ensures membrane targeting and cytoplasmic splicing of XBP1u mRNA[END_REF][START_REF] Ron | Signal integration in the endoplasmic reticulum unfolded protein response[END_REF]Howell, 2013).

CrIRE1 plays an essential role in ER stress tolerance in Chlamydomonas, similar to IRE1s of other organisms, but differs from other IRE1s in the C-terminal tandem zinc-finger domain [START_REF] Yamaoka | Identification and functional study of the endoplasmic reticulum stress sensor IRE1 in Chlamydomonas reinhardtii[END_REF]. The conserved kinase and RNase domains of CrIRE1 suggest that CrIRE1, like other IRE1 proteins, splices the mRNA of a transcription factor; however, the identity of this transcription factor was hitherto unknown in Chlamydomonas.

In this study, we identified a transcription factor, CrbZIP1, that governs the ER stress response in Chlamydomonas, and revealed the underlying molecular mechanism. In addition to providing insight into the mode of action of CrbZIP1, this study highlights the importance of membrane lipid remodeling in ER stress management.

RESULTS

Chlamydomonas bZIP1 mRNA is spliced under ER stress

We searched for the mRNA targeted by CrIRE1 from among Chlamydomonas basic leucine zipper (bZIP) proteins, since IRE1 proteins of diverse organisms activate bZIP transcription factors by splicing their mRNA [START_REF] Yanagitani | Translational pausing ensures membrane targeting and cytoplasmic splicing of XBP1u mRNA[END_REF][START_REF] Ron | Signal integration in the endoplasmic reticulum unfolded protein response[END_REF]Howell, 2013). Among the 19 genes that contain a bZIP domain in the Chlamydomonas genome, we decided to study Cre05.g238250 (CrbZIP1) as the potential target gene of CrIRE1, as it was the closest relative of Arabidopsis AtbZIP60 (Figure 1A), the established target of AtIRE1 (Nagashima et al., 2011;Deng et al., 2011).

To test whether CrbZIP1 was indeed the target gene of CrIRE1, we first tested whether the CrbZIP1 transcript was spliced under ER stress conditions. Under normal conditions, there was only one product of CrbZIP1 transcription, but under the ER stress induced by tunicamycin, there were two different CrbZIP1 transcripts in the Chlamydomonas strains CC-4533 (cw15 mt -, cell wall-deficient) and C-9 (CC-5098, mt -) (Figure 1B). However, in the CrIRE1 knock-down mutants crire1-1 and crire1-2, whose parental strain is CC-4533 [START_REF] Yamaoka | Identification and functional study of the endoplasmic reticulum stress sensor IRE1 in Chlamydomonas reinhardtii[END_REF], the smaller transcript was barely detectable (Figure 1B).

Sequencing of the transcripts revealed that 32 nucleotides were spliced out of the CrbZIP1 transcript under ER stress, resulting in a frameshift and generating a new stop codon (Figure 1C). We designated the un-spliced form of CrbZIP1 as CrbZIP1u and the spliced form of CrbZIP1 as CrbZIP1s. The conserved nucleotide sequence commonly found at the splicing site in the stem-loop structure of IRE1-target mRNA (AtbZIP60, HAC1, XBP1 shown in Supplemental Figure 1) did not exist in the double stem-loop of CrbZIP1 mRNA predicted using CentroidFold (Supplemental Figure 1 CrbZIP1; http://rtools.cbrc.jp/centroidfold/), suggesting that Chlamydomonas IRE1 uses a different mechanism to recognize and splice the target mRNA.

We then compared the predicted structures of proteins encoded by CrbZIP1u and CrbZIP1s. Both were predicted to contain a bZIP domain, but only CrbZIP1u contained a transmembrane domain (Figure 1D). The new stop codon generated by splicing was predicted to delete the transmembrane domain (Figures 1C and 1D). In Arabidopsis, AtbZIP60 mRNA is spliced by IRE1A/IRE1B at the site of the double stem-loop structure (Supplemental Figure 1), which causes a frameshift and consequent deletion of a transmembrane domain in AtbZIP60 (Nagashima et al., 2011;Deng et al., 2011). Then, the transcription factor is no longer located at the ER membrane, but enters the nucleus and regulates the expression of ER-stress-related genes. To test whether CrbZIP1 undergoes similar modification under ER stress, we analyzed the size and amount of CrbZIP1u and CrbZIP1s proteins under normal conditions and the ER stress conditions induced by treatment with 1 µg/mL tunicamycin by immunoblotting with anti-CrbZIP1 antibody (Figure 1E, Supplemental Figure 2A). Under normal conditions, CrbZIP1 protein bands were barely detectable, but after ER stress treatment, both CrbZIP1u and CrbZIP1s proteins were detectable in the protein preparations from both CC-4533 and C-9 cells (Figure 1E, Supplemental Figure 2A). In addition to tunicamycin, treatment with another ER stress agent, dithiothreitol (DTT), also induced both CrbZIP1u and CrbZIP1s proteins in the CC-4533 and C-9 cells (Figure 1E, Supplemental Figure 2A). However, in the crire1 mutants, only a CrbZIP1u protein band, but not a CrbZIP1s protein band, was detected (Figure 1E, Supplemental Figure 2A). Thus, the CrbZIP1 spliced under ER stress might be the downstream target mRNA of CrIRE1.

CrbZIP1 protein levels increased under the ER stress conditions imposed by both tunicamycin and DTT (Figure 1E, Supplemental Figure 2A); the CrbZIP1u protein level, which was very low or absent under normal conditions (0 h in Figure 1E and Supplemental Figure 2A), increased after treatment with the ER stress inducers (2 and 6 h in Figure 1E and Supplemental Figure 2A). This rapid and large induction of CrbZIP1 proteins was in contrast to the stable level of CrbZIP1u transcript (Figure 1B) and suggested that ER stress induces the translation of CrbZIP1u protein. Similar regulation of translation was reported in animal cells; the translation of the XBP1u mRNA, the target mRNA of human IRE1, is paused at the N terminus of XBP1 under normal conditions but resumes rapidly under ER stress [START_REF] Yanagitani | Cotranslational targeting of XBP1 protein to the membrane promotes cytoplasmic splicing of its own mRNA[END_REF][START_REF] Yanagitani | Translational pausing ensures membrane targeting and cytoplasmic splicing of XBP1u mRNA[END_REF].

Spliced CrbZIP1s is translocated to the nucleus from the ER

Next, to test whether the splicing of CrbZIP1u mRNA, which was predicted to remove the transmembrane domain, altered the subcellular localization of CrbZIP1s protein, we transiently expressed CrbZIP1 proteins fused with superfolder GFP (sGFP) at their C-termini in Nicotiana benthamiana epidermal cells. CrbZIP1u-sGFP colocalized with the ER marker AtBiP1-tagRFP, whereas CrbZIP1s-sGFP colocalized with the nucleus marker DAPI (Figure 1F). Together, these results suggest that ER stress activates the RNase activity of CrIRE1, which splices CrbZIP1u transcript to CrbZIP1s, and the resulting CrbZIP1s localizes to the nucleus, consistent with its function as a transcription factor.

crbzip1 mutants are hypersensitive to ER stress

To characterize the function of CrbZIP1 in Chlamydomonas, we used two independent lines of crbzip1 mutants: crbzip1-1, which was isolated from mutant pools of Chlamydomonas strain C-9, and crbzip1-2, which was obtained from the Chlamydomonas mutant library (https://www.chlamylibrary.org), derived from the CC-4533 parental line. The APHVIII cassette, for selection on paromomycin, was inserted into the first exon of CrbZIP1 in both mutants (Figure 2A). Immunoblot analysis showed that crbzip1-1 and crbzip1-2 did not contain a detectable level of CrbZIP1 proteins, indicating that both are crbzip1 knockout mutants (Supplemental Figure 3B). We also generated complementation lines for the crbzip1-1 and crbzip1-2 mutants, named crbzip1-1C and crbzip1-2C, respectively, by introducing CrbZIP1 genomic DNA into each mutant background and confirmed that the complementation lines expressed CrbZIP1s transcript and produced CrbZIP1 proteins (Supplemental Figure 3).

In the absence of tunicamycin, there was no difference in growth among crbzip1-1, crbzip1-2, their parental strains, and the complementation lines (Control in Figures 2B and2C). However, after 48 h of treatment with 1 µg/mL tunicamycin, the two crbzip1 knockout lines exhibited severe chlorosis, while their parental strains maintained green color (Figure 2B). Under ER stress conditions, growth was restored in the complementation lines to the level of the parental lines (Figures 2B and2C), indicating that the mutations in CrbZIP1 in the crbzip1 mutants were responsible for their tunicamycin-sensitive phenotype. Whereas the cell numbers of the parental strains and complemented strains were slightly increased after 48 h of tunicamycin treatment, those of the crbzip1 mutants were unchanged (Figure 2C). However, at 24 h of treatment, almost all crbzip1 mutant cells were stained by propidium iodide, a dye that permeates dead or damaged cells (Figure 2C), suggesting serious damage or death of the cells. Next, we observed the lipid droplets inside the cells using the lipid-specific fluorescent dye, Nile red. crbzip1 mutants accumulated lipid droplets under ER stress, although parental strains and complemented strains barely displayed the Nile red fluorescence (Figure 2D). The lipid droplets in crbzip1 mutants seemed to be present in extraplastidic regions (Figure 2D).

CrbZIP1 is necessary for the expression of UPR genes under ER stress

If CrbZIP1 has important functions in the ER stress response, the crbzip1 mutants might differ from their parental lines in the activation of genes that confer ER stress resistance.

Indeed, under tunicamycin treatment, the expression of genes encoding the chaperones BiP1 and CAL2, membrane-trafficking protein SAR1, translocon SEC61G, and disulfide forming proteins ERO1, PDI6, and Pb60/PDI1A, which were reported to be involved in the ER stress response (Perez-Martin et al., 2014), was upregulated only in the parental lines, and not in the crbzip1 mutants (Figure 3). Interestingly, we found that the fold induction of expression by ER stress of CrbZIP1s and many UPR genes (CAL2, SAR1, SEC61G, ERO1, and PDI6) was higher in CC-4533 than in C-9 cells (Figure 3, Supplemental Figure 3A). This was because the expression levels of these genes were lower in CC-4533 cells than in C-9 cells at the 0-h time point (Supplemental Table 1). The difference in basal expression level of the ER-stress-related genes might be due to the large variations in genome sequences among individual Chlamydomonas strains [START_REF] Siaut | Oil accumulation in the model green alga Chlamydomonas reinhardtii: characterization, variability between common laboratory strains and relationship with starch reserves[END_REF]Gallaher et al., 2015).

Lipid remodeling is altered in the crbzip1 mutants upon ER stress

Lipid remodeling is known to be essential for cell survival under cold stress (Moellering et al., 2010), but it is not clear if it also plays a role in the cell's response to ER stress. To investigate the mechanisms underlying ER stress management, we analyzed the entire glycerolipid profiles of the crbzip1 mutants and their parental strains under ER stress. After tunicamycin treatment, the mol% of TAG content did not increase very much from the starting value (0 h) in the parental lines; however, this value dramatically increased in the crbzip1-1 and crbzip1-2 mutants and was 5.8-and 9.4-fold that of each parental strain, respectively (Figure 4A, red boxes). Consistent with this result, the expression level of the gene encoding a key enzyme involved in TAG biosynthesis, ER-type DGTT1, was up-regulated only in the crbzip1 mutants after tunicamycin treatment (Figures 4B and4C), but the expression of genes encoding other acyltransferases was not up-regulated in the crbzip1 mutants (Supplemental Figure 4).

To determine the source of the fatty acids used for TAG biosynthesis in the crbzip1 mutants, we compared the major glycerolipid content of crbzip1 cells with that of their parental lines. After 12 h of treatment, the most striking difference was the mol% of a major extraplastidic membrane lipid, DGTS (Giroud et al., 1988), which decreased in crbzip1 mutants, but increased in parental lines (Figure 4A, green boxes). This result suggests that the fatty acids in the TAGs formed in crbzip1 cells during ER stress were derived from ER membrane lipid. To determine how DGTS levels increased in the parental lines, we assayed the expression level of BTA1, which encodes a key enzyme in DGTS biosynthesis in Chlamydomonas (Riekhof et al., 2005). BTA1 expression increased under ER stress, in the parental lines and complementation lines, but not in the crbzip1 mutants (Figures 4D and4E, Supplemental Figure 5). Furthermore, CrbZIP1s protein directly bound to the promoter region of BTA1 in a yeast one-hybrid assay (Figure 4F). These results suggest that CrbZIP1s up-regulates BTA1 expression and thus the production of DGTS synthesis under ER stress in Chlamydomonas.

CrbZIP1 expression is necessary for ER stress-induced biosynthesis of pinolenic acid, which enhances ER stress tolerance

Next, we analyzed the fatty acid composition of the crbzip1 mutants and their parental strains.

During ER stress, the abundance of pinolenic acid (18:3Δ5,9,12) and coniferonic acid (18:4Δ5,9,12,15) in the total fatty acids was increased in the parental strains but was reduced in the crbzip1 mutants (Figure 4A, blue boxes). Subsequent analysis of fatty acid composition of individual glycerolipids revealed that the pinolenic acid content in DGTS differed dramatically between the crbzip1 mutants and their parental lines (Figure 4A, Supplemental Figure 6). In DGTS, the pinolenic acid content slightly increased in parental strains but decreased greatly in the crbzip1 mutants after tunicamycin treatment (Figure 4A, yellow boxes). Pinolenic acid biosynthesis in the ER requires the desaturases FAD2 and CrDES in Chlamydomonas (Kajikawa et al., 2006). FAD2 and CrDES expression increased significantly in the parental strains, but not in the crbzip1 mutants, upon treatment with tunicamycin (Figure 5B), suggesting that CrbZIP1 induces these desaturases. This possibility was supported by a yeast one-hybrid assay, which revealed that CrbZIP1s interacted directly with the promoter region of CrDES (Figure 5C). By contrast, the FAD7 transcript level was induced both in the parental lines and in the crbzip1 mutants (Figure 5B), indicating that the production of alpha-linolenic acid (18:3Δ9,12,15) or coniferonic acid (18:4Δ5,9,12,15) is not the downstream target of CrbZIP1.

We then hypothesized that the induction of pinolenic acid in Chlamydomonas cells might protect the cells from ER stress. To address this possibility, we isolated crdes1-1 and crdes1-2 mutants in the C-9 and CC-4533 background, respectively (Figure 6A, Supplemental Figure 7). The crdes1-1 mutant did not contain detectable amounts of pinolenic acid (18:3Δ5,9,12) or coniferonic acid (18:4 Δ5,9,12,15; Figure 6B, blue arrows in Total FAs), and the crdes1-2 mutant contained lower levels of the two compounds than the parental line (Figure 6B, green arrows in Total FAs, yellow boxes in DGTS; Supplemental Figure 8, yellow boxes in PE and TAG), suggesting that crdes1-1 is a knockout mutant and crdes1-2 is a knockdown mutant. The fatty acid composition of other major glycerolipid species was not altered much in the crdes mutants (Supplemental Figure 8). Although the pinolenic acid content of PE and DGTS was reduced relative to the parental lines, the DGTS and PE contents were maintained in the crdes mutants (Figure 6B, Glycerolipids). The increase in alpha-linolenic acid (18:3Δ9,12,15) appeared to compensate for the loss of pinolenic acid in PE and DGTS in both the crdes mutants (Figures 6B, Supplemental Next, we investigated whether the two crdes mutants differed from their parental lines with respect to ER stress resistance. No difference in growth between any of the Chlamydomonas strains was apparent when the cells were cultured on normal TAP medium (Figure 6C, TAP). However, when the strains were spotted on TAP medium containing 0.2 µg/mL tunicamycin, crdes mutants exhibited hypersensitivity to ER stress, similarly to crbzip1 mutants (Figure 6C, TAP + Tm). These data suggest that CrbZIP1-mediated pinolenic acid biosynthesis improves the survival of Chlamydomonas exposed to ER stress.

DISCUSSION

CrbZIP1 functions downstream of CrIRE1 and plays a critical role in the ER stress response

In microalgae, no transcription factor has been identified that functions downstream of the ER stress sensor. In this study, we established that the Chlamydomonas transcription factor CrbZIP1 functions downstream of CrIRE1, and activates the expression of many genes that contribute to survival under ER stress. The following observations support this conclusion.

First, CrbZIP1 mRNA was spliced under ER stress in Chlamydomonas cells, but this splicing activity was very low in crire1 knockdown mutants (Figure 1B). Second, two independent mutant alleles of CrbZIP1, crbzip1-1 and crbzip1-2, which did not exhibit any growth defect under normal conditions, exhibited increased sensitivity to ER stress: they were completely bleached within 2 days of exposure to a concentration of tunicamycin that did not induce chlorosis in their parental lines (Figure 2B). Third, CrbZIP1 upregulated the expression of ER stress response genes, including chaperone genes and lipid biosynthesis genes (Figure 3). We also obtained data that are consistent with CrbZIP1 having a role in the ER stress response:

CrbZIP1 levels increased under ER stress (Figure 1E), and the CrbZIP1s protein encoded by the spliced form of CrbZIP1 was localized in the nucleus (Figure 1F).

Increase in pinolenic acid content protects the cells during ER stress

Pinolenic acid levels increased in the parental strains, but dramatically decreased in the crbzip1 mutants under ER stress (Figure 4A). Pinolenic acid (18:3Δ5,9,12), a Δ5-desaturated metabolite of linoleic acid (18:2Δ9,12), is found in the oil of Korean pine (Pinus koraiensis) nuts and needles and in some microalgae. A polymethylene group within the carbon skeleton of pinolenic acid makes it structurally distinct from its positional isomer gamma-linolenic acid (18:3Δ6,9,12). Pinolenic acid was demonstrated to provide specific health benefits not imparted by several other 18:3 fatty acid species (Asset et al., 2000;Asset et al., 2001;[START_REF] Song | A novel family of cysrich membrane proteins mediates cadmium resistance in Arabidopsis[END_REF]Pasman et al 2008). Chlamydomonas was reported to produce pinolenic acid, but the physiological role of this fatty acid in this alga remained unknown (Giroud et al., 1988).

In this study, we isolated two independent mutants of CrDES, a desaturase required for pinolenic acid production, and found that the crdes mutants were much more impaired in growth in medium containing tunicamycin than were their parental lines (Figure 6C). Notably, the crdes1-1 knockout mutant displayed ER stress hypersensitivity comparable to that of the crbzip1-1 mutant (Figure 6C), suggesting that the upregulation of CrDES is one of the major routes through which CrbZIP1 confers ER stress resistance in Chlamydomonas. Furthermore, the yeast one-hybrid assay suggested that CrbZIP1s directly regulates CrDES expression (Figure 5C). Therefore, CrbZIP1-dependent pinolenic acid biosynthesis seems to be necessary for effective protection of this microalga from ER stress. Since pinolenic acid is produced only in a limited number of organisms, these results also indicate that ER stress tolerance mechanisms differ among various organisms.

How does pinolenic acid enhance tolerance to ER stress in Chlamydomonas?

Previous reports revealed that reactive oxygen species (ROS) are induced by ER stress in Chlamydomonas cells (Pérez-Martín et al., 2014), and that polyunsaturated fatty acids in membrane lipids act as sinks for ROS in plastids (Mene-Saffrane et al., 2009;Zeoeller et al., 2012). Thus, pinolenic acid might be able to mitigate ROS toxicity. However, this general effect of polyunsaturated fatty acids does not explain the effect of pinolenic acid in ER stress resistance in Chlamydomonas, since the levels of another polyunsaturated fatty acid, alpha-linolenic acid (18:3Δ6,9,12), increased in DGTS and PE in crdes mutants (Figure 6B, Supplemental Figure 8), but failed to protect the cells from ER stress (Figure 6C). Another possibility is that pinolenic acid functions as a signaling molecule, much like jasmonic acid in many terrestrial plants. Trees in the Pinus family (Pinaceae) are among the longest living plants on Earth (Rogers and Clifford, 2006;[START_REF] Withington | COMPARISONS OF STRUCTURE AND LIFE SPAN IN ROOTS AND LEAVES AMONG TEMPERATE TREES[END_REF]. Thus, it is tempting to speculate that pinolenic acid in the leaves of these trees confers ER stress resistance and consequently contributes to the longevity of the plant. Further studies are needed to understand the protective function of pinolenic acid in Chlamydomonas subjected to ER stress, and it would be interesting to test whether pinolenic acid confers ER stress resistance in other organisms, too.

Increase of DGTS under ER stress

Some algae, including Chlamydomonas, have been reported to contain DGTS but not phosphatidylcholine (PC) in the ER membrane (Kato et al., 1996). Our study revealed that CrbZIP1 directly upregulated the expression of the DGTS synthase gene BTA1, and that DGTS content significantly increased in the parental lines but not in the crbzip1 mutants under ER stress (Figure 4A, Glycerolipids). An increase in ER membrane lipids was also observed in other organisms exposed to ER stress. In animals, XBP1 plays an important role in the biosynthesis of PC, the main ER phospholipid, which allows ER membrane expansion under ER stress [START_REF] Sriburi | Coordinate regulation of phospholipid biosynthesis and secretory pathway gene expression in XBP-1(S)-induced endoplasmic reticulum biogenesis[END_REF]. Furthermore, in yeast, the increase in ER size by upregulation of PC biosynthesis improves ER stress tolerance [START_REF] Schuck | Membrane expansion alleviates endoplasmic reticulum stress independently of the unfolded protein response[END_REF]. By analogy with these studies in animals and yeast, we hypothesized that the increase in DGTS levels in Chlamydomonas cells might contribute to their survival under ER stress by expanding the ER membrane. In DGTS, the polar betaine trimethylglycine is linked by an ether bond at the sn-3 position of the glycerol moiety. Trimethylglycine is an osmolyte that stabilizes proteins under many stress conditions (Auton et al., 2011). Covering the surface of the ER lumen with trimethylglycine might mediate protein folding under ER stress in Chlamydomonas. Thus, it would be interesting to study the biological function of this betaine lipid further. crbzip1 mutations induce hypersensitivity to ER stress and promote the formation of lipid droplets crbzip1 mutants accumulated lipid droplets under the ER stress induced by tunicamycin (Figure 2D). Lipid droplets were also observed in crire1 mutants treated with the same concentration (1 µg/mL) of tunicamycin [START_REF] Yamaoka | Identification and functional study of the endoplasmic reticulum stress sensor IRE1 in Chlamydomonas reinhardtii[END_REF]. These results imply that mutations in the CrIRE1/CrbZIP1 pathway disrupt the regular UPR under ER stress, resulting in the accumulation of lipid droplets and rendering these mutants more vulnerable to stress than their parental lines. The biosynthesis of the lipids accumulated in the lipid droplets seemed to be catalyzed by an acyltransferase, DGTT1, previously shown to play a major role in TAG accumulation under nitrogen starvation stress (Boyle et al., 2012), since the gene encoding this protein was expressed at higher levels in crbzip1 mutants than in their parental strains under ER stress (Figure 4C).

In summary, we established CrbZIP1 as the target of CrIRE1 under ER stress, and provided insight into the CrIRE1/CrbZIP1-mediated response to ER stress in Chlamydomonas (Figure 7). Under the ER stress induced by 1 µg/mL tunicamycin, CrbZIP1 is activated by cleavage and upregulates the expression of general UPR genes and of genes encoding DGTS synthase and pinolenic acid synthase, and thereby overcomes the stress.

However, in the absence of functional CrIRE1/CrbZIP1 proteins, Chlamydomonas cells exhibited another stress response strategy and stress symptom, namely the induction of acyltransferase and the formation of lipid droplets. Our data clearly indicate that lipid modifications contribute to the survival of the Chlamydomonas cells exposed to ER stress. It would be very interesting to test whether similar lipid modifications occur during the ER stress response in other organisms. We predict that some aspects of the lipid modification revealed here might be common to the ER stress response in many organisms, since the IRE1-mediated ER stress response pathway is highly conserved (Howell 2013;[START_REF] Ron | Signal integration in the endoplasmic reticulum unfolded protein response[END_REF][START_REF] Yamaoka | Identification and functional study of the endoplasmic reticulum stress sensor IRE1 in Chlamydomonas reinhardtii[END_REF].

METHODS

Strains and culture conditions

The Chlamydomonas strain C-9 (CC-5098, mt -), originally provided by the IAM Culture Collection at The University of Tokyo and available from the Chlamydomonas Resource Center, was used as the parental strain. The crbzip1-1 and crdes1-1 mutants, containing the APHVIII and APHVII insertions, respectively, were isolated in accordance with a previous report [START_REF] Yamano | Characterization of cooperative bicarbonate uptake into chloroplast stroma in the green alga Chlamydomonas reinhardtii[END_REF]. The Chlamydomonas strain CC-4533 (cw15 mt -) and the crbzip1-2 (LMJ.RY0402.143080) and credes1-2 (LMJ.RY0402.087433) mutants were obtained from the Chlamydomonas Genetics Center (USA) (Li et al., 2016). Chlamydomonas cells were cultured in an Erlenmeyer flask containing Tris-acetate-phosphate (TAP) medium (Harris, 2009) at 23°C under continuous light (75 μmol photons m -2 s -1 ) supplied by tubular fluorescent lamps (color temperature 4000K) with shaking at 190 rpm. Cells in the exponential growth phase (3x10 6 cells/mL) were treated with the indicated concentration of tunicamycin (Sigma-Aldrich, T7765) diluted from a 5 mg/mL stock in 0.05 N NaOH or DTT (dithiothreitol; Duchefa Biochemie, Netherlands) diluted from a 1 M stock in water. Dead cells were stained with 2 µg/mL propidium iodide for 15 min in darkness, as described in Kim et al. (2015), and cells immobilized in 1% (w/v) low-gelling agarose (Sigma-Aldrich) were counted using a hemocytometer under a fluorescence microscope (Nikon Optiphot microscope) with a TRITC filter.

Phylogenetic tree analyses

Amino acid sequences were obtained from Phytozome v12.1 (https://phytozome.jgi.doe.gov), TAIR (https://www.arabidopsis.org), and the Saccharomyces Genome Database (http://www.yeastgenome.org) and were aligned using the Clustal Omega algorithm [START_REF] Sievers | Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega[END_REF]. The phylogenetic tree was constructed using MEGA 7.0 (Kumar et al., 2016) with 1,000 bootstrap replicates.

Immunoblot analysis

Anti-CrbZIP1 antibody was generated against a synthetic peptide containing the 23-amino acid fragment located at the N-terminus of CrbZIP1 (GPSLLDDELFSEDLMQFLETIEG).

Rabbits were injected with the peptide for the production of anti-CrbZIP1 polyclonal antibody (Young In Frontier, South Korea). To detect CrbZIP1 protein levels, total protein was extracted from Chlamydomonas cells and 40 µg of total protein was subjected to immunoblot analysis as described previously [START_REF] Song | A novel family of cysrich membrane proteins mediates cadmium resistance in Arabidopsis[END_REF].

Subcellular localization of CrbZIP1 in Nicotiana benthamiana epidermal cells

CrbZIP1u and CrbZIP1s cDNA were introduced into pGWB5 (Nakagawa et al., 2007). Arabidopsis BiP1 cDNA were introduced into pGWB560 (Nakagawa et al., 2008). N. benthamiana leaves were infiltrated as described previously [START_REF] Sparkes | Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants[END_REF].

Microscopy analysis

To visualize lipid droplets, cells were stained with 1 μg/mL Nile red (Sigma-Aldrich) diluted from a 0.1 mg/mL stock solution in acetone for 20 min in darkness at room temperature, and the cells were immobilized in 1% (w/v) low-gelling agarose (Sigma-Aldrich). To visualize the nucleus, the DAPI staining was performed as described by [START_REF] Yamaoka | PHOSPHATIDYLSERINE SYNTHASE1 is required for microspore development in Arabidopsis thaliana[END_REF].

Fluorescence was observed using a confocal laser-scanning microscope (Olympus).

RNA extraction and quantification

Total RNA was extracted and quantified as described previously (Jang et al., 2015), with a few modifications. Total RNA was isolated using homemade TRIzol, and cDNA was synthesized using a RevertAid First Strand cDNA Synthesis Kit (K1621, Thermo Scientific) and 3 µg of total RNA. Quantitative RT-PCR was performed using SYBR Premix Ex Taq (Takara) and the primer sets are listed in Supplemental Table 1. The quantitative RT-PCR results were normalized by the level of RPL17 expression [START_REF] Yamaoka | Identification and functional study of the endoplasmic reticulum stress sensor IRE1 in Chlamydomonas reinhardtii[END_REF].

Isolation of complementation strains

The promoter region of the Chlamydomonas expression vector pChlamy_1 (Thermo Scientific) was removed, and the CrbZIP1 promoter region fused with the 5′-UTR (2135 bp) and the CrbZIP1 genomic DNA with the 3′-UTR (2911 bp) was inserted as a XbaI fragment (pChlamy-CrbZIP1). crbizp1-1 and crbzip1-2 were transformed with pChlamy-CrbZIP1, and the transformants were selected by hygromycin tolerance. CrbZIP1 proteins from 96 transformants were individually analyzed by immunoblot assay, and the transformants that contained similar amounts of CrbZIP1 proteins to the parental strains were selected as complementation lines of crbzip1 mutants.

Lipid extraction and quantification

Lipids were extracted and analyzed as described previously (Kim et al., 2013) with modifications. Briefly, 40 mL of Chlamydomonas cell culture (approximately 5 x 10 6 cells/mL) was harvested by centrifugation at 2,000 g for 5 min at room temperature and used for total lipid extraction. To quantify TAGs, 1 mg of the total lipid extract was separated on a thin-layer chromatography (TLC) plate using the following solvent mixture: hexane:diethyl ether:acetic acid (80:30:1, by volume). To separate membrane lipids, 1 mg of the total lipid extract was separated on a TLC plate using the following solvent mixture: chloroform:methanol:acetic acid:water (75:13:9:3, by volume). Lipid spots were visualized under UV after spraying with 0.01% (w/v) primuline (Sigma) dissolved in acetone:H 2 O (4:1, v/v). Lipid bands were recovered from the plate and quantified by a gas chromatograph (GC-FID, SHIMADZU GC-2010) equipped with an HP-INNOWax capillary column (30 m × 0.25 mm, 0.25 μm film thickness, Agilent Technologies) after transesterification.

Yeast one-hybrid assay

Yeast one-hybrid assays were carried out using the Matchmaker™ Yeast One-HybridSystem (Clontech, USA), as described in the manufacturer's protocol. The promoters of BTA1 (655 bp) and CrDES (736 bp) were cloned into the pHISi vector, and codon-optimized CrbZIP1s for yeast expression was subcloned into the pGAD424 vector using the primers listed in Table S1.

Yeast cells were spotted on medium containing SD-Leu-Ura-His with or without 3-AT with a series of 1:10 dilutions.

Accession Numbers

Chlamydomonas genes analyzed in this study were identified in Phytozome v12 (http://phytozome.jgi.doe.gov/pz/portal.html) under the following accession numbers: CrbZIP1, Cre05.g238250.t1.2; CrIRE1, Cre08.g371052.t1.1; BiP1, Cre02.g080700.t1.2; CAL2, Cre01.g038400.t1.2; SAR1, Cre11.g468300.t1.2; SEC61, Cre16.g680230.t1.1; ERO1, Cre17.g723150.t1.3; PDI6, Cre12.g518200.t1.3; Rb60, Cre02.g088200.t1.2; DGTT1; Cre12.g557750.t1.1; BTA1, Cre07.g324200.t1.2; FAD2, Cre17.g711150.t1.1; CrDES, Cre10.g453600.t2.1; FAD7, Cre01.g038600.t1.2; PDAT, Cre02.g106400.t1.1; DGAT, Cre01.g045903.t1.1; DGTT2, Cre09.g386912.t1.1; DGTT3, Cre06.g299050.t1.2; DGTT4, Cre03.g205050.t1.2. Arabidopsis genes analyzed in this study were identified in TAIR (E) Expression level of BTA1 in Chlamydomonas cells subjected to ER stress. Experimental conditions and data processing were the same as in (C).

(F) Yeast one-hybrid assay showing that CrbZIP1s interacts directly with the promoter region of BTA1. Yeast cells were spotted on medium containing SD-Leu-Ura-His with or without 3-AT with a series of 1:10 dilutions. (A) C18 FA biosynthesis pathway in Chlamydomonas.

(B) Relative transcript abundance of desaturase genes after tunicamycin (Tm) treatment.

Cells were treated with 1 µg/mL Tm for the indicated periods. The RT-qPCR results were normalized by the level of RPL17 expression and the fold changes of expression levels were determined relative to the levels at 0 h. Error bars represent standard errors based on four biological replicates for 0, 2, and 6 h. Statistical analysis was carried out using a Student's t-test (*, p < 0.05; **, p < 0.01) and the Wilcoxon rank sum test (Supplemental Dataset 1).

(C) Yeast one-hybrid assay showing that CrbZIP1s interacts directly with the promoter region of CrDES. Yeast cells were spotted on medium containing SD-Leu-Ura-His with or without 3-AT with a series of 1:10 dilutions. Right: The crbzip1 mutation upregulates TAG biosynthesis from the fatty acids hydrolyzed from a major ER membrane lipid, DGTS, resulting in the formation of lipid droplets, which store TAGs.

  Figure 8, red boxes).

(

  https://www.arabidopsis.org) under the following accession numbers: AtbZIP60, AT1G42990.1; AtBiP1, AT5G28540.1. The yeast gene analyzed in this study was identified in the Saccharomyces Genome Database (http://www.yeastgenome.org) under the following accession number: Hac1, S000001863. boxes, gray boxes, and lines, respectively. (B) Photographs of cultures grown in control TAP medium or TAP medium containing 1 µg/mL Tm for the indicated periods. (C) Cell numbers and viability of cells grown in the presence or absence of Tm. Propidium iodide (PI) was used to stain dead and damaged cells, and cells not stained with PI were considered healthy. Averages from four biological replicates from two individual experiments and their standard errors are shown. (D) Confocal microscopy images of Nile red-stained cells grown in medium with 1 µg/mL Tm for 0, 6, 12, or 24 h. Images of chlorophyll autofluorescence were merged with those of Nile red fluorescence, which indicated lipid droplets.

Figure 3 .

 3 Figure 3. Expression levels of ER stress marker genes in crbzip1 mutants and their parental strains. Cells were treated with 1 µg/mL tunicamycin (Tm) for the indicated periods. The RT-qPCR results were normalized by the level of RPL17 expression and the fold changes of expression levels at 2 h and 6 h were determined relative to the levels at 0 h. Error bars represent standard errors based on four biological replicates from two individual experiments. Statistical analysis was carried out using a Student's t-test (*, p < 0.05; **, p < 0.01) and the Wilcoxon rank sum test (Supplemental Dataset 1).

Figure 4 .

 4 Figure 4. ER-stress-induced lipid remodeling differs between the crbzip1 mutant and their parental lines. (A) Levels of fatty acids (FAs) in glycerolipids and the fatty acid composition of total fatty acids and of diacylglyceryltrimethylhomo-Ser (DGTS). Lipids extracted from crbzip1 mutants and their parental strains treated with 1 µg/mL tunicamycin (Tm) for the indicated periods were analyzed using GC-FID. Error bars represent standard errors based on three biological replicates. Red box indicates TAG accumulation in crbzip1 cells. Green box represents the induction of DGTS only in the parental strains. Blue boxes indicate the decreases of pinolenic acid in total FAs in crbzip1 cells. Yellow boxes indicate the dramatic reductions of pinolenic acid in DGTS. phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), sulfoquinovosyldiacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), and triacylglycerol (TAG). Statistical analysis was carried out using a Student's t-test (*, p < 0.05; **, p < 0.01, Supplemental Dataset 2), and the significant differences between the mutant and

Figure 5 .

 5 Figure 5. CrbZIP1s promotes the biosynthesis of pinolenic acid (18:3Δ5,9,12) under ER stress.

Figure 6 .

 6 Figure 6. crdes knockdown mutants are hypersensitive to the ER stress imposed by tunicamycin treatment. (A) Schematic representation of insertion sites of the APHVII and APHVIII cassettes in the genomic sequence of CrDES in crdes mutants. The positions of UTRs, exons, and introns are

Figure 7 .

 7 Figure 7. Proposed model for a mechanism of ER stress responses in crbzip1 mutants and their parental lines in Chlamydomonas. Left: In the parental lines, activated CrIRE1 splices CrbZIP1u mRNA to produce CrbZIP1s, which is translated to CrbZIP1s. CrbZIP1s enters the nucleus, where it induces the expression of UPR genes and ER membrane lipid biosynthesis genes, such as BTA1 and CrDES. The dimerization and phosphorylation of CrIRE1 are drawn based on reports in other organisms but have not been revealed in Chlamydomonas.

388 389 ACKNOWLEDGMENTS This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC-2015M3A6A2065746) awarded to Y. Lee, the Japan Society for the Promotion of Science KAKENHI (16H04805) and the Japan Science and Technology Agency, Advanced Low Carbon Technology Research and Development Program (ALCA, JPMJAL1105) awarded to H. Fukuzawa, Agence Nationale de la Recherche (ANR-MUsCA) awarded to Y. Li-Beisson, and the Korea Research Fellowship Program funded by the Ministry of Science, ICT and Future Planning through the National Research Foundation of Korea (2016H1D3A1909463) to Y. Yamaoka. We thank the Chlamydomonas Mutant Library Group at the Carnegie Institution for Science and the Chlamydomonas Resource Center at the University of Minnesota for providing the indexed Chlamydomonas insertional mutants.

Supplemental Table

 1. Expression levels of ER stress sensor and ER stress marker genes in crbzip1 mutants and their parental strains.

Supplemental Table 2. Primer sequences.

Supplemental