Youngsook Lee, ylee@postech.ac.kr. 24 25The authors declare there is no conflict of interests. 26 27Running title: CrbZIP1 modulates membrane lipids under ER stress 28 29One-Sentence Summary: The mRNA of a Chlamydomonas bZIP transcription factor is 30 spliced by CrIRE1 under ER stress, and the resulting protein protects Chlamydomonas cells 31 from ER stress by modulating lipid remodeling. 32 33 34Keywords: bZIP; Chlamydomonas; DGTS; ER stress; pinolenic acid 35 36Word count: 7958 words 37 38 39 2 ABSTRACT 40Endoplasmic reticulum (ER) stress is caused by the stress-induced accumulation of unfolded 41 proteins in the ER. Here, we identified proteins and lipids that function downstream of the 42 ER stress sensor inositol-requiring enzyme1 (CrIRE1), which contributes to ER stress 43 tolerance in Chlamydomonas reinhardtii. Treatment with the ER stress inducer tunicamycin 44 resulted in the splicing of a 32-nucleotide fragment of a bZIP transcription factor (CrbZIP1) 45 mRNA by CrIRE1, which resulted in the loss of the transmembrane domain in CrbZIP1, and 46 the translocation of CrbZIP1 from the ER to the nucleus. Mutants deficient in CrbZIP1 failed 47to induce the expression of the unfolded protein response genes and grew poorly under ER 48 stress. Levels of diacylglyceryltrimethylhomo-Ser (DGTS) and pinolenic acid (18:3Δ5,9,12) 49 increased in the parental strains but decreased in the crbzip1 mutants under ER stress. A yeast 50one-hybrid assay revealed that CrbZIP1 activated the expression of enzymes catalyzing the 51 biosynthesis of DGTS and pinolenic acid. Moreover, two independent alleles of crdes mutant, 52 which failed to synthesize pinolenic acid, were more sensitive to ER stress than were their 53 parental lines. Together, these results indicate that CrbZIP1 is a critical component of the ER 54 stress response mediated by CrIRE1 in Chlamydomonas that acts via lipid remodeling. 55 57Chlamydomonas reinhardtii is a model microalga widely used for studies of photosynthesis, 58 flagella biogenesis, and lipid metabolism (Harris, 2001). Similar to many other microalgae, 59Chlamydomonas produces a large amount of triacylglycerol (TAG) under stress conditions 60 (Merchant et al., 2012), and this prompted intensive studies of the mechanisms underlying 61 stress responses in this organism. The endoplasmic reticulum (ER) stress response is an 62 important stress response that is common to many eukaryotes. ER stress can be induced by 63 biotic and abiotic factors, such as pathogen infection, temperature, high salinity, wounding, 64 and ER stress inducing agents (Howell, 2013), and results in the accumulation of unfolded 65 proteins in the ER. In mammals, the unfolded proteins accumulated in the ER are sensed by 66 proteins in the ER membrane that transmit this information to the nucleus, inducing the 67 expression of genes and restoring normal protein metabolism. This process is called the 68 unfolded protein response (UPR). UPR genes encode molecular chaperones, folding enzymes, 69 lipid biosynthesis enzymes, and componen...

Photosynthetic organisms are exposed to various environmental sources of oxidative stress. Land plants have diverse mechanisms to withstand oxidative stress, but how microalgae do so remains unclear. Here, we characterized the Chlamydomonas reinhardtii bZIP transcription factor BLZ8, which is highly induced by oxidative stress. Oxidative stress tolerance increased with increasing BLZ8 expression levels. BLZ8 regulated the expression of genes likely involved in the carbon-concentrating mechanism (CCM): HIGH-LIGHT ACTIVATED 3 (HLA3), CARBONIC ANHYDRASE 7 (CAH7), and CARBONIC ANHYDRASE 8 (CAH8). BLZ8 expression increased the photosynthetic affinity for inorganic carbon under alkaline stress conditions, suggesting that BLZ8 induces the CCM. BLZ8 expression also increased the photosynthetic linear electron transfer rate, reducing the excitation pressure of the photosynthetic electron transport chain and in turn suppressing reactive oxygen species (ROS) production under oxidative stress conditions. A carbonic anhydrase inhibitor, ethoxzolamide, abolished the enhanced tolerance to alkaline stress conferred by BLZ8 overexpression. BLZ8 directly regulated the expression of the three target genes and required bZIP2 as a dimerization partner in activating CAH8 and HLA3. Our results suggest that a CCM-mediated increase in the CO2 supply for photosynthesis is critical to minimize oxidative damage in microalgae, since slow gas diffusion in aqueous environments limits CO2 availability for photosynthesis, which can trigger ROS formation.

In many eukaryotes, endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR) via the transmembrane endoribonuclease IRE1 to maintain ER homeostasis. The ER stress response in microalgae has not been studied in detail. Here, we identified Chlamydomonas reinhardtii IRE1 (CrIRE1) and characterized two independent knock-down alleles of this gene. CrIRE1 is similar to IRE1s identified in budding yeast, plants, and humans, in terms of conserved domains, but differs in having the tandem zinc-finger domain at the C terminus. CrIRE1 was highly induced under ER stress conditions, and the expression of a chimeric protein consisting of the luminal N-terminal region of CrIRE1 fused to the cytosolic C-terminal region of yeast Ire1p rescued the yeast ∆ire1 mutant. Both allelic ire1 knock-down mutants ire1-1 and ire1-2 were much more sensitive than their parental strain CC-4533 to the ER stress inducers tunicamycin, dithiothreitol and brefeldin A. Treatment with a low concentration of tunicamycin resulted in growth arrest and cytolysis in ire1 mutants, but not in CC-4533 cells. Furthermore, in the mutants, ER stress marker gene expression was reduced, and reactive oxygen species (ROS) marker gene expression was increased. The survival of ire1 mutants treated with tunicamycin improved in the presence of the ROS scavenger glutathione, suggesting that ire1 mutants failed to maintain ROS levels under ER stress. Together, these results indicate that CrIRE1 functions as an important component of the ER stress response in Chlamydomonas, and suggest that the ER stress sensor IRE1 is highly conserved during the evolutionary history.

Phosphatidylserine (PS) is involved in various cellular processes in yeast and animals. However, PS functions in plants remain unclear. In Arabidopsis, PS is relatively enriched in flower and root tissues, and the genetic disturbance of PS biosynthesis in PHOSPHATIDYLSERINE SYNTHASE1 (PSS1)/pss1 heterozygotes induces sporophytic and gametophytic defects during pollen maturation. This study functionally characterized PS in Arabidopsis roots and observed that pss1 seedlings exhibited a short-root phenotype by reducing the meristem size and cell elongation capacity. Confocal microscopy imaging analyses of PS with GFP-LactC2 and the endocytic activity with FM 4-64 revealed that although GFP-LactC2 (or PS) was localized in the plasma membrane and endocytic membranes, the lack of PS in pss1 roots did not affect the constitutive endocytosis. Instead, a fluorescence imaging analysis of the cytokinetic phases in the dividing zone of pss1-2 roots revealed a significant delay in telophase progression, requiring active cargo vesicle trafficking for cell plate formation. Confocal microscopy imaging analysis of transgenic GFP-LactC2 root cells with developing cell plates indicated that GFP-LactC2 was localized at the cell plate. Moreover, confocal microscopy images of transgenic pss1-2 and PSS1 roots expressing the cell plate-specific syntaxin construct ProKNOLLE:eGFP-KNOLLE showed abnormal cell plate development in pss1-2 ProKNOLLE:eGFP-KNOLLE roots. These results suggested that PS is required for root cytokinesis, possibly because it helps mediate the cargo vesicular trafficking required for cell plate formation.

A-type ATP-binding cassette (ABCA) proteins transport lipids and lipid-based molecules in humans, and their malfunction is associated with various inherited diseases. Although plant genomes encode many ABCA transporters, their molecular and physiological functions remain largely unknown. Seeds are rapidly developing organs that rely on the biosynthesis and transport of large quantities of lipids to generate new membranes and storage lipids. In this study, we characterized the Arabidopsis (Arabidopsis thaliana) ABCA10 transporter, which is selectively expressed in female gametophytes and early developing seeds. By 3 days after flowering (DAF), seeds from the abca10 loss-of-function mutant exhibited a smaller chalazal endosperm than those of the wild type. By 4 DAF, their endosperm nuclei occupied a smaller area than those of the wild type. The endosperm nuclei of the mutants also failed to distribute evenly inside the seed coat and stayed aggregated instead, possibly due to inadequate expansion of abca10 endosperm. This endosperm defect might have retarded abca10 embryo development. At 7 DAF, a substantial portion of abca10 embryos remained at the globular or earlier developmental stages, whereas wild-type embryos were at the torpedo or later stages. ABCA10 is likely involved in lipid metabolism, as ABCA10 overexpression induced the overaccumulation of triacylglycerol but did not change the carbohydrate or protein contents in seeds. In agreement, ABCA10 localized to the endoplasmic reticulum (ER), the major site of lipid biosynthesis. Our results reveal that ABCA10 plays an essential role in early seed development, possibly by transporting substrates for lipid metabolism to the ER.