Overexpression of a MYB1 Transcription Factor Enhances Triacylglycerol and Starch Accumulation and Biomass Production in the Green Microalga Chlamydomonas reinhardtii.

Microalgae are promising platforms for biofuel production. Transcription factors (TFs) are emerging as key regulators of lipid metabolism for biofuel production in microalgae. We previously identified a novel TF MYB1, which mediates lipid accumulation in the green microalga Chlamydomonas under nitrogen depletion. However, the function of MYB1 on lipid metabolism in microalgae under standard growth conditions remains poorly understood. Here, we examined the effects of MYB1 overexpression (MYB1-OE) on lipid metabolism and physiological changes in Chlamydomonas. Under standard growth conditions, MYB1-OE transformants accumulated 1.9 to 3.2-fold more triacylglycerols (TAGs) than that in the parental line (PL), and total fatty acids (FAs) also significantly increased. Moreover, saturated FA (C16:0) was enriched in TAGs and total FAs in MYB1-OE transformants. Notably, starch and protein content and biomass production also significantly increased in MYB1-OE transformants compared with that in PL. Furthermore, RT-qPCR results showed that the expressions of key genes involved in TAG, FA, and starch biosynthesis were upregulated. In addition, MYB1-OE transformants showed higher biomass production without a compromised cell growth rate and photosynthetic activity. Overall, our results indicate that MYB1 overexpression not only enhanced lipid content but also improved starch and protein content and biomass production under standard growth conditions. TF MYB1 engineering is a promising genetic engineering tool for biofuel production in microalgae.

Sterol Biosynthesis Contributes to Brefeldin-A-Induced Endoplasmic Reticulum Stress Resistance in Chlamydomonas reinhardtii.

The endoplasmic reticulum (ER) stress response is an evolutionarily conserved mechanism in most eukaryotes. In this response, sterols in the phospholipid bilayer play a crucial role in controlling membrane fluidity and homeostasis. Despite the significance of both the ER stress response and sterols in maintaining ER homeostasis, their relationship remains poorly explored. Our investigation focused on Chlamydomonas strain CC-4533 and revealed that free sterol biosynthesis increased in response to ER stress, except in mutants of the ER stress sensor IRE1. Transcript analysis of Chlamydomonas experiencing ER stress unveiled the regulatory role of the IRE1/bZIP1 pathway in inducing the expression of ERG5, which encodes C-22 sterol desaturase. Through the isolation of three erg5 mutant alleles, we observed a defect in the synthesis of Chlamydomonas' sterol end products, ergosterol and 7-dehydroporiferasterol. Furthermore, these erg5 mutants also exhibited increased sensitivity to ER stress induced by brefeldin A (BFA, an inhibitor of ER-Golgi trafficking), whereas tunicamycin (Tm, an inhibitor of N-glycosylation) and dithiothreitol (DTT, an inhibitor of disulfide-bond formation) had no such effect. Intriguingly, the sterol biosynthesis inhibitors fenpropimorph (Fp) and fenhexamid (Fh), which impede steps upstream of the ERG5 enzyme in sterol biosynthesis, rescued BFA hypersensitivity in CC-4533 cells. Collectively, our findings support the conclusion that the accumulation of intermediates in the sterol biosynthetic pathway influences ER stress in a complex manner. This study highlights the significance and complexity of regulating sterol biosynthesis during the ER stress response in microalgae.

Co-Expression of Lipid Transporters Simultaneously Enhances Oil and Starch Accumulation in the Green Microalga Chlamydomonas reinhardtii under Nitrogen Starvation.

Lipid transporters synergistically contribute to oil accumulation under normal conditions in microalgae; however, their effects on lipid metabolism under stress conditions are unknown. Here, we examined the effect of the co-expression of lipid transporters, fatty acid transporters, (FAX1 and FAX2) and ABC transporter (ABCA2) on lipid metabolism and physiological changes in the green microalga Chlamydomonas under nitrogen (N) starvation. The results showed that the TAG content in FAX1-FAX2-ABCA2 over-expressor (OE) was 2.4-fold greater than in the parental line. Notably, in FAX1-FAX2-ABCA2-OE, the major membrane lipids and the starch and cellular biomass content also significantly increased compared with the control lines. Moreover, the expression levels of genes directly involved in TAG, fatty acid, and starch biosynthesis were upregulated. FAX1-FAX2-ABCA2-OE showed altered photosynthesis activity and increased ROS levels during nitrogen (N) deprivation. Our results indicated that FAX1-FAX2-ABCA2 overexpression not only enhanced cellular lipids but also improved starch and biomass contents under N starvation through modulation of lipid and starch metabolism and changes in photosynthesis activity. The strategy developed here could also be applied to other microalgae to produce FA-derived energy-rich and value-added compounds.

Effect of Common ER Stress-Inducing Drugs on the Growth and Lipid Phenotypes of Chlamydomonas and Arabidopsis.

Endoplasmic reticulum (ER) stress is caused by the stress-induced accumulation of unfolded proteins in the ER. Several compounds are used to induce the unfolded protein response (UPR) in animals, with different modes of action, but which ER stress-inducing drugs induce ER stress in microalgae or land plants is unclear. In this study, we examined the effects of seven chemicals that were reported to induce ER stress in animals on the growth, UPR gene expression and fatty acid profiles of Chlamydomonas reinhardtii (Chlamydomonas) and Arabidopsis thaliana (Arabidopsis): 2-deoxyglucose, dithiothreitol (DTT), tunicamycin (TM), thapsigargin, brefeldin A (BFA), monensin (MON) and eeyarestatin I. In both model photosynthetic organisms, DTT, TM, BFA and MON treatment induced ER stress, as indicated by the induction of spliced bZIP1 and bZIP60, respectively. In Chlamydomonas, DTT, TM and BFA treatment induced the production of transcripts related to lipid biosynthesis, but MON treatment did not. In Arabidopsis, DTT, TM, BFA and MON inhibited seed germination and seedling growth with the activation of bZIP60. These findings lay the foundation for using four types of ER stress-inducing drugs in photosynthetic organisms, and they help uncover the mode of action of each compound.

Biotechnological Approaches for Biomass and Lipid Production Using Microalgae Chlorella and Its Future Perspectives.

Heavy reliance on fossil fuels has been associated with increased climate disasters. As an alternative, microalgae have been proposed as an effective agent for biomass production. Several advantages of microalgae include faster growth, usage of non-arable land, recovery of nutrients from wastewater, efficient CO2 capture, and high amount of biomolecules that are valuable for humans. Microalgae Chlorella spp. are a large group of eukaryotic, photosynthetic, unicellular microorganisms with high adaptability to environmental variations. Over the past decades, Chlorella has been used for the large-scale production of biomass. In addition, Chlorella has been actively used in various food industries for improving human health because of its antioxidant, antidiabetic, and immunomodulatory functions. However, the major restrictions in microalgal biofuel technology are the cost-consuming cultivation, processing, and lipid extraction processes. Therefore, various trials have been performed to enhance the biomass productivity and the lipid contents of Chlorella cells. This study provides a comprehensive review of lipid enhancement strategies mainly published in the last five years and aimed at regulating carbon sources, nutrients, stresses, and expression of exogenous genes to improve biomass production and lipid synthesis.

The Chlamydomonas transcription factor MYB1 mediates lipid accumulation under nitrogen depletion.

Microalgae accumulate high levels of oil under stress, but the underlying biosynthetic pathways are not fully understood. We sought to identify key regulators of lipid metabolism under stress conditions. We found that the Chlamydomonas reinhardtii gene encoding the MYB-type transcription factor MYB1 is highly induced under stress conditions. Two myb1 mutants accumulated less total fatty acids and storage lipids than their parental strain upon nitrogen (N) depletion. Transcriptome analysis revealed that genes involved in lipid metabolism are highly enriched in the wild-type but not in the myb1-1 mutant after 4 h of N depletion. Among these genes were several involved in the transport of fatty acids from the chloroplast to the endoplasmic reticulum (ER): acyl-ACP thioesterase (FAT1), Fatty Acid EXporters (FAX1, FAX2), and long-chain acyl-CoA synthetase1 (LACS1). Furthermore, overexpression of FAT1 in the chloroplast increased lipid production. These results suggest that, upon N depletion, MYB1 promotes lipid accumulation by facilitating fatty acid transport from the chloroplast to the ER. This study identifies MYB1 as an important positive regulator of lipid accumulation in C. reinhardtii upon N depletion, adding another player to the established regulators of this process, including NITROGEN RESPONSE REGULATOR 1 (NRR1) and TRIACYLGLYCEROL ACCUMULATION REGULATOR 1 (TAR1).

Early seed development requires the A-type ATP-binding cassette protein ABCA10.

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 d 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.

The Chlamydomonas bZIP transcription factor BLZ8 confers oxidative stress tolerance by inducing the carbon-concentrating mechanism.

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 basic leucine zipper (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.

Phosphatidylserine Is Required for the Normal Progression of Cell Plate Formation in Arabidopsis Root Meristems.

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 pss1-2ProKNOLLE: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.

Arabidopsis seedling establishment under waterlogging requires ABCG5-mediated formation of a dense cuticle layer.

Germination requires sufficient water absorption by seeds, but excessive water in the soil inhibits plant growth. We therefore hypothesized that tolerance mechanisms exist that help young seedlings survive and develop in waterlogged conditions. Many ATP-BINDING CASSETTE TRANSPORTER subfamily G (ABCG) proteins protect terrestrial plants from harsh environmental conditions. To establish whether any of these proteins facilitate plant development under waterlogged conditions, we observed the early seedling growth of many ABCG transporter mutants under waterlogged conditions. abcg5 seedlings exhibited severe developmental problems under waterlogged conditions: the shoot apical meristem was small, and the seedling failed to develop true leaves. The seedlings had a high water content and reduced buoyancy on water, suggesting that they were unable to retain air spaces on and inside the plant. Supporting this possibility, abcg5 cotyledons had increased cuticle permeability, reduced cuticular wax contents, and a much less dense cuticle layer than the wild-type. These results indicate that proper development of plants under waterlogged conditions requires the dense cuticle layer formed by ABCG5 activity.

The phosphatidylethanolamine-binding protein DTH1 mediates degradation of lipid droplets in Chlamydomonas reinhardtii.

Lipid droplets (LDs) are intracellular organelles found in a wide range of organisms and play important roles in stress tolerance. During nitrogen (N) starvation, Chlamydomonas reinhardtii stores large amounts of triacylglycerols (TAGs) inside LDs. When N is resupplied, the LDs disappear and the TAGs are degraded, presumably providing carbon and energy for regrowth. The mechanism by which cells degrade LDs is poorly understood. Here, we isolated a mutant (dth1-1, Delayed in TAG Hydrolysis 1) in which TAG degradation during recovery from N starvation was compromised. Consequently, the dth1-1 mutant grew poorly compared to its parental line during N recovery. Two additional independent loss-of-function mutants (dth1-2 and dth1-3) also exhibited delayed TAG remobilization. DTH1 transcript levels increased sevenfold upon N resupply, and DTH1 protein was localized to LDs. DTH1 contains a putative lipid-binding domain (DTH1LBD) with alpha helices predicted to be structurally similar to those in apolipoproteins E and A-I. Recombinant DTH1LBD bound specifically to phosphatidylethanolamine (PE), a major phospholipid coating the LD surface. Overexpression of DTH1LBD in Chlamydomonas phenocopied the dth1 mutant's defective TAG degradation, suggesting that the function of DTH1 depends on its ability to bind PE. Together, our results demonstrate that the lipid-binding DTH1 plays an essential role in LD degradation and provide insight into the molecular mechanism of protein anchorage to LDs at the LD surface in photosynthetic cells.

Caenorhabditis elegans Lipin 1 moderates the lifespan-shortening effects of dietary glucose by maintaining ω-6 polyunsaturated fatty acids.

Excessive glucose causes various diseases and decreases lifespan by altering metabolic processes, but underlying mechanisms remain incompletely understood. Here, we show that Lipin 1/LPIN-1, a phosphatidic acid phosphatase and a putative transcriptional coregulator, prevents life-shortening effects of dietary glucose on Caenorhabditis elegans. We found that depletion of lpin-1 decreased overall lipid levels, despite increasing the expression of genes that promote fat synthesis and desaturation, and downregulation of lipolysis. We then showed that knockdown of lpin-1 altered the composition of various fatty acids in the opposite direction of dietary glucose. In particular, the levels of two ω-6 polyunsaturated fatty acids (PUFAs), linoleic acid and arachidonic acid, were increased by knockdown of lpin-1 but decreased by glucose feeding. Importantly, these ω-6 PUFAs attenuated the short lifespan of glucose-fed lpin-1-inhibited animals. Thus, the production of ω-6 PUFAs is crucial for protecting animals from living very short under glucose-rich conditions.

Non-intrinsic ATP-binding cassette proteins ABCI19, ABCI20 and ABCI21 modulate cytokinin response at the endoplasmic reticulum in Arabidopsis thaliana.

KEY MESSAGE: The non-intrinsic ABC proteins ABCI20 and ABCI21 are induced by light under HY5 regulation, localize to the ER, and ameliorate cytokinin-driven growth inhibition in young Arabidopsis thaliana seedlings. The plant ATP-binding cassette (ABC) I subfamily (ABCIs) comprises heterogeneous proteins containing any of the domains found in other ABC proteins. Some ABCIs are known to function in basic metabolism and stress responses, but many remain functionally uncharacterized. ABCI19, ABCI20, and ABCI21 of Arabidopsis thaliana cluster together in a phylogenetic tree, and are suggested to be targets of the transcription factor ELONGATED HYPOCOTYL 5 (HY5). Here, we reveal that these three ABCIs are involved in modulating cytokinin responses during early seedling development. The ABCI19, ABCI20 and ABCI21 promoters harbor HY5-binding motifs, and ABCI20 and ABCI21 expression was induced by light in a HY5-dependent manner. abci19 abci20 abci21 triple and abci20 abci21 double knockout mutants were hypersensitive to cytokinin in seedling growth retardation assays, but did not show phenotypic differences from the wild type in either control medium or auxin-, ABA-, GA-, ACC- or BR-containing media. ABCI19, ABCI20, and ABCI21 were expressed in young seedlings and the three proteins interacted with each other, forming a large protein complex at the endoplasmic reticulum (ER) membrane. These results suggest that ABCI19, ABCI20, and ABCI21 fine-tune the cytokinin response at the ER under the control of HY5 at the young seedling stage.

MDT-15/MED15 permits longevity at low temperature via enhancing lipidostasis and proteostasis.

Low temperatures delay aging and promote longevity in many organisms. However, the metabolic and homeostatic aspects of low-temperature-induced longevity remain poorly understood. Here, we show that lipid homeostasis regulated by Caenorhabditis elegans Mediator 15 (MDT-15 or MED15), a transcriptional coregulator, is essential for low-temperature-induced longevity and proteostasis. We find that inhibition of mdt-15 prevents animals from living long at low temperatures. We show that MDT-15 up-regulates fat-7, a fatty acid desaturase that converts saturated fatty acids (SFAs) to unsaturated fatty acids (UFAs), at low temperatures. We then demonstrate that maintaining a high UFA/SFA ratio is essential for proteostasis at low temperatures. We show that dietary supplementation with a monounsaturated fatty acid, oleic acid (OA), substantially mitigates the short life span and proteotoxicity in mdt-15(-) animals at low temperatures. Thus, lipidostasis regulated by MDT-15 appears to be a limiting factor for proteostasis and longevity at low temperatures. Our findings highlight the crucial roles of lipid regulation in maintaining normal organismal physiology under different environmental conditions.

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

Endoplasmic reticulum (ER) stress is caused by the stress-induced accumulation of unfolded proteins in the ER. Here, we identified proteins and lipids that function downstream of the ER stress sensor INOSITOL-REQUIRING ENZYME1 (CrIRE1) that contributes to ER stress tolerance in Chlamydomonas (Chlamydomonas reinhardtii). Treatment with the ER stress inducer tunicamycin resulted in the splicing of a 32-nucleotide fragment of a basic leucine zipper 1 (bZIP1) transcription factor (CrbZIP1) mRNA by CrIRE1 that, in turn, resulted in the loss of the transmembrane domain in CrbZIP1, and the translocation of CrbZIP1 from the ER to the nucleus. Mutants deficient in CrbZIP1 failed to induce the expression of the unfolded protein response genes and grew poorly under ER stress. Levels of diacylglyceryltrimethylhomoserine (DGTS) and pinolenic acid (18:3Δ5,9,12) increased in the parental strains but decreased in the crbzip1 mutants under ER stress. A yeast one-hybrid assay revealed that CrbZIP1 activated the expression of enzymes catalyzing the biosynthesis of DGTS and pinolenic acid. Moreover, two lines harboring independent mutant alleles of Chlamydomonas desaturase (CrDES) failed to synthesize pinolenic acid and were more sensitive to ER stress than were their parental lines. Together, these results indicate that CrbZIP1 is a critical component of the ER stress response mediated by CrIRE1 in Chlamydomonas that acts via lipid remodeling.

Molecular Genetic Tools and Emerging Synthetic Biology Strategies to Increase Cellular Oil Content in Chlamydomonas reinhardtii.

Microalgae constitute a highly diverse group of eukaryotic and photosynthetic microorganisms that have developed extremely efficient systems for harvesting and transforming solar energy into energy-rich molecules such as lipids. Although microalgae are considered to be one of the most promising platforms for the sustainable production of liquid oil, the oil content of these organisms is naturally low, and algal oil production is currently not economically viable. Chlamydomonas reinhardtii (Chlamydomonas) is an established algal model due to its fast growth, high transformation efficiency, and well-understood physiology and to the availability of detailed genome information and versatile molecular tools for this organism. In this review, we summarize recent advances in the development of genetic manipulation tools for Chlamydomonas, from gene delivery methods to state-of-the-art genome-editing technologies and fluorescent dye-based high-throughput mutant screening approaches. Furthermore, we discuss practical strategies and toolkits that enhance transgene expression, such as choice of expression vector and background strain. We then provide examples of how advanced genetic tools have been used to increase oil content in Chlamydomonas. Collectively, the current literature indicates that microalgal oil content can be increased by overexpressing key enzymes that catalyze lipid biosynthesis, blocking lipid degradation, silencing metabolic pathways that compete with lipid biosynthesis and modulating redox state. The tools and knowledge generated through metabolic engineering studies should pave the way for developing a synthetic biological approach to enhance lipid productivity in microalgae.

Identification and functional study of the endoplasmic reticulum stress sensor IRE1 in Chlamydomonas reinhardtii.

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.

Identification of a Chlamydomonas plastidial 2-lysophosphatidic acid acyltransferase and its use to engineer microalgae with increased oil content.

Despite a strong interest in microalgal oil production, our understanding of the biosynthetic pathways that produce algal lipids and the genes involved in the biosynthetic processes remains incomplete. Here, we report that Chlamydomonas reinhardtii Cre09.g398289 encodes a plastid-targeted 2-lysophosphatidic acid acyltransferase (CrLPAAT1) that acylates the sn-2 position of a 2-lysophosphatidic acid to form phosphatidic acid, the first common precursor of membrane and storage lipids. In vitro enzyme assays showed that CrLPAAT1 prefers 16:0-CoA to 18:1-CoA as an acyl donor. Fluorescent protein-tagged CrLPAAT1 was localized to the plastid membrane in C. reinhardtii cells. Furthermore, expression of CrLPAAT1 in plastids led to a > 20% increase in oil content under nitrogen-deficient conditions. Taken together, these results demonstrate that CrLPAAT1 is an authentic plastid-targeted LPAAT in C. reinhardtii, and that it may be used as a molecular tool to genetically increase oil content in microalgae.

Plant ABC Transporters Enable Many Unique Aspects of a Terrestrial Plant's Lifestyle.

Terrestrial plants have two to four times more ATP-binding cassette (ABC) transporter genes than other organisms, including their ancestral microalgae. Recent studies found that plants harboring mutations in these transporters exhibit dramatic phenotypes, many of which are related to developmental processes and functions necessary for life on dry land. These results suggest that ABC transporters multiplied during evolution and assumed novel functions that allowed plants to adapt to terrestrial environmental conditions. Examining the literature on plant ABC transporters from this viewpoint led us to propose that diverse ABC transporters enabled many unique and essential aspects of a terrestrial plant's lifestyle, by transporting various compounds across specific membranes of the plant.