Yasuyo Yamaoka

AtABCA9 transporter supplies fatty acids for lipid synthesis to the endoplasmic reticulum

Fatty acids, the building blocks of biological lipids, are synthesized in plastids and then transported to the endoplasmic reticulum (ER) for assimilation into specific lipid classes. The mechanism of fatty acid transport from plastids to the ER has not been identified. Here we report that AtABCA9, an ABC transporter in Arabidopsis thaliana, mediates this transport. AtABCA9 was localized to the ER, and atabca9 null mutations reduced seed triacylglycerol (TAG) content by 35% compared with WT. Developing atabca9 seeds incorporated 35% less 14C-oleoyl-CoA into TAG compared with WT seeds. Furthermore, overexpression of AtABCA9 enhanced TAG deposition by up to 40%. These data strongly support a role for AtABCA9 as a supplier of fatty acid substrates for TAG biosynthesis at the ER during the seed-filling stage. AtABCA9 may be a powerful tool for increasing lipid production in oilseeds.

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 plants lifestyle, by transporting various compounds across specific membranes of the plant.

Rapid induction of lipid droplets in Chlamydomonas reinhardtii and Chlorella vulgaris by Brefeldin A.

Algal lipids are the focus of intensive research because they are potential sources of biodiesel. However, most algae produce neutral lipids only under stress conditions. Here, we report that treatment with Brefeldin A (BFA), a chemical inducer of ER stress, rapidly triggers lipid droplet (LD) formation in two different microalgal species, Chlamydomonas reinhardtii and Chlorella vulgaris. LD staining using Nile red revealed that BFA-treated algal cells exhibited many more fluorescent bodies than control cells. Lipid analyses based on thin layer chromatography and gas chromatography revealed that the additional lipids formed upon BFA treatment were mainly triacylglycerols (TAGs). The increase in TAG accumulation was accompanied by a decrease in the betaine lipid diacylglyceryl N,N,N-trimethylhomoserine (DGTS), a major component of the extraplastidic membrane lipids in Chlamydomonas, suggesting that at least some of the TAGs were assembled from the degradation products of membrane lipids. Interestingly, BFA induced TAG accumulation in the Chlamydomonas cells regardless of the presence or absence of an acetate or nitrogen source in the medium. This effect of BFA in Chlamydomonas cells seems to be due to BFA-induced ER stress, as supported by the induction of three homologs of ER stress marker genes by the drug. Together, these results suggest that ER stress rapidly triggers TAG accumulation in two green microalgae, C. reinhardtii and C. vulgaris. A further investigation of the link between ER stress and TAG synthesis may yield an efficient means of producing biofuel from algae.

The Role of Arabidopsis ABCG9 and ABCG31 ATP Binding Cassette Transporters in Pollen Fitness and the Deposition of Steryl Glycosides on the Pollen Coat

This work identified two ABC transporters important for normal pollen coat deposition and, thus, critical for pollen fitness. The transporters are probably involved in the transfer of pollen coat material from maternal tissues to the pollen surface. The pollen coat protects pollen grains from harmful environmental stresses such as drought and cold. Many compounds in the pollen coat are synthesized in the tapetum. However, the pathway by which they are transferred to the pollen surface remains obscure. We found that two Arabidopsis thaliana ATP binding cassette transporters, ABCG9 and ABCG31, were highly expressed in the tapetum and are involved in pollen coat deposition. Upon exposure to dry air, many abcg9 abcg31 pollen grains shriveled up and collapsed, and this phenotype was restored by complementation with ABCG9pro:GFP:ABCG9. GFP-tagged ABCG9 or ABCG31 localized to the plasma membrane. Electron microscopy revealed that the mutant pollen coat resembled the immature coat of the wild type, which contained many electron-lucent structures. Steryl glycosides were reduced to about half of wild-type levels in the abcg9 abcg31 pollen, but no differences in free sterols or steryl esters were observed. A mutant deficient in steryl glycoside biosynthesis, ugt80A2 ugt80B1, exhibited a similar phenotype. Together, these results indicate that steryl glycosides are critical for pollen fitness, by supporting pollen coat maturation, and that ABCG9 and ABCG31 contribute to the accumulation of this sterol on the surface of pollen.

SREBP and MDT-15 protect C. elegans from glucose-induced accelerated aging by preventing accumulation of saturated fat

Glucose-rich diets shorten the life spans of various organisms. However, the metabolic processes involved in this phenomenon remain unknown. Here, we show that sterol regulatory element-binding protein (SREBP) and mediator-15 (MDT-15) prevent the life-shortening effects of a glucose-rich diet by regulating fat-converting processes in Caenorhabditis elegans. Up-regulation of the SREBP/MDT-15 transcription factor complex was necessary and sufficient for alleviating the life-shortening effect of a glucose-rich diet. Glucose feeding induced key enzymes that convert saturated fatty acids (SFAs) to unsaturated fatty acids (UFAs), which are regulated by SREBP and MDT-15. Furthermore, SREBP/MDT-15 reduced the levels of SFAs and moderated glucose toxicity on life span. Our study may help to develop strategies against elevated blood glucose and free fatty acids, which cause glucolipotoxicity in diabetic patients.

Isozyme-Specific Modes of Activation of CTP:Phosphorylcholine Cytidylyltransferase in Arabidopsis thaliana at Low Temperature

Arabidopsis thaliana increases cellular phosphatidylcholine (PC) content during cold acclimation by up-regulating PC biosynthesis. The A. thaliana genes CCT1 and CCT2 encode CTP:phosphorylcholine cytidylyltransferases (CCTs; EC 2.7.7.15), which regulate PC biosynthesis via the CDP-choline pathway. We isolated the T-DNA-tagged knockout mutants cct1 and cct2 of A. thaliana (Wassilevskaja; WS). CCT activity in cct1 and cct2 plants accounted for 29 and 79% to the cellular CCT activity of WS plants, respectively. When plants were exposed to 2 degrees C for 7 d, CCT activity increased in both cct1 and cct2 plants, and immunoblot analyses revealed that cct1 contained an increased level of CCT2 protein whereas cct2 exhibited little increase in CCT1 level. For each mutant grown at 23 degrees C, CCT activity was mainly enriched in the particulate (15,000 x g pellet) and microsomal (150,000 x g pellet) fractions from rosette leaf homogenates. After exposure to cold, the particulate and microsomal fractions of cct1 plants had higher total CCT activity due to increased levels of CCT2; in contrast, the levels of CCT1 in cct2 plants remained unchanged in particulate and microsomal fractions despite a significant increase in the total CCT activity. We conclude that the CDP-choline pathway of A. thaliana is up-regulated at low temperature via isogene-specific modes: enhanced expression of CCT2 and post-translational activation/inactivation of CCT1 in membranes. PC levels were similarly maintained in both mutants and WS plants after 14 d at 2 degrees C, suggesting that either of the CCT genes is sufficient for PC biosynthesis at low temperature.

The small molecule fenpropimorph rapidly converts chloroplast membrane lipids to triacylglycerols in Chlamydomonas reinhardtii

Concern about global warming has prompted an intense interest in developing economical methods of producing biofuels. Microalgae provide a promising platform for biofuel production, because they accumulate high levels of lipids, and do not compete with food or feed sources. However, current methods of producing algal oil involve subjecting the microalgae to stress conditions, such as nitrogen deprivation, and are prohibitively expensive. Here, we report that the fungicide fenpropimorph rapidly causes high levels of neutral lipids to accumulate in Chlamydomonas reinhardtii cells. When treated with fenpropimorph (10 μg mL-1) for 1 h, Chlamydomonas cells accumulated at least fourfold the amount of triacylglycerols (TAGs) present in the untreated control cells. Furthermore, the quantity of TAGs present after 1 h of fenpropimorph treatment was over twofold higher than that formed after 9 days of nitrogen starvation in medium with no acetate supplement. Biochemical analysis of lipids revealed that the accumulated TAGs were derived mainly from chloroplast polar membrane lipids. Such a conversion of chloroplast polar lipids to TAGs is desirable for biodiesel production, because polar lipids are usually removed during the biodiesel production process. Thus, our data exemplified that a cost and time effective method of producing TAGs is possible using fenpropimorph or similar drugs.

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

Summary 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.

Characterization of a Chlamydomonas reinhardtii Mutant Defective in a Maltose Transporter

Microalgae are potential sources of energy and high-value materials. To decipher the process of energy metabolism in green algae, we created a mutant pool of strain CC-503 of the model green microalga Chlamydomonas reinhardtii, by random insertion of an antibiotic resistance gene, and screened the pool for lines with altered carbon metabolism. We identified a mutant that harbored the antibiotic resistance gene in CrMEX1, a putative Maltose Exporter-Like protein 1 (Cre12.g486600.t1.2). The mutant had reduced levels of CrMEX1 expression and, similarly to the Arabidopsis mex1 knockout mutant, which cannot export maltose from the chloroplast, it over-accumulated starch granules in the chloroplast. The mutant’s lipid levels were slightly higher than those of the wild type, and its initial growth kinetics were not significantly different from those of the wild type, but the mutant culture did not reach the same high cell density as the wild type in acetate-containing culture medium under continuous light. These results suggest that CrMEX1 encodes a maltose transporter protein, and that export of photoassimilates from chloroplasts is necessary for normal Chlamydomonas growth, even under continuous light with an ample supply of carbon in the form of acetate.

Epigenetic floral homeotic mutation in pD991-AP3-derived T-DNA-tagged lines for CTP:Phosphorylcholine cytidylyltransferase (CCT) Genes: The homeotic mutation of the cct1-1 allele is enhanced by the cct2 allele and alleviated by CCT1 overexpression

The apetala3 (ap3)-like homeotic mutation (ap3-HM) is recognized among pD991-AP3-derived Arabidopsis thaliana T-DNA-tagged lines carrying the -448 to +47 region of AP3 in their T-DNA. In the corresponding mutant lines for CTP:phosphorylcholine cytidylyltransferase genes, cct1-1 and cct2 (Inatsugi et al. 2009), some flowers of cct1-1 (F4) and many flowers of cct1-1 cct2 (F3) showed ap3-HM, and all flowers of cct1-1 (F5) and cct1-1 cct2 (F4) became increasingly homeotic. In contrast, cct2 flowers were normal for all generations tested. These results demonstrated that ap3-HM is linked to the cct1-1 allele and is enhanced by the cct2 allele. The ap3-HM in cct mutants was inversely correlated with AP3 transcript levels in enriched flower buds. Bisulfite sequencing revealed severe methylation within endogenous AP3 promoter regions in cct1-1 (F3; -317 to -2) and cct1-1 cct2 (F3; -473 to -2), but wild-type (Wassilevskaja) and cct2 plants showed no corresponding methylation. The ap3-HM in cct1-1 cct2 mutants was fully rescued by expressing a PISTILLATA promoter–AP3 construct, and was better alleviated in the F1 offspring of a cross with the CCT1-overexpressing mutant cct1-2 (Columbia) than with the wild type. We discuss possible links between expression of CCT and suppression of ap3-HM.