INHIBITION OF ASTAXANTHIN SYNTHESIS UNDER HIGH IRRADIANCE DOES NOT ABOLISH TRIACYLGLYCEROL ACCUMULATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE)1

Under stress conditions, Haematococcus pluvialis Flotow accumulates fatty acid–esterified astaxanthin, in extraplastidial lipid globules. The enhanced accumulation of fatty acids, mainly in triacylglycerols (TAG), among which oleic acid predominates, is linearly correlated with that of astaxanthin. We used inhibitors of either carotenoid or lipid biosynthesis to assess the interrelationship between carotenogenesis and TAG accumulation under high light irradiance as the stress factor. The two carotenogenesis inhibitors used—norflurazon, an inhibitor of phytoene desaturase, and diphenylamine (DPA), an inhibitor of β‐carotene C‐4 oxygenase—suppressed the accumulation of astaxanthin in a concentration‐dependent manner. Concurrently, the accumulation of neutral lipids was significantly less affected. The lipid biosynthesis inhibitor sethoxydim, which inhibits acetyl‐CoA carboxylase, significantly decreased de novo fatty acid synthesis and, in concert, drastically inhibited astaxanthin formation. In the presence of various concentrations of the three inhibitors, the inhibition of astaxanthin was not accompanied by a proportional decrease in oleic acid, which was used as a marker for TAG fatty acids. When astaxanthin synthesis was completely inhibited, the volumetric content of oleic acid was about 60% of the control value when the two carotenogenesis inhibitors (0.05 μM norflurazon or 20 μM DPA) were used and 27% of the control when the lipid‐synthesis inhibitor (50 μM) was used. We suggest therefore that TAG accumulation under high irradiance is not tightly coupled with astaxanthin accumulation, although the correlation between these two processes was demonstrated earlier. Furthermore, we propose that the accumulation of a certain amount of TAG is a prerequisite for the initiation of fatty acid–esterified astaxanthin accumulation in lipid globules.     

脱落酸对丛枝菌根真菌侵染和产孢的调控效应研究

【目的】揭示脱落酸(ABA)对丛枝菌根(AM)真菌侵染和产孢的影响,建立利用外源ABA促进孢子产量的高效菌剂扩繁方法。【方法】利用番茄毛状根和AM真菌Rhizophagus irregularis DAOM 197198建立双重培养体系,通过外源施用ABA、赤霉素(GA)或者使用ABA、GA的缺陷突变体,染色观察菌根侵染,荧光定量PCR测定丛枝发育和脂质合成运输相关基因的表达,统计丛枝和孢子的数量,从而揭示ABA对AM真菌侵染和产孢的影响。【结果】ABA缺陷突变体not中的F%(侵染频率)、a%(丛枝丰度)、丛枝数量,以及丛枝发育特异性相关基因EXO70A1-like (LOC101253481)、脂质合成运输相关基因RAM2和STR2的表达均显著低于其野生型MT;外源施用ABA显著促进了F%、M%(侵染强度)、丛枝数量、孢子产量,以及脂质合成运输相关基因RAM2和STR2的表达,外源添加ABA处理的孢子产量约为不添加处理的4.5倍;外源GA处理极显著抑制了菌根侵染的所有指标和孢子产量;GA缺陷突变体gib3与其野生型MM的AM真菌侵染之间没有显著差异,但gib3的孢子产量显著高于MM。【结论】ABA通过促进脂质的合成和运输,提高AM真菌的侵染和丛枝形成,进而增加AM真菌的孢子产量。     

Fungal lipid accumulation and development of mycelial structures by two arbuscular mycorrhizal fungi

We monitored the development of intraradical and extraradical mycelia of the arbuscular mycorrhizal (AM) fungi Scutellospora calospora and Glomus intraradices when colonizing Plantago lanceolata. The occurrence of arbuscules (branched hyphal structures) and vesicles (lipid storage organs) was compared with the amounts of signature fatty acids. The fatty acid 16:1omega5 was used as a signature for both AM fungal phospholipids (membrane constituents) and neutral lipids (energy storage) in roots (intraradical mycelium) and in soil (extraradical mycelium). The formation of arbuscules and the accumulation of AM fungal phospholipids in intraradical mycelium followed each other closely in both fungal species. In contrast, the neutral lipids of G. intraradices increased continuously in the intraradical mycelium, while vesicle occurrence decreased after initial rapid root colonization by the fungus. S. calospora does not form vesicles and accumulated more neutral lipids in extraradical than in intraradical mycelium, while the opposite pattern was found for G. intraradices. G. intraradices allocated more of its lipids to storage than did S. calospora. Thus, within a species, the fatty acid 16:1omega5 is a good indicator for AM fungal development. The phospholipid fatty acid 16:1omega5 is especially suitable for indicating the frequency of arbuscules in the symbiosis. We propose that the ratio of neutral lipids to phospholipids is more important than is the presence of vesicles in determining the storage status of AM fungi.     

Fungal Lipid Accumulation and Development of Mycelial Structures by Two Arbuscular Mycorrhizal Fungi

We monitored the development of intraradical and extraradical mycelia of the arbuscular mycorrhizal (AM) fungi Scutellospora calospora and Glomus intraradices when colonizing Plantago lanceolata. The occurrence of arbuscules (branched hyphal structures) and vesicles (lipid storage organs) was compared with the amounts of signature fatty acids. The fatty acid 16:1ω5 was used as a signature for both AM fungal phospholipids (membrane constituents) and neutral lipids (energy storage) in roots (intraradical mycelium) and in soil (extraradical mycelium). The formation of arbuscules and the accumulation of AM fungal phospholipids in intraradical mycelium followed each other closely in both fungal species. In contrast, the neutral lipids of G. intraradices increased continuously in the intraradical mycelium, while vesicle occurrence decreased after initial rapid root colonization by the fungus. S. calospora does not form vesicles and accumulated more neutral lipids in extraradical than in intraradical mycelium, while the opposite pattern was found for G. intraradices. G. intraradices allocated more of its lipids to storage than did S. calospora. Thus, within a species, the fatty acid 16:1ω5 is a good indicator for AM fungal development. The phospholipid fatty acid 16:1ω5 is especially suitable for indicating the frequency of arbuscules in the symbiosis. We propose that the ratio of neutral lipids to phospholipids is more important than is the presence of vesicles in determining the storage status of AM fungi.     

Deficit and excess of soil water impact on plant growth of Lotus tenuis by affecting nutrient uptake and arbuscular mycorrhizal symbiosis

The impact of deficit and excess of soil water on plant growth, morphological plant features, N and P plant nutrition, soil properties, Rhizobium nodulation and the symbiosis between arbuscular mycorrhizal (AM) fungi and Lotus tenuis Waldst. & Kit. were studied in a saline-sodic soil. Water excess treatment decreased root growth by 36% and increased shoot growth by 13% whereas water deficit treatment decreased both root and shoot growth (26 and 32%, respectively). Differences between stress conditions on shoot growth were due to the ability of L. tenuis to tolerate low oxygen concentration in the soil and the sufficiency of nutrients in soil to sustain shoot growth demands. Water excess treatment decreased pH, and increased available P and labile C in soil. Water deficit treatment decreased available P and also increased labile C. In general, N and P acquisition were affected more by water excess than water deficit. The number of nodules per gram of fresh roots only increased in water excess roots (97%). Under both stress conditions there was a significant proportion of roots colonized by AM fungi. Compared to control treatment, arbuscule formation decreased by 55 and 14% under water excess and water deficit, respectively. Vesicle formation increased 256% in water excess treatment and did not change under water deficit treatment. L. tenuis plants subjected to water deficit or excess treatments could grow, nodulated and maintained a symbiotic association with AM fungi by different strategies. Under water excess, L. tenuis plants decreased root growth and increased shoot growth to facilitate water elimination by transpiration. Under water deficit, L. tenuis plants decreased root growth but also shoot growth which in turn significant decreased the shoot/root ratio. In the present study, under water excess conditions AM fungi reduced nutrient transfer structures (arbuscules), the number of entry points and spore, and hyphal densities in soil, but increased resistance structures (vesicles). At water deficit, however, AM fungi reduced external hyphae and arbuscules to some extent, investing more in maintaining a similar proportion of vesicles in roots and spores in soil compared to control treatment.     

A purple acid phosphatase,GmPAP33, participates in arbuscule degeneration during arbuscular mycorrhizal symbiosis in soybean

Arbuscules are the central structures of the symbiotic association between terrestrial plants and arbuscular mycorrhizal (AM) fungi. However, arbuscules are also ephemeral structures, and following development, these structures are soon digested and ultimately disappear. Currently, little is known regarding the mechanism underlying the digestion of senescent arbuscules. Here, biochemical and functional analyses were integrated to test the hypothesis that a purple acid phosphatase, GmPAP33, controls the hydrolysis of phospholipids during arbuscule degeneration. The expression of GmPAP33 was enhanced by AM fungal inoculation independent of the P conditions in soybean roots. Promoter‐β‐glucuronidase (GUS) reporter assays revealed that the expression of GmPAP33 was mainly localized to arbuscule‐containing cells during symbiosis. The recombinant GmPAP33 exhibited high hydrolytic activity towards phospholipids, phosphatidylcholine, and phosphatidic acid. Furthermore, soybean plants overexpressing GmPAP33 exhibited increased percentages of large arbuscules and improved yield and P content compared with wild‐type plants when inoculated with AM fungi. Mycorrhizal RNAi plants had high phospholipid levels and a large percentage of small arbuscules. These results in combination with the subcellular localization of GmPAP33 at the plasma membrane indicate that GmPAP33 participates in arbuscule degeneration during AM symbiosis via involvement in phospholipid hydrolysis.     

Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development

Arbuscular mycorrhiza (AM) fungi form nutrient‐acquiring symbioses with the majority of higher plants. Nutrient exchange occurs via arbuscules, highly branched hyphal structures that are formed within root cortical cells. With a view to identifying host genes involved in AM development, we isolated Lotus japonicus AM‐defective mutants via a microscopic screen of an ethyl methanesulfonate‐mutagenized population. A standardized mapping procedure was developed that facilitated positioning of the defective loci on the genetic map of L. japonicus, and, in five cases, allowed identification of mutants of known symbiotic genes. Two additional mutants representing independent loci did not form mature arbuscules during symbiosis with two divergent AM fungal species, but exhibited signs of premature arbuscule arrest or senescence. Marker gene expression patterns indicated that the two mutants are affected in distinct steps of arbuscule development. Both mutants formed wild‐type‐like root nodules upon inoculation with Mesorhizobium loti, indicating that the mutated loci are essential during AM but not during root nodule symbiosis.     

Gibberellin regulates infection and colonization of host roots by arbuscular mycorrhizal fungi

Arbuscular mycorrhiza (AM) is established by the entry of AM fungi into the host plant roots and the formation of symbiotic structures called arbuscules. The host plant supplies photosynthetic products to the AM fungi, which in return provide phosphate and other minerals to the host through the arbuscules. Both partners gain great advantages from this symbiotic interaction, and both regulate AM development. Our recent work revealed that gibberellic acids (GAs) are required for AM development in the legume Lotus japonicus. GA signaling interact with symbiosis signaling pathways, directing AM fungal colonization in host roots. Expression analysis showed that genes for GA biosynthesis and metabolism were induced in host roots around AM fungal hyphae, suggesting that the GA signaling changes with both location and time during AM development. The fluctuating GA concentrations sometimes positively and sometimes negatively affect the expression of AM-induced genes that regulate AM fungal infection and colonization.     

The Lotus japonicus acyl‐acyl carrier protein thioesterase FatM is required for mycorrhiza formation and lipid accumulation of Rhizophagus irregularis

Arbuscular mycorrhiza (AM) fungi establish symbiotic interactions with plants, providing the host plant with minerals, i.e. phosphate, in exchange for organic carbon. Arbuscular mycorrhiza fungi of the order Glomerales produce vesicles which store lipids as an energy and carbon source. Acyl‐acyl carrier protein (ACP) thioesterases (Fat) are essential components of the plant plastid‐localized fatty acid synthase and determine the chain length of de novo synthesized fatty acids. In addition to the ubiquitous FatA and FatB thioesterases, AM‐competent plants contain an additional, AM‐specific, FatM gene. Here, we characterize FatM from Lotus japonicus by phenotypically analyzing fatm mutant lines and by studying the biochemical function of the recombinant FatM protein. Reduced shoot phosphate content in fatm indicates compromised symbiotic phosphate uptake due to reduced arbuscule branching, and the fungus shows reduced lipid accumulation accompanied by the occurrence of smaller and less frequent vesicles. Lipid profiling reveals a decrease in mycorrhiza‐specific phospholipid forms, AM fungal signature fatty acids (e.g. 16:1ω5, 18:1ω7 and 20:3) and storage lipids. Recombinant FatM shows preference for palmitoyl (16:0)‐ACP, indicating that large amounts of 16:0 fatty acid are exported from the plastids of arbuscule‐containing cells. Stable isotope labeling with [¹³C₂]acetate showed reduced incorporation into mycorrhiza‐specific fatty acids in the fatm mutant. Therefore, colonized cells reprogram plastidial de novo fatty acid synthesis towards the production of extra amounts of 16:0, which is in agreement with previous results that fatty acid‐containing lipids are transported from the plant to the fungus.     

Medicago truncatula mtpt4 mutants reveal a role for nitrogen in the regulation of arbuscule degeneration in arbuscular mycorrhizal symbiosis

Plants acquire essential mineral nutrients such as phosphorus (P) and nitrogen (N) directly from the soil, but the majority of the vascular plants also gain access to these mineral nutrients through endosymbiotic associations with arbuscular mycorrhizal (AM) fungi. In AM symbiosis, the fungi deliver P and N to the root through branched hyphae called arbuscules. Previously we identified MtPT4, a Medicago truncatula phosphate transporter located in the periarbuscular membrane that is essential for symbiotic phosphate transport and for maintenance of the symbiosis. In mtpt4 mutants arbuscule degeneration occurs prematurely and symbiosis fails. Here, we show that premature arbuscule degeneration occurs in mtpt4 mutants even when the fungus has access to carbon from a nurse plant. Thus, carbon limitation is unlikely to be the primary cause of fungal death. Surprisingly, premature arbuscule degeneration is suppressed if mtpt4 mutants are deprived of nitrogen. In mtpt4 mutants with a low N status, arbuscule lifespan does not differ from that of the wild type, colonization of the mtpt4 root system occurs as in the wild type and the fungus completes its life cycle. Sulphur is another essential macronutrient delivered to the plant by the AM fungus; however, suppression of premature arbuscule degeneration does not occur in sulphur-deprived mtpt4 plants. The mtpt4 arbuscule phenotype is strongly correlated with shoot N levels. Analyses of an mtpt4-2 sunn-1 double mutant indicates that SUNN, required for N-mediated autoregulation of nodulation, is not involved. Together, the data reveal an unexpected role for N in the regulation of arbuscule lifespan in AM symbiosis.     

The effect of cadmium on lipid and fatty acid biosynthesis in tomato leaves

This research aims to examine the effect of cadmium uptake on lipid composition and fatty acid biosynthesis, in young leaves of tomato treated seedlings (Lycopersicon esculentum cv. Ibiza F1). Results in membrane lipids investigations revealed that high cadmium concentrations affect the main lipid classes, leading to strong changes in their composition and fatty acid content. Thus, the exposure of tomato plants to cadmium caused a concentration-related decrease in the unsaturated fatty acid content, resulting in a lower degree of fatty acid unsaturation. The level of lipid peroxides was significantly enhanced at high Cd concentrations. Studies of the lipid metabolism using radioactive labelling with [1-¹⁴C]acetate as a major precursor of lipid biosynthesis, showed that levels of radioactivity incorporation in total lipids as well as in all lipid classes were lowered by Cd doses. In total lipid fatty acids, [1-¹⁴C]acetate incorporation was reduced in tri-unsaturated fatty acids (C16:3 and C18:3); While it was enhanced in the palmitic (C16:0), palmitoleic (C16:1), stearic (C18:0) and linoleic (C18:2) acids. [1-¹⁴C]acetate incorporation into C16:3 and C18:3 of galactolipids [monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG)] and some phospholipids [phosphatidylcholine (PC) and phosphatidylglycerol (PG)] was inhibited by Cd stress. Our results showed that in tomato plants, cadmium stress provoked an inhibition of polar lipid biosynthesis and reduced fatty acid desaturation process.     

Two Medicago truncatula Half-ABC Transporters Are Essential for Arbuscule Development in Arbuscular Mycorrhizal Symbiosis

In the symbiotic association of plants and arbuscular mycorrhizal (AM) fungi, the fungal symbiont resides in the root cortical cells where it delivers mineral nutrients to its plant host through branched hyphae called arbuscules. Here, we report a Medicago truncatula mutant, stunted arbuscule (str), in which arbuscule development is impaired and AM symbiosis fails. In contrast with legume symbiosis mutants reported previously, str shows a wild-type nodulation phenotype. STR was identified by positional cloning and encodes a half-size ATP binding cassette (ABC) transporter of a subfamily (ABCG) whose roles in plants are largely unknown. STR is a representative of a novel clade in the ABCG subfamily, and its orthologs are highly conserved throughout the vascular plants but absent from Arabidopsis thaliana. The STR clade is unusual in that it lacks the taxon-specific diversification that is typical of the ABCG gene family. This distinct phylogenetic profile enabled the identification of a second AM symbiosis-induced half-transporter, STR2. Silencing of STR2 by RNA interference results in a stunted arbuscule phenotype identical to that of str. STR and STR2 are coexpressed constitutively in the vascular tissue, and expression is induced in cortical cells containing arbuscules. STR heterodimerizes with STR2, and the resulting transporter is located in the peri-arbuscular membrane where its activity is required for arbuscule development and consequently a functional AM symbiosis.     

Arbuscules of vesicular-arbuscular mycorrhizal fungi inhabit an acidic compartment within plant roots

 The most widespread type of mycorrhiza is the so-called vesicular-arbuscular mycorrhiza. In this endomycorrhiza, fungal hyphae penetrate plant cell walls in the root cortex. There they form densely branched arbuscules. Fungus and plant plasma membrane are separated by a common interfacial apoplast. The pH of the compartment between the symbionts is of pivotal importance for nutrient transfer. Histochemical experiments were conducted to check for an acidic nature of the interface in the model system Glomus versiforme (Karst.) Berch-Allium porrum L. Two chemically different acidotropic dyes (neutral red and LysoSensor Green DND-189) stained the arbuscules intensely. The staining of arbuscules could be eliminated by addition of the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) or treatments leading to membrane rupture. Therefore, the staining of the arbuscules was based on the ion-trap mechanism, which indicates acidic, membrane-bound compartments. Microscopic examination of stained arbuscules at high optical resolution revealed a peripheral accumulation of the dye. Since plasmolysis rapidly destained the arbuscules, it is concluded that the dyes accumulate in the arbuscular interface, indicating the highly acidic nature of this compartment. The findings are discussed with respect to their relevance for the nutrient transfer in mycorrhizas. In addition, evidence for a discontinuity in the arbuscular interface between the stem and the branches of the arbuscule is given. Received: 15 September 1999 / Accepted: 20 February 2000     

植物激素调控丛枝菌根发育的作用机制研究进展

丛枝菌根（arbuscular mycorrhiza，AM）是土壤中AM真菌和绝大多数维管植物根系长期进化过程中相互识别、相互作用形成的互利共生体。AM的发育与功能效应依赖AM真菌-寄主植物之间精准的“分子对话”，同时受到环境条件特别是土壤养分水平、干旱和盐渍化的制约。植物激素作为低浓度的小分子有机物，是参与调控AM共生过程的重要信号分子。其中，主要有9种植物激素参与AM发育过程且分工各有不同：独脚金内酯（strigolactones，SLs）参与AM真菌-寄主植物之间最初的共生识别，脱落酸（abscisic acid，ABA）和油菜素内酯（brassinosteroid，BR）促进前期的菌丝入侵，但水杨酸（salicylic acid，SA）和乙烯（ethylene，ET）抑制前期的菌丝入侵，生长素（auxin，Aux）、ABA和BR促进随后的丛枝形成而ET和赤霉素（gibberellin，GA）的作用则相反，茉莉酸（jasmonic acid，JA）对菌丝入侵与丛枝形成均可能存在正调控或负调控作用。目前细胞分裂素（cytokinin，CTK）在AM发育中的作用尚不明确。更为复杂的是，通常植物激素信号之间的交叉互作决定AM的发育进程。本文针对AM发育过程总结了不同植物激素的调控作用特点和不同植物激素信号之间的互作（协同或拮抗），以及胁迫条件下不同植物激素信号的可能调控机制。深入研究和系统阐明植物激素调控AM真菌-寄主植物共生的生理/分子机制，将有助于促进生物共生学理论研究及菌根技术的应用。     

A theoretical study of lipid accumulation in the liver—implications for nonalcoholic fatty liver disease

A hallmark of the nonalcoholic fatty liver disease is the accumulation of lipids. We developed a mathematical model of the hepatic lipid dynamics to simulate the fate of fatty acids in hepatocytes. Our model involves fatty acid uptake, lipid oxidation, and lipid export. It takes into account that storage of triacylglycerol within hepatocytes leads to cell enlargement reducing the sinusoids radius and impairing hepatic microcirculation. Thus oxygen supply is reduced, which impairs lipid oxidation. The analysis of our model revealed a bistable behavior (two stable steady states) of the system, in agreement with histological observations showing distinct areas of lipid accumulation in lobules. The first (healthy) state is characterized by intact lipid oxidation and a low amount of stored lipids. The second state in our model may correspond to the steatotic cell; it is marked by a high amount of stored lipids and a reduced lipid oxidation caused by impaired oxygen supply. Our model stresses the role of insufficient oxygen supply for the development of steatosis. We discuss implications of our results in regard to the experimental design aimed at exploring lipid metabolism reactions under steatotic conditions. Moreover, the model helps to understand the reversibility of lipid accumulation and predicts the reversible switch to show hysteresis. The system can switch from the steatotic state back to the healthy state by reduction of fatty acid uptake below the threshold at which steatosis started. The reversibility corresponds to the observation that caloric restriction can reduce the lipid content in the liver.     

Medicago truncatula Vapyrin is a novel protein required for arbuscular mycorrhizal symbiosis

Arbuscular mycorrhizal (AM) symbiosis is a widespread mutualism formed between vascular plants and fungi of the Glomeromycota. In this endosymbiosis, fungal hyphae enter the roots, growing through epidermal cells to the cortex where they establish differentiated hyphae called arbuscules in the cortical cells. Reprogramming of the plant epidermal and cortical cells occurs to enable intracellular growth of the fungal symbiont; however, the plant genes underlying this process are largely unknown. Here, through the use of RNAi, we demonstrate that the expression of a Medicago truncatula gene named Vapyrin is essential for arbuscule formation, and also for efficient epidermal penetration by AM fungi. Vapyrin is induced transiently in the epidermis coincident with hyphal penetration, and then in the cortex during arbuscule formation. The Vapyrin protein is cytoplasmic, and in cells containing AM fungal hyphae, the protein accumulates in small puncta that move through the cytoplasm. Vapyrin is a novel protein composed of two domains that mediate protein–protein interactions: an N‐terminal VAMP‐associated protein (VAP)/major sperm protein (MSP) domain and a C‐terminal ankyrin‐repeat domain. Putative Vapyrin orthologs exist widely in the plant kingdom, but not in Arabidopsis, or in non‐plant species. The data suggest a role for Vapyrin in cellular remodeling to support the intracellular development of fungal hyphae during AM symbiosis.     

Effect of pH on neutral lipid and biomass accumulation in microalgal strains native to the Canadian prairies and the Athabasca oil sands

Algal biodiesel has been a subject of growing importance in the realm of renewable energy due to carbon capture properties and its potential for photosynthetic efficiency with high lipid output. This study identified five isolates of freshwater green algae, belonging to the Chlorellaceae, and measured the lipid classes and fatty acid profiles of these species to determine suitability for biodiesel production. To induce the greater accumulation of lipids, especially in the form of triacylglycerols (TAGs) desired for biodiesel, we examined the lipid accumulation in cells stressed by nitrogen limitation, sulfur deficiency, or pH stress. Increases in biomass were monitored in order to determine if adjusting pH incrementally over the course of the experiment had any effect on growth and lipid accumulation of several isolates. TAG accumulation was visually screened by Nile Red fluorescence and further assessed by gas chromatography. Lipid amounts were comparably equal or better for pH stress treatments than for standard nutrient-deprivation treatments. Incrementally adjusted pH over the course of growth triggered lipid accumulation comparable to constant pH stress treatments, yet biomass accumulation was equivalent to unstressed growth. One isolate obtained from the Athabasca oil-sands region of Alberta, OS4-2, is a good candidate for biodiesel production, having accumulated over 45 % of its dry weight as lipid, with over 80 % of the lipid as triacylglycerols, and contains an abundance of 18:1 fatty acids. This class of fatty acids improves the cold flow and oxidative stability of biodiesel and is ideal for biofuel used in a Canadian climate.