Starchless Mutant Research Articles

Comparison of the effects of nitrogen-, sulfur- and combined nitrogen- and sulfur-deprivations on cell growth, lipid bodies and gene expressions in Chlamydomonas reinhardtii cc5373-sta6

BackgroundBiofuel research that aims to optimize growth conditions in microalgae is critically important. Chlamydomonas reinhardtii is a green microalga that offers advantages for biofuel production research. This study compares the effects of nitrogen-, sulfur-, and nitrogen and sulfur- deprivations on the C. reinhardtii starchless mutant cc5373-sta6. Specifically, it compares growth, lipid body accumulation, and expression levels of acetyl-CoA carboxylase (ACC) and phosphoenolpyruvate carboxylase (PEPC).ResultsAmong nutrient-deprived cells, TAP-S cells showed significantly higher total chlorophyll, cell density, and protein content at day 6 (p < 0.05). Confocal analysis showed a significantly higher number of lipid bodies in cells subjected to nutrient deprivation than in the control over the course of six days; N deprivation for six days significantly increased the size of lipid bodies (p < 0.01). In comparison with the control, significantly higher ACC expression was observed after 8 and 24 h of NS deprivation and only after 24 h with N deprivation. On the other hand, ACC and PEPC expression at 8 and 24 h of S deprivation was not significantly different from that in the control. A significantly lower PEPC expression was observed after 8 h of N and NS deprivation (p < 0.01), but a significantly higher PEPC expression was observed after 24 h (p < 0.01).ConclusionsBased on our findings, it would be optimum to cultivate cc5373-sta6 cells in nutrient deprived conditions (-N, -S or –NS) for four days; whereby there is cell growth, and both a high number of lipid bodies and a larger size of lipid bodies produced.

Flocculation of oleaginous green algae with Mortierella alpina fungi

Microalgae are promising sources of valuable bioproducts such as biofuels, food, and nutraceuticals. However, harvesting microalgae is challenging due to their small size and low biomass concentrations. To address this challenge, bio-flocculation of starchless mutants of Chlamydomonas reinhardtii (sta6/sta7) was investigated with Mortierella alpina, an oleaginous fungus with high concentrations of arachidonic acid (ARA). Triacylglycerides (TAG) reached 85 % of total lipids in sta6 and sta7 through a nitrogen regime. Scanning electron microscopy determined cell-wall attachment and extra polymeric substances (EPS) to be responsible for flocculation. An algal-fungal biomass ratio around 1:1 (three membranes) was optimal for bio-flocculation (80–85 % flocculation efficiency in 24 h). Nitrogen-deprived sta6/sta7 were flocculated with strains of M. alpina (NVP17b, NVP47, and NVP153) with aggregates exhibiting fatty acid profiles similar to C. reinhardtii, with ARA (3–10 % of total fatty acids). This study showcases M. alpina as a strong bio-flocculation candidate for microalgae and advances a mechanistic understanding of algal-fungal interaction.

Species variation in the hydrogen isotope composition of leaf cellulose is mostly driven by isotopic variation in leaf sucrose.

Experimental approaches to isolate drivers of variation in the carbon-bound hydrogen isotope composition (δ2 H) of plant cellulose are rare and current models are limited in their application. This is in part due to a lack in understanding of how 2 H-fractionations in carbohydrates differ between species. We analysed, for the first time, the δ2 H of leaf sucrose along with the δ2 H and δ18 O of leaf cellulose and leaf and xylem water across seven herbaceous species and a starchless mutant of tobacco. The δ2 H of sucrose explained 66% of the δ2 H variation in cellulose (R2 = 0.66), which was associated with species differences in the 2 H enrichment of sucrose above leaf water ( : -126% to -192‰) rather than by variation in leaf water δ2 H itself. was positively related to dark respiration (R2 = 0.27), and isotopic exchange of hydrogen in sugars was positively related to the turnover time of carbohydrates (R2 = 0.38), but only when was fixed to the literature accepted value of ‰. No relation was found between isotopic exchange of hydrogen and oxygen, suggesting large differences in the processes shaping post-photosynthetic fractionation between elements. Our results strongly advocate that for robust applications of the leaf cellulose hydrogen isotope model, parameterization utilizing δ2 H of sugars is needed.

Differences in Glycerolipid Response of Chlamydomonas reinhardtii Starchless Mutant to High Light and Nitrogen Deprivation Stress Under Three Carbon Supply Regimes.

Carbon source serves as a crucial factor for microalgal lipid biosynthesis. The supplied exogenous inorganic or organic carbon affects lipid accumulation in microalgae under stress conditions. However, the impacts of different carbon availability on glycerolipid metabolism, triacylglycerol (TAG) metabolism in particular, still remain elusive in microalgae. Chlamydomonas starchless mutant BAFJ5 has emerged as a model system to study TAG metabolism, due to its property of hyper-accumulating TAG. In this study, the glycerolipidomic response of the starchless BAFJ5 to high light and nitrogen-deprived (HL-N) stress was deciphered in detail to distinguish glycerolipid metabolism under three carbon supply regimes. The results revealed that the autotrophically and mixotrophically grown BAFJ5 cells aerated with air containing 2% CO2 presented similar changes in growth, photosynthetic activity, biochemical components, and glycerolipid metabolism under HL-N conditions. But the mixotrophically grown BAFJ5 aerated with air containing 0.04% CO2 exhibited more superior accumulation in TAG, which was esterified with a significantly higher proportion of C18:1n9 and prominently the lower proportions of polyunsaturated fatty acids. In addition, these cells increased the relative levels of C18:2n6 in the membrane lipids, i.e., monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), in priority, and decreased that of C18:3n3 and C18:4n3 in the betaine lipid, N,N,N-trimethylhomoserine diacylglycerol (DGTS), subsequently, to adapt to the HL-N stress conditions, compared to the cells under the other two conditions. Thus, it was suggested that C. reinhardtii starchless mutant appeared to present distinct metabolism for TAG biosynthesis involving membrane lipid remodeling under distinct carbon supply regimes. This study provides insights into how the different carbon supply regimes affect lipid metabolism in Chlamydomonas starchless cells, which will benefit the optimized production of storage lipids in microalgae.

The disassembly of lipid droplets in Chlamydomonas.

Lipid droplets (LDs) are ubiquitous and specialized organelles in eukaryotic cells. Consisting of a triacylglycerol core surrounded by a monolayer of membrane lipids, LDs are decorated with proteins and have myriad functions, from carbon/energy storage to membrane lipid remodeling and signal transduction. The biogenesis and turnover of LDs are therefore tightly coordinated with cellular metabolic needs in a fluctuating environment. Lipid droplet turnover requires remodeling of the protein coat, lipolysis, autophagy and fatty acid β-oxidation. Several key components of these processes have been identified in Chlamydomonas (Chlamydomonas reinhardtii), including the major lipid droplet protein, a CXC-domain containing regulatory protein, the phosphatidylethanolamine-binding DTH1 (DELAYED IN TAG HYDROLYSIS1), two lipases and two enzymes involved in fatty acid β-oxidation. Here, we review LD turnover and discuss its physiological significance in Chlamydomonas, a major model green microalga in research on algal oil.

Evaluation of diurnal responses of Tetradesmus obliquus under nitrogen limitation

Tetradesmus obliquus is an oleaginous microalga with high potential for triacylglycerol production. We characterized the biochemical composition and the transcriptional landscape of T. obliquus wild-type and the starchless mutant (slm1), adapted to 16:8 h light dark (LD) cycles under nitrogen limitation. In comparison to the nitrogen replete conditions, the diurnal RNA samples from both strains also displayed a cyclic pattern, but with much less variation which could be related to a reduced transcription activity in at least the usually highly active processes. During nitrogen limitation, the wild-type continued to use starch as the preferred storage compound to store energy and carbon. Starch was accumulated to an average content of 0.25 g·gDW−1, which is higher than the maximum observed under nitrogen replete conditions. Small oscillations were observed, indicating that starch was being used as a diurnal energy storage compound, but to a lesser extent than under nitrogen replete conditions. For the slm1 mutant, TAG content was higher than for the wild-type (average steady state value was 0.26 g·gDW−1 for slm1 compared to 0.06 g·gDW−1 for the wild-type). Despite the higher TAG content in the slm1, the conversion efficiency of photons into biomass components for the slm1 was only half of the one obtained for the wild-type. This is related to the observed decrease in biomass productivity (from 1.29 gDW·L−1·day−1 for the wild-type to 0.52 gDW·L−1·day−1 for the slm1). While the transcriptome of slm1 displayed clear signs of energy generation by degrading TAG and amino-acids during the dark period, no significant variation of these metabolites could be measured. When looking through the diurnal cycle, the photosynthetic efficiency was lower for the slm1 mutant compared to the wild-type especially during the second half of the light period, where starch accumulation occurred in the wild-type.

Galactolipid DGDG and Betaine Lipid DGTS Direct De Novo Synthesized Linolenate into Triacylglycerol in a Stress-Induced Starchless Mutant of Chlamydomonas reinhardtii.

The increasing demand for triacylglycerol (TAG) enriching polyunsaturated fatty acids (PUFAs) has led to a surge of interest in microalgal TAG metabolism. Polar membrane lipids serve as the desaturation carrier for PUFA, and the functional group of PUFA can be incorporated into TAG. Monogalactoglycerolipid has been found to provide the de novo synthesized oleate acyl group or the nascent polyunsaturated diacylglycerol backbone for TAG biosynthesis in the model green alga, Chlamydomonas reinhardtii. However, whether other membrane lipids take part in the formation of PUFA-attached TAG has not been clearly discovered. A time course study of glycerolipidomics in the starchless mutant of C. reinhardtii, BAFJ5, which hyper-accumulates TAG, revealed that digalactosyldiacylglycerol (DGDG) and diacylglycerol-N,N,N-trimethylhomoserine (DGTS) turned into the main components of membrane lipids, accounting for 62% of the total polar lipids, under nitrogen deprivation combined with high light conditions. In addition, the membrane lipid molecules DGDG 18:3n3/16:0 and DGTS 16:0/18:3n6 were presumed to be involved in the consecutive integration of the de novo synthesized linolenates into TAG. Based on the stoichiometry calculation, DGDG and DGTS were demonstrated to provide a major contribution to the accumulation of linolenate-attached TAG. Our study gives insights into the potential PUFA-attached TAG formation pathway mediated by the turnover of de novo synthesized DGDG and DGTS in the starchless mutant of Chlamydomonas.

Melatonin facilitates the coordination of cell growth and lipid accumulation in nitrogen-stressed Chlamydomonas reinhardtii for biodiesel production

In microalgae, the lipid accumulation under nitrogen stress is always accompanied by low photosynthetic activity and low growth rate, which adversely affects the total biomass and the lipid productivity. It is highly desirable to develop a practicable strategy for promoting lipid accumulation meanwhile minimizing the adverse effects under nitrogen stress. In this research, we investigate whether melatonin (MT, an antioxidant that protects against abiotic stress in plant cells) can mitigate the oxidative stress and enhance the lipid production in Chlamydomonas reinhardtii starchless mutants under nitrogen stress. The results of physiological and biochemical analysis indicate that exogenous MT weakens the nitrogen stress-induced oxidative damage by delaying the chlorophyll loss, activating antioxidases, reducing the lipid peroxidation. In addition, MT stimulates the biosynthesis of functional membrane lipids (monogalactosyldiacylglycerol, digalactosyldiacylglycerol and diacylglyceryl-trimethylhomoserine) and polyunsaturated fatty acids, increasing the cell response to nitrogen stress and enhancing the whole lipid biosynthesis. The 5 μM MT induces the increase of the total fatty acid methyl ester-lipid content by 35.4% (from 49.2% to 66.7% of dry weight), the biomass by 7.4% (to 0.92 g L−1), and the lipid productivity by 42.7% compared with the control groups without MT treatment. Moreover, we investigated the biodiesel properties of the lipids derived from MT-treated nitrogen-stressed cells, and the results suggest that the variation in fatty acid proportion induced by MT has little impact on the lipid's biodiesel properties. Thus, exogenous MT can facilitate the accumulation of lipids by enhancing the cellular antioxidant capability and mitigating the adverse effects of nitrogen stress.

Unraveling enhanced membrane lipid biosynthesis in Chlamydomonas reinhardtii starchless mutant sta6 by using an electrospray ionization mass spectrometry-based lipidomics method

The unicellular green alga Chlamydomonas reinhardtii, a well-established model organism, has been widely used in dissecting glycerolipid metabolism in oxygenating photosynthetic organisms. In previous studies, it has been found that shunting carbon precursors from the starch synthesis pathway can lead to a 10-fold increase in TAG content as compared to the wild type, but it is unknown whether inactivation of AGPase may affect membrane lipids biosynthesis. The study aims to investigate global changes in lipid metabolism and homeostasis in the starchless mutant C. reinhardtii sta6. By utilizing an electrospray ionization/mass spectrometry (ESI/MS)-based lipidomics approach, a total of 105 membrane lipid molecules of C. reinhardtii were resolved, including 16 monogalactosyldiacylglycerol (MGDG), 16 digalactosyldiacylglycerol (DGDG), 11 phosphatidylglycerol (PG), 6 sulfoquinovosyldiacylglycerol (SQDG), 49 diacylglyceryl-N,N,N-trimethylhomoserine (DGTS), 2 phosphatidylethanolamine (PE), and 5 phosphatidylinositol (PI) molecules. The quantitative results indicated that the membrane lipid profiles were similar between the two C. reinhardtii strains grown under both low- and high-light conditions, but the cellular contents of a great number of lipids were altered in sta6 due to the defect in starch biosynthesis. Under low-light conditions, sta6 accumulated more PI, MGDG, DGDG but less amounts of DGTS as compared to WT. Under high light, sta6 cells contained higher content membrane lipids than cc-124, except for PG, which is more or less similar in both strains. Our results demonstrate that the cellular membrane lipid homeostasis underwent profound changes in the starchless mutant, and thereby its physiological impact remains to be explored.

Metabolic plasticity of the starchless mutant of Chlorella sorokiniana and mechanisms underlying its enhanced lipid production revealed by comparative metabolomics analysis

The unicellular green alga Chlorella sorokiniana is of great industrial interest for biofuels production due to its rapid growth, high lipid content, and capabilities in tolerating broad environment and cultivation conditions. C. sorokiniana can grow under both heterotrophic and photoautotrophic conditions, which offers opportunities to produce lipids from this species/strain by employing coupled heterotrophic and phototrophic cultivation mode. In this study, a starchless mutant SLM2 was successfully obtained by high-throughput screening of the chemical mutagenesis pool of a C. sorokiniana strain that has the potential to achieve high cell density under heterotrophic conditions, aiming to enhance its lipid content and productivity. Unexpectedly, enhanced lipid production was only observed in photoautrophically-grown SLM2 cells subjected to stress conditions (i.e. high-light and nutrient limitation). When the heterotrophically-grown cells were subjected to the same stress conditions that can trigger lipid accumulation, less differences in lipid productivities were observed between wild type (WT) and SLM2. To understand metabolic basis underlying such a discrepancy, comparative time-course metabolomics analysis was performed for SLM2 and WT from different cultivation conditions. The results highlighted up-regulated metabolic flux related to the increased lipid biosynthesis in the phototrophically-grown starchless mutant cells, which included enhanced oxidative pentose phosphate pathway in a mode favoring NADPH production, and the augmented glycolysis pathway providing precursors for both fatty acids and glycerol backbone synthesis. In SLM2, the classical TCA cycle is down-regulated, while its bypass the gamma-aminobutyric acid (GABA) shunt is triggered, which is speculated to contribute to enhanced lipid accumulation in the starchless mutant as well. In addition to revealing the metabolic plasticity possessed by starchless mutant, it is suggested that photoautotrophic cultivation mode is more suitable than coupled heterotrophic and photoautotrophic cultivation mode for production of lipids by using the starchless mutant SLM2 obtained in this study.

Diversion of Carbon Flux from Sugars to Lipids Improves the Growth of an Arabidopsis Starchless Mutant.

Inactivation of ADP-glucose pyrophosphorylase1 (ADG1) causes a starchless phenotype in Arabidopsis. Mutants defective in ADG1 show severe growth retardation in day/night conditions but exhibit similar growth to wild type under continuous light, implying that starch plays an important role in supporting respiration, metabolism and growth at night. In addition to carbohydrates, lipids and proteins can serve as alternative respiratory substrates for the energy production in mature plants. To test the role of lipids in plant growth, we generated transgenic plants overexpressing phospholipid:diacylglycerol acyltransferase1 (PDAT1) in adg1. We found that PDAT1 overexpression caused an increase in both fatty acid synthesis and turnover and increased the accumulation of triacylglycerol (TAG) at the expense of sugars, and enhanced the growth of adg1. We demonstrated that unlike sugars, which were metabolized within a few hours of darkness, TAG breakdown was slow, occurring throughout the entire dark period. The slow pace of TAG hydrolysis provided a sustained supply of fatty acids for energy production, thereby alleviating energy deficiency at night and thereby improving the growth of the starchless mutants. We conclude that lipids can contribute to plant growth by providing a constant supply of fatty acids as an alternative energy source in mature starchless mutant plants.

Impairment of starch biosynthesis results in elevated oxidative stress and autophagy activity in Chlamydomonas reinhardtii

Autophagy is a self-degradation system wherein cellular materials are recycled. Although autophagy has been extensively studied in yeast and mammalian systems, integrated stress responses in microalgae remain poorly understood. Accordingly, we carried out a comparative study on the oxidative stress responses of Chlamydomonas reinhardtii wild-type and a starchless (sta6) mutant previously shown to accumulate high lipid content under adverse conditions. To our surprise, the sta6 mutant exhibited significantly higher levels of lipid peroxidation in the same growth conditions compared to controls. The sta6 mutant was more sensitive to oxidative stress induced by H2O2, whereas the wild-type was relatively more resistant. In addition, significantly up-regulated autophagy-related factors including ATG1, ATG101, and ATG8 were maintained in the sta6 mutant regardless of nitrogen availability. Also, the sta6 mutant exhibited relatively higher ATG8 protein level compared to wild-type under non-stress condition, and quickly reached a saturation point of autophagy when H2O2 was applied. Our results indicate that, in addition to the impact of carbon allocation, the increased lipid phenotype of the sta6 mutant may result from alterations in the cellular oxidative state, which in turn activates autophagy to clean up oxidatively damaged components and fuel lipid production.

The diurnal transcriptional landscape of the microalga Tetradesmus obliquus

Tetradesmus obliquus is a promising oleaginous microalga. We functionally annotated its genome and characterized the transcriptional landscape of T. obliquus adapted to 16:8 h light dark (LD) cycles in turbidostat culture conditions at very high temporal resolution (1 h intervals). Revealing a cycle of cellular events, six distinct expression profiles were obtained, each with transcriptional phenotypes correlating with measurements of biochemical composition.The impact of starch deficiency was studied using the starchless mutant slm1. Significant changes in the transcriptional landscape were observed. Starch deficiency resulted in incapacity to supply energy during the dark period, resulting in a shift of energy demanding processes to an earlier or later time point. Our study provides new perspectives on the role of starch and the adaptation to LD cycles of oleaginous microalgae.

Lipid productivity in TALEN-induced starchless mutants of the unicellular green alga Coccomyxa sp. strain Obi

Starchless mutants of several green algae including Chlamydomonas reinhardtii and Scenedesmus obliquus showed improved lipid productivity. In this study, we isolated mutants of the unicellular green alga, Coccomyxa sp. strain Obi, defective in the gene for the large subunit of ATP:α-d-glucose-1-phosphate adenylyltransferase (AGPL) by means of a transcription activator-like effector nuclease (TALEN) as follows. First, two expression cassettes encoding left and right arms of TALEN for AGPL mutagenesis were introduced into strain Obi, and from two independent transformants of strain Obi carrying both expression cassettes, seven derivatives bearing different mutations in AGPL were isolated. When these mutants were grown in 1/2-diluted and 1/3-diluted minimal media, their lipid contents were significantly higher than those of their parental strains. The growth in 1/3-diluted medium was similar between the mutants and their parental strains, while the growth in 1/2-diluted medium was significantly lower for the mutants than for their parental strains. The lipid productivity, which is a crucial factor for commercial production of biofuels, was not statistically different between the parental and mutant strains. We concluded that the suppression of starch biosynthesis to increase lipid productivity in microalgae is a risky strategy as it could result in low biomass productivity.

Diurnal periodicity of assimilate transport shapes resource allocation and whole-plant carbon balance.

Whole-plant carbon balance comprises diurnal fluctuations of photosynthetic carbon gain and respiratory losses, as well as partitioning of assimilates between phototrophic and heterotrophic organs. Because it is difficult to access, the root system is frequently neglected in growth models, or its metabolism is rated based on generalizations from other organs. Here, whole-plant cuvettes were used for investigating total-plant carbon exchange with the environment over full diurnal cycles. Dynamics of primary metabolism and diurnally resolved phloem exudation profiles, as proxy of assimilate transport, were combined to obtain a full picture of resource allocation. This uncovered a strong impact of periodicity of inter-organ transport on the efficiency of carbon gain. While a sinusoidal fluctuation of the transport rate, with minor diel deflections, minimized respiratory losses in Arabidopsis wild-type plants, triangular or rectangular patterns of transport, found in mutants defective in either starch or sucrose metabolism, increased root respiration at the end or beginning of the day, respectively. Power spectral density and cross-correlation analysis revealed that only the rate of starch synthesis was strictly correlated to the rate of net photosynthesis in wild-type, while in a sucrose-phosphate synthase mutant (spsa1), this applied also to carboxylate synthesis, serving as an alternative carbon pool. In the starchless mutant of plastidial phospho-gluco mutase (pgm), none of these rates, but concentrations of sucrose and glucose in the root, followed the pattern of photosynthesis, indicating direct transduction of shoot sugar levels to the root. The results demonstrate that starch metabolism alone is insufficient to buffer diurnal fluctuations of carbon exchange.

The impact of day length on cell division and efficiency of light use in a starchless mutant of Tetradesmus obliquus

Large scale microalgal production will be primarily done under natural sunlight conditions, where microalgae will be exposed to diurnal cycles of light and dark (LD) and to differences in the length of both periods (photoperiod). Tetradesmus obliquus (formerly known as Scenedesmus obliquus), a strain with potential for biofuel production, and the starchless mutant slm1 were grown under 3 different LD periods: 16:8 h, 14:10 h and 12:12 h. Cell division started a fix number of hours after the light went on (sunrise), independently of the length of the photoperiod. For the wild-type, cell division started approximately 14 h after the beginning of the day and occurred mainly at night. For the starchless mutant slm1, timing of cell division was also independent of the photoperiod length (starting 10–12 h after sunrise). However, as opposed to the wild-type, cell division always started during the day. For both strains, growth rate increased with increased length of the light period. The slm1 mutant is capable of surviving long dark periods (up to 12 h) despite the lack of starch. In general, the slm1 mutant has a lower photosynthetic efficiency than the wild-type, with the 12:12 h LD resulting into even less efficiency than the other two LD cycles.

The role of starch as transient energy buffer in synchronized microalgal growth in Acutodesmus obliquus

Photosynthetic organisms have evolved to use light efficiently by scheduling their cellular processes, such as growth and cell division, at specific times of the day. During the day, fixed carbon is used for growth and is partially stored as carbohydrates (e.g., starch). It is commonly assumed that this accumulated starch is essential for fuelling up cell division at night. To test this hypothesis, this study investigates growth, cell division and presence of a transitory energy storage (TES) in both the wild-type and starchless mutant strain of Acutodesmus obliquus under light/dark (LD) cycles and nitrogen replete conditions.A. obliquus (formerly known as Scenedesmus obliquus) wild-type utilized light 20% more efficiently under LD regimes compared with continuous light. When exposed to LD regimes, the wild-type scheduled cell division in a 4-hour period starting 2h before ‘sunset’. Starch acted as the major transitory energy storage (TES) compound: it was accumulated during the last part of the light period and was consumed throughout the entire dark period. The slm1 mutant, with a blocked starch synthesis pathway, showed diurnal rhythms in growth and cell division. However, no other carbohydrates nor triacylglycerols took over the role of TES compound in slm1. Therefore, in contrast to what is generally acknowledged, this study shows that neither starch nor any other major alternative TES is required for synchronized growth and cell division in A. obliquus. The starchless mutant did show a reduced growth and cell division rate compared to the wild-type. Starch, thus, plays a major role in efficient harnessing of light energy over LD cycles, likely because the ability to accumulate starch enhances biomass production capacity and accelerates cell division rate in A. obliquus.

Continuous versus batch production of lipids in the microalgae Acutodesmus obliquus

This work provides a novel quantitative comparison of batch versus continuous microalgal lipid production in the wild type and starchless mutant strain of Acutodesmus obliquus. Both strains showed higher TAG yields on light under batch operation compared to continuous nitrogen limitation. The starchless mutant showed 0.20gTAGmolph−1 for batch and 0.12gTAGmolph−1 for continuous operation, while the wildtype only showed 0.16gTAGmolph−1 for batch and 0.08gTAGmolph−1 for continuous operation. Also, higher TAG contents were found under batch starvation (26% of dry weight for the wildtype and 43% of dry weight for starchless mutant) compared to continuous cultivations (16% of dry weight for the wildtype and 33% of dry weight for starchless mutant). Starch acts as the favoured storage metabolite during nitrogen limitation in A. obliquus, whereas TAG is only accumulated after starch reaches a cellular maximum of 40% of dry weight.

The Search for a Lipid Trigger: The Effect of Salt Stress on the Lipid Profile of the Model Microalgal Species Chlamydomonas reinhardtii for Biofuels Production.

Background: Algal cells produce neutral lipid when stressed and this can be used to generate biodiesel.Objective: Salt stressed cells of the model microalgal species Chlamydomonas reinhardtii were tested for their suitability to produce lipid for biodiesel.Methods: The starchless mutant of C. reinhardtii (CC-4325) was subjected to salt stress (0.1, 0.2 and 0.3 M NaCl) and transesterification and GC analysis were used to determine fatty acid methyl ester (FAME) content and profile.Results: Fatty acid profile was found to vary under salt stress conditions, with a clear distinction between 0.1 M NaCl, which the algae could tolerate, and the higher levels of NaCl (0.2 and 0.3 M), which caused cell death. Lipid content was increased under salt conditions, either through long-term exposure to 0.1 M NaCl, or short-term exposure to 0.2 and 0.3 M NaCl. Palmitic acid (C16:0) and linolenic acid (C18:3n3) were found to increase significantly at the higher salinities.Conclusion: Salt increase can act as a lipid trigger for C. reinhardtii.

Subcellular reprogramming of metabolism during cold acclimation in Arabidopsis thaliana.

Metabolite changes in plant leaves during exposure to low temperatures involve re-allocation of a large number of metabolites between sub-cellular compartments. Therefore, metabolite determination at the whole cell level may be insufficient for interpretation of the functional significance of cellular compounds. To investigate the cold-induced metabolite dynamics at the level of individual sub-cellular compartments, an integrative platform was developed that combines quantitative metabolite profiling by gas chromatography coupled to mass spectrometry (GC-MS) with the non-aqueous fractionation technique allowing separation of cytosol, vacuole and the plastidial compartment. Two mutants of Arabidopsis thaliana representing antipodes in the diversion of carbohydrate metabolism between sucrose and starch were compared to Col-0 wildtype before and after cold acclimation to investigate interactions of cold acclimation with subcellular re-programming of metabolism. A multivariate analysis of the data set revealed dominant effects of compartmentation on metabolite concentrations that were modulated by environmental condition and genetic determinants. While for both, the starchless mutant of plastidial phospho-gluco mutase (pgm) and a mutant defective in sucrose-phosphate synthase A1, metabolic constraints, especially at low temperature, could be uncovered based on subcellularly resolved metabolite profiles, only pgm had lowered freezing tolerance. Metabolic profiles of pgm point to redox imbalance as a possible reason for reduced cold acclimation capacity.