Chlamydomonas starchless mutant defective in ADP-glucose pyrophosphorylase hyper-accumulates triacylglycerol

Many microalgae and plants have the ability to synthesize large amounts of triacylglycerol (TAG) that can be used to produce biofuels. Presently, TAG-based biofuel production is limited by the feedstock supply. Metabolic engineering of lipid synthesis pathways to overproduce TAGs in oleaginous microalgae and oil crop plants has achieved only modest success. We demonstrate that inactivation of ADP-glucose pyrophosphorylase in a Chlamydomonas starchless mutant led to a 10-fold increase in TAG, suggesting that shunting of photosynthetic carbon partitioning from starch to TAG synthesis may represent a more effective strategy than direct manipulation of the lipid synthesis pathway to overproduce TAG.     

Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant

Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP‐glucose pyrophosphorylase. This mutant is unable to convert glucose‐1–phosphate to ADP‐glucose, the precursor of starch biosynthesis. During nutrient‐replete culturing, sta6 does not re‐direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC–MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO₂) decrease in sta6 due to attenuated rates of NADPH re‐oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system‐wide consequences of slower NADPH re‐oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high‐light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient‐replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.     

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.     

Photosynthetic carbon partitioning and lipid production in the oleaginous microalga Pseudochlorococcum sp. (Chlorophyceae) under nitrogen-limited conditions

Photosynthetic carbon partitioning into starch and neutral lipid was investigated in the oleaginous green microalga Pseudochlorococcum sp. When grown under low light and nitrogen-replete conditions, the algal cells possessed a basal level of starch. When grown under high light and nitrogen-limited conditions, starch synthesis was transiently up-regulated. After nitrogen depletion, starch content decreased while neutral lipid rapidly increased to 52.1% of cell dry weight, with a maximum neutral lipid productivity of 0.35 g L⁻¹ D⁻¹. These results suggest that Pseudochlorococcum used starch as a primary carbon and energy storage product. As nitrogen was depleted for an extended period of time, cells shift the carbon partitioning into neutral lipid as a secondary storage product. Partial inhibition of starch synthesis and degradation enzymes resulted in a decrease in neutral lipid content, indicating that conversion of starch to neutral lipid may contribute to overall neutral lipid accumulation. Biotechnological application of Pseudochlorococcum sp. as a production strain for biofuel was assessed.     

Genetic and transgenic perturbations of carbon reserve production in Arabidopsis seeds reveal metabolic interactions of biochemical pathways

The biosynthesis of seed oil and starch both depend on the supply of carbon from the maternal plant. The biochemical interactions between these two pathways are not fully understood. In the Arabidopsis mutant shrunken seed 1 (sse1)/pex16, a reduced rate of fatty acid synthesis leads to starch accumulation. To further understand the metabolic impact of the decrease in oil synthesis, we compared soluble metabolites in sse1 and wild type (WT) seeds. Sugars, sugar phosphates, alcohols, pyruvate, and many other organic acids accumulated in sse1 seeds as a likely consequence of the reduced carbon demand for lipid synthesis. The enlarged pool size of hexose-P, the metabolites at the crossroad of sugar metabolism, glycolysis, and starch synthesis, was likely a direct cause of the increased flow into starch. Downstream of glycolysis, more carbon entered the TCA cycle as an alternative to the fatty acid pathway, causing the total amount of TCA cycle intermediates to rise while moving the steady state of the cycle away from fumarate. To convert the excess carbon metabolites into starch, we introduced the Escherichia coli starch synthetic enzyme ADP-glucose pyrophosphorylase (AGPase) into sse1 seeds. Expression of AGPase enhanced net starch biosynthesis in the mutant, resulting in starch levels that reached 37% of seed weight. However, further increases above this level were not achieved and most of the carbon intermediates remained high in comparison with the WT, indicating that additional mechanisms limit starch deposition in Arabidopsis seeds.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Isolation and characterization of a starchless mutant of Chlorella pyrenoidosa STL-PI with a high growth rate, and high protein and polyunsaturated fatty acid content

The growth characteristics, biochemical composition and ultrastructure of a novel starchless mutant of Chlorella pyrenoidosa, designated as STL‐PI, are compared to the same characteristics of its parental strain, C. pyrenoidosa 82T. The STL‐PI mutant had a 22 ± 5% higher growth rate, and 24.5 ± 4.2% more protein than the parental strain, 82T. Furthermore, the STL‐PI mutant accumulated 20.4% more polyunsaturated fatty acids and 18% less saturated fatty acids than the parental 82T. When the parental 82T was cultured in a nitrogen‐free media, their starch content increased from 6.8 ± 2.8% to 22.5 ± 3.1%. In contrast, the STL‐PI mutant produced no starch, regardless of the growth conditions. Instead, the mutant cells responded to nitrogen limitation by further increasing their lipid content from 25.2 ± 1.2% to 38.0 ± 2.3% per dry weight. Transmission electron micrography revealed that nitrogen limitation typically stimulates the formation of starch granules in the chloroplast of 82T cells. Yet no starch granules were observed in the STL‐PI cells. Instead, only the formation of large lipid globules was observed in the mutant cells. These results demonstrate that the starchless mutant STL‐P1 possesses novel physiological and phytochemical properties distinct from the 82T cells: their cells were deficient in starch synthesis and showed higher growth rates and productivity than 82T cells.     

Oil accumulation is controlled by carbon precursor supply for fatty acid synthesis in Chlamydomonas reinhardtii

Microalgal oils have attracted much interest as potential feedstocks for renewable fuels, yet our understanding of the regulatory mechanisms controlling oil biosynthesis and storage in microalgae is rather limited. Using Chlamydomonas reinhardtii as a model system, we show here that starch, rather than oil, is the dominant storage sink for reduced carbon under a wide variety of conditions. In short-term treatments, significant amounts of oil were found to be accumulated concomitantly with starch only under conditions of N starvation, as expected, or in cells cultured with high acetate in otherwise standard growth medium. Time-course analysis revealed that oil accumulation under N starvation lags behind that of starch and rapid oil synthesis occurs only when carbon supply exceeds the capacity of starch synthesis. In the starchless mutant BAFJ5, blocking starch synthesis results in significant increases in the extent and rate of oil accumulation. In the parental strain, but not the starchless mutant, oil accumulation under N starvation was strictly dependent on the available external acetate supply and the amount of oil increased steadily as the acetate concentration increased to the levels several-fold higher than that of the standard growth medium. Additionally, oil accumulation under N starvation is saturated at low light intensities and appears to be largely independent of de novo protein synthesis. Collectively, our results suggest that carbon availability is a key metabolic factor controlling oil biosynthesis and carbon partitioning between starch and oil in Chlamydomonas.     

Carbon partitioning in Arabidopsis thaliana is a dynamic process controlled by the plants metabolic status and its circadian clock

Plant growth involves the coordinated distribution of carbon resources both towards structural components and towards storage compounds that assure a steady carbon supply over the complete diurnal cycle. We used ¹⁴CO₂ labelling to track assimilated carbon in both source and sink tissues. Source tissues exhibit large variations in carbon allocation throughout the light period. The most prominent change was detected in partitioning towards starch, being low in the morning and more than double later in the day. Export into sink tissues showed reciprocal changes. Fewer and smaller changes in carbon allocation occurred in sink tissues where, in most respects, carbon was partitioned similarly, whether the sink leaf assimilated it through photosynthesis or imported it from source leaves. Mutants deficient in the production or remobilization of leaf starch exhibited major alterations in carbon allocation. Low‐starch mutants that suffer from carbon starvation at night allocated much more carbon into neutral sugars and had higher rates of export than the wild type, partly because of the reduced allocation into starch, but also because of reduced allocation into structural components. Moreover, mutants deficient in the plant's circadian system showed considerable changes in their carbon partitioning pattern suggesting control by the circadian clock.     

Growth and reproduction of Arabidopsis thaliana in relation to storage of starch and nitrate in the wild-type and in starch-deficient and nitrate-uptake-deficient mutants

We have investigated the interactions between resource assimilation and storage in rosette leaves, and their impact on the growth and reproduction of the annual species Arabidopsis thaliana. The resource balance was experimentally perturbed by changing (i) the external nutrition, by varying the nitrogen supply; (ii) the assimilation and reallocation of resources from rosette leaves to reproductive organs, by cutting or covering rosette leaves at the time of early flower bud formation, and (iii) the internal carbon and nitrogen balance of the plants, by using isogenic mutants either lacking starch formation (PGM mutant) or with reduced nitrate uptake (NU mutant). When plants were grown on high nitrogen, they had higher concentrations of carbohydrates and nitrate in their leaves during the rosette phase than during flowering. However, these storage pools did not significantly contribute to the bulk flow of resources to seeds. The pool size of stored resources in rosette leaves at the onset of seed filling was very low compared to the total amount of carbon and nitrogen needed for seed formation. Instead, the rosette leaves had an important function in the continued assimilation of resources during seed ripening, as shown by the low seed yield of plants whose leaves were covered or cut off. When a key resource became limiting, such as nitrogen in the NU mutants and in plants grown on a low nitrogen supply, stored resources in the rosette leaves (e.g. nitrogen) were remobilized, and made a larger contribution to seed biomass. A change in nutrition resulted in a complete reversal of the plant response: plants shifted from high to low nutrition exhibited a seed yield similar to that of plants grown continuously on a low nitrogen supply, and vice versa. This demonstrates that resource assimilation during the reproductive phase determines seed production. The PGM mutant had a reduced growth rate and a smaller biomass during the rosette phase as a result of changes in respiration caused by a high turnover of soluble sugars ( Caspar et al. 1986 ; W. Schulze et al. 1991 ). During flowering, however, the vegetative growth rate in the PGM mutant increased, and exceeded that of the wild-type. By the end of the flowering stage, the biomass of the PGM mutant did not differ from that of the wild-type. However, in contrast to the wild-type, the PGM mutant maintained a high vegetative growth rate during seed formation, but had a low rate of seed production. These differences in allocation in the PGM mutant result in a significantly lower seed yield in the starchless mutants. This indicates that starch formation is not only an important factor during growth in the rosette phase, but is also important for whole plant allocation during seed formation. The NU mutant resembled the wild-type grown on a low nitrogen supply, except that it unexpectedly showed symptoms of carbohydrate shortage as well as nitrogen deficiency. In all genotypes and treatments, there was a striking correlation between the concentrations of nitrate and organic nitrogen and shoot growth on the one hand, and sucrose concentration and root growth on the other. In addition, nitrate reductase activity (NRA) was correlated with the total carbohydrate concentration: low carbohydrate levels in starchless mutants led to low NRA even at high nitrate supply. Thus the concentrations of stored carbohydrates and nitrate are directly or indirectly involved in regulating allocation.     

Gravitropism in roots of intermediate-starch mutants of Arabidopsis

Gravitropism was studied in roots of wild type (WT) Arabidopsis thaliana (L.) Heynh. (strain Wassilewskija) and three starch-deficient mutants that were generated, by T-DNA insertional mutagenesis. One of these mutants was starchless while the other two were intermediate mutants, which had 51% and 60%, respectively, of the WT amount of starch as. determined by light and electron microscopy. The four parameters used to assay gravitropism were: orientation during vertical growth, time course of curvature, induction, and intermittent stimulation experiments. WT roots were much more responsive to gravity than were roots of the slarchless mutant, and the intermediate starch mutants exhibited an intermediate graviresponse. Our data suggest that lowered starch content in the mutants primarily affects gravitropism rather than differential growth because both phototropic curvature and growth rates were approximately equal among all four genotypes. Since responses of intermediate-starch mutants were closer to the WT response than to that of the starchless mutant, it appears that 51–60% of the WT level of starch is near the threshold amount needed for full gravitropic sensitivity. While other interpretations are possible, the data are consistent with the starch statolith hypothesis for gravity perception in that the degree of graviresponsiveness is proportional to the total mass of plastids per cell.     

High temperature enhances lipid accumulation in nitrogen-deprived <Emphasis Type="Italic">Scenedesmus obtusus</Emphasis> XJ-15

This study investigated the changes in lipid and starch contents, lipid fraction, and lipid profile in the nitrogen-starved Scenedesmus obtusus XJ-15 at different temperatures (17, 25, and 33 °C). The optimal temperature for both growth and lipid accumulation under nitrogen-sufficient condition was found to be 25 °C. However, under nitrogen deprivation, the total and neutral lipids increased with increasing temperature, and achieved the highest lipid content of 47.60 % of dry cell weight and the highest TAG content of 79.66 % of total lipid at 33 °C. In the meantime, the stored cellular starch content decreased with the increasing temperature. Thus, high temperature induced carbon flux from starch toward TAG accumulation in microalgae during nitrogen starvation. In addition, the decreased polar lipids may also serve for TAG synthesis under high temperature, and high temperature further reduced the degree of the fatty acid unsaturation and favored a better biodiesel production. These results suggested that high-temperature stress can be a good strategy for enhancing biofuel production in oleaginous microalgae during nitrogen deficiency.     

Decrease in leaf sucrose synthesis leads to increased leaf starch turnover and decreased RuBP regeneration-limited photosynthesis but not Rubisco-limited photosynthesis in Arabidopsis null mutants of SPSA1

We investigated the individual effect of null mutations of each of the four sucrose‐phosphate synthase (SPS) genes in Arabidopsis (SPSA1, SPSA2, SPSB and SPSC) on photosynthesis and carbon partitioning. Null mutants spsa1 and spsc led to decreases in maximum SPS activity in leaves by 80 and 13%, respectively, whereas null mutants spsa2 and spsb had no significant effect. Consistently, isoform‐specific antibodies detected only the SPSA1 and SPSC proteins in leaf extracts. Leaf photosynthesis at ambient [CO₂] was not different among the genotypes but was 20% lower in spsa1 mutants when measured under saturating [CO₂] levels. Carbon partitioning at ambient [CO₂] was altered only in the spsa1 null mutant. Cold treatment of plants (4 °C for 96 h) increased leaf soluble sugars and starch and increased the leaf content of SPSA1 and SPSC proteins twofold to threefold, and of the four null mutants, only spsa1 reduced leaf non‐structural carbohydrate accumulation in response to cold treatment. It is concluded that SPSA1 plays a major role in photosynthetic sucrose synthesis in Arabidopsis leaves, and decreases in leaf SPS activity lead to increased starch synthesis and starch turnover and decreased Ribulose 1,5‐bisphosphate regeneration‐limited photosynthesis but not ribulose 1·5‐bisphosphate carboxylase/oxygenase (Rubisco)‐limited photosynthesis, indicating a limitation of triose‐phosphate utilization (TPU).     

High‐throughput fluorescence‐activated cell sorting for lipid hyperaccumulating Chlamydomonas reinhardtii mutants

The genetically tractable microalga Chlamydomonas reinhardtii has many advantages as a model for renewable bioproducts and/or biofuels production. However, one limitation of C. reinhardtii is its relatively low‐lipid content compared with some other algal species. To overcome this limitation, we combined ethane methyl sulfonate mutagenesis with fluorescence‐activated cell sorting (FACS) of cells stained with the lipophilic stain Nile Red to isolate lipid hyperaccumulating mutants of C. reinhardtii. By manipulating the FACS gates, we sorted mutagenized cells with extremely high Nile Red fluorescence signals that were rarely detected in nonmutagenized populations. This strategy successfully isolated several putative lipid hyperaccumulating mutants exhibiting 23% to 58% (dry weight basis) higher fatty acid contents than their progenitor strains. Significantly, for most mutants, nitrogen starvation was not required to attain high‐lipid content nor was there a requirement for a deficiency in starch accumulation. Microscopy of Nile Red stained cells revealed that some mutants exhibit an increase in the number of lipid bodies, which correlated with TLC analysis of triacyglycerol content. Increased lipid content could also arise through increased biomass production. Collectively, our findings highlight the ability to enhance intracellular lipid accumulation in algae using random mutagenesis in conjunction with a robust FACS and lipid yield verification regime. Our lipid hyperaccumulating mutants could serve as a genetic resource for stacking additional desirable traits to further increase lipid production and for identifying genes contributing to lipid hyperaccumulation, without lengthy lipid‐induction periods.     

A predicted plastid rhomboid protease affects phosphatidic acid metabolism in Arabidopsis thaliana

The thylakoid membranes of the chloroplast harbor the photosynthetic machinery that converts light into chemical energy. Chloroplast membranes are unique in their lipid makeup, which is dominated by the galactolipids mono‐ and digalactosyldiacylglycerol (MGDG and DGDG). The most abundant galactolipid, MGDG, is assembled through both plastid and endoplasmic reticulum (ER) pathways in Arabidopsis, resulting in distinguishable molecular lipid species. Phosphatidic acid (PA) is the first glycerolipid formed by the plastid galactolipid biosynthetic pathway. It is converted to substrate diacylglycerol (DAG) for MGDG Synthase (MGD1) which adds to it a galactose from UDP‐Gal. The enzymatic reactions yielding these galactolipids have been well established. However, auxiliary or regulatory factors are largely unknown. We identified a predicted rhomboid‐like protease 10 (RBL10), located in plastids of Arabidopsis thaliana, that affects galactolipid biosynthesis likely through intramembrane proteolysis. Plants with T‐DNA disruptions in RBL10 have greatly decreased 16:3 (acyl carbons:double bonds) and increased 18:3 acyl chain abundance in MGDG of leaves. Additionally, rbl10‐1 mutants show reduced [¹⁴C]–acetate incorporation into MGDG during pulse?chase labeling, indicating a reduced flux through the plastid galactolipid biosynthesis pathway. While plastid MGDG biosynthesis is blocked in rbl10‐1 mutants, they are capable of synthesizing PA, as well as producing normal amounts of MGDG by compensating with ER‐derived lipid precursors. These findings link this predicted protease to the utilization of PA for plastid galactolipid biosynthesis potentially revealing a regulatory mechanism in chloroplasts.     

Comparison of CO2 and bicarbonate as inorganic carbon sources for triacylglycerol and starch accumulation in Chlamydomonas reinhardtii

Microalgae are capable of accumulating high levels of lipids and starch as carbon storage compounds. Investigation into the metabolic activities involved in the synthesis of these compounds has escalated since these compounds can be used as precursors for food and fuel. Here, we detail the results of a comprehensive analysis of Chlamydomonas reinhardtii using high or low inorganic carbon concentrations and speciation between carbon dioxide and bicarbonate, and the effects these have on inducing lipid and starch accumulation during nitrogen depletion. High concentrations of CO₂ (5%; v/v) produced the highest amount of biofuel precursors, transesterified to fatty acid methyl esters, but exhibited rapid accumulation and degradation characteristics. Low CO₂ (0.04%; v/v) caused carbon limitation and minimized triacylglycerol (TAG) and starch accumulation. High bicarbonate caused a cessation of cell cycling and accumulation of both TAG and starch that was more stable than the other experimental conditions. Starch accumulated prior to TAG and then degraded as maximum TAG was reached. This suggests carbon reallocation from starch‐based to TAG‐based carbon storage. Biotechnol. Bioeng. 2013; 110: 87–96. © 2012 Wiley Periodicals, Inc.     

Relationship between starch degradation and carbon demand for maintenance and growth in Arabidopsis thaliana in different irradiance and temperature regimes

Experiments were designed to compare the relationship between starch degradation and the use of carbon for maintenance and growth in Arabidopsis in source‐limited and sink‐limited conditions. It is known that starch degradation is regulated by the clock in source‐limited plants, which degrade their starch in a linear manner such that it is almost but not completely exhausted at dawn. We asked whether this response is maintained under an extreme carbon deficit. Arabidopsis was subjected to a sudden combination of a day of low irradiance, to decrease starch at dusk, and a warm night. Starch was degraded in a linear manner through the night, even though the plants became acutely carbon starved. We conclude that starch degradation is not increased to meet demand in carbon‐limited plants. This network property will allow stringent control of starch turnover in a fluctuating environment. In contrast, in sink‐limited plants, which do not completely mobilize their starch during the night, starch degradation was accelerated in warm nights to meet the increased demand for maintenance and growth. Across all conditions, the rate of growth at night depends on the rate of starch degradation, whereas the rate of maintenance respiration decreases only when starch degradation is very slow.     

Domestication and the storage starch biosynthesis pathway: signatures of selection from a whole sorghum genome sequencing strategy

Next‐generation sequencing of complete genomes has given researchers unprecedented levels of information to study the multifaceted evolutionary changes that have shaped elite plant germplasm. In conjunction with population genetic analytical techniques and detailed online databases, we can more accurately capture the effects of domestication on entire biological pathways of agronomic importance. In this study, we explore the genetic diversity and signatures of selection in all predicted gene models of the storage starch synthesis pathway of Sorghum bicolor, utilizing a diversity panel containing lines categorized as either ‘Landraces’ or ‘Wild and Weedy’ genotypes. Amongst a total of 114 genes involved in starch synthesis, 71 had at least a single signal of purifying selection and 62 a signal of balancing selection and others a mix of both. This included key genes such as STARCH PHOSPHORYLASE 2 (SbPHO2, under balancing selection), PULLULANASE (SbPUL, under balancing selection) and ADP‐glucose pyrophosphorylases (SHRUNKEN2, SbSH2 under purifying selection). Effectively, many genes within the primary starch synthesis pathway had a clear reduction in nucleotide diversity between the Landraces and wild and weedy lines indicating that the ancestral effects of domestication are still clearly identifiable. There was evidence of the positional rate variation within the well‐characterized primary starch synthesis pathway of sorghum, particularly in the Landraces, whereby low evolutionary rates upstream and high rates downstream in the metabolic pathway were expected. This observation did not extend to the wild and weedy lines or the minor starch synthesis pathways.