ABSTRACT
Triacylglycerol (TAG) is amongst the most energy dense storage form of reduced carbon in living systems. TAG metabolism plays critical roles in cellular energy balance, lipid homeostasis, cell growth and stress responses. In higher plants, microalgae and fungi, TAG is assembled by acyl-CoA-dependent and -independent pathways catalyzed by diacylglycerol:acyltransferase (DGAT) and phospholipid:diacylglycerol acyltransferase (PDAT), respectively. This review contains a summary of the current understanding of the physiological functions of PDATs. Emphasis is placed on their role in lipid remodeling and lipid homeostasis in response to abiotic stress or perturbations in lipid metabolism.
ABSTRACT
Two parallel pathways compartmentalized in the chloroplast and the endoplasmic reticulum contribute to thylakoid lipid synthesis in plants, but how these two pathways are coordinated during thylakoid biogenesis and remodeling remains unknown. We report here the molecular characterization of a homologous ADIPOSE TRIGLYCERIDE LIPASE-LIKE gene, previously referred to as ATGLL. The ATGLL gene is ubiquitously expressed throughout development and rapidly upregulated in response to a wide range of environmental cues. We show that ATGLL is a chloroplast non-regioselective lipase with a hydrolytic activity preferentially towards 16:0 of diacylglycerol (DAG). Comprehensive lipid profiling and radiotracer labeling studies revealed a negative correlation of ATGLL expression and the relative contribution of the chloroplast lipid pathway to thylakoid lipid biosynthesis. Additionally, we show that genetic manipulation of ATGLL expression resulted in changes in triacylglycerol levels in leaves. We propose that ATGLL, through affecting the level of prokaryotic DAG in the chloroplast, plays important roles in balancing the two glycerolipid pathways and in maintaining lipid homeostasis in plants.
Subject(s)
Arabidopsis Proteins , Arabidopsis , Arabidopsis/genetics , Arabidopsis/metabolism , Lipoprotein Lipase/metabolism , Chloroplasts/metabolism , Thylakoids/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Plants/metabolism , LipidsABSTRACT
Autophagy is a catabolic process in which cytoplasmic components are delivered to vacuoles or lysosomes for degradation and nutrient recycling. Autophagy-mediated degradation of membrane lipids provides a source of fatty acids for the synthesis of energy-rich, storage lipid esters such as triacylglycerol (TAG). In eukaryotes, storage lipids are packaged into dynamic subcellular organelles, lipid droplets. In times of energy scarcity, lipid droplets can be degraded via autophagy in a process termed lipophagy to release fatty acids for energy production via fatty acid ß-oxidation. On the other hand, emerging evidence suggests that lipid droplets are required for the efficient execution of autophagic processes. Here, we review recent advances in our understanding of metabolic interactions between autophagy and TAG storage, and discuss mechanisms of lipophagy. Free fatty acids are cytotoxic due to their detergent-like properties and their incorporation into lipid intermediates that are toxic at high levels. Thus, we also discuss how cells manage lipotoxic stresses during autophagy-mediated mobilization of fatty acids from lipid droplets and organellar membranes for energy generation.
Subject(s)
Autophagy , Lipid Droplets , Fatty Acids/metabolism , Lipid Droplets/metabolism , Lipid Metabolism , Triglycerides/metabolism , Vacuoles/metabolismABSTRACT
Lipid droplets (LDs) are intracellular organelles critical for energy storage and lipid metabolism. They are typically composed of an oil core coated by a monolayer of phospholipids and proteins such as oleosins. The mechanistic details of LD biogenesis remain poorly defined. However, emerging evidence suggest that their formation is a spatiotemporally regulated process, occurring at specific sites of the endoplasmic reticulum defined by a specific set of lipids and proteins. Here, we show that sterols are required for formation of oleosin-coated LDs in Arabidopsis. Analysis of sterol pathway mutants revealed that deficiency in several ∆5-sterols accounts for the phenotype. Importantly, mutants deficient in these sterols also display reduced LD number, increased LD size and reduced oil content in seeds. Collectively, our data reveal a role of sterols in coordinating the synthesis of oil and oleosins and their assembly into LDs, highlighting the importance of membrane lipids in regulating LD biogenesis.
Subject(s)
Lipid Droplets/metabolism , Phytosterols/metabolism , Seeds/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Lipid Metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mutation , Phytosterols/genetics , Plant Leaves/metabolism , Plants, Genetically Modified , Seeds/genetics , Seeds/growth & development , Triglycerides/metabolismABSTRACT
Lipid remodeling, defined herein as post-synthetic structural modifications of membrane lipids, play crucial roles in regulating the physicochemical properties of cellular membranes and hence their many functions. Processes affected by lipid remodeling include lipid metabolism, membrane repair, cellular homeostasis, fatty acid trafficking, cellular signaling and stress tolerance. Glycerolipids are the major structural components of cellular membranes and their composition can be adjusted by modifying their head groups, their acyl chain lengths and the number and position of double bonds. This review summarizes recent advances in our understanding of mechanisms of membrane lipid remodeling with emphasis on the lipases and acyltransferases involved in the modification of phosphatidylcholine and monogalactosyldiacylglycerol, the major membrane lipids of extraplastidic and photosynthetic membranes, respectively. We also discuss the role of triacylglycerol metabolism in membrane acyl chain remodeling. Finally, we discuss emerging data concerning the functional roles of glycerolipid remodeling in plant stress responses. Illustrating the molecular basis of lipid remodeling may lead to novel strategies for crop improvement and other biotechnological applications such as bioenergy production.
Subject(s)
Enzymes/metabolism , Membrane Lipids/metabolism , Plants/metabolism , Gene Expression Regulation, Plant , Membrane Lipids/chemistry , Membrane Lipids/genetics , Plant Cells , Plant Proteins/metabolism , Triglycerides/metabolismABSTRACT
Lipids metabolism is comprised of networks of reactions occurred in different subcellular compartments. Isotopic labeling is a good way to track the transformations and movements of metabolites without perturbing overall cellular metabolism. Fatty acids, the building blocks of membrane lipids and storage triacylglycerols, are synthesized in plastids. The immediate precursor for fatty acid synthesis is acetyl-CoA. Exogenous acetate is rapidly incorporated into fatty acids in leaves and isolated plastids because it can diffuse freely through cellular membranes, enter the plastid where it is rapidly metabolized to acetyl-CoA. Therefore, isotope-labeled acetate is often used as a tracer for the investigation of fatty acid synthesis and complex lipid metabolism in plants and other organisms. The basic principle of isotope labeling and its recent technological advances have been reviewed ( Allen et al., 2015 ). The present protocol describes the use of 14C-labeled acetate to determine rates of fatty acid synthesis and degradation and to track the metabolism of glycerolipids in leaves. This method, which is often referred to as acetate pulse-chase labeling, has been widely used to probe various aspects of lipid metabolism ( Allen et al., 2015 ), including the role of autophagy in membrane lipid turnover ( Fan et al., 2019 ) and the interplay between lipid and starch metabolism pathways ( Yu et al., 2018 ).
ABSTRACT
Reprogramming metabolism, in addition to modifying the structure and function of the photosynthetic machinery, is crucial for plant acclimation to changing light conditions. One of the key acclimatory responses involves reorganization of the photosynthetic membrane system including changes in thylakoid stacking. Glycerolipids are the main structural component of thylakoids and their synthesis involves two main pathways localized in the plastid and the endoplasmic reticulum (ER); however, the role of lipid metabolism in light acclimation remains poorly understood. We found that fatty acid synthesis, membrane lipid content, the plastid lipid biosynthetic pathway activity, and the degree of thylakoid stacking were significantly higher in plants grown under low light compared with plants grown under normal light. Plants grown under high light, on the other hand, showed a lower rate of fatty acid synthesis, a higher fatty acid flux through the ER pathway, higher triacylglycerol content, and thylakoid membrane unstacking. We additionally demonstrated that changes in rates of fatty acid synthesis under different growth light conditions are due to post-translational regulation of the plastidic acetyl-CoA carboxylase activity. Furthermore, Arabidopsis mutants defective in one of the two glycerolipid biosynthetic pathways displayed altered growth patterns and a severely reduced ability to remodel thylakoid architecture, particularly under high light. Overall, this study reveals how plants fine-tune fatty acid and glycerolipid biosynthesis to cellular metabolic needs in response to long-term changes in light conditions, highlighting the importance of lipid metabolism in light acclimation.
Subject(s)
Acclimatization/physiology , Arabidopsis Proteins/metabolism , Chloroplasts/metabolism , Light , Membrane Lipids/metabolism , Plant Leaves/metabolism , Thylakoids/metabolism , Arabidopsis/genetics , Arabidopsis/metabolism , Genetic Variation , GenotypeABSTRACT
Neutral lipids in the form of triacylglycerol (TAG) have emerged as critical regulators of cellular energy balance, lipid homeostasis, growth, development and stress response in organisms ranging from plants to yeast. Although TAGs are mostly recognized as the main storage component in cytoplasmic lipid droplets (LDs), TAG-rich LDs with similar structural and functional characteristics to those found in the cytoplasm also exist in chloroplasts of microalgae and higher plants. Chloroplasts contain up to 70% of total lipids in photosynthetic cells, yet how organisms maintain chloroplast lipid homeostasis remains an under-investigated area of research. Here we summarize the current state of knowledge about the metabolism of TAG and its function in chloroplasts, with a focus on the enzymes catalyzing the final steps of TAG assembly and the role of TAG synthesis in protection against lipotoxicity. We also discuss emerging data regarding connections between cytoplasmic and chloroplast TAG metabolism and the role of autophagy in the degradation of chloroplast storage lipids.
Subject(s)
Chloroplasts/metabolism , Cytoplasm/metabolism , Triglycerides/metabolism , Animals , Autophagy , Lipid Droplets/metabolism , Lipid Metabolism , Mammals/metabolism , Plants/metabolismABSTRACT
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.
ABSTRACT
Autophagy is a major catabolic pathway whereby cytoplasmic constituents including lipid droplets (LDs), storage compartments for neutral lipids, are delivered to the lysosome or vacuole for degradation. The autophagic degradation of cytosolic LDs, a process termed lipophagy, has been extensively studied in yeast and mammals, but little is known about the role for autophagy in lipid metabolism in plants. Organisms maintain a basal level of autophagy under favorable conditions and upregulate the autophagic activity under stress including starvation. Here, we demonstrate that Arabidopsis (Arabidopsis thaliana) basal autophagy contributes to triacylglycerol (TAG) synthesis, whereas inducible autophagy contributes to LD degradation. We found that disruption of basal autophagy impedes organellar membrane lipid turnover and hence fatty acid mobilization from membrane lipids to TAG. We show that lipophagy is induced under starvation as indicated by colocalization of LDs with the autophagic marker and the presence of LDs in vacuoles. We additionally show that lipophagy occurs in a process morphologically resembling microlipophagy and requires the core components of the macroautophagic machinery. Together, this study provides mechanistic insight into lipophagy and reveals a dual role for autophagy in regulating lipid synthesis and turnover in plants.
Subject(s)
Arabidopsis/cytology , Arabidopsis/metabolism , Autophagy , Lipid Metabolism , Arabidopsis/genetics , Arabidopsis Proteins/metabolism , Biosynthetic Pathways , Chloroplasts/metabolism , Endoplasmic Reticulum/metabolism , Lipid Droplets/metabolism , Lipid Droplets/ultrastructure , Membrane Lipids/metabolism , Models, Biological , Mutation/genetics , Plant Leaves/cytology , Plant Leaves/growth & development , Plant Leaves/metabolism , Plant Leaves/ultrastructure , Plants, Genetically Modified , Triglycerides/biosynthesis , Vacuoles/metabolism , Vacuoles/ultrastructureABSTRACT
Peroxisomal ß-oxidation is the sole pathway for metabolic breakdown of fatty acids to generate energy and carbon skeletons in plants, is essential for oilseed germination and plays an important role in growth, development and cellular homeostasis. Yet, this process also produces cytotoxic reactive oxygen species (ROS) as byproducts. We recently showed that disruption of fatty acid ß-oxidation enhance plant survival under carbon starvation conditions. Here, we extend these findings by demonstrating that blocking fatty acid import into peroxisomes reduces ROS accumulation and increases plant tolerance to salt stress, whereas increasing fatty acid flux into the ß-oxidation pathway has opposite effects. Together, these results support the view that peroxisomal ß-oxidation of fatty acids enhances stress-induced ROS production, thereby negatively impacting plant survival under adverse environmental conditions.
Subject(s)
Arabidopsis/metabolism , Fatty Acids/metabolism , Arabidopsis/drug effects , Oxidation-Reduction/drug effects , Peroxisomes/drug effects , Peroxisomes/metabolism , Reactive Oxygen Species/metabolism , Salt Stress , Sodium Chloride/pharmacologyABSTRACT
Starch and lipids represent two major forms of carbon and energy storage in plants and play central roles in diverse cellular processes. However, whether and how starch and lipid metabolic pathways interact to regulate metabolism and growth are poorly understood. Here, we show that lipids can partially compensate for the lack of function of transient starch during normal growth and development in Arabidopsis (Arabidopsis thaliana). Disruption of starch synthesis resulted in a significant increase in fatty acid synthesis via posttranslational regulation of the plastidic acetyl-coenzyme A carboxylase and a concurrent increase in the synthesis and turnover of membrane lipids and triacylglycerol. Genetic analysis showed that blocking fatty acid peroxisomal ß-oxidation, the sole pathway for metabolic breakdown of fatty acids in plants, significantly compromised or stunted the growth and development of mutants defective in starch synthesis under long days or short days, respectively. We also found that the combined disruption of starch synthesis and fatty acid turnover resulted in increased accumulation of membrane lipids, triacylglycerol, and soluble sugars and altered fatty acid flux between the two lipid biosynthetic pathways compartmentalized in either the chloroplast or the endoplasmic reticulum. Collectively, our findings provide insight into the role of fatty acid ß-oxidation and the regulatory network controlling fatty acid synthesis, and they reveal the mechanistic basis by which starch and lipid metabolic pathways interact and undergo cross talk to modulate carbon allocation, energy homeostasis, and plant growth.
Subject(s)
Lipid Metabolism , Membrane Lipids/biosynthesis , Plant Leaves/metabolism , Starch/metabolism , Acetyl-CoA Carboxylase/genetics , Acetyl-CoA Carboxylase/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Fatty Acids/biosynthesis , Gene Expression Regulation, Developmental , Gene Expression Regulation, Plant , Plant Leaves/genetics , Plant Leaves/growth & development , Plants, Genetically Modified , Triglycerides/biosynthesisABSTRACT
Neutral lipid metabolism is a key aspect of intracellular homeostasis and energy balance and plays a vital role in cell survival under adverse conditions, including nutrient deprivation in yeast and mammals, but the role of triacylglycerol (TAG) metabolism in plant stress response remains largely unknown. By thoroughly characterizing mutants defective in SUGAR-DEPENDENT1 (SDP1) triacylglycerol lipase or PEROXISOMAL ABC TRANSPORTER 1 (PXA1), here we show that TAG is a key intermediate in the mobilization of fatty acids from membrane lipids for peroxisomal ß-oxidation under prolonged dark treatment. Disruption of SDP1 increased TAG accumulation in cytosolic lipid droplets and markedly enhanced plant tolerance to extended darkness. We demonstrate that blocking TAG hydrolysis enhances plant tolerance to dark treatment via two distinct mechanisms. In pxa1 mutants, in which free fatty acids accumulated rapidly under extended darkness, SDP1 disruption resulted in a marked decrease in levels of cytotoxic lipid intermediates such as free fatty acids and phosphatidic acid, suggesting a buffer function of TAG accumulation against lipotoxicity under fatty acid overload. In the wild type, in which free fatty acids remained low and unchanged under dark treatment, disruption of SDP1 caused a decrease in reactive oxygen species production and hence the level of lipid peroxidation, indicating a role of TAG in protection against oxidative damage. Overall, our findings reveal a crucial role for TAG metabolism in membrane lipid breakdown, fatty acid turnover, and plant survival under extended darkness.
Subject(s)
Arabidopsis/physiology , Darkness , Fatty Acids/metabolism , Membrane Lipids/metabolism , Triglycerides/metabolism , Adaptation, Physiological , Arabidopsis Proteins/metabolism , Lipid Peroxidation , Mutation/genetics , Oxidation-Reduction , Oxidative Stress , Phosphatidic Acids/metabolism , Phosphatidylcholines/metabolism , Phosphatidylethanolamines/metabolism , Plant Leaves/metabolism , Plant Leaves/ultrastructureABSTRACT
Eukaryotic cells are characterized by compartmentalization and specialization of metabolism within membrane-bound organelles. Nevertheless, many fundamental processes extend across multiple subcellular compartments. Here, we describe and assess the pathways and cellular organization of triacylglycerol biosynthesis in microalgae. In particular, we emphases the dynamic interplay among the endoplasmic reticulum, lipid droplets and chloroplasts in acyl remodeling and triacylglycerol accumulation under nitrogen starvation in the model alga Chlamydomonas reinhardtii.
Subject(s)
Microalgae/metabolism , Triglycerides/biosynthesis , Acyl Coenzyme A/metabolism , Diglycerides/biosynthesis , Subcellular Fractions/metabolism , Triglycerides/metabolismABSTRACT
The biogenesis of photosynthetic membranes in the plastids of higher plants requires an extensive supply of lipid precursors from the endoplasmic reticulum (ER). Four TRIGALACTOSYLDIACYLGLYCEROL (TGD) proteins (TGD1,2,3,4) have thus far been implicated in this lipid transfer process. While TGD1, TGD2, and TGD3 constitute an ATP binding cassette transporter complex residing in the plastid inner envelope, TGD4 is a transmembrane lipid transfer protein present in the outer envelope. These observations raise questions regarding how lipids transit across the aqueous intermembrane space. Here, we describe the isolation and characterization of a novel Arabidopsis thaliana gene, TGD5. Disruption of TGD5 results in similar phenotypic effects as previously described in tgd1,2,3,4 mutants, including deficiency of ER-derived thylakoid lipids, accumulation of oligogalactolipids, and triacylglycerol. Genetic analysis indicates that TGD4 is epistatic to TGD5 in ER-to-plastid lipid trafficking, whereas double mutants of a null tgd5 allele with tgd1-1 or tgd2-1 show a synergistic embryo-lethal phenotype. TGD5 encodes a small glycine-rich protein that is localized in the envelope membranes of chloroplasts. Coimmunoprecipitation assays show that TGD5 physically interacts with TGD1, TGD2, TGD3, and TGD4. Collectively, these results suggest that TGD5 facilitates lipid transfer from the outer to the inner plastid envelope by bridging TGD4 with the TGD1,2,3 transporter complex.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Arabidopsis/genetics , Arabidopsis/ultrastructure , Arabidopsis Proteins/genetics , Biological Transport , Carrier Proteins/genetics , Carrier Proteins/metabolism , Endoplasmic Reticulum/metabolism , Epistasis, Genetic , Intracellular Membranes/metabolism , Lipid Metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Mesophyll Cells , Mutation , Phenotype , Plastids/metabolism , Protein Binding , Thylakoids/metabolismABSTRACT
Triacylglycerol (TAG) metabolism is a key aspect of intracellular lipid homeostasis in yeast and mammals, but its role in vegetative tissues of plants remains poorly defined. We previously reported that PHOSPHOLIPID:DIACYLGLYCEROL ACYLTRANSFERASE1 (PDAT1) is crucial for diverting fatty acids (FAs) from membrane lipid synthesis to TAG and thereby protecting against FA-induced cell death in leaves. Here, we show that overexpression of PDAT1 enhances the turnover of FAs in leaf lipids. Using the trigalactosyldiacylglycerol1-1 (tgd1-1) mutant, which displays substantially enhanced PDAT1-mediated TAG synthesis, we demonstrate that disruption of SUGAR-DEPENDENT1 (SDP1) TAG lipase or PEROXISOMAL TRANSPORTER1 (PXA1) severely decreases FA turnover, leading to increases in leaf TAG accumulation, to 9% of dry weight, and in total leaf lipid, by 3-fold. The membrane lipid composition of tgd1-1 sdp1-4 and tgd1-1 pxa1-2 double mutants is altered, and their growth and development are compromised. We also show that two Arabidopsis thaliana lipin homologs provide most of the diacylglycerol for TAG synthesis and that loss of their functions markedly reduces TAG content, but with only minor impact on eukaryotic galactolipid synthesis. Collectively, these results show that Arabidopsis lipins, along with PDAT1 and SDP1, function synergistically in directing FAs toward peroxisomal ß-oxidation via TAG intermediates, thereby maintaining membrane lipid homeostasis in leaves.
Subject(s)
Acyltransferases/metabolism , Arabidopsis Proteins/metabolism , Carboxylic Ester Hydrolases/metabolism , Fatty Acids/metabolism , Homeostasis , Membrane Lipids/metabolism , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/metabolism , Acyltransferases/genetics , Adenosine Triphosphatases , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis Proteins/genetics , Carboxylic Ester Hydrolases/genetics , Gene Expression Regulation, Plant , Lipase/genetics , Lipase/metabolism , Lipid Droplets/metabolism , Lipid Droplets/ultrastructure , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Microscopy, Electron, Transmission , Microscopy, Fluorescence , Models, Biological , Mutation , Oxidation-Reduction , Peroxisomes/metabolism , Plant Leaves/genetics , Plant Leaves/metabolism , Plant Leaves/ultrastructure , Plants, Genetically Modified , Reverse Transcriptase Polymerase Chain Reaction , Triglycerides/metabolism , beta-Glucosidase/genetics , beta-Glucosidase/metabolismABSTRACT
Microalgae are diverse groups of eukaryotic organisms capable of efficiently converting sunlight into chemical energy through photosynthesis with carbohydrates and oils as major storage products. Like other eukaryotes, microalgae store oils in dynamic subcellular organelles named oil droplets. In this chapter, we present a detailed description of basic procedures that can be followed for the isolation of mutants defective in oil droplet biogenesis and for the imaging and analysis of oil droplets in the model unicellular green alga Chlamydomonas reinhardtii. Several commonly used methods for isolating and purifying oil droplets in microalgae are discussed.
Subject(s)
Inclusion Bodies/metabolism , Microalgae/metabolism , Oils/metabolism , Organelles/metabolism , Carbohydrates/chemistry , Chlamydomonas reinhardtii/genetics , Chlamydomonas reinhardtii/metabolism , Inclusion Bodies/chemistry , Microalgae/chemistry , Mutation , Organelles/chemistry , Photosynthesis/genetics , Sunlight , Triglycerides/chemistry , Triglycerides/metabolismABSTRACT
Phospholipid:diacylglycerol acyltransferase (PDAT) and diacylglycerol:acyl CoA acyltransferase play overlapping roles in triacylglycerol (TAG) assembly in Arabidopsis, and are essential for seed and pollen development, but the functional importance of PDAT in vegetative tissues remains largely unknown. Taking advantage of the Arabidopsis tgd1-1 mutant that accumulates oil in vegetative tissues, we demonstrate here that PDAT1 is crucial for TAG biosynthesis in growing tissues. We show that disruption of PDAT1 in the tgd1-1 mutant background causes serious growth retardation, gametophytic defects and premature cell death in developing leaves. Lipid analysis data indicated that knockout of PDAT1 results in increases in the levels of free fatty acids (FFAs) and diacylglycerol. In vivo ¹4C-acetate labeling experiments showed that, compared with wild-type, tgd1-1 exhibits a 3.8-fold higher rate of fatty acid synthesis (FAS), which is unaffected by disruption or over-expression of PDAT1, indicating a lack of feedback regulation of FAS in tgd1-1. We also show that detached leaves of both pdat1-2 and tgd1-1 pdat1-2 display increased sensitivity to FFA but not to diacylglycerol. Taken together, our results reveal a critical role for PDAT1 in mediating TAG synthesis and thereby protecting against FFA-induced cell death in fast-growing tissues of plants.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/enzymology , Diacylglycerol O-Acyltransferase/metabolism , Gene Expression Regulation, Enzymologic , Membrane Transport Proteins/metabolism , Triglycerides/biosynthesis , Acyltransferases/genetics , Acyltransferases/metabolism , Arabidopsis/drug effects , Arabidopsis/genetics , Arabidopsis/physiology , Arabidopsis Proteins/genetics , Cell Death/drug effects , Diacylglycerol O-Acyltransferase/genetics , Fatty Acids, Nonesterified/pharmacology , Gene Expression Regulation, Plant , Gene Knockout Techniques , Lipids/analysis , Membrane Transport Proteins/genetics , Models, Biological , Mutation , Plant Leaves/drug effects , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Leaves/physiology , Seedlings/drug effects , Seedlings/enzymology , Seedlings/genetics , Seedlings/physiology , Seeds/drug effects , Seeds/enzymology , Seeds/genetics , Seeds/physiologyABSTRACT
There is growing interest in engineering green biomass to expand the production of plant oils as feed and biofuels. Here, we show that phospholipid:diacylglycerol acyltransferase1 (PDAT1) is a critical enzyme involved in triacylglycerol (TAG) synthesis in leaves. Overexpression of PDAT1 increases leaf TAG accumulation, leading to oil droplet overexpansion through fusion. Ectopic expression of oleosin promotes the clustering of small oil droplets. Coexpression of PDAT1 with oleosin boosts leaf TAG content by up to 6.4% of the dry weight without affecting membrane lipid composition and plant growth. PDAT1 overexpression stimulates fatty acid synthesis (FAS) and increases fatty acid flux toward the prokaryotic glycerolipid pathway. In the trigalactosyldiacylglycerol1-1 mutant, which is defective in eukaryotic thylakoid lipid synthesis, the combined overexpression of PDAT1 with oleosin increases leaf TAG content to 8.6% of the dry weight and total leaf lipid by fourfold. In the plastidic glycerol-3-phosphate acyltransferase1 mutant, which is defective in the prokaryotic glycerolipid pathway, PDAT1 overexpression enhances TAG content at the expense of thylakoid membrane lipids, leading to defects in chloroplast division and thylakoid biogenesis. Collectively, these results reveal a dual role for PDAT1 in enhancing fatty acid and TAG synthesis in leaves and suggest that increasing FAS is the key to engineering high levels of TAG accumulation in green biomass.
Subject(s)
Arabidopsis/enzymology , Diacylglycerol O-Acyltransferase/metabolism , Gene Expression Regulation, Plant , Phospholipids/metabolism , Triglycerides/metabolism , Arabidopsis/genetics , Arabidopsis/growth & development , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Diacylglycerol O-Acyltransferase/genetics , Fatty Acids/metabolism , Galactolipids/metabolism , Gene Expression , Membrane Lipids/metabolism , Mutation , Phenotype , Plant Leaves/enzymology , Plant Leaves/genetics , Plant Leaves/growth & development , Plant Oils/metabolism , Plants, Genetically Modified , Thylakoids/metabolismABSTRACT
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.