Mechanisms of Spirodela polyrhiza tolerance to FGD wastewater-induced heavy-metal stress: Lipidomics, transcriptomics, and functional validation.

Unlike terrestrial angiosperm plants, the freshwater aquatic angiosperm duckweed (Spirodela polyrhiza) grows directly in water and has distinct responses to heavy-metal stress. Plantlets accumulate metabolites, including lipids and carbohydrates, under heavy-metal stress, but how they balance metabolite levels is unclear, and the gene networks that mediate heavy-metal stress responses remain unknown. Here, we show that heavy-metal stress induced by flue gas desulfurization (FGD) wastewater reduces chlorophyll contents, inhibits growth, reduces membrane lipid biosynthesis, and stimulates membrane lipid degradation in S. polyrhiza, leading to triacylglycerol and carbohydrate accumulation. In FGD wastewater-treated plantlets, the degraded products of monogalactosyldiacylglycerol, primarily polyunsaturated fatty acids (18:3), were incorporated into triacylglycerols. Genes involved in early fatty acid biosynthesis, ß-oxidation, and lipid degradation were upregulated while genes involved in cuticular wax biosynthesis were downregulated by treatment. The transcription factor gene WRINKLED3 (SpWRI3) was upregulated in FGD wastewater-treated plantlets, and its ectopic expression increased tolerance to FGD wastewater in transgenic Arabidopsis (Arabidopsis thaliana). Transgenic Arabidopsis plants showed enhanced glutathione and lower malondialdehyde contents under stress, suggesting that SpWRI3 functions in S. polyrhiza tolerance of FGD wastewater-induced heavy-metal stress. These results provide a basis for improving heavy metal-stress tolerance in plants for industrial applications.

Physiological Functions of Phospholipid: Diacylglycerol Acyltransferases.

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.

The van der Waals interaction and absorption and electron circular dichroism spectra of two-dimensional bilayer stacked structures.

van der Waals (vdW) heterojunctions based on two-dimensional (2D) materials, graphene and transition metal dichalcogenides (TMDs), are a research hotspot for future optoelectronic and exciton devices. Bond-free vdW interactions are key to 2D material heterojunction device reliability and stability. However, most of the current research on 2D stacked materials heterostructures mainly focuses on optical properties and electronic structure. Furthermore, vdW interaction in 2D heterostructures is studied and understood on the basis of qualitative description and energy ranges from the literature. There are few studies on the nature of vdW interaction based on practical calculations of the quantitative strength and microscopic mechanism of vdW interaction between 2D stacked materials. Therefore, this paper explores the vdW interaction between 2D material stacked bilayer structures, including bilayer graphene, graphene/MoS2 and graphene/WS2 heterostructures, focusing on quantitative analysis of the energy components of the vdW interaction. We first visually observed the weak interactions in the three stacked bilayer structures through noncovalent interaction (NCI) analysis, and found that the interactions are concentrated in the binding region between the two-layer structures. We mainly decomposed the weak interaction energy in the three 2D material bilayer heterostructures through energy decomposition analysis based on the force field (EDA-FF) method and obtained the energy values and proportions of the three components-electrostatic energy, exchange repulsion energy and dispersion energy of the total binding energy between the 2D stacked bilayer structures. The vdW interaction energy is the sum of the exchange repulsion energy and dispersion energy, and the dispersion energy of the vdW interaction accounts for more than 60% of the binding energy of the weak interaction between the 2D bilayer stacked structures. The vdW strengths in the bilayer structures are on the order of 177.07, 123.85, and 133.93 kJ/mol, approxmately 1-2 orders of magnitude larger than the classically defined vdW energies of 0.1-10 kJ/mol. Furthermore, we calculate the density of states of the three 2D stacked structures, and further obtained HOMO-LOMO information; to further understand the electronic structures of the graphene/MoS2 and graphene/WS2 heterostructures, we calculated their optical absorption spectra and electron circular dichroism (ECD) spectra. According to the calculation results, the two heterostructures have strong absorption peaks in the visible region, and the charge transfer forms at the strong absorption peak can be determined according to the charge transfer diagram. The ECD spectra indicate that the configurations of the graphene/MoS2 and graphene/WS2 heterostructures have large chirality. Our work contributes to a deeper understanding of the nature of the weak interactions and optical properties in 2D stacked materials, which plays a fundamental role in promoting the construction of stable 2D heterostructure configurations and the development of multifunctional 2D devices. The research is conducive to further promoting the basic research and practical development of strong optoelectronic and excitonic 2D heterojunctions devices.

A chloroplast diacylglycerol lipase modulates glycerolipid pathway balance in Arabidopsis.

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.

QTL Analysis Reveals Conserved and Differential Genetic Regulation of Maize Lateral Angles above the Ear.

Improving the density tolerance and planting density has great importance for increasing maize production. The key to promoting high density planting is breeding maize with a compact canopy architecture, which is mainly influenced by the angles of the leaves and tassel branches above the ear. It is still unclear whether the leaf angles of different stem nodes and tassel branches are controlled by similar genetic regulatory mechanisms, which limits the ability to breed for density-tolerant maize. Here, we developed a population with 571 double haploid lines derived from inbred lines, PHBA6 and Chang7-2, showing significant differences in canopy architecture. Phenotypic and QTL analyses revealed that the genetic regulation mechanism was largely similar for closely adjacent leaves above the ears. In contrast, the regulation mechanisms specifying the angles of distant leaves and the angles of leaves vs. tassel branches are largely different. The liguless1 gene was identified as a candidate gene for QTLs co-regulating the angles of different leaves and the tassel branch, consistent with its known roles in regulating plant architecture. Our findings can be used to develop strategies for the improvement of leaf and tassel architecture through the introduction of trait-specific or pleiotropic genes, thus benefiting the breeding of maize with increased density tolerance in the future.

Purple acid phosphatase2 stimulates a futile cycle of lipid synthesis and degradation, and mitigates the negative growth effects of triacylglycerol accumulation in vegetative tissues.

Storage lipids (mostly triacylglycerols, TAGs) serve as an important energy and carbon reserve in plants, and hyperaccumulation of TAG in vegetative tissues can have negative effects on plant growth. Purple acid phosphatase2 (PAP2) was previously shown to affect carbon metabolism and boost plant growth. However, the effects of PAP2 on lipid metabolism remain unknown. Here, we demonstrated that PAP2 can stimulate a futile cycle of fatty acid (FA) synthesis and degradation, and mitigate negative growth effects associated with high accumulation of TAG in vegetative tissues. Constitutive expression of PAP2 in Arabidopsis thaliana enhanced both lipid synthesis and degradation in leaves and led to a substantial increase in seed oil yield. Suppressing lipid degradation in a PAP2-overexpressing line by disrupting sugar-dependent1 (SDP1), a predominant TAG lipase, significantly elevated vegetative TAG content and improved plant growth. Diverting FAs from membrane lipids to TAGs in PAP2-overexpressing plants by constitutively expressing phospholipid:diacylglycerol acyltransferase1 (PDAT1) greatly increased TAG content in vegetative tissues without compromising biomass yield. These results highlight the potential of combining PAP2 with TAG-promoting factors to enhance carbon assimilation, FA synthesis and allocation to TAGs for optimized plant growth and storage lipid accumulation in vegetative tissues.

Links between autophagy and lipid droplet dynamics.

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.

Sterols are required for the coordinated assembly of lipid droplets in developing seeds.

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.

The Role of Sugar Signaling in Regulating Plant Fatty Acid Synthesis.

Photosynthates such as glucose, sucrose, and some of their derivatives play dual roles as metabolic intermediates and signaling molecules that influence plant cell metabolism. Such sugars provide substrates for de novo fatty acid (FA) biosynthesis. However, compared with the well-defined examples of sugar signaling in starch and anthocyanin synthesis, until recently relatively little was known about the role of signaling in regulating FA and lipid biosynthesis. Recent research progress shows that trehalose 6-phosphate and 2-oxoglutarate (2-OG) play direct signaling roles in the regulation of FA biosynthesis by modulating transcription factor stability and enzymatic activities involved in FA biosynthesis. Specifically, mechanistic links between sucrose non-fermenting-1-related protein kinase 1 (SnRK1)-mediated trehalose 6-phosphate (T6P) sensing and its regulation by phosphorylation of WRI1 stability, diacylglycerol acyltransferase 1 (DGAT1) enzyme activity, and of 2-OG-mediated relief of inhibition of acetyl-CoA carboxylase (ACCase) activity by protein PII are exemplified in detail in this review.

Mechanisms and functions of membrane lipid remodeling in plants.

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.

Using 14C-acetate Pulse-chase Labeling to Study Fatty Acid and Glycerolipid Metabolism in Plant Leaves.

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 ).

Chloroplast lipid biosynthesis is fine-tuned to thylakoid membrane remodeling during light acclimation.

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.

Metabolic and functional connections between cytoplasmic and chloroplast triacylglycerol storage.

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.

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.

Dual Role for Autophagy in Lipid Metabolism in Arabidopsis.

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.

Peroxisomal fatty acid ß-oxidation negatively impacts plant survival under salt stress.

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.

Starch Deficiency Enhances Lipid Biosynthesis and Turnover in Leaves.

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.

Cytokinin Signaling in Mycobacterium tuberculosis.

It was recently reported that the human-exclusive pathogen Mycobacterium tuberculosis secretes cytokinins, which had only been known as plant hormones. While cytokinins are well-established, adenine-based signaling molecules in plants, they have never been shown to participate in signal transduction in other kingdoms of life. M. tuberculosis is not known to interact with plants. Therefore, we tested the hypothesis that cytokinins trigger transcriptional changes within this bacterial species. Here, we show cytokinins induced the strong expression of the M. tuberculosis gene Rv0077c. We found that Rv0077c expression is repressed by a TetR-like transcriptional repressor, Rv0078. Strikingly, cytokinin-induced expression of Rv0077c resulted in a loss of acid-fast staining of M. tuberculosis While acid-fast staining is thought to be associated with changes in the bacterial cell envelope and virulence, Rv0077c-induced loss of acid-fastness did not affect antibiotic susceptibility or attenuate bacterial growth in mice, consistent with an unaltered mycolic acid profile of Rv0077c-expressing cells. Collectively, these findings show cytokinins signal transcriptional changes that can affect M. tuberculosis acid-fastness and that cytokinin signaling is no longer limited to the kingdom Plantae.IMPORTANCE Cytokinins have only previously been known as plant hormones. The discovery that they can be used as signaling molecules outside of plants broadens the repertoire of small molecules that can potentially affect gene expression in all domains of life.

Gravity-directed separation of both immiscible and emulsified oil/water mixtures utilizing coconut shell layer.

Pressure-driven and lower flux of superwetting ultrafiltration membranes in various emulsions separation are long-standing issues and major barriers for their large-scale utilization. Even though currently reported membranes have achieved great success in emulsions separeation, they still suffer from low flux and complex fabrication process resulting from their smaller nanoscale pore size. Herein, utilizition of coconut shell as a novel biomaterial for developing into a layer through the simple smashing, cleaning and stacking procedures, which not only could avoid the complexity of film making process, but also could realize efficient gravity-directed separation of both immiscible oil/water mixtures and water-in-oil emulsions with high flux. Specifically, the layer acted as "water-removing" type filtrate material with excellent underwater superoleophobicity, exhibiting high efficiency for various immiscible oil/water mixtures separation and larger oil intrusion pressure. More importantly, the layer could also serve as adsorbent material with underoil superhydrophilicity, achieving gravity-directed kinds of water-in-oil emulsions separation with high separation efficiency (above 99.99%) and higher flux (above 1620L/m2h), even when their pore sizes are larger than that of emulsified droplets. We deeply believe that this study would open up a new strategy for both immiscible oil/water mixtures and water-in-oil emulsions separation.

Sugar Potentiation of Fatty Acid and Triacylglycerol Accumulation.

Photosynthetically derived sugar provides carbon skeletons for lipid biosynthesis. We used mutants of Arabidopsis (Arabidopsis thaliana) and the expression of oleogenic factors to investigate relationships among sugar availability, lipid synthesis, and the accumulation of triacylglycerol (TAG) in leaf tissue. The adg1 mutation disables the small subunit of ADP-glucose pyrophosphorylase, the first step in starch synthesis, and the suc2 mutation disables a sucrose/proton symporter that facilitates sucrose loading from leaves into phloem. The adg1suc2 double mutant increases glucose plus sucrose content in leaves 80-fold relative to the wild type, total fatty acid (FA) content 1.8-fold to 8.3% dry weight, and TAG more than 10-fold to 1.2% dry weight. The WRINKLED1 transcription factor also accumulates to higher levels in these leaves, and the rate of FA synthesis increases by 58%. Adding tt4, which disables chalcone synthase, had little effect, but adding the tgd1 mutation, which disables an importer of lipids into plastids to create adg1suc2tt4tgd1, increased total leaf FA to 13.5% dry weight and TAG to 3.8% dry weight, demonstrating a synergistic effect upon combining these mutations. Combining adg1suc2 with the sdp1 mutation, deficient in the predominant TAG lipase, had little effect on total FA content but increased the TAG accumulation by 66% to 2% dry weight. Expression of the WRINKLED1 transcription factor, along with DIACYLGLYCEROL ACYLTRANSFERASE1 and the OLEOSIN1 oil body-associated protein, in the adg1suc2 mutant doubled leaf FA content and increased TAG content to 2.3% dry weight, a level 4.6-fold higher than that resulting from expression of the same factors in the wild type.