The Root Cap Cuticle: A Cell Wall Structure for Seedling Establishment and Lateral Root Formation.

The root cap surrounding the tip of plant roots is thought to protect the delicate stem cells in the root meristem. We discovered that the first layer of root cap cells is covered by an electron-opaque cell wall modification resembling a plant cuticle. Cuticles are polyester-based protective structures considered exclusive to aerial plant organs. Mutations in cutin biosynthesis genes affect the composition and ultrastructure of this cuticular structure, confirming its cutin-like characteristics. Strikingly, targeted degradation of the root cap cuticle causes a hypersensitivity to abiotic stresses during seedling establishment. Furthermore, lateral root primordia also display a cuticle that, when defective, causes delayed outgrowth and organ deformations, suggesting that it facilitates lateral root emergence. Our results show that the previously unrecognized root cap cuticle protects the root meristem during the critical phase of seedling establishment and promotes the efficient formation of lateral roots.

Dividing cells regulate their lipid composition and localization.

Although massive membrane rearrangements occur during cell division, little is known about specific roles that lipids might play in this process. We report that the lipidome changes with the cell cycle. LC-MS-based lipid profiling shows that 11 lipids with specific chemical structures accumulate in dividing cells. Using AFM, we demonstrate differences in the mechanical properties of live dividing cells and their isolated lipids relative to nondividing cells. In parallel, systematic RNAi knockdown of lipid biosynthetic enzymes identified enzymes required for division, which highly correlated with lipids accumulated in dividing cells. We show that cells specifically regulate the localization of lipids to midbodies, membrane-based structures where cleavage occurs. We conclude that cells actively regulate and modulate their lipid composition and localization during division, with both signaling and structural roles likely. This work has broader implications for the active and sustained participation of lipids in basic biology.

Discovery, structure and mechanism of a tetraether lipid synthase.

Archaea synthesize isoprenoid-based ether-linked membrane lipids, which enable them to withstand extreme environmental conditions, such as high temperatures, high salinity, and low or high pH values1-5. In some archaea, such as Methanocaldococcus jannaschii, these lipids are further modified by forming carbon-carbon bonds between the termini of two lipid tails within one glycerophospholipid to generate the macrocyclic archaeol or forming two carbon-carbon bonds between the termini of two lipid tails from two glycerophospholipids to generate the macrocycle glycerol dibiphytanyl glycerol tetraether (GDGT)1,2. GDGT contains two 40-carbon lipid chains (biphytanyl chains) that span both leaflets of the membrane, providing enhanced stability to extreme conditions. How these specialized lipids are formed has puzzled scientists for decades. The reaction necessitates the coupling of two completely inert sp3-hybridized carbon centres, which, to our knowledge, has not been observed in nature. Here we show that the gene product of mj0619 from M. jannaschii, which encodes a radical S-adenosylmethionine enzyme, is responsible for biphytanyl chain formation during synthesis of both the macrocyclic archaeol and GDGT membrane lipids6. Structures of the enzyme show the presence of four metallocofactors: three [Fe4S4] clusters and one mononuclear rubredoxin-like iron ion. In vitro mechanistic studies show that Csp3-Csp3 bond formation takes place on fully saturated archaeal lipid substrates and involves an intermediate bond between the substrate carbon and a sulfur of one of the [Fe4S4] clusters. Our results not only establish the biosynthetic route for tetraether formation but also improve the use of GDGT in GDGT-based paleoclimatology indices7-10.

Identification of two archaeal GDGT lipid-modifying proteins reveals diverse microbes capable of GMGT biosynthesis and modification.

Archaea produce unique membrane-spanning lipids (MSLs), termed glycerol dialkyl glycerol tetraethers (GDGTs), which aid in adaptive responses to various environmental challenges. GDGTs can be modified through cyclization, cross-linking, methylation, hydroxylation, and desaturation, resulting in structurally distinct GDGT lipids. Here, we report the identification of radical SAM proteins responsible for two of these modifications-a glycerol monoalkyl glycerol tetraether (GMGT) synthase (Gms), responsible for covalently cross-linking the two hydrocarbon tails of a GDGT to produce GMGTs, and a GMGT methylase (Gmm), capable of methylating the core hydrocarbon tail. Heterologous expression of Gms proteins from various archaea in Thermococcus kodakarensis results in the production of GMGTs in two isomeric forms. Further, coexpression of Gms and Gmm produces mono- and dimethylated GMGTs and minor amounts of trimethylated GMGTs with only trace GDGT methylation. Phylogenetic analyses reveal the presence of Gms homologs in diverse archaeal genomes spanning all four archaeal superphyla and in multiple bacterial phyla with the genetic potential to synthesize fatty acid-based MSLs, demonstrating that GMGT production may be more widespread than previously appreciated. We demonstrate GMGT production in three Gms-encoding archaea, identifying an increase in GMGTs in response to elevated temperature in two Archaeoglobus species and the production of GMGTs with up to six rings in Vulcanisaeta distributa. The occurrence of such highly cyclized GMGTs has been limited to environmental samples and their detection in culture demonstrates the utility of combining genetic, bioinformatic, and lipid analyses to identify producers of distinct archaeal membrane lipids.

GDGT cyclization proteins identify the dominant archaeal sources of tetraether lipids in the ocean.

Glycerol dibiphytanyl glycerol tetraethers (GDGTs) are distinctive archaeal membrane-spanning lipids with up to eight cyclopentane rings and/or one cyclohexane ring. The number of rings added to the GDGT core structure can vary as a function of environmental conditions, such as changes in growth temperature. This physiological response enables cyclic GDGTs preserved in sediments to be employed as proxies for reconstructing past global and regional temperatures and to provide fundamental insights into ancient climate variability. Yet, confidence in GDGT-based paleotemperature proxies is hindered by uncertainty concerning the archaeal communities contributing to GDGT pools in modern environments and ambiguity in the environmental and physiological factors that affect GDGT cyclization in extant archaea. To properly constrain these uncertainties, a comprehensive understanding of GDGT biosynthesis is required. Here, we identify 2 GDGT ring synthases, GrsA and GrsB, essential for GDGT ring formation in Sulfolobus acidocaldarius Both proteins are radical S-adenosylmethionine proteins, indicating that GDGT cyclization occurs through a free radical mechanism. In addition, we demonstrate that GrsA introduces rings specifically at the C-7 position of the core GDGT lipid, while GrsB cyclizes at the C-3 position, suggesting that cyclization patterns are differentially controlled by 2 separate enzymes and potentially influenced by distinct environmental factors. Finally, phylogenetic analyses of the Grs proteins reveal that marine Thaumarchaeota, and not Euryarchaeota, are the dominant source of cyclized GDGTs in open ocean settings, addressing a major source of uncertainty in GDGT-based paleotemperature proxy applications.

Lipidomic and transcriptomic profiling of developing nodules reveals the essential roles of active glycolysis and fatty acid and membrane lipid biosynthesis in soybean nodulation.

Symbiotic rhizobia-legume interactions are energy-demanding processes, and the carbon supply from host cells that is critically required for nodulation and nitrogen fixation is not fully understood. Investigation of the lipidomic and carbohydrate profiles with the transcriptome of developing nodules revealed highly activated glycolysis, fatty acid (FA), 2-monoacylglycerol (2-MAG), and membrane lipid biosynthesis and transport during nodule development. RNA-sequence profiling of metabolic genes in roots and developing nodules highlighted the enhanced expression of genes involved in the biosynthesis and transport of FAs, membrane lipids, and 2-MAG in rhizobia-soybean symbioses via the RAML-WRI-FatM-GPAT-STRL pathway, which is similar to that in legume-arbuscular mycorrhizal fungi symbiosis. The essential roles of the metabolic pathway during soybean nodulation were further supported by analysis of transgenic hairy roots overexpressing soybean GmWRI1b-OE and GmLEC2a-OE. GmLEC2a-OE hairy roots produced fewer nodules, in contrast to GmWRI1b-OE hairy roots. GmLEC2a-OE hairy roots displayed different or even opposite expression patterns of the genes involved in glycolysis and the synthesis of FAs, 2-MAG, TAG, and membrane lipids compared to GmWRI1b-OE hairy roots. Glycolysis, FA and membrane lipid biosynthesis were repressed in GmLEC2a-OE but increased in GmWRI1b-OE hairy roots, which may account for the reduced nodulation in GmLEC2a-OE hairy roots but increased nodulation in GmWRI1b-OE hairy roots. These data show that active FA, 2-MAG and membrane lipid biosynthesis are essential for nodulation and rhizobia-soybean symbioses. These data shed light on essential and complex lipid metabolism for soybean nodulation and nodule development, laying the foundation for the future detailed investigation of soybean nodulation.

The Endoplasmic Reticulum Pathway for Membrane Lipid Synthesis Has a Significant Contribution toward Shoot Removal-Induced Root Chloroplast Development in Arabidopsis.

Chloroplast lipids are synthesized via two distinct pathways: the plastidic pathway and endoplasmic reticulum (ER) pathway. We previously reported that the contribution of the two pathways toward chloroplast development is different between mesophyll cells and guard cells in Arabidopsis leaf tissues and that the ER pathway plays a major role in guard cell chloroplast development. However, little is known about the contribution of the two pathways toward chloroplast development in other tissue cells, and in this study, we focused on root cells. Chloroplast development is normally repressed in roots but can be induced when the roots are detached from the shoots (root greening). We found that, similar to guard cells, root cells exhibit a higher proportion of glycolipid from the ER pathway. Root greening was repressed in the gles1 mutant, which has a defect in ER-to-plastid lipid transportation via the ER pathway, while normal root greening was observed in the ats1 mutant, whose plastidic pathway is blocked. Lipid analysis revealed that the gles1 mutation caused drastic decrease in the ER-derived glycolipids in roots. Furthermore, the gles1 detached roots showed smaller chloroplasts containing less starch than WT. These results suggest that the ER pathway has a significant contribution toward chloroplast development in the root cells.

Redirection of lipid flux toward phospholipids in yeast increases fatty acid turnover and secretion.

Bio-based production of fatty acids and fatty acid-derived products can enable sustainable substitution of petroleum-derived fuels and chemicals. However, developing new microbial cell factories for producing high levels of fatty acids requires extensive engineering of lipid metabolism, a complex and tightly regulated metabolic network. Here we generated a Saccharomyces cerevisiae platform strain with a simplified lipid metabolism network with high-level production of free fatty acids (FFAs) due to redirected fatty acid metabolism and reduced feedback regulation. Deletion of the main fatty acid activation genes (the first step in ß-oxidation), main storage lipid formation genes, and phosphatidate phosphatase genes resulted in a constrained lipid metabolic network in which fatty acid flux was directed to a large extent toward phospholipids. This resulted in simultaneous increases of phospholipids by up to 2.8-fold and of FFAs by up to 40-fold compared with wild-type levels. Further deletion of phospholipase genes PLB1 and PLB2 resulted in a 46% decrease in FFA levels and 105% increase in phospholipid levels, suggesting that phospholipid hydrolysis plays an important role in FFA production when phospholipid levels are increased. The multiple deletion mutant generated allowed for a study of fatty acid dynamics in lipid metabolism and represents a platform strain with interesting properties that provide insight into the future development of lipid-related cell factories.

Molecular Cloning and Functional Analysis of GmLACS2-3 Reveals Its Involvement in Cutin and Suberin Biosynthesis along with Abiotic Stress Tolerance.

Cutin and wax are the main precursors of the cuticle that covers the aerial parts of plants and provide protection against biotic and abiotic stresses. Long-chain acyl-CoA synthetases (LACSs) play diversified roles in the synthesis of cutin, wax, and triacylglycerol (TAG). Most of the information concerned with LACS functions is obtained from model plants, whereas the roles of LACS genes in Glycine max are less known. Here, we have identified 19 LACS genes in Glycine max, an important crop plant, and further focused our attention on 4 LACS2 genes (named as GmLACS2-1, 2, 3, 4, respectively). These GmLACS2 genes display different expression patterns in various organs and also show different responses to abiotic stresses, implying that these genes might play diversified functions during plant growth and against stresses. To further identify the role of GmLACS2-3, greatly induced by abiotic stresses, we transformed a construct containing its full length of coding sequence into Arabidopsis. The expression of GmLACS2-3 in an Arabidopsis atlacs2 mutant greatly suppressed its phenotype, suggesting it plays conserved roles with that of AtLACS2. The overexpression of GmLACS2-3 in wild-type plants significantly increased the amounts of cutin and suberin but had little effect on wax amounts, indicating the specific role of GmLACS2-3 in the synthesis of cutin and suberin. In addition, these GmLACS2-3 overexpressing plants showed enhanced drought tolerance. Taken together, our study deepens our understanding of the functions of LACS genes in different plants and also provides a clue for cultivating crops with strong drought resistance.

RRx-001 Increases Erythrocyte Preferential Adhesion to the Tumor Vasculature.

Red blood cells (RBCs) serve a variety of functions beyond mere oxygen transport both in health and pathology. Notably, RRx-001, a minimally toxic pleiotropic anticancer agent with macrophage activating and vascular normalization properties currently in Phase III trials, induces modification to RBCs which could promote vascular adhesion similar to sickle cells. This study assessed whether RBCs exposed to RRx-001 adhere to the tumor microvasculature and whether this adhesion alters tumor viability. We next investigated the biomechanics of RBC adhesion in the context of local inflammatory cytokines after treatment with RRx-001 as a potential mechanism for preferential tumor aggregation. Human HEP-G2 and HT-29 tumor cells were subcutaneously implanted into nu/nu mice and were infused with RRx-001-treated and Technetium-99m (99mTc)-labeled blood. RBC adhesion was quantified in an in vitro human umbilical vein endothelial cell (HUVEC) assay under both normoxic and hypoxic conditions with administration of either lipopolysaccharide (LPS) or Tumor necrosis alpha (TNFα) to mimic the known inflammation in the tumor microenvironment. One hour following administration of 99mTc labeled RBCs treated with 10 mg/kg RRx-001, we observed an approximate 2.0-fold and 1.5-fold increase in 99mTc-labeled RBCs compared to vehicle control in HEPG2 and HT-29 tumor models, respectively. Furthermore, we observed an approximate 40% and 36% decrease in HEP-G2 and HT-29 tumor weight, respectively, following treatment with RRx-001. To quantify RBC adhesive potential, we determined τ50, or the shear stress required for 50% disassociation of RBCs from HUVECs. After administration of TNF-α under normoxia, τ50 was determined to be 4.5 dynes/cm2 (95% CI: 4.3-4.7 dynes/cm2) for RBCs treated with 10 µM RRx-001, which was significantly different (p < 0.05) from τ50 in the absence of treatment. Under hypoxic conditions, the difference of τ50 with (4.8 dynes/cm2; 95% CI: 4.6-5.1 dynes/cm2) and without (2.6 dynes/cm2; 95% CI: 2.4-2.8 dynes/cm2) 10 µM RRx-001 treatment was exacerbated (p = 0.05). In conclusion, we demonstrated that RBCs treated with RRx-001 preferentially aggregate in HEP-G2 and HT-29 tumors, likely due to interactions between RRx-001 and cysteine residues within RBCs. Furthermore, RRx-001 treated RBCs demonstrated increased adhesive potential to endothelial cells upon introduction of TNF-α and hypoxia suggesting that RRx-001 may induce preferential adhesion in the tumor but not in other tissues with endothelial dysfunction due to conditions prevalent in older cancer patients such as heart disease or diabetic vasculopathy.

Adaptations in chloroplast membrane lipid synthesis from synthesis in ancestral cyanobacterial endosymbionts.

Cyanobacteria and chloroplasts are believed to share a common ancestor, but synthetic pathways for membrane lipids are different. Lyso-phosphatidic acid (lyso-PA) is the precursor for the synthesis of all membrane lipids and synthesized by an acyl-ACP dependent glycerol-3-phosphate acyltransferase (GPAT) in chloroplasts. In cyanobacteria, GPAT genes are not found and, instead, genes coding for enzymes in the acyl-phosphate dependent lyso-PA synthetic pathway (plsX and plsY) are conserved. We report that the PlsX/Y dependent lyso-PA synthetic pathway is essential in cyanobacteria, but can be replaced by acyl-ACP dependent GPAT from Escherichia coli (plsB) and Arabidopsis thaliana (ATS1). Cyanobacteria thus display the capacity to accept enzymes from other organisms to synthesize essential components. This ability may have enabled them to evolve into current chloroplasts from their ancestral origins.

Genome-wide analysis of glycerol-3-phosphate O-acyltransferase gene family and functional characterization of two cutin group GPATs in Brassica napus.

MAIN CONCLUSION: Genome-wide identification, spatio-temporal expression analysis and functional characterization of selected Brassica napus GPATs highlight their roles in cuticular wax biosynthesis and defense against fungal pathogens. Glycerol-3-phosphate 1-O-acyltransferase (GPAT) is a key enzyme in the biosynthesis of glycerolipids, a major component of cellular membranes and extracellular protective layers, such as cuticles in plants. Brassica napus is an economically important crop and cultivated worldwide mostly for its edible oil. The B. napus GPATs (BnGPATs) are insufficiently characterized. Here, we performed genome-wide analysis to identify putative GPATs in B. napus and its diploid progenitors B. rapa and B oleracea. The 32 B. napus BnGPATs are phylogenetically divided into three major groups, cutin, suberin, and diverse ancient groups. Analysis of transcriptomes of different tissues and seeds at different developmental stages revealed the spatial and temporal expression profiles of BnGPATs. The yield and oil quality of B. napus are adversely affected by the necrotrophic fungus, Sclerotinia sclerotiorum. We showed that several BnGPATs, including cutin-related BnGPAT19 and 21, were upregulated in the S. sclerotiorum resistant line. RNAi-mediated suppression of BnGPAT19 and 21 in B. napus resulted in thinner cuticle, leading to rapid water and chlorophyll loss in toluidine blue staining and leaf bleaching assays. In addition, the RNAi plants also developed severe necrotic lesions following fungal inoculation compared to the wild-type plants, indicating that BnGPAT19 and 21 are likely involved in cuticular wax biosynthesis that is critical for initial pathogen defense. Taken together, we provided a comprehensive account of GPATs B. napus and characterized BnGPAT19 and 21 for their potential roles in cuticular wax biosynthesis and defense against fungal pathogens.

A Multilevel Study of Melon Fruit Reticulation Provides Insight into Skin Ligno-Suberization Hallmarks.

The skin of fleshy fruit is typically covered by a thick cuticle. Some fruit species develop different forms of layers directly above their skin. Reticulation, for example, is a specialized suberin-based coating that ornaments some commercially important melon (Cucumis melo) fruit and is an important quality trait. Despite its importance, the structural, molecular, and biochemical features associated with reticulation are not fully understood. Here, we performed a multilevel investigation of structural attributes, chemical composition, and gene expression profiles on a set of reticulated and smooth skin melons. High-resolution microscopy, surface profiling, and histochemical staining assays show that reticulation comprises cells with heavily suberized walls accumulating large amounts of typical suberin monomers, as well as lignified cells localized underneath the specialized suberized cell layer. Reticulated skin was characterized by induced expression of biosynthetic genes acting in the core phenylpropanoid, suberin, lignin, and lignan pathways. Transcripts of genes associated with lipid polymer assembly, cell wall organization, and loosening were highly enriched in reticulated skin tissue. These signatures were exclusive to reticulated structures and absent in both the smooth surfaces observed in between reticulated regions and in the skin of smooth fruit. Our data provide important insights into the molecular and metabolic bases of reticulation and its tight association with skin ligno-suberization during melon fruit development. Moreover, these insights are likely to contribute to melon breeding programs aimed at improving postharvest qualities associated with fleshy fruit surface layers.

The R2R3-MYB transcription factor AtMYB49 modulates salt tolerance in Arabidopsis by modulating the cuticle formation and antioxidant defence.

Salt stress activates defence responses in plants, including changes in leaf surface structure. Here, we showed that the transcriptional activation of cutin deposition and antioxidant defence by the R2R3-type MYB transcription factor AtMYB49 contributed to salt tolerance in Arabidopsis thaliana. Characterization of loss-of-function myb49 mutants, and chimeric AtMYB49-SRDX-overexpressing SRDX49 transcriptional repressor and AtMYB49-overexpressing (OX49) overexpressor plants demonstrated a positive role of AtMYB49 in salt tolerance. Transcriptome analysis revealed that many genes belonging to the category "cutin, suberin and wax biosyntheses" were markedly up-regulated and down-regulated in OX49 and SRDX49 plants, respectively, under normal and/or salt stress conditions. Some of these differentially expressed genes, including MYB41, ASFT, FACT and CYP86B1, were also shown to be the direct targets of AtMYB49 and activated by AtMYB49. Biochemical analysis indicated that AtMYB49 modulated cutin deposition in the leaves. Importantly, cuticular transpiration, chlorophyll leaching and toluidine blue-staining assays revealed a link between increased AtMYB49-mediated cutin deposition in leaves and enhanced salt tolerance. Additionally, increased AtMYB49 expression elevated Ca2+ level in leaves and improved antioxidant capacity by up-regulating genes encoding peroxidases and late embryogenesis abundant proteins. These results suggest that genetic manipulation of AtMYB49 may provide a novel way to improve salt tolerance in plants.

Regulation and remodeling of intermediate metabolite and membrane lipid during NaCl-induced stress in freshwater microalga Micractinium sp. XJ-2 for biofuel production.

Microalgae can accumulate a large fraction of reduced carbon as lipids under NaCl stress. This study investigated the mechanism of carbon allocation and reduction and triacylglycerol (TAG) accumulation in microalgae under NaCl-induced stress. Micractinium sp. XJ-2 was exposed to NaCl stress and cells were subjected to physiological, biochemical, and metabolic analyses to elucidate the stress-responsive mechanism. Lipid increased with NaCl concentration after 0-12 hr, then stabilized after 12-48 hr, and finally decreased after 48-72 hr, whereas TAG increased (0-48 hr) and then decreased (48-72 hr). Under NaCl-induced stress at lower concentrations, TAG accumulation, at first, mainly shown to rely on the carbon fixation through photosynthetic fixation, carbohydrate degradation, and membrane lipids remodeling. Moreover, carbon compounds generated by the degradation of some amino acids were reallocated and enhanced fatty acid synthesis. The remodeling of the membrane lipids of NaCl-induced microalgae relied on the following processes: (a) Increase in the amount of phospholipids and reduction in the amount of glycolipids and (b) extension of long-chain fatty acids. This study enhances our understanding of TAG production under NaCl stress and thus will provide a theoretical basis for the industrial application of NaCl-induced in the microalgal biodiesel industry.

Lipid landscapes and pipelines in membrane homeostasis.

The lipid composition of cellular organelles is tailored to suit their specialized tasks. A fundamental transition in the lipid landscape divides the secretory pathway in early and late membrane territories, allowing an adaptation from biogenic to barrier functions. Defending the contrasting features of these territories against erosion by vesicular traffic poses a major logistical problem. To this end, cells evolved a network of lipid composition sensors and pipelines along which lipids are moved by non-vesicular mechanisms. We review recent insights into the molecular basis of this regulatory network and consider examples in which malfunction of its components leads to system failure and disease.

Integrated Metabolomic and Transcriptomic Analysis to Characterize Cutin Biosynthesis between Low- and High-Cutin Genotypes of Capsicum chinense Jacq.

Habanero peppers constantly face biotic and abiotic stresses such as pathogen/pest infections, extreme temperature, drought and UV radiation. In addition, the fruit cutin lipid composition plays an important role in post-harvest water loss rates, which in turn causes shriveling and reduced fruit quality and storage. In this study, we integrated metabolome and transcriptome profiling pertaining to cutin in two habanero genotypes: PI 224448 and PI 257145. The fruits were selected by the waxy or glossy phenotype on their surfaces. Metabolomics analysis showed a significant variation in cutin composition, with about 6-fold higher cutin in PI 257145 than PI 224448. It also revealed that 10,16-dihydroxy hexadecanoic acid is the most abundant monomer in PI 257145. Transcriptomic analysis of high-cutin PI 257145 and low-cutin PI 224448 resulted in the identification of 2703 statistically significant differentially expressed genes, including 1693 genes upregulated and 1010 downregulated in high-cutin PI 257145. Genes and transcription factors such as GDSL lipase, glycerol-3 phosphate acyltransferase 6, long-chain acyltransferase 2, cytochrome P450 86A/77A, SHN1, ANL2 and HDG1 highly contributed to the high cutin content in PI 257145. We predicted a putative cutin biosynthetic pathway for habanero peppers based on deep transcriptome analysis. This is the first study of the transcriptome and metabolome pertaining to cutin in habanero peppers. These analyses improve our knowledge of the molecular mechanisms regulating the accumulation of cutin in habanero pepper fruits. These resources can be built on for developing cultivars with high cutin content that show resistance to biotic and abiotic stresses with superior postharvest appearance.

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.

Reduced Biosynthesis of Digalactosyldiacylglycerol, a Major Chloroplast Membrane Lipid, Leads to Oxylipin Overproduction and Phloem Cap Lignification in Arabidopsis.

DIGALACTOSYLDIACYLGLYCEROL SYNTHASE1 (DGD1) is a chloroplast outer membrane protein responsible for the biosynthesis of the lipid digalactosyldiacylglycerol (DGDG) from monogalactosyldiacylglycerol (MGDG). The Arabidopsis thaliana dgd1 mutants have a greater than 90% reduction in DGDG content, reduced photosynthesis, and altered chloroplast morphology. However, the most pronounced visible phenotype is the extremely short inflorescence stem, but how deficient DGDG biosynthesis causes this phenotype is unclear. We found that, in dgd1 mutants, phloem cap cells were lignified and jasmonic acid (JA)-responsive genes were highly upregulated under normal growth conditions. The coronative insensitive1 dgd1 and allene oxide synthase dgd1 double mutants no longer exhibited the short inflorescence stem and lignification phenotypes but still had the same lipid profile and reduced photosynthesis as dgd1 single mutants. Hormone and lipidomics analyses showed higher levels of JA, JA-isoleucine, 12-oxo-phytodienoic acid, and arabidopsides in dgd1 mutants. Transcript and protein level analyses further suggest that JA biosynthesis in dgd1 is initially activated through the increased expression of genes encoding 13-lipoxygenases (LOXs) and phospholipase A-Iγ3 (At1g51440), a plastid lipase with a high substrate preference for MGDG, and is sustained by further increases in LOX and allene oxide cyclase mRNA and protein levels. Our results demonstrate a link between the biosynthesis of DGDG and JA.

Modification of membrane lipid compositions in single-celled organisms - From basics to applications.

All intact cells, and their organelles, are surrounded by a ∼30 Šhydrophobic film that typically separates the interior from the environment. This film is composed of lipid bilayers that form from a pool of structurally highly diverse, amphipathic lipids. The specific composition and nature of these lipids strongly contributes to many different processes in the cell by influencing membrane structures, membrane protein sorting and functionalities. In this review, we discuss strategies to alter membrane lipid compositions of organelles and plasma membranes in different organisms, focusing on microbial cells. Reflecting the many essential roles of lipids in cellular regulation, we delineate diverse cellular processes affected by membrane lipid modifications and discuss possible applications in a biotechnological and biomedical context. A major motivation for membrane lipid engineering has been the improvement of expression, translocation and activity of heterologous membrane proteins, which can facilitate the biochemical and structural characterization of this challenging class of proteins. Additionally, better expression of membrane proteins or membrane lipid engineering - or a combination of both - led to improved production of high-value compounds and food additives, e.g. polyunsaturated fatty acids and glycolipids, in diverse hosts. More recently it has been shown that diverse cellular pathologies such as cancer and Alzheimer's disease are associated with lipid alterations. Hence, the progress in our understanding of membrane structure, function and protein-lipid interactions, and the resulting possibilities regarding the engineering of membrane lipid composition clearly enable novel nutraceutical and pharmaceutical interventions to be developed. Significant progress in this important area of research is highlighted in this review.