Differential impact of impaired steryl ester biosynthesis on the metabolome of tomato fruits and seeds.

Steryl esters (SE) are a storage pool of sterols that accumulates in cytoplasmic lipid droplets and helps to maintain plasma membrane sterol homeostasis throughout plant growth and development. Ester formation in plant SE is catalyzed by phospholipid:sterol acyltransferase (PSAT) and acyl-CoA:sterol acyltransferase (ASAT), which transfer long-chain fatty acid groups to free sterols from phospholipids and acyl-CoA, respectively. Comparative mass spectrometry-based metabolomic analysis between ripe fruits and seeds of a tomato (Solanum lycopersicum cv Micro-Tom) mutant lacking functional PSAT and ASAT enzymes (slasat1xslpsat1) shows that disruption of SE biosynthesis has a differential impact on the metabolome of these organs, including changes in the composition of free and glycosylated sterols.

sp. nov., isolated at Marimurtra Botanical Garden in Blanes, Catalonia, Spain.

In the framework of the research project called fitomatics, we have isolated and characterized a bacterial plant-endophyte from the rhizomes of , hereafter referred to as strain FIT81. The bacterium is Gram negative, rod-shaped with lophotrichous flagella, and catalase- and oxidase-positive. The optimal growth temperature of strain FIT81 is 28 °C, although it can grow within a temperature range of 4-32 °C.

Effects of impaired steryl ester biosynthesis on tomato growth and developmental processes.

Steryl esters (SE) are stored in cytoplasmic lipid droplets and serve as a reservoir of sterols that helps to maintain free sterols (FS) homeostasis in cell membranes throughout plant growth and development, and provides the FS needed to meet the high demand of these key plasma membrane components during rapid plant organ growth and expansion. SE are also involved in the recycling of sterols and fatty acids released from membranes during plant tissues senescence. SE are synthesized by sterol acyltransferases, which catalyze the transfer of long-chain fatty acid groups to the hydroxyl group at C3 position of FS.

Down-regulation of tomato STEROL GLYCOSYLTRANSFERASE 1 perturbs plant development and facilitates viroid infection.

Potato spindle tuber viroid (PSTVd) is a plant pathogen naturally infecting economically important crops such as tomato (Solanum lycopersicum). Here, we aimed to engineer tomato plants highly resistant to PSTVd and developed several S. lycopersicum lines expressing an artificial microRNA (amiRNA) against PSTVd (amiR-PSTVd).

Phytosterol metabolism in plant positive-strand RNA virus replication.

The genome of most plant viruses consists of a single positive-strand of RNA (+ ssRNA). Successful replication of these viruses is fully dependent on the endomembrane system of the infected cells, which experiences a massive proliferation and a profound reshaping that enables assembly of the macromolecular complexes where virus genome replication occurs. Assembly of these viral replicase complexes (VRCs) requires a highly orchestrated interplay of multiple virus and co-opted host cell factors to create an optimal microenvironment for efficient assembly and functioning of the virus genome replication machinery.

Structural and functional analysis of tomato sterol C22 desaturase.

Background: Sterols are structural and functional components of eukaryotic cell membranes. Plants produce a complex mixture of sterols, among which β-sitosterol, stigmasterol, campesterol, and cholesterol in some Solanaceae, are the most abundant species. Many reports have shown that the stigmasterol to β-sitosterol ratio changes during plant development and in response to stresses, suggesting that it may play a role in the regulation of these processes.

Inactivation of UDP-Glucose Sterol Glucosyltransferases Enhances Resistance to .

Free and glycosylated sterols are both structural components of the plasma membrane that regulate their biophysical properties and consequently different plasma membrane-associated processes such as plant adaptation to stress or signaling. Several reports relate changes in glycosylated sterols levels with the plant response to abiotic stress, but the information about the role of these compounds in the response to biotic stress is scarce. In this work, we have studied the response to the necrotrophic fungus in an mutant that is severely impaired in steryl glycosides biosynthesis due to the inactivation of the two sterol glucosyltransferases (UGT80A2 and UGT80B1) reported in this plant.

Distinct metabolic pathways drive monoterpenoid biosynthesis in a natural population of Pelargonium graveolens.

Pelargonium graveolens is a wild predecessor to rose-scented geranium hybrids prized for their essential oils used as fragrances and flavorings. However, little is known about their biosynthesis. Here we present metabolic evidence that at least two distinct monoterpene biosynthetic pathways contribute to their volatile profiles, namely, cyclic p-menthanes such as (-)-isomenthone and acyclic monoterpene alcohols such as geraniol and (-)-citronellol and their derivatives (referred to here as citronelloid monoterpenes).

Identification and Characterization of Sterol Acyltransferases Responsible for Steryl Ester Biosynthesis in Tomato.

Steryl esters (SEs) serve as a storage pool of sterols that helps to maintain proper levels of free sterols (FSs) in cell membranes throughout plant growth and development, and participates in the recycling of FSs and fatty acids released from cell membranes in aging tissues. SEs are synthesized by sterol acyltransferases, a family of enzymes that catalyze the transfer of fatty acil groups to the hydroxyl group at C-3 position of the sterol backbone. Sterol acyltransferases are categorized into acyl-CoA:sterol acyltransferases (ASAT) and phospholipid:sterol acyltransferases (PSAT) depending on whether the fatty acyl donor substrate is a long-chain acyl-CoA or a phospolipid.

Nerolidol production in agroinfiltrated tobacco: Impact of protein stability and membrane targeting of strawberry (Fragraria ananassa) NEROLIDOL SYNTHASE1.

The sesquiterpene alcohol nerolidol, synthesized from farnesyl diphosphate (FDP), mediates plant-insect interactions across multiple trophic levels with major implications for pest management in agriculture. We compared nerolidol engineering strategies in tobacco using agroinfiltration to transiently express strawberry (Fragraria ananassa) linalool/nerolidol synthase (FaNES1) either at the endoplasmic reticulum (ER) or in the cytosol as a soluble protein. Using solid phase microextraction and gas chromatography-mass spectrometry (SPME-GCMS), we have determined that FaNES1 directed to the ER via fusion to the transmembrane domain of squalene synthase or hydroxymethylglutaryl - CoA reductase displayed significant improvements in terms of transcript levels, protein accumulation, and volatile production when compared to its cytosolic form.

Complex interplays between phytosterols and plastid development.

Isoprenoids comprise the largest class of natural compounds and are found in all kinds of organisms. In plants, they participate in both primary and specialized metabolism, playing essential roles in nearly all aspects of growth and development. The enormous diversity of this family of compounds is extensively exploited for biotechnological and biomedical applications as biomaterials, biofuels or drugs.

Emerging roles for conjugated sterols in plants.

In plants, sterols are found in free form (free sterols, FSs) and conjugated as steryl esters (SEs), steryl glycosides (SGs) and acyl steryl glycosides (ASGs). Conjugated sterols are ubiquitously found in plants but their relative contents highly differ among species and their profile may change in response to developmental and environmental cues. SEs play a central role in membrane sterol homeostasis and also represent a storage pool of sterols in particular plant tissues.

Tomato UDP-Glucose Sterol Glycosyltransferases: A Family of Developmental and Stress Regulated Genes that Encode Cytosolic and Membrane-Associated Forms of the Enzyme.

Sterol glycosyltransferases (SGTs) catalyze the glycosylation of the free hydroxyl group at C-3 position of sterols to produce sterol glycosides. Glycosylated sterols and free sterols are primarily located in cell membranes where in combination with other membrane-bound lipids play a key role in modulating their properties and functioning. In contrast to most plant species, those of the genus contain very high levels of glycosylated sterols, which in the case of tomato may account for more than 85% of the total sterol content.

Elucidation of the biosynthesis of carnosic acid and its reconstitution in yeast.

Rosemary extracts containing the phenolic diterpenes carnosic acid and its derivative carnosol are approved food additives used in an increasingly wide range of products to enhance shelf-life, thanks to their high anti-oxidant activity. We describe here the elucidation of the complete biosynthetic pathway of carnosic acid and its reconstitution in yeast cells. Cytochrome P450 oxygenases (CYP76AH22-24) from Rosmarinus officinalis and Salvia fruticosa already characterized as ferruginol synthases are also able to produce 11-hydroxyferruginol.

Suppressing Farnesyl Diphosphate Synthase Alters Chloroplast Development and Triggers Sterol-Dependent Induction of Jasmonate- and Fe-Related Responses.

Farnesyl diphosphate synthase (FPS) catalyzes the synthesis of farnesyl diphosphate from isopentenyl diphosphate and dimethylallyl diphosphate. Arabidopsis (Arabidopsis thaliana) contains two genes (FPS1 and FPS2) encoding FPS. Single fps1 and fps2 knockout mutants are phenotypically indistinguishable from wild-type plants, while fps1/fps2 double mutants are embryo lethal.

Strategies and Methodologies for the Co-expression of Multiple Proteins in Plants.

The first transgenes were introduced in a plant genome more than 30 years ago. Since then, the capabilities of the plant scientific community to engineer the genome of plants have progressed at an unparalleled speed. Plant genetic engineering has become a central technology that has dramatically incremented our basic knowledge of plant biology and has enabled the translation of this knowledge into a number of increasingly complex and sophisticated biotechnological applications, which in most cases rely on the simultaneous co-expression of multiple recombinant proteins from different origins.

Profile of blinatumomab and its potential in the treatment of relapsed/refractory acute lymphoblastic leukemia.

The CD19 marker is expressed on the surface of normal and malignant immature or mature B-cells. On the other hand, immunotherapy involving T-cells is a promising modality of treatment for many neoplastic diseases including leukemias and lymphomas. The CD19/CD3-bispecific T-cell-engaging (BiTE(®)) monoclonal antibody blinatumomab can transiently engage cytotoxic T-cells to CD19+ target B-cells inducing serial perforin-mediated lysis.

Towards Elucidating Carnosic Acid Biosynthesis in Lamiaceae: Functional Characterization of the Three First Steps of the Pathway in Salvia fruticosa and Rosmarinus officinalis.

Carnosic acid (CA) is a phenolic diterpene with anti-tumour, anti-diabetic, antibacterial and neuroprotective properties that is produced by a number of species from several genera of the Lamiaceae family, including Salvia fruticosa (Cretan sage) and Rosmarinus officinalis (Rosemary). To elucidate CA biosynthesis, glandular trichome transcriptome data of S. fruticosa were mined for terpene synthase genes.

Arabidopsis Squalene Epoxidase 3 (SQE3) Complements SQE1 and Is Important for Embryo Development and Bulk Squalene Epoxidase Activity.

The existence of multigenic families in the mevalonate pathway suggests divergent functional roles for pathway components involved in the biosynthesis of plant sterols. Squalene epoxidases (SQEs) are key components of this pathway, and Squalene Epoxidase 1 (SQE1) has been identified as a fundamental enzyme in this biosynthetic step. In the present work, we extended the characterization of the remaining SQE family members, phylogenetically resolving between true SQEs and a subfamily of SQE-like proteins that is exclusive to Brassicaceae.

Farnesyl diphosphate synthase assay.

Farnesyl diphosphate synthase (FPS) catalyzes the sequential head-to-tail condensation of isopentenyl diphosphate (IPP, C5) with dimethylallyl diphosphate (DMAPP, C5) and geranyl diphosphate (GPP, C10) to produce farnesyl diphosphate (FPP, C15). This short-chain prenyl diphosphate constitutes a key branch-point of the isoprenoid biosynthetic pathway from which a variety of bioactive isoprenoids that are vital for normal plant growth and survival are produced. Here we describe a protocol to obtain highly purified preparations of recombinant FPS and a radiochemical assay method for measuring FPS activity in purified enzyme preparations and plant tissue extracts.

Determination of 3-hydroxy-3-methylglutaryl CoA reductase activity in plants.

The enzyme 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase catalyzes the NADPH-mediated reductive deacylation of HMG-CoA to mevalonic acid, which is the first committed step of the mevalonate pathway for isoprenoid biosynthesis. In agreement with its key regulatory role in the pathway, plant HMG-CoA reductase is modulated by many diverse external stimuli and endogenous factors and can be detected to variable levels in every plant tissue. A fine determination of HMG-CoA reductase activity levels is required to understand its contribution to plant development and adaptation to changing environmental conditions.

Elucidation and in planta reconstitution of the parthenolide biosynthetic pathway.

Parthenolide, the main bioactive compound of the medicinal plant feverfew (Tanacetum parthenium), is a promising anti-cancer drug. However, the biosynthetic pathway of parthenolide has not been elucidated yet. Here we report on the isolation and characterization of all the genes from feverfew that are required for the biosynthesis of parthenolide, using a combination of 454 sequencing of a feverfew glandular trichome cDNA library, co-expression analysis and metabolomics.

Characterization of two genes for the biosynthesis of abietane-type diterpenes in rosemary (Rosmarinus officinalis) glandular trichomes.

Rosemary (Rosmarinus officinalis) produces the phenolic diterpenes carnosic acid and carnosol, which, in addition to their general antioxidant activities, have recently been suggested as potential ingredients for the prevention and treatment of neurodegenerative diseases. Little is known about the biosynthesis of these diterpenes. Here we show that the biosynthesis of phenolic diterpenes in rosemary predominantly takes place in the glandular trichomes of young leaves, and used this feature to identify the first committed steps.

The SUD1 gene encodes a putative E3 ubiquitin ligase and is a positive regulator of 3-hydroxy-3-methylglutaryl coenzyme a reductase activity in Arabidopsis.

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity.