Farnesyl Diphosphate Synthase Assay

Arró, Montserrat; Manzano, David; Ferrer, Albert

doi:10.1007/978-1-4939-0606-2_4

Cited by 5 publications

(8 citation statements)

References 16 publications

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“…In order to verify that the cloned cDNAs actually encode functional SGTs, the corresponding ORFs were cloned in frame downstream of the glutathione S -transferase (GST) coding sequence into pGEX-3X-NotI expression vector (Arró et al, 2014). The resulting recombinant plasmids were introduced into E. coli cells and expression of the GST-SlSGT fusion proteins was induced with IPTG.…”

Section: Resultsmentioning

confidence: 99%

“…A Bam HI restriction site was included in the sequence of reverse primers used for SlSGT1, SlSGT2, and SlSGT3 ORF amplification whereas a Sma I restriction site was included in the reverse primer for amplification of the SlSGT3 ORF. The resulting products were digested with either Bam HI or Sma I and cloned into the corresponding sites of the pGEX-3X-NotI vector previously digested with Not I and blunt-ended by treatment with nuclease S1, as described in Arró et al (2014). All constructs were sequenced to confirm the in-frame sequence fusions.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting recombinant plasmids and the pGEX-3X-NotI empty vector were transformed into competent E. coli BL21 (DE3) cells harboring plasmid pUBS520, which carries the gene coding for the tRNA of rare arginine codons AGG and AGA. The correct fusion between the GST and SlSGT ORFs was confirmed by sequencing with the pGEX 5′ primer (Arró et al, 2014). To express the GST-SlSGT fusion proteins, overnight cultures of the transformed E. coli cells (3 mL) were grown at 37°C in LB medium supplemented with ampicillin (100 mg/L) and kanamycin (25 mg/L).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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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 Solanum contain very high levels of glycosylated sterols, which in the case of tomato may account for more than 85% of the total sterol content. In this study, we report the identification and functional characterization of the four members of the tomato (Solanum lycopersicum cv. Micro-Tom) SGT gene family. Expression of recombinant SlSGT proteins in E. coli cells and N. benthamiana leaves demonstrated the ability of the four enzymes to glycosylate different sterol species including cholesterol, brassicasterol, campesterol, stigmasterol, and β-sitosterol, which is consistent with the occurrence in their primary structure of the putative steroid-binding domain found in steroid UDP-glucuronosyltransferases and the UDP-sugar binding domain characteristic for a superfamily of nucleoside diphosphosugar glycosyltransferases. Subcellular localization studies based on fluorescence recovery after photobleaching and cell fractionation analyses revealed that the four tomato SGTs, like the Arabidopsis SGTs UGT80A2 and UGT80B1, localize into the cytosol and the PM, although there are clear differences in their relative distribution between these two cell fractions. The SlSGT genes have specialized but still largely overlapping expression patterns in different organs of tomato plants and throughout the different stages of fruit development and ripening. Moreover, they are differentially regulated in response to biotic and abiotic stress conditions. SlSGT4 expression increases markedly in response to osmotic, salt, and cold stress, as well as upon treatment with abscisic acid and methyl jasmonate. Stress-induced SlSGT2 expression largely parallels that of SlSGT4. On the contrary, SlSGT1 and SlSGT3 expression remains almost unaltered under the tested stress conditions. Overall, this study contributes to broaden the current knowledge on plant SGTs and provides support to the view that tomato SGTs play overlapping but not completely redundant biological functions involved in mediating developmental and stress responses.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

et al. 2017

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“…Seedling extracts for HMGR and FPS activity assays were obtained as described by Campos et al (2014) and Arró et al (2014), respectively. HMGR activity was determined as described by Campos et al (2014) and is reported as picomoles of 3-HMG-CoA converted into MVA per minute per milliliter of protein extract at 37°C.…”

Section: Western-blot Analysis and Enzyme Activity Assaysmentioning

confidence: 99%

“…HMGR activity was determined as described by Campos et al (2014) and is reported as picomoles of 3-HMG-CoA converted into MVA per minute per milliliter of protein extract at 37°C. FPS activity was measured as described by Arró et al (2014) and is reported as picomoles of IPP incorporated into acid-labile products per minute per milliliter of protein extract at 37°C. Immunoblot analysis was performed as described by Keim et al (2012) in the same protein extracts used for enzyme activity assays.…”

Section: Western-blot Analysis and Enzyme Activity Assaysmentioning

confidence: 99%

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

et al. 2016

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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. To assess the effect of FPS down-regulation at postembryonic developmental stages, we generated Arabidopsis conditional knockdown mutants expressing artificial microRNAs devised to simultaneously silence both FPS genes. Induction of silencing from germination rapidly caused chlorosis and a strong developmental phenotype that led to seedling lethality. However, silencing of FPS after seed germination resulted in a slight developmental delay only, although leaves and cotyledons continued to show chlorosis and altered chloroplasts. Metabolomic analyses also revealed drastic changes in the profile of sterols, ubiquinones, and plastidial isoprenoids. RNA sequencing and reverse transcription-quantitative polymerase chain reaction transcriptomic analysis showed that a reduction in FPS activity levels triggers the misregulation of genes involved in biotic and abiotic stress responses, the most prominent one being the rapid induction of a set of genes related to the jasmonic acid pathway. Down-regulation of FPS also triggered an iron-deficiency transcriptional response that is consistent with the irondeficient phenotype observed in FPS-silenced plants. The specific inhibition of the sterol biosynthesis pathway by chemical and genetic blockage mimicked these transcriptional responses, indicating that sterol depletion is the primary cause of the observed alterations. Our results highlight the importance of sterol homeostasis for normal chloroplast development and function and reveal important clues about how isoprenoid and sterol metabolism is integrated within plant physiology and development.Isoprenoids are the largest class of all known natural products in living organisms, with tens of thousands of different compounds. In plants, isoprenoids perform essential biological functions, including maintenance of proper membrane structure and function (sterols), electron transport (ubiquinones and plastoquinones), posttranslational protein modification (dolichols serving as glycosylation cofactors and prenyl groups), photosynthesis (chlorophylls and carotenoids), and regulation of growth and development (abscisic acid, brassinosteroids, cytokinins, and GAs [Croteau et al., 2000] and strigolactones [Al-Babili and Bouwmeester, 2015]). A large number of isoprenoids also play prominent roles as mediators of interactions between plants and their environment, including a variety of defense responses against biotic and abiotic stresses (Tholl and Lee, 2011). In fact, there is hardly any aspect of plant growth, development, and reproduction not relying on isoprenoids or isoprenoid-derived compounds. In addition, many plant isoprenoids are of great economic importan...

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A Simple In Vitro Assay to Measure the Activity of Geranylgeranyl Diphosphate Synthase and Other Short-Chain Prenyltransferases

Barja

Rodríguez‐Concepción

2019

Methods in Molecular Biology

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Most carotenoids are C40 metabolites produced from C20 geranylgeranyl diphosphate (GGPP). The enzymes that produce this precursor, GGPP synthases (GGPPS), are members of the short-chain prenyltransferase (SC-PT) family. SC-PTs are enzymes that catalyze the sequential head-to-tail addition of one or more C5 molecules of isopentenyl diphosphate (IPP) to dimethylallyl diphosphate (DMAPP) with the concomitant release of pyrophosphate (PPi). SC-PTs produce linear isoprenyl diphosphates of up to C20 (GGPP) that serve as precursors for many groups of isoprenoids with a wide range of essential biological functions in Eucarya, Bacteria and Archaea. Enzymatic analysis of SC-PT activity normally requires complex, laborious and expensive methods such as radioactivity-based assays or liquid chromatography-mass spectrometry (LC-MS). Here we describe a fast and inexpensive spectrophotometric protocol for determining the kinetic parameters of SC-PTs in purified enzyme preparations, using an adapted assay for PPi quantification. We developed the method using the Arabidopsis thaliana GGPPS11 enzyme, which produces geranylgeranyl diphosphate for the synthesis of carotenoids in the chloroplast.

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Farnesyl Diphosphate Synthase Assay

Cited by 5 publications

References 16 publications

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

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

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

A Simple In Vitro Assay to Measure the Activity of Geranylgeranyl Diphosphate Synthase and Other Short-Chain Prenyltransferases

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