Overexpression and Inhibition of 3-Hydroxy-3-Methylglutaryl-CoA Synthase Affect Central Metabolic Pathways in Tobacco.

Little has been established on the relationship between the mevalonate (MVA) pathway and other metabolic pathways except for the sterol and glucosinolate biosynthesis pathways. In the MVA pathway, 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to form 3-hydroxy-3-methylglutaryl-coenzyme A. Our previous studies had shown that, while the recombinant Brassica juncea HMGS1 (BjHMGS1) mutant S359A displayed 10-fold higher enzyme activity than wild-type (wt) BjHMGS1, transgenic tobacco overexpressing S359A (OE-S359A) exhibited higher sterol content, growth rate and seed yield than OE-wtBjHMGS1. Herein, untargeted proteomics and targeted metabolomics were employed to understand the phenotypic effects of HMGS overexpression in tobacco by examining which other metabolic pathways were affected. Sequential window acquisition of all theoretical mass spectra quantitative proteomics analysis on OE-wtBjHMGS1 and OE-S359A identified the misregulation of proteins in primary metabolism and cell wall modification, while some proteins related to photosynthesis and the tricarboxylic acid cycle were upregulated in OE-S359A. Metabolomic analysis indicated corresponding changes in carbohydrate, amino acid and fatty acid contents in HMGS-OEs, and F-244, a specific inhibitor of HMGS, was applied successfully on tobacco to confirm these observations. Finally, the crystal structure of acetyl-CoA-liganded S359A revealed that improved activity of S359A likely resulted from a loss in hydrogen bonding between Ser359 and acyl-CoA, which is evident in wtBjHMGS1. This work suggests that regulation of plant growth by HMGS can influence the central metabolic pathways. Furthermore, this study demonstrates that the application of the HMGS-specific inhibitor (F-244) in tobacco represents an effective approach for studying the HMGS/MVA pathway.

The specific molecular architecture of plant 3-hydroxy-3-methylglutaryl-CoA lyase.

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase (HMGL) is involved in branched-chain amino acid catabolism leading to acetyl-CoA production. Here, using bioinformatics analyses and protein sequence alignments, we found that in Arabidopsis thaliana a single gene encodes two HMGL isoforms differing in size (51 kDa, HMGL51 and 46 kDa, HMGL46). Similar to animal HMGLs, both isoforms comprised a C-terminal type 1 peroxisomal retention motif, and HMGL51 contained a mitochondrial leader peptide. We observed that only a shortened HMGL (35 kDa, HMGL35) is conserved across all kingdoms of life. Most notably, all plant HMGLs also contained a specific N-terminal extension (P100) that is located between the N-terminal mitochondrial targeting sequence TP35 and HMGL35 and is absent in bacteria and other eukaryotes. Interestingly, using HMGL enzyme assays, we found that rather than HMGL46, homodimeric recombinant HMGL35 is the active enzyme catalyzing acetyl-CoA and acetoacetate synthesis when incubated with (S)-HMG-CoA. This suggested that the plant-specific P100 peptide may inactivate HMGL according to specific physiological requirements. Therefore, we investigated whether the P100 peptide in HMGL46 alters its activity, possibly by modifying the HMGL46 structure. We found that induced expression of a cytosolic HMGL35 version in A. thaliana delays germination and leads to rapid wilting and chlorosis in mature plants. Our results suggest that in plants, P100-mediated HMGL inactivation outside of peroxisomes or mitochondria is crucial, protecting against potentially cytotoxic effects of HMGL activity while it transits to these organelles.

Overexpression of HMG-CoA synthase promotes Arabidopsis root growth and adversely affects glucosinolate biosynthesis.

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyses the second step of the mevalonate (MVA) pathway. An HMGS inhibitor (F-244) has been reported to retard growth in wheat, tobacco, and Brassica juncea, but the mechanism remains unknown. Although the effects of HMGS on downstream isoprenoid metabolites have been extensively reported, not much is known on how it might affect non-isoprenoid metabolic pathways. Here, the mechanism of F-244-mediated inhibition of primary root growth in Arabidopsis and the relationship between HMGS and non-isoprenoid metabolic pathways were investigated by untargeted SWATH-MS quantitative proteomics, quantitative real-time PCR, and target metabolite analysis. Our results revealed that the inhibition of primary root growth caused by F-244 was a consequence of reduced stigmasterol, auxin, and cytokinin levels. Interestingly, proteomic analyses identified a relationship between HMGS and glucosinolate biosynthesis. Inhibition of HMGS activated glucosinolate biosynthesis, resulting from the induction of glucosinolate biosynthesis-related genes, suppression of sterol biosynthesis-related genes, and reduction in sterol levels. In contrast, HMGS overexpression inhibited glucosinolate biosynthesis, due to down-regulation of glucosinolate biosynthesis-related genes, up-regulation of sterol biosynthesis-related genes, and increase in sterol content. Thus, HMGS might represent a target for the manipulation of glucosinolate biosynthesis, given the regulatory relationship between HMGS in the MVA pathway and glucosinolate biosynthesis.

More than 40 Years Active in Steroid and Isoprenoid Research-A Personal Note on W. David Nes' Career and His Multiple Achievements in this Field.

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Improved fruit α-tocopherol, carotenoid, squalene and phytosterol contents through manipulation of Brassica juncea 3-HYDROXY-3-METHYLGLUTARYL-COA SYNTHASE1 in transgenic tomato.

3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) in the mevalonate (MVA) pathway generates isoprenoids including phytosterols. Dietary phytosterols are important because they can lower blood cholesterol levels. Previously, the overexpression of Brassica juncea wild-type (wt) and mutant (S359A) BjHMGS1 in Arabidopsis up-regulated several genes in sterol biosynthesis and increased sterol content. Recombinant S359A had earlier displayed a 10-fold higher in vitro enzyme activity. Furthermore, tobacco HMGS overexpressors (OEs) exhibited improved sterol content, plant growth and seed yield. Increased growth and seed yield in tobacco OE-S359A over OE-wtBjHMGS1 coincided with elevations in NtSQS expression and sterol content. Herein, the overexpression of wt and mutant (S359A) BjHMGS1 in a crop plant, tomato (Solanum lycopersicum), caused an accumulation of MVA-derived squalene and phytosterols, as well as methylerythritol phosphate (MEP)-derived α-tocopherol (vitamin E) and carotenoids, which are important to human health as antioxidants. In tomato HMGS-OE seedlings, genes associated with the biosyntheses of C10, C15 and C20 universal precursors of isoprenoids, phytosterols, brassinosteroids, dolichols, methylerythritol phosphate, carotenoid and vitamin E were up-regulated. In OE-S359A tomato fruits, increased squalene and phytosterol contents over OE-wtBjHMGS1 were attributed to heightened SlHMGR2, SlFPS1, SlSQS and SlCYP710A11 expression. In both tomato OE-wtBjHMGS1 and OE-S359A fruits, the up-regulation of SlGPS and SlGGPPS1 in the MEP pathway that led to α-tocopherol and carotenoid accumulation indicated cross-talk between the MVA and MEP pathways. Taken together, the manipulation of BjHMGS1 represents a promising strategy to simultaneously elevate health-promoting squalene, phytosterols, α-tocopherol and carotenoids in tomato, an edible fruit.

S-carvone suppresses cellulase-induced capsidiol production in Nicotiana tabacum by interfering with protein isoprenylation.

S-Carvone has been described as a negative regulator of mevalonic acid (MVA) production by interfering with 3-hydroxy-3-methyl glutaryl coenzyme A reductase (HMGR) activity, a key player in isoprenoid biosynthesis. The impact of this monoterpene on the production of capsidiol in Nicotiana tabacum, an assumed MVA-derived sesquiterpenoid phytoalexin produced in response to elicitation by cellulase, was investigated. As expected, capsidiol production, as well as early stages of elicitation such as hydrogen peroxide production or stimulation of 5-epi-aristolochene synthase activity, were repressed. Despite the lack of capsidiol synthesis, apparent HMGR activity was boosted. Feeding experiments using (1-13C)Glc followed by analysis of labeling patterns by 13C-NMR, confirmed an MVA-dependent biosynthesis; however, treatments with fosmidomycin, an inhibitor of the MVA-independent 2-C-methyl-D-erythritol 4-phosphate (MEP) isoprenoid pathway, unexpectedly down-regulated the biosynthesis of this sesquiterpene as well. We postulated that S-carvone does not directly inhibit the production of MVA by inactivating HMGR, but possibly targets an MEP-derived isoprenoid involved in the early steps of the elicitation process. A new model is proposed in which the monoterpene blocks an MEP pathway-dependent protein geranylgeranylation necessary for the signaling cascade. The production of capsidiol was inhibited when plants were treated with some inhibitors of protein prenylation or by further monoterpenes. Moreover, S-carvone hindered isoprenylation of a prenylable GFP indicator protein expressed in N. tabacum cell lines, which can be chemically complemented with geranylgeraniol. The model was further validated using N. tabacum cell extracts or recombinant N. tabacum protein prenyltransferases expressed in Escherichia coli. Our study endorsed a reevaluation of the effect of S-carvone on plant isoprenoid metabolism.

Past achievements, current status and future perspectives of studies on 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) in the mevalonate (MVA) pathway.

KEY MESSAGE: HMGS functions in phytosterol biosynthesis, development and stress responses. F-244 could specifically-inhibit HMGS in tobacco BY-2 cells and Brassica seedlings. An update on HMGS from higher plants is presented. 3-Hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) is the second enzyme in the mevalonate pathway of isoprenoid biosynthesis and catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to produce S-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA). Besides HMG-CoA reductase (HMGR), HMGS is another key enzyme in the regulation of cholesterol and ketone bodies in mammals. In plants, it plays an important role in phytosterol biosynthesis. Here, we summarize the past investigations on eukaryotic HMGS with particular focus on plant HMGS, its enzymatic properties, gene expression, protein structure, and its current status of research in China. An update of the findings on HMGS from animals (human, rat, avian) to plants (Brassica juncea, Hevea brasiliensis, Arabidopsis thaliana) will be discussed. Current studies on HMGS have been vastly promoted by developments in biochemistry and molecular biology. Nonetheless, several limitations have been encountered, thus some novel advances in HMGS-related research that have recently emerged will be touched on.

Methyl-Jasmonate Functions as a Molecular Switch Promoting Cross-Talk between Pathways for the Biosynthesis of Isoprenoid Backbones Used to Modify Proteins in Plants.

In plants, the plastidial mevalonate (MVA)-independent pathway is required for the modification with geranylgeranyl groups of CaaL-motif proteins, which are substrates of protein geranylgeranyltransferase type-I (PGGT-I). As a consequence, fosmidomycin, a specific inhibitor of 1-deoxy-d-xylulose (DX)-5 phosphate reductoisomerase/DXR, the second enzyme in this so-called methylerythritol phosphate (MEP) pathway, also acts as an effective inhibitor of protein prenylation. This can be visualized in plant cells by confocal microscopy by expressing GFP-CaM-CVIL, a prenylation sensor protein. After treatment with fosmidomycin, the plasma membrane localization of this GFP-based sensor is altered, and a nuclear distribution of fluorescence is observed instead. In tobacco cells, a visual screen of conditions allowing membrane localization in the presence of fosmidomycin identified jasmonic acid methyl esther (MeJA) as a chemical capable of gradually overcoming inhibition. Using Arabidopsis protein prenyltransferase loss-of-function mutant lines expressing GFP-CaM-CVIL proteins, we demonstrated that in the presence of MeJA, protein farnesyltransferase (PFT) can modify the GFP-CaM-CVIL sensor, a substrate the enzyme does not recognize under standard conditions. Similar to MeJA, farnesol and MVA also alter the protein substrate specificity of PFT, whereas DX and geranylgeraniol have limited or no effect. Our data suggest that MeJA adjusts the protein substrate specificity of PFT by promoting a metabolic cross-talk directing the origin of the prenyl group used to modify the protein. MVA, or an MVA-derived metabolite, appears to be a key metabolic intermediate for this change in substrate specificity.

Phosphoproteome exploration reveals a reformatting of cellular processes in response to low sterol biosynthetic capacity in Arabidopsis.

Sterols are membrane-bound isoprenoid lipids that are required for cell viability and growth. In plants, it is generally assumed that 3-hydroxy-3-methylglutaryl-CoA-reductase (HMGR) is a key element of their biosynthesis, but the molecular regulation of that pathway is largely unknown. In an attempt to identify regulators of the biosynthetic flux from acyl-CoA toward phytosterols, we compared the membrane phosphoproteome of wild-type Arabidopsis thaliana and of a mutant being deficient in HMGR1. We performed a N-terminal labeling of microsomal peptides with a trimethoxyphenyl phosphonium (TMPP) derivative, followed by a quantitative assessment of phosphopeptides with a spectral counting method. TMPP derivatization of peptides resulted in an improved LC-MS/MS detection due to increased hydrophobicity in chromatography and ionization efficiency in electrospray. The phosphoproteome coverage was 40% higher with this methodology. We further found that 31 proteins were in a different phosphorylation state in the hmgr1-1 mutant as compared with the wild-type. One-third of these proteins were identified based on novel phosphopeptides. This approach revealed that phosphorylation changes in the Arabidopsis membrane proteome targets major cellular processes such as transports, calcium homeostasis, photomorphogenesis, and carbohydrate synthesis. A reformatting of these processes appears to be a response of a genetically reduced sterol biosynthesis.

Overexpression of Brassica juncea wild-type and mutant HMG-CoA synthase 1 in Arabidopsis up-regulates genes in sterol biosynthesis and enhances sterol production and stress tolerance.

Brassica juncea 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) is encoded by four isogenes (BjHMGS1-BjHMGS4). In vitro enzyme assays had indicated that the recombinant BjHMGS1 H188N mutant lacked substrate inhibition by acetoacetyl-CoA (AcAc-CoA) and showed 8-fold decreased enzyme activity. The S359A mutant demonstrated 10-fold higher activity, while the H188N/S359A double mutant displayed a 10-fold increased enzyme activity and lacked inhibition by AcAc-CoA. Here, wild-type and mutant BjHMGS1 were overexpressed in Arabidopsis to examine their effects in planta. The expression of selected genes in isoprenoid biosynthesis, isoprenoid content, seed germination and stress tolerance was analysed in HMGS overexpressors (OEs). Those mRNAs encoding enzymes 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), sterol methyltransferase 2 (SMT2), delta-24 sterol reductase (DWF1), C-22 sterol desaturase (CYP710A1) and brassinosteroid-6-oxidase 2 (BR6OX2) were up-regulated in HMGS-OEs. The total sterol content in leaves and seedlings of OE-wtBjHMGS1, OE-S359A and OE-H188N/S359A was significantly higher than OE-H188N. HMGS-OE seeds germinated earlier than wild-type and vector-transformed controls. HMGS-OEs further displayed reduced hydrogen peroxide (H(2) O(2) )-induced cell death and constitutive expression of salicylic acid (SA)-dependent pathogenesis-related genes (PR1, PR2 and PR5), resulting in an increased resistance to Botrytis cinerea, with OE-S359A showing the highest and OE-H188N the lowest tolerance. These results suggest that overexpression of HMGS up-regulates HMGR, SMT2, DWF1, CYP710A1 and BR6OX2, leading to enhanced sterol content and stress tolerance in Arabidopsis.

Distinct triterpene synthases in the laticifers of Euphorbia lathyris.

Euphorbia lathyris was proposed about fifty years ago as a potential agroenergetic crop. The tremendous amounts of triterpenes present in its latex has driven investigations for transforming this particular biological fluid into an industrial hydrocarbon source. The huge accumulation of terpenes in the latex of many plant species represent a challenging question regarding cellular homeostasis. In fact, the enzymes, the mechanisms and the controllers that tune the amount of products accumulated in specialized compartments (to fulfill ecological roles) or deposited at important sites (as essential factors) are not known. Here, we have isolated oxidosqualene cyclases highly expressed in the latex of Euphorbia lathyris. This triterpene biosynthetic machinery is made of distinct paralogous enzymes responsible for the massive accumulation of steroidal and non-steroidal tetracyclic triterpenes. More than eighty years after the isolation of butyrospermol from shea butter (Heilbronn IM, Moffet GL, and Spring FS J. Chem. Soc. 1934, 1583), a butyrospermol synthase is characterized in this work using yeast and in folia heterologous expression assays.

A serine involved in actin-dependent subcellular localization of a stress-induced tobacco BY-2 hydroxymethylglutaryl-CoA reductase isoform.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is unique in the first part of the cytoplasmic isoprenoid pathway, as it contains a membrane domain that includes ER-specific retention motifs. When fused to GFP, this domain targets two tobacco BY-2 HMGR isoforms differentially. While the first isoform is ER-localized, a second stress-induced one forms globular structures connected by tubular structures. A serine positioned upstream of the ER retention motif seems to be implicated in this specific subcellular localization. Surprisingly, these structures are closely connected to F-actin, and their intactness is dependent upon the integrity of the filaments or the action of a calmodulin antagonist.

Isoprenoid biosynthesis as a target for antibacterial and antiparasitic drugs: phosphonohydroxamic acids as inhibitors of deoxyxylulose phosphate reducto-isomerase.

Isoprenoid biosynthesis via the methylerythritol phosphate pathway is a target against pathogenic bacteria and the malaria parasite Plasmodium falciparum. 4-(Hydroxyamino)-4-oxobutylphosphonic acid and 4-[hydroxy(methyl)amino]-4-oxobutyl phosphonic acid, two novel inhibitors of DXR (1-deoxy-D-xylulose 5-phosphate reducto-isomerase), the second enzyme of the pathway, have been synthesized and compared with fosmidomycin, the best known inhibitor of this enzyme. The latter phosphonohydroxamic acid showed a high inhibitory activity towards DXR, much like fosmidomycin, as well as significant antibacterial activity against Escherichia coli in tests on Petri dishes.

The potential of the mevalonate pathway for enhanced isoprenoid production.

The cytosol-localised mevalonic acid (MVA) pathway delivers the basic isoprene unit isopentenyl diphosphate (IPP). In higher plants, this central metabolic intermediate is also synthesised by the plastid-localised methylerythritol phosphate (MEP) pathway. Both MVA and MEP pathways conspire through exchange of intermediates and regulatory interactions. Products downstream of IPP such as phytosterols, carotenoids, vitamin E, artemisinin, tanshinone and paclitaxel demonstrate antioxidant, cholesterol-reducing, anti-ageing, anticancer, antimalarial, anti-inflammatory and antibacterial activities. Other isoprenoid precursors including isoprene, isoprenol, geraniol, farnesene and farnesol are economically valuable. An update on the MVA pathway and its interaction with the MEP pathway is presented, including the improvement in the production of phytosterols and other isoprenoid derivatives. Such attempts are for instance based on the bioengineering of microbes such as Escherichia coli and Saccharomyces cerevisiae, as well as plants. The function of relevant genes in the MVA pathway that can be utilised in metabolic engineering is reviewed and future perspectives are presented.

Farnesol-mediated shift in the metabolic origin of prenyl groups used for protein prenylation in plants.

Little is known about how plant cells regulate the exchange of prenyl diphosphates between the two compartmentalized isoprenoid biosynthesis pathways. Prenylation of proteins is a suitable model to study such interactions between the plastidial methylerythritol phosphate (MEP) and the cytosolic mevalonate (MVA) pathways because prenyl moieties used to modify proteins rely on both origins. Tobacco cells expressing a prenylatable GFP were treated with specific MEP and/or MVA pathways inhibitors to block the formation of prenyl diphosphates and therefore the possibility to modify the proteins. Chemical complementation assays using prenyl alcohol precursors restore the prenylation. Indeed, geranylgeraniol (C20 prenyl alcohol) and to a lesser but significant level C15-farnesol restored the prenylation of a protein bearing a geranylgeranylation CaaX motif, which under standard conditions is modified by a MEP-derived prenyl group. However, the restoration takes place in different ways. While geranylgeraniol operates directly as a metabolic precursor, the C15-prenyl alcohol functions indirectly as a signal that leads to shift the metabolic origin of prenyl groups in modified proteins, here from the plastidial MEP pathway in favor of the cytosolic MVA pathway. Furthermore, farnesol interferes negatively with the MEP pathway in an engineered Escherichia coli strain synthesizing isoprenoids either starting from MVA or from MEP. Following the cellular uptake of a fluorescent analog of farnesol, we showed its close interaction with tobacco plastids and modification of plastid homeostasis. As a consequence, in tobacco farnesol supposedly inhibits the plastidial MEP pathway and activates the cytosolic MVA pathway, leading to the shift in the metabolic origin and thereby acts as a potential regulator of crosstalk between the two pathways. Together, those results suggest a new role for farnesol (or a metabolite thereof) as a central molecule for the regulation of isoprenoid biosynthesis in plants.

Brassica juncea 3-hydroxy-3-methylglutaryl (HMG)-CoA synthase 1: expression and characterization of recombinant wild-type and mutant enzymes.

3-hydroxy-3-methylglutaryl (HMG)-CoA synthase (HMGS; EC 2.3.3.10) is the second enzyme in the cytoplasmic mevalonate pathway of isoprenoid biosynthesis, and catalyses the condensation of acetyl-CoA with acetoacetyl-CoA (AcAc-CoA) to yield S-HMG-CoA. In this study, we have first characterized in detail a plant HMGS, Brassica juncea HMGS1 (BjHMGS1), as a His6-tagged protein from Escherichia coli. Native gel electrophoresis analysis showed that the enzyme behaves as a homodimer with a calculated mass of 105.8 kDa. It is activated by 5 mM dithioerythreitol and is inhibited by F-244 which is specific for HMGS enzymes. It has a pH optimum of 8.5 and a temperature optimum of 35 degrees C, with an energy of activation of 62.5 J x mol(-1). Unlike cytosolic HMGS from chicken and cockroach, cations like Mg2+, Mn2+, Zn2+ and Co2+ did not stimulate His6-BjHMGS1 activity in vitro; instead all except Mg2+ were inhibitory. His6-BjHMGS1 has an apparent K(m-acetyl-CoA) of 43 microM and a V(max) of 0.47 micromol x mg(-1) x min(-1), and was inhibited by one of the substrates (AcAc-CoA) and by both products (HMG-CoA and HS-CoA). Site-directed mutagenesis of conserved amino acid residues in BjHMGS1 revealed that substitutions R157A, H188N and C212S resulted in a decreased V(max), indicating some involvement of these residues in catalytic capacity. Unlike His6-BjHMGS1 and its soluble purified mutant derivatives, the H188N mutant did not display substrate inhibition by AcAc-CoA. Substitution S359A resulted in a 10-fold increased specific activity. Based on these kinetic analyses, we generated a novel double mutation H188N/S359A, which resulted in a 10-fold increased specific activity, but still lacking inhibition by AcAc-CoA, strongly suggesting that His-188 is involved in conferring substrate inhibition on His6-BjHMGS1. Substitution of an aminoacyl residue resulting in loss of substrate inhibition has never been previously reported for any HMGS.

Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cells.

Tobacco BY-2 cell suspensions are our preferred model for studying isoprenoid biosynthesis pathways, due to their easy genetic transformation and the efficient absorption of metabolic precursors, intermediates, and/or inhibitors. Using this model system, we have analyzed the effects of chemical and genetic blockage of cycloartenol synthase (CAS, EC 5.4.99.8), an oxidosqualene cyclase that catalyzes the first committed step in the sterol pathway of plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Short-term treatments (24 h) resulted in accumulation of oxidosqualene with no changes in the final sterol products. Interestingly, long-term treatments (6 days) induced down-regulation in gene expression not only of CAS but also of the SMT2 gene coding sterol methyltransferase 2 (EC 2.1.1.41). This explains some of the increase in 24-methyl sterols at the expense of the 24-ethyl sterols stigmasterol and sitosterol. In our alternative strategy, CAS gene expression was partially blocked by using an inducible artificial microRNA. The limited effectiveness of this approach might be explained by some dependence of the machinery for RNAi formation on an operating MVA/sterol pathway. For comparison we checked the effect of RO 48-8071 on a green cell suspension of Arabidopsis and on seedlings, containing a small spectrum of triterpenes besides phytosterols. Triterpenes remained essentially unaffected, but phytosterol accumulation was clearly diminished.

Development of an image-based screening system for inhibitors of the plastidial MEP pathway and of protein geranylgeranylation.

We have recently established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, which involves expressing a dexamethasone-inducible GFP fused to the prenylable, carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with oxoclomazone and fosmidomycin, as well as inhibition of protein geranylgeranyl transferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect this localization. Furthermore, complementation assays with pathway-specific intermediates confirmed that the precursors for the cytosolic isoprenylation of this fusion protein are predominantly provided by the MEP pathway. In order to optimize this visualization system from a more qualitative assay to a statistically trustable medium or a high-throughput screening system, we established new conditions that permit culture and analysis in 96-well microtiter plates, followed by fluorescence microscopy. For further refinement, the existing GFP-BD-CVIL cell line was transformed with an estradiol-inducible vector driving the expression of a RFP protein, C-terminally fused to a nuclear localization signal (NLS-RFP). We are thus able to quantify the total number of viable cells versus the number of inhibited cells after various treatments. This approach also includes a semi-automatic counting system, based on the freely available image processing software. As a result, the time of image analysis as well as the risk of user-generated bias is reduced to a minimum. Moreover, there is no cross-induction of gene expression by dexamethasone and estradiol, which is an important prerequisite for this test system.

Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cell Suspensions: A Proteomic Analysis.

The effect of an inhibitor of cycloartenol synthase (CAS, EC 5.4.99.8) on the proteome of tobacco BY-2 cells has been examined. CAS catalyzes the first committed step in phytosterol synthesis in plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Proteins were separated by two-dimensional electrophoresis and spots, that clearly looked differentially accumulated after visual inspection, were cut, in-gel trypsin digested, and peptides were analyzed by nano-HPLC-MS/MS. Distinct peptides were compared to sequences in the data banks and attributed to corresponding proteins and genes. Inhibition of CAS induced proteins that appear to mitigate the negative effects of the chemical exposure. However, as all enzymes that are directly involved in phytosterol biosynthesis are low-abundant proteins, significant changes in their levels could not be observed. Differences could be seen with enzymes involved in primary metabolism (glycolysis, pentose phosphate pathway etc.), in proteins of the chaperonin family, and those, like actin, that participate in formation and strengthening of the cytoskeleton and have some impact on cell growth and division.

A sensitive radiometric assay to measure D-xylulose kinase activity.

A radiometric test system for D-xylulose kinase (XK) was developed for the measurement of enzyme activity in crude cell extracts and to minimize the volume of reaction mixtures besides increasing the sensitivity. [U-14C]xylulose 5-phosphate was produced from commercially available [U-14C]xylose in a coupled assay system containing D-xylose isomerase, which yields [U-14C]xylulose, the substrate of ATP-dependent D-xylulose kinase. Separation of products and substrates was achieved by thin layer chromatography, identification of radioactive spots by radioscanning followed by quantitative scintillation counting. The protocol was validated through determination of kinetic constants of a purified His-tagged enzyme from Escherichia coli and comparison with the spectrophotometric method. The radiometric assay was applied to determine xylulose kinase activity in crude cell extracts from a variety of eukaryotic and prokaryotic organisms.