SummaryTo investigate the contribution of farnesyl diphosphate synthase (FPS) to the overall control of the mevalonic acid pathway in plants, we have generated transgenic Arabidopsis thaliana overexpressing the Arabidopsis FPS1S isoform. Despite high levels of FPS activity in transgenic plants (8-to 12-fold as compared to wild-type plants), the content of sterols and the levels of 3-hydroxy-3-methylglutaryl-CoA reductase activity in leaves were similar to those in control plants. Plants overexpressing FPS1S showed a cell death/senescence-like phenotype and grew less vigorously than wild-type plants. The onset and the severity of these phenotypes directly correlated with the levels of FPS activity. In leaves of plants with increased FPS activity, the expression of the senescence activated gene SAG12 was prematurely induced. Transgenic plants grown in the presence of either mevalonic acid (MVA) or the cytokinin 2-isopentenyladenine (2-iP) recovered the wild-type phenotype. Quanti®cation of endogenous cytokinins demonstrated that FPS1S overexpression speci®cally reduces the levels of endogenous zeatin-type cytokinins in leaves. Altogether these results support the notion that increasing FPS activity without a concomitant increase of MVA production leads to a reduction of IPP and DMAPP available for cytokinin biosynthesis. The reduced cytokinin levels would be, at least in part, responsible for the phenotypic alterations observed in the transgenic plants. The ®nding that wild-type and transgenic plants accumulated similar increased amounts of sterols when grown in the presence of exogenous MVA suggests that FPS1S is not limiting for sterol biosynthesis.

Overexpression of Arabidopsis thaliana farnesyl diphosphate synthase isoform 1S (FPS1S) in transgenic A. thaliana (L.) Heynh. leads to necrotic lesion formation in leaves in planta and to premature senescence in detached leaves [A. Masferrer et al. (2002) Plant J 30:123-132]. Here we report that leaves of plants overexpressing FPS1S with symptoms of necrosis show increased H2O2 formation and induction of both the pathogenesis-related 1 (PR-1) and the alternative oxidase 1a (AOX1a) genes. These findings indicate that plants overexpressing FPS1S should be considered as lesion-mimic mutants and lead us to propose that H2O2 is the main inducing agent of necrosis in these plants. The onset of necrosis appears in a developmentally regulated manner that correlates with the developmental decline of endogenous 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) activity. Accordingly, constitutive overexpression of HMGR in plants overexpressing FPS1S prevents both necrosis and premature senescence. These observations demonstrate that both phenotypes are due to an insufficient supply of mevalonic acid and support the notion that the metabolic imbalance associated with FPS1S overexpression is, in fact, triggered by the developmental decline of HMGR activity. We also show that overexpression of FPS1S alleviates growth inhibition caused by overexpression of the catalytic domain of isoform HMGR1S. Overall, our results reinforce the view that the levels of specific intermediates of the mevalonic acid pathway must be strictly controlled, particularly those located at branch-point positions, in order to avoid deleterious effects on plant growth and development.

A wide diversity of isoprenoids is produced in different plant compartments. Most groups of isoprenoids synthesized in plastids, and some produced elsewhere in the plant cell derive from geranylgeranyl diphosphate (GGPP) synthesized by GGPP synthase (GGPPS) enzymes. In Arabidopsis (Arabidopsis thaliana), five genes appear to encode GGPPS isoforms localized in plastids (two), the endoplasmic reticulum (two), and mitochondria (one). However, the loss of function of the plastid-targeted GGPPS11 isoform (referred to as G11) is sufficient to cause lethality. Here, we show that the absence of a strong transcription initiation site in the G11 gene results in the production of transcripts of different lengths. The longer transcripts encode an isoform with a functional plastid import sequence that produces GGPP for the major groups of photosynthesis-related plastidial isoprenoids. However, shorter transcripts are also produced that lack the first translation initiation codon and rely on a second in-frame ATG codon to produce an enzymatically active isoform lacking this N-terminal domain. This short enzyme localizes in the cytosol and is essential for embryo development. Our results confirm that the production of differentially targeted enzyme isoforms from the same gene is a central mechanism to control the biosynthesis of isoprenoid precursors in different plant cell compartments.Plants produce tens of thousands of isoprenoid compounds, including some that are essential for respiration, photosynthesis, and regulation of growth and development. Despite their structural and functional diversity, all isoprenoids derive from the same fivecarbon precursors, the double-bond isomers isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which can be interconverted by IPP/ DMAPP isomerase (IDI) enzymes. Plants use two unrelated pathways to synthesize these units (Fig. 1). The mevalonic acid (MVA) pathway synthesizes IPP in the cytosol, whereas the methylerythritol 4-phosphate (MEP) pathway supplies both IPP and DMAPP in the plastid (Bouvier et al., 2005;Vranová et al., 2013; Rodriguez-Concepción and Boronat, 2015). IPP and DMAPP units can be exchanged between cell compartments to a certain level. For example, MVA-derived IPP can be imported by mitochondria for the biosynthesis of ubiquinone (Lütke-Brinkhaus et al., 1984;Disch et al., 1998). However, this limited exchange of common isoprenoid precursors is not active enough to rescue a genetic or pharmacological blockage of one of the pathways with IPP/DMAPP produced by the noninhibited pathway (Bouvier et al., 2005;Vranová et al., 2013; Rodriguez-Concepción and Boronat, 2015). Addition of IPP units to DMAPP generates longer prenyl diphosphate molecules, including C10 geranyl diphosphate (GPP), C15 farnesyl diphosphate (FPP), and C20 geranylgeranyl diphosphate (GGPP), which are then used in specific downstream pathways to produce particular isoprenoids (Fig. 1). FPP and GGPP pools represent nodes of the major metabolic branch points in the isoprenoid biosynthesis ...

The role of RNA metabolism in chromatin silencing is now widely recognized. We have studied the Arabidopsis RNA-binding protein FCA that down-regulates an endogenous floral repressor gene through a chromatin mechanism involving histone demethylase activity. This mechanism needs FCA to interact with an RNA 3 processing/polyadenylation factor (FY/Pfs2p), but the subsequent events leading to chromatin changes are unknown. Here, we show that this FCA-FY interaction is required for general chromatin silencing roles where hairpin transgenes induce DNA methylation of an endogenous gene. We also show 2 conserved RNA processing factors, AtCPSF100 and AtCPSF160, but not FCA, are stably associated with FY in vivo and form a range of different-sized complexes. A hypomorphic fy allele producing a shorter protein, able to provide some FY functions but unable to interact with FCA, reduces abundance of some of the larger MW complexes. Suppressor mutants, which specifically disrupt the FY motif through which FCA interacts, also lacked these larger complexes. Our data support a model whereby FCA, perhaps after recognition of a specific RNA feature, transiently interacts with FY, an integral component of the canonical RNA 3 processing machinery, changing the interactions of the different RNA processing components. These altered interactions would appear to be a necessary step in this RNA-mediated chromatin silencing.flowering ͉ gene silencing ͉ RNA processing N oncoding RNAs are now thought to play major roles in directing chromatin silencing (1). We have pursued the mechanistic basis of RNA-mediated silencing of the Arabidopsis floral regulator FLOWERING LOCUS C (FLC) by the autonomous pathway (2-4). This pathway has a widespread role in chromatin silencing in the Arabidopsis genome (5) and involves both RNA-binding (FCA, FPA, FLK)/RNA 3Ј processing proteins (FY) and putative chromatin regulators (FVE, FLD and LD) (6). The RRM proteins FCA and FPA at least partially require one of the chromatin regulators, FVE-an MSI homologue-or FLD, an LSD1 homologue) to effect silencing; however, the requirements for specific chromatin regulators differ at different loci (7). Our current view is that this pathway functions as part of a transcriptome surveillance mechanism, transcriptionally down-regulating loci producing ''aberrant'' transcripts.The RNA-binding protein FCA was used in affinity purification experiments to clone FY, the Arabidopsis homologue of the essential yeast RNA 3Ј processing factor Pfs2p (8). fy null alleles were found to be embryo lethal (9); however, the initial fy alleles ( fy-1, fy-2) were viable, just late-flowering because of production of a truncated peptide lacking the FCA interaction domain (9). These results suggested that FY might be a component of the Arabidopsis core RNA processing machinery, with the floweringspecific defects resulting from the loss of the FCA-FY interaction. FCA would then regulate the recruitment of specific transcripts to the core processing machinery. This classic posttranscriptional fun...

Arabidopsis thaliana contains two genes encoding farnesyl diphosphate (FPP) synthase (FPS), the prenyl diphoshate synthase that catalyzes the synthesis of FPP from isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). In this study, we provide evidence that the two Arabidopsis short FPS isozymes FPS1S and FPS2 localize to the cytosol. Both enzymes were expressed in E. coli, purified and biochemically characterized. Despite FPS1S and FPS2 share more than 90% amino acid sequence identity, FPS2 was found to be more efficient as a catalyst, more sensitive to the inhibitory effect of NaCl, and more resistant to thermal inactivation than FPS1S. Homology modelling for FPS1S and FPS2 and analysis of the amino acid differences between the two enzymes revealed an increase in surface polarity and a greater capacity to form surface salt bridges of FPS2 compared to FPS1S. These factors most likely account for the enhanced thermostability of FPS2. Expression analysis of FPS::GUS genes in seeds showed that FPS1 and FPS2 display complementary patterns of expression particularly at late stages of seed development, which suggests that Arabidopsis seeds have two spatially segregated sources of FPP. Functional complementation studies of the Arabidopsis fps2 knockout mutant seed phenotypes demonstrated that under normal conditions FPS1S and FPS2 are functionally interchangeable. A putative role for FPS2 in maintaining seed germination capacity under adverse environmental conditions is discussed.

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...

To investigate the role of mitochondrial farnesyl diphosphate synthase (FPS) in plant isoprenoid biosynthesis we characterized transgenic Arabidopsis thaliana plants overexpressing FPS1L isoform. This overexpressed protein was properly targeted to mitochondria yielding a mature and active form of the enzyme of 40 kDa. Leaves from transgenic plants grown under continuous light exhibited symptoms of chlorosis and cell death correlating to H(2)O(2) accumulation, and leaves detached from the same plants displayed accelerated senescence. Overexpression of FPS in mitochondria also led to altered leaf cytokinin profile, with a reduction in the contents of physiologically active trans-zeatin- and isopentenyladenine-type cytokinins and their corresponding riboside monophosphates as well as enhanced levels of cis-zeatin 7-glucoside and storage cytokinin O-glucosides. Overexpression of 3-hydroxy-3-methylglutaryl coenzyme A reductase did not prevent chlorosis in plants overexpressing FPS1L, but did rescue accelerated senescence of detached leaves and restored wild-type levels of cytokinins. We propose that the overexpression of FPS1L leads to an enhanced uptake and metabolism of mevalonic acid-derived isopentenyl diphosphate and/or dimethylallyl diphosphate by mitochondria, thereby altering cytokinin homeostasis and causing a mitochondrial dysfunction that renders plants more sensitive to the oxidative stress induced by continuous light.

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.