Dedicated Roles of Plastid Transketolases during the Early Onset of Isoprenoid Biogenesis in Pepper Fruits

Isopentenyl diphosphate (IPP), which is produced from mevalonic acid or other nonmevalonic substrates, is the universal precursor of isoprenoids in nature. Despite the presence of several isoprenoid compounds in plastids, enzymes of the mevalonate pathway leading to IPP formation have never been isolated or identified to our knowledge. We now describe the characterization of two pepper (Capsicum annuum L.) cDNAs, CapTKT1 and CapTKT2, that encode transketolases having distinct and dedicated specificities. CapTKT1 is primarily involved in plastidial pentose phosphate and glycolytic cycle integration, whereas CapTKT2 initiates the synthesis of isoprenoids in plastids via the nonmevalonic acid pathway. From pyruvate and glyceraldehyde-3-phosphate, CapTKT2 catalyzes the formation of 1-deoxy-xylulose-5-phosphate, the IPP precursor. CapTKT1 is almost constitutively expressed during the chloroplast-to-chromoplast transition, whereas CapTKT2 is overexpressed during this period, probably to furnish the IPP necessary for increased carotenoid biosynthesis. Because deoxy-xylulose phosphate is shared by the plastid pathways of isoprenoid, thiamine (vitamin B₁), and pyridoxine (vitamin B₆) biosynthesis, our results may explain why albino phenotypes usually occur in thiamine-deficient plants.     

Distinct light-mediated pathways regulate the biosynthesis and exchange of isoprenoid precursors during Arabidopsis seedling development

Plants synthesize an astonishing diversity of isoprenoids, some of which play essential roles in photosynthesis, respiration, and the regulation of growth and development. Two independent pathways for the biosynthesis of isoprenoid precursors coexist within the plant cell: the cytosolic mevalonic acid (MVA) pathway and the plastidial methylerythritol phosphate (MEP) pathway. In at least some plants (including Arabidopsis), common precursors are exchanged between the cytosol and the plastid. However, little is known about the signals that coordinate their biosynthesis and exchange. To identify such signals, we arrested seedling development by specifically blocking the MVA pathway with mevinolin (MEV) or the MEP pathway with fosmidomycin (FSM) and searched for MEV-resistant Arabidopsis mutants that also could survive in the presence of FSM. Here, we show that one such mutant, rim1, is a new phyB allele (phyB-m1). Although the MEV-resistant phenotype of mutant seedlings is caused by the upregulation of MVA synthesis, its resistance to FSM most likely is the result of an enhanced intake of MVA-derived isoprenoid precursors by the plastid. The analysis of other light-hyposensitive mutants showed that distinct light perception and signal transduction pathways regulate these two differential mechanisms for resistance, providing evidence for a coordinated regulation of the activity of the MVA pathway and the crosstalk between cell compartments for isoprenoid biosynthesis during the first stages of seedling development.     

Evidence of isoprenoid precursor toxicity in Bacillus subtilis

The mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways for isoprenoid biosynthesis both culminate in the production of the two-five carbon prenyl diphosphates: dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). These are the building blocks for higher isoprenoids, including many that have industrial and pharmaceutical applications. With growing interest in producing commercial isoprenoids through microbial engineering, reports have appeared of toxicity associated with the accumulation of prenyl diphosphates in Escherichia coli expressing a heterologous MVA pathway. Here we explored whether similar prenyl diphosphate toxicity, related to MEP pathway flux, could also be observed in the bacterium Bacillus subtilis. After genetic and metabolic manipulations of the endogenous MEP pathway in B. subtilis, measurements of cell growth, MEP pathway flux, and DMAPP contents suggested cytotoxicity related to prenyl diphosphate accumulation. These results have implications as to understanding the factors impacting isoprenoid biosynthesis in microbial systems.     

Two independent biochemical pathways for isopentenyl diphosphate and isoprenoid biosynthesis in higher plants

In the early times of isoprenoid research, a single pathway was found for the formation of the C₅ monomer, isopentenyl diphosphate (IPP), and this acetate/mevalonate pathway was supposed to occur ubiquitously in all living organisms. Now, 40 years later, a totally different IPP biosynthesis route has been detected in eubacteria, green algae and higher plants. In this new pathway glyceraldehyde 3-phosphate (GAP) and pyruvate are precursors of isopentenyl diphosphate, but not acetyl-CoA and mevalonic acid. In green tissues of three higher plants it was shown that all chloroplastbound isoprenoids (β-carotene, phytyl chains of chlorophylls and nona-prenyl chain of plastoquinone-9) are formed via the GAP/pyruvate pathway, whereas the cytoplasmic sterols are formed via the acetate/mevalonate pathway. Also, isoprene, emitted by various plants at high light conditions by action of the plastid-bound isoprene synthase, is formed via the new GAP/pyruvate pathway. Thus, in higher plants, there exist two separate and biochemically different IPP biosynthesis pathways: (1) the novel alternative GAP/pyruvate pathway apparently bound to the plastidic compartment and (2) the classical cytoplasmic acetate/mevalonate pathway. This new GAP/pyruvate pathway for IPP formation allows a reasonable interpretation of previous odd results concerning the biosynthesis of chloroplast isoprenoids, which, so far, had mainly been interpreted assuming compartmentation differences. The novel GAP/pyruvate pathway for IPP formation in plastids appears as a heritage of their prokaryotic, endosymbiotic ancestors.     

Monoterpene and sesquiterpene biosynthesis in glandular trichomes of peppermint (Mentha x piperita) rely exclusively on plastid-derived isopentenyl diphosphate

The subcellular compartmentation of isopentenyl diphosphate (IPP) synthesis was examined in secretory cells isolated from glandular trichomes of peppermint (Mentha x piperita L. cv. Black Mitcham). As a consequence of their anatomy and the conditions of their isolation, the isolated secretory cells are non-specifically permeable to low-molecular-weight water-soluble metabolites. Thus, the cytoplasm is readily accessible to the exogenous buffer whereas the selective permeability of subcellular organelles is maintained. With the appropriate choice of exogenous substrates, this feature allows the assessment of cytoplasmic and organellar (e.g. plastidic) metabolism in situ. Glycolytic substrates such as [¹⁴C]glucose-6-phosphate and [¹⁴C]pyruvic acid are incorporated into both monoterpenes and sesquiterpenes with a monoterpene:sesquiterpene ratio that closely mimics that observed in vivo, indicating that the correct subcellular partitioning of these substrates is maintained in this model system. Additionally, exogenous [¹⁴C]mevalonic acid and [¹⁴C]IPP, which are both intitially metabolized in the cytoplasm, produce an abnormally high proportion of sesquiterpenes. In contrast, incubation with either [¹⁴C]citrate or [¹⁴C]acetyl-CoA results in the accumulation of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) with no detectable isoprenoids formed. Taken together, these results indicate that the cytoplasmic mevalonic acid pathway is blocked at HMG-CoA reductase and that the IPP utilized for both monoterpene and sesquiterpene biosynthesis is synthesized exclusively in the plastids.     

Metabolic engineering of the mevalonate and non-mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids in tomato

The genetic manipulation of both the mevalonic acid (MVA) and methylerythritol-4-phosphate (MEP) pathways, leading to the formation of isopentenyl diphosphate (IPP), has been achieved in tomato using 3-hydroxymethylglutaryl CoA (hmgr-1) and 1-deoxy-d-xylulose-5-phosphate synthase (dxs) genes, respectively. Transgenic plants containing an additional hmgr-1 from Arabidopsis thaliana, under the control of the cauliflower mosaic virus (CaMV) 35S constitutive promoter, contained elevated phytosterols (up to 2.4-fold), but IPP-derived isoprenoids in the plastid were unaltered. Transgenic lines containing a bacterial dxs targeted to the plastid with the tomato dxs transit sequence resulted in an increased carotenoid content (1.6-fold), which was inherited in the next generation. Phytoene and beta-carotene exhibited the greatest increases (2.4- and 2.2-fold, respectively). Extra-plastidic isoprenoids were unaffected in these lines. These data are discussed with respect to the regulation, compartmentalization and manipulation of isoprenoid biosynthetic pathways and their relevance to plant biotechnology.     

Evidence of Isoprenoid Precursor Toxicity in Bacillus subtilis

The mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways for isoprenoid biosynthesis both culminate in the production of the two-five carbon prenyl diphosphates: dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP). These are the building blocks for higher isoprenoids, including many that have industrial and pharmaceutical applications. With growing interest in producing commercial isoprenoids through microbial engineering, reports have appeared of toxicity associated with the accumulation of prenyl diphosphates in Escherichia coli expressing a heterologous MVA pathway. Here we explored whether similar prenyl diphosphate toxicity, related to MEP pathway flux, could also be observed in the bacterium Bacillus subtilis. After genetic and metabolic manipulations of the endogenous MEP pathway in B. subtilis, measurements of cell growth, MEP pathway flux, and DMAPP contents suggested cytotoxicity related to prenyl diphosphate accumulation. These results have implications as to understanding the factors impacting isoprenoid biosynthesis in microbial systems.     

Chlorophyta exclusively use the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway for the biosynthesis of isoprenoids

The biosynthesis of the C5 building block of isoprenoids, isopentenyl diphosphate (IPP), proceeds in higher plants via two basically different pathways; in the cytosolic compartment sterols are formed via mevalonate (MVA), whereas in the plastids the isoprenoids are formed via the 1-deoxyxylulose 5-phosphate/2-C-methylerythritol 4-phosphate pathway (DOXP/MEP pathway). In the present investigation, we found for the Charophyceae, being close relatives to land plants, and in the original green flagellate Mesostignma virilde the same IPP biosynthesis pattern as in higher plants: sterols are formed via MVA, and the phytol-moiety of chlorophylls via the DOXP/MEP pathway. In contrast, representatives of four classes of the Chlorophyta (Chlorophyceae, Ulvophyceae, Trebouxiophyceae, Prasinophyceae) did not incorporate MVA into sterols or phytol. Instead, they incorporated [1-2H1]-1-deoxy-D-xylulose into phytol and sterols. The results indicate that the entire Chlorophyta lineage, which is well separated from the land plant/Charophyceae lineage, is devoid of the acetate/ MVA pathway and uses the DOXP/MEP pathway not only for plastidic, but also for cytosolic isoprenoid formation.     

Early Steps in Isoprenoid Biosynthesis: Multilevel Regulation of the Supply of Common Precursors in Plant Cells

Isoprenoids are produced in all organisms but are especially abundant and diverse in plants. Two separate pathways operate in plant cells to synthesize prenyl diphosphate precursors common to all isoprenoids. Cytosolic and mitochondrial precursors are produced by the mevalonic acid (MVA) pathway whereas the recently discovered methylerythritol phosphate (MEP) pathway is located in plastids. However, both pathways may participate in the synthesis of at least some isoprenoids under certain circumstances. Although genes encoding all the enzymes from both pathways have already been cloned, little is known about the regulatory mechanisms that control the supply of isoprenoid precursors. Genetic approaches are providing valuable information on the regulation of both pathways. Thus, recent data from overexpression experiments in transgenic plants show that several enzymes share control over the metabolic flux through the MEP pathway, whereas a single regulatory step has been proposed for the MVA pathway. Identification of Arabidopsis thaliana mutants that are resistant to the inhibition of the MVA and the MEP pathways is a promising approach to uncover mechanisms involved in the crosstalk between pathways. The characterization of some of these mutants impaired in light perception and signaling has recently provided genetic evidence for a role of light as a key factor to modulate the availability of isoprenoid precursors in Arabidopsis seedlings. The picture emerging from recent data supports that a complex regulatory network appears to be at work in plant cells to ensure the supply of isoprenoid precursors when needed.     

Isoprenoid biosynthesis via 1-deoxy-D-xylulose 5-phosphate/2-C-methyl-D-erythritol 4-phosphate (DOXP/MEP) pathway

Higher plants, several algae, bacteria, some strains of Streptomyces and possibly malaria parasite Plasmodium falciparum contain the novel, plastidic DOXP/MEP pathway for isoprenoid biosynthesis. This pathway, alternative with respect to the classical mevalonate pathway, starts with condensation of pyruvate and glyceraldehyde-3-phosphate which yields 1-deoxy-D-xylulose 5-phosphate (DOXP); the latter product can be converted to isopentenyl diphosphate (IPP) and eventually to isoprenoids or thiamine and pyridoxal. Subsequent reactions of this pathway involve transformation of DOXP to 2-C-methyl-D-erythritol 4-phosphate (MEP) which after condensation with CTP forms 4-diphosphocytidyl-2-amethyl-D-erythritol (CDP-ME). Then CDP-ME is phosphorylated to 4-diphosphocytidyl-2-amethyl-D-erythritol 2-phosphate (CDP-ME2P) and to 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (ME-2,4cPP) which is the last known intermediate of the DOXP/MEP pathway. For- mation of IPP and dimethylallyl diphosphate (DMAPP) from ME-2,4cPP still requires clarification. This novel pathway appears to be involved in biosynthesis of carotenoids, phytol (side chain of chlorophylls), isoprene, mono-, di-, tetraterpenes and plastoquinone whereas the mevalonate pathway is responsible for formation of sterols, sesquiterpenes and triterpenes. Several isoprenoids were found to be of mixed origin suggesting that some exchange and/or cooperation exists between these two pathways of different biosynthetic origin. Contradictory results described below could indicate that these two pathways are operating under different physiological conditions of the cell and are dependent on the developmental state of plastids.     

1-脱氧木酮糖-5-磷酸合成酶(DXS)及其编码基因

萜类物质是广泛分布于生物界的一类天然产物,也是重要生命物质。萜类物质通过甲羟戊酸(MVA)途径和2-C-甲基-D-赤藻糖醇-4-磷酸(MEP)途径合成,古细菌、真菌和动物及人的萜类物质主要通过MVA途径合成,而多数真细菌(即通常而言的细菌)则利用MEP途径。植物同时拥有两种途径但分别定位于细胞质和质体。1-脱氧木酮糖-5-磷酸合成酶(DXS)是MEP途径的第一个酶,也是该途径的关键调控位点。现从DXS在MEP途径中的作用、DXS结构、亚细胞定位和酶活性、编码基因及突变体等方面对DXS进行全面阐述。拟南芥DXS基因插入突变体cla1-1发生白化,DXS基因表达与类胡萝卜素等萜类物质积累密切相关,在转基因生物体中过度表达DXS可促进萜类物质合成。植物DXS具有典型的质体转运肽序列,决定了DXS的质体定位。完备的DXS活性分析体系为DXS抑制剂开发筛选等研究奠定良好基础。DXS由一至多个基因编码,随生物种类而异,根据同源性,植物DXS基因可分成两类。DXS基因家族不同成员具有不同的表达模式,但通常有一个成员在多种组织中广泛表达。     

CHLOROPLAST BIOGENESIS genes act cell and noncell autonomously in early chloroplast development

In order to identify nuclear genes required for early chloroplast development, a collection of photosynthetic pigment mutants of Arabidopsis was assembled and screened for lines with extremely low levels of chlorophyll. Nine chloroplast biogenesis (clb) mutants that affect proplastid growth and thylakoid membrane formation and result in an albino seedling phenotype were identified. These mutations identify six new genes as well as a novel allele of cla1. clb mutants have less than 2% of wild-type chlorophyll levels, and little or no expression of nuclear and plastid-encoded genes required for chloroplast development and function. In all but one mutant, proplastids do not differentiate enough to form elongated stroma thylakoid membranes. Analysis of mutants during embryogenesis allows differentiation between CLB genes that act noncell autonomously, where partial maternal complementation of chloroplast development is observed in embryos, and those that act cell autonomously, where complementation during embryogenesis is not observed. Molecular characterization of the noncell autonomous clb4 mutant established that the CLB4 gene encodes for hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (HDS), the next to the last enzyme of the methylerythritol 4-phosphate (MEP) pathway for the synthesis of plastidic isoprenoids. The noncell autonomous nature of the clb4 mutant suggests that products of the MEP pathway can travel between tissues, and provides in vivo evidence that some movement of MEP intermediates exists from the cytoplasm to the plastid. The isolation and characterization of clb mutants represents the first systematic study of genes required for early chloroplast development in Arabidopsis.     

Genetic evidence for the role of isopentenyl diphosphate isomerases in the mevalonate pathway and plant development in Arabidopsis

Isopentenyl/dimethylallyl diphosphate isomerase (IPI) catalyzes the interconversion of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the universal C(5) units of isoprenoids. In plants, IPP and DMAPP are synthesized via the cytosolic mevalonate (MVA) and plastidic methylerythritol phosphate (MEP) pathways, respectively. However, the role of IPI in each pathway and in plant development is unknown due to a lack of genetic studies using IPI-defective mutants. Here, we show that the atipi1atipi2 double mutant, which is defective in two Arabidopsis IPI isozymes, exhibits dwarfism and male sterility under long-day conditions and decreased pigmentation under continuous light, whereas the atipi1 and atipi2 single mutants are phenotypically normal. We also show that the sterol and ubiquinone levels in the double mutant are <50% of those in wild-type plants, and that the male-sterile phenotype is chemically complemented by squalene, a sterol precursor. In vivo isotope labeling experiments using the atipi1atipi2 double mutant revealed a decrease in the incorporation of MVA (in its lactone form) into sterols, with no decrease in the incorporation of MEP pathway intermediates into tocopherol. These results demonstrate a critical role for IPI in isoprenoid biosynthesis via the MVA pathway, and they imply that IPI is essential for the maintenance of appropriate levels of IPP and DMAPP in different subcellular compartments in plants.     

Fruit carotenoid‐deficient mutants in tomato reveal a function of the plastidial isopentenyl diphosphate isomerase (IDI1) in carotenoid biosynthesis

Isoprenoids consist of a large class of compounds that are present in all living organisms. They are derived from the 5C building blocks isopentenyl diphosphate (IDP) and its isomer dimethylallyl diphosphate (DMADP). In plants, IDP is synthesized in the cytoplasm from mevalonic acid via the MVA pathway, and in plastids from 2‐C‐methyl‐d ‐erythritol‐4‐phosphate through the MEP pathway. The enzyme IDP isomerase (IDI) catalyzes the interconversion between IDP and DMADP. Most plants contain two IDI enzymes, the functions of which are characteristically compartmentalized in the cells. Carotenoids are isoprenoids that play essential roles in photosynthesis and provide colors to flowers and fruits. They are synthesized in the plastids via the MEP pathway. Fruits of Solanum lycopersicum (tomato) accumulate high levels of the red carotene lycopene. We have identified mutations in tomato that reduce overall carotenoid accumulation in fruits. Four alleles of a locus named FRUIT CAROTENOID DEFICIENT 1 (fcd1) were characterized. Map‐based cloning of fcd1 indicated that this gene encodes the plastidial enzyme IDI1. Lack of IDI1 reduced the concentration of carotenoids in fruits, flowers and cotyledons, but not in mature leaves. These results indicate that the plastidial IDI plays an important function in carotenoid biosynthesis, thus highlighting its role in optimizing the ratio between IDP and DMADP as precursors for different downstream isoprenoid pathways.     

Metabolic engineering of biosynthetic pathways leading to isoprenoids: Mono- and sesquiterpenes in plastids and cytosol

Abstract

Isoprenoids are a large and structurally diverse family of compounds that play essential roles in plants as hormones, photosynthetic pigments, electron carriers, and membrane components as well as serving in communication and defense. Now it is unequivocally proved that two distinct and independent biosynthetic routes exist to isopentenyl diphosphate (IPP) and its allylic isomer dimethylallyl diphosphate (DMAPP), the two building blocks for isoprenoids in plants. The cytosolic pathway is triggered by Acetyl Coenzyme A where classical intermediate mevalonic acid is formed which, in turn, converts into IPP and DMAPP. These further combine to elongate into sesquiterpenes (C₁₅) and triterpenes (C₃₀); whereas the plastidial pathway provides precursors for the biosynthesis of isoprene (C₅), monoterpenes (C₁₀), diterpenes (C₂₀), and tetraterpenes (C₄₀). The pathway is initiated by the transketolase-type condensation of pyruvate (C-2 and C-3) and glyceraldehyde-3-phosphate to 1-deoxyxylulose-5-phosphate (DXP), followed by the isomerization and reduction of this intermediate to 2-C-methylerythritol-4-phosphate (MEP), formation of the cytidine 5'-diphosphate (CDP) derivative, phosphorylation at C2, and cyclization to 2-C-methylerythritol-2,4-cyclodiphosphate (CDP-Me2P as the last defined step). The genes encoding each enzyme of the plastid pathway up to formation of the cyclic diphosphate have been isolated from plants and from eubacteria where the pathway exists. Studies on the complete biosynthetic pathways using radio-labeled substrates will help in characterizing and identifying the enzymes involved in each and every step of cyclization, isomerization, chain elongation, hydrogen shifts, oxidation and hydroxylation during the formation of many isoprenoid compounds present in food and flavor substances and are highly useful to human beings.     

Impact of the stringency of cell selection on plastid segregation in protoplast fusion-derived Nicotiana regenerates

Summary Vegetative segregation of a mixed plastid population in protoplast fusion-derived cell lines can be directed by a selection favouring the multiplication of one of the parental plastid types. This report defines some of the critical conditions leading to a homogeneous plastid population in cybrid plants generated by protoplast fusion between Nicotiana plumbaginifolia and an albino and streptomycin-resistant N. tabacum plastid mutant. Light (1,500 lx) conferred a strong selective advantage to chloroplasts versus albino plastids, while the lack of this effect in dim light (300 lx) indicated that a sufficient light intensity is essential to the phenomenon. Selection on streptomycin-containing medium in the dark, however, led to the preferential multiplication of resistant plastids. Streptomycin selection of resistant chloroplasts in the light, consequently, results in a plastid selection of doubled stringency. In another experiment a definite, but leaky, selection for chloroplast recombination (selection for greening on streptomycin-containing medium in dim light) was used to reveal various recombination products. Protoplast fusion in fact resulted in cybrid plants showing only simple chimeric segregation of unchanged parental plastids. These results demonstrate the essential requirement for stringent plastid selection, as defined by cell culture conditions, to precede the formation of shoots expected to possess the desired plastid genetic composition.     

1-Deoxy-D-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening

The recently discovered 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of plastid isoprenoids (including carotenoids) is not fully elucidated yet despite its central importance for plant life. It is known, however, that the first reaction completely specific to the pathway is the conversion of 1-deoxy-D-xylulose 5-phosphate (DXP) into MEP by the enzyme DXP reductoisomerase (DXR). We have identified a tomato cDNA encoding a protein with homology to DXR and in vivo activity, and show that the levels of the corresponding DXR mRNA and encoded protein in fruit tissues are similar before and during the massive accumulation of carotenoids characteristic of fruit ripening. The results are consistent with a non-limiting role of DXR, and support previous work proposing DXP synthase (DXS) as the first regulatory enzyme for plastid isoprenoid biosynthesis in tomato fruit. Inhibition of DXR activity by fosmidomycin showed that plastid isoprenoid biosynthesis is required for tomato fruit carotenogenesis but not for other ripening processes. In addition, dormancy was reduced in seeds from fosmidomycin-treated fruit but not in seeds from the tomato yellow ripe mutant (defective in phytoene synthase-1, PSY1), suggesting that the isoform PSY2 might channel the production of carotenoids for abscisic acid biosynthesis. Furthermore, the complete arrest of tomato seedling development using fosmidomycin confirms a key role of the MEP pathway in plant development.