Butenyl 4-diphosphate Reductase Research Articles

Enhanced metabolic flux of methylerythritol phosphate (MEP) pathway by overexpression of Ginkgo biloba 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate Reductase 1 (GbHDR1) gene in poplar

Terpenoids are of great interests in a broad range of health-beneficial biological activities and various industrial applications. In plants, terpenoids are synthesized by two distinct pathways, methylerythritol phosphate (MEP) and mevalonate pathways in a separate location. MEP pathway supplies isoprene precursors isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP) of terpenoid biosynthesis in plant plastids. The MEP pathway has been an engineering target to increase the metabolic flux towards higher terpenoid production in plants. 1-Hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (HDR) is the terminal step of the MEP pathway to regulate the terpenoid biosynthesis and is encoded by three paralogous genes in Ginkgo biloba. In this study, we assessed the effect of overexpression of GbHDR1 on terpenoid metabolism in poplar plants. Overexpression of GbHDR1 in poplar plants accelerated growth and delayed winter-bud formation. Transcript levels of gibberellin, chlorophylls, and carotenoid biosynthetic genes in GbHDR1-overexpressing (GbHDR1ox) poplars were up-regulated, suggesting metabolic flux enhancement. Moreover, enhanced contents of chlorophylls and carotenoids in the leaves of the GbHDR1ox plants resulted in a higher photosynthetic rate as a consequence. Therefore, we expect the GbHDR1 overexpression will be a desirable engineering point of the MEP pathway for enhancing terpenoid metabolic flux and production in plants.

Effects of lovastin, fosmidomycin and methyl jasmonate on andrographolide biosynthesis in the Andrographis paniculata

Andrographolide is a diterpene secondary metabolite product of Andrographis paniculata. It has been known to be a pharmaceutically important compound synthesized via the cytosolic mevalonate (MVA) and the plastidial 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. To understand the biosynthetic pathway of andrographolide biosynthesis in Andrographis paniculata, lovastatin, fosmidomycin and methyl jasmonate (MeJA) were used to inhibit the key enzymes 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), and 1-deoxy-d-xylulose-5-phosphate reducto-isomerase (DXR) involved in the synthesis of andrographolide in the MVA and MEP pathways, respectively. The inhibition of andrographolide accumulation was linked with the expression level of the studied regulatory genes, 3-hydroxy-3-methyl glutaryl coenzyme A synthase (hmgs), 3-hydroxy-3-methyl glutaryl coenzyme A reductase (hmgr), 1-deoxyxylulose-5-phosphate synthase (dxs), 1-deoxyxylulose-5-phosphate reductoisomerase (dxr), 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase (hds),1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate reductase (hdr), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase(isph), isopentenyl diphosphate isomerase (ipp), geranylgeranyl diphosphatesynthase (ggps) of the MVA and MEP pathways. The pathways associated transcript expression level, and andrographolide biosynthesis was significantly modulated by the inhibitors indicating that the andrographolide biosynthesis is strongly responsive at the transcriptional level. The results demonstrated that both pathways can contribute to the biosynthesis of andrographolide in A. paniculata. Both hmgr and dxr played a critical role consistent with some crossover between MVA and MEP pathways in andrographolide biosynthesis.

Cloning and bioinformatics analysis of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase（PcHDR1）gene in Phlegmarirus carinatus

The open reading frame of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) was cloned from Phlegmarirus carinatus by RT-PCR method and the sequence was analyzed by bioinformatics tools. After searching the transcriptome dataset of P. carinatus, one unique sequence encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase was discovered. The primers were designed according to the cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from the dataset. And then, the open reading frame (ORF) of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, named as PcHDR1 (GenBank Accession number：JQ957845), was cloned by RT-PCR strategy with the template of mixed RNA extracted from roots, stem and leaf of P. carinatus. The bioinformatic analysis of this gene and its corresponding protein was performed. The ORF of PcHDR1 consisted of 1 437 base pairs (bp), encoding one polypeptide with 478 amino acids. The sequence comparison showed that PcHDR1 is closest with GbHDR (Ginkgo biloba),and the sequence homology was up to 78%. Bioinformatics prediction and analysis indicated that PcHDR1 protein contained a conserved domain of LytB, without transmembrane region and signal peptides. This study cloned and analyzed 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from P. carinatus. The result will provide a foundation for exploring the function of PcHDR1 involved in terpene biosynthesis in P. carinatus plants.

Organ- and Growing Stage-Specific Expression of Solanesol Biosynthesis Genes in Nicotiana tabacum Reveals Their Association with Solanesol Content.

Solanesol is a noncyclic terpene alcohol that is composed of nine isoprene units and mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum L.). In the present study, RNA-seq analyses of tobacco leaves, stems, and roots were used to identify putative solanesol biosynthesis genes. Six 1-deoxy-d-xylulose 5-phosphate synthase (DXS), two 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), two 2-C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD), four 4-diphosphocytidyl-2-C-methyl-d-erythritol kinase (IspE), two 2-C-methyl-d-erythritol 2,4-cyclo-diphosphate synthase (IspF), four 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG), two 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IspH), six isopentenyl diphosphate isomerase (IPI), and two solanesyl diphosphate synthase (SPS) candidate genes were identified in the solanesol biosynthetic pathway. Furthermore, the two N. tabacum SPS proteins (NtSPS1 and NtSPS2), which possessed two conserved aspartate-rich DDxxD domains, were highly homologous with SPS enzymes from other solanaceous plant species. In addition, the solanesol contents of three organs and of leaves from four growing stages of tobacco plants corresponded with the distribution of chlorophyll. Our findings provide a comprehensive evaluation of the correlation between the expression of different biosynthesis genes and the accumulation of solanesol, thus providing valuable insight into the regulation of solanesol biosynthesis in tobacco.

Parthenolide accumulation and expression of genes related to parthenolide biosynthesis affected by exogenous application of methyl jasmonate and salicylic acid in Tanacetum parthenium.

Up-regulation of germacrene A synthase and down-regulation of parthenolide hydroxylase genes play key role in parthenolide accumulation of feverfew plants treated with methyl jasmonate and salicylic acid. Parthenolide is an important sesquiterpene lactone due to its anti-migraine and anti-cancer properties. Parthenolide amount was quantified by high-performance liquid chromatography after foliar application of methyl jasmonate (100µM) or salicylic acid (1.0mM) on feverfew leaves in time course experiment (3-96h). Results indicate that exogenous application of methyl jasmonate or salicylic acid activated parthenolide biosynthesis. Parthenolide content reached its highest amount at 24h after methyl jasmonate or salicylic acid treatments, which were 3.1- and 1.96-fold higher than control plants, respectively. Parthenolide transiently increased due to methyl jasmonate or salicylic acid treatments until 24h, but did not show significant difference compared with control plants at 48 and 96h time points in both treatments. Also, the transcript levels of early pathway (upstream) genes of terpene biosynthesis including 3-hydroxy-3-methylglutaryl-coenzyme A reductase, 1-deoxy-D-xylulose-5-phosphate reductoisomerase and hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase and the biosynthetic genes of parthenolide including germacrene A synthase, germacrene A oxidase, costunolide synthase and parthenolide synthase were increased by methyl jasmonate and salicylic acid treatments, but with different intensity. The transcriptional levels of these genes were higher in methyl jasmonate-treated plants than salicylic acid-treated plants. Parthenolide content measurements along with expression pattern analysis of the aforementioned genes and parthenolide hydroxylase as side branch gene of parthenolide suggest that the expression patterns of early pathway genes were not directly consistent with parthenolide accumulation pattern; hence, parthenolide accumulation is probably further modulated by the expression of its biosynthetic genes, especially germacrene A synthase and also its side branch gene, parthenolide hydroxylase.

The 2-C-methylerythritol 4-phosphate pathway in melon is regulated by specialized isoforms for the first and last steps.

The 2-C-methyl-d-erythritol-4-phosphate (MEP) pathway provides the precursors for the biosynthesis of plastidial isoprenoids, which include the carotenoid pigments of many fruits. We have analysed the genes encoding the seven enzymes of the MEP pathway in melon (Cucumis melo L.) and determined that the first one, 1-deoxyxylulose 5-phosphate synthase (DXS), and the last one, 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR), are represented in the genome as a small gene family and paralogous pair, respectively. In the case of DXS, three genes encode functional DXS activities which fall into previously established type I (CmDXS1) and II (CmDXS2a and CmDXS2b) categories, while a fourth DXS-like gene belonging to the type III group did not encode a protein with DXS activity. Their expression patterns and phylogenies suggest that CmDXS1 is functionally specialized for developmental and photosynthetic processes, while CmDXS2a and CmDXS2b are induced in flowers and ripening fruit of orange- (but not white-) fleshed varieties, coinciding with β-carotene accumulation. This is the first instance connecting type II DXS genes to specialized isoprenoid biosynthesis in the fruit of an agronomically important species. Two HDR paralogues were shown to encode functional enzymes, although only CmHDR1 was highly expressed in the tissues and developmental stages tested. Phylogenetic analysis showed that in cucurbits such as melon, these HDR paralogues probably arose through individual gene duplications in a common angiosperm ancestor, mimicking a prior division in gymnosperms, while other flowering plants, including apple, soy, canola, and poplar, acquired HDR duplicates recently as homoeologues through large-scale genome duplications. We report the influence of gene duplication history on the regulation of the MEP pathway in melon and the role of specialized MEP-pathway isoforms in providing precursors for β-carotene production in orange-fleshed melon varieties.

The HDR gene involved in the TIA pathway from Rauvolfia verticillata: Cloning, characterization and functional identification

1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR, EC 1.17.1.2) catalyzes the last reaction of the methylerythritol phosphate (MEP) pathway. The full-length cDNA sequence of HDR was cloned and characterized from terpenoid-indole-alkaloid-producing Rauvolfia verticillata. The new cDNA was named as RvHDR and submitted to GenBank® to be assigned with an accession number: EU034699. The full-length cDNA of RvHDR was 1679-bp containing a 1389-bp open reading frame (ORF) encoding a polypeptide of 462-amino acids with a calculated molecular mass of 52 kDa and an isoelectric point of 5.26. Comparative and bioinformatic analyses revealed that RvHDR had extensive homology with HDRs from other plant species and contained a conserved transit peptide for plastids. The phylogenetic indicated that all HDRs could be divided into three groups and RvHDR belonged to plant HDRs family.RvHDR was found to be expressed in all tested tissues including roots, stems, leaves, flowers and fruits but at different levels. The highest expression level was found in flowers, and higher expression level in leaves and fruits; the expression level was low in roots and lowest in stems. Expression profiling analyses revealed that RvHDR expression was induced by exogenous elicitors including methyl jasmonate, acetyl salicylic acid, abscisic acid and UV, and showed the transcription levels were all up-regulated compared to the control. Finally,RvHDR was transformed into the E. coli HDR mutant strain MG1655 ara HDR, which was able to rescue the lethal phenotype of the E. coli HDR mutant. This confirmed that RvHDR had the typically function of HDR gene. The cloning, characterization and functional identification of RvHDR will be helpful to understand more about the function of HDR at the level of molecular genetics and help to unveil the biosynthetic mechanism of TIAs precursor and provides a candidate gene for metabolic engineering of the TIAs pathway in R. verticillata. Key words: Rauvolfia verticillata, HDR gene, cloning, expression profile, functional complementation.

Molecular characterization of the Oncidium orchid HDR gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, the last step of the methylerythritol phosphate pathway

Two pathways are used by higher plants for the biosynthesis of isoprenoid precursors: the mevalonate pathway in the cytosol and a 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway in the plastids, with 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) catalyzing the last step in the MEP pathway. In order to understand the contribution of MEP pathway in isoprenoid biosynthesis of Oncidium orchid, a full-length cDNA corresponding to HDR from the flower tissues of Oncidium Gower Ramsey was cloned. The deduced OncHDR amino acid sequence contains a plastid signal peptide at the N-terminus and four conserved cysteine residues. RT-PCR analysis of HDR in Oncidium flowering plants revealed ubiquitous expression in organs and tissues, with preferential expression in the floral organs. Phylogenetic analysis revealed evolutionary conservation of the encoding HDR protein sequence. The genomic sequence of the HDR in Oncidium is similar to that in Arabidopsis, grape, and rice in structure. Successful complementation by OncHDR of an E. coli hdr(-) mutant confirmed its function. Transgenic tobacco carrying the OncHDR promoter-GUS gene fusion showed expression in most tissues, as well as in reproductive organs, as revealed by histochemical staining. Light induced strong GUS expression driven by the OncHDR promoter in transgenic tobacco seedlings. Taken together, our data suggest a role for OncHDR as a light-activated gene.

Regulation of resin acid synthesis in Pinus densiflora by differential transcription of genes encoding multiple 1-deoxy-d-xylulose 5-phosphate synthase and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase genes

Pinus densiflora Siebold et Zucc. is the major green canopy species in the mountainous area of Korea. To assess the response of resin acid biosynthetic genes to mechanical and chemical stimuli, we cloned cDNAs of genes encoding enzymes involved in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway (1-deoxy-d-xylulose 5-phosphate synthase (PdDXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PdDXR) and 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (PdHDR)) by the rapid amplification of cDNA ends (RACE) technique. In addition, we cloned the gene encoding abietadiene synthase (PdABS) as a marker for the site of pine resin biosynthesis. PdHDR and PdDXS occurred as two gene families. In the phylogenetic trees, PdDXSs, PdDXR and PdHDRs each formed a separate clade from their respective angiosperm homologs. PdDXS2, PdHDR2 and PdDXR were most actively transcribed in stem wood, whereas PdABS was specifically transcribed. The abundance of PdDXS2 transcripts in wood in the resting state was generally 50-fold higher than the abundance of PdDXS1 transcripts, and PdHDR2 transcripts were more abundant by an order of magnitude in wood than in other tissues, with the ratio of PdHDR2 to PdHDR1 transcripts in wood being about 1. Application of 1 mM methyl jasmonate (MeJA) selectively enhanced the transcript levels of PdDXS2 and PdHDR2 in wood. The ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 reached 900 and 20, respectively, on the second day after MeJA treatment, whereas the transcript level of PdABS increased twofold by 3 days after MeJA treatment. Wounding of the stem differentially enhanced the transcript ratios of PdDXS2 to PdDXS1 and PdHDR2 to PdHDR1 to 300 and 70, respectively. The increase in the transcript levels of the MEP pathway genes in response to wounding was accompanied by two orders of magnitude increase in PdABS transcripts. These observations indicated that resin acid biosynthesis activity, represented by PdABS transcription, was correlated with the selective transcriptions of PdDXS2 and PdHDR2. Introduction of PdDXS2, PdHDR1 and PdHDR2 rescued their respective knockout Escherichia coli mutants, confirming that at least these three genes were functionally active. Intracellular targeting of the green fluorescent protein fused to the N-terminal 100 amino acid residues of these genes in the Arabidopsis transient expression system showed that the proteins were all targeted to the chloroplasts. Our results suggest that the MEP pathway regulates resin biosynthesis in the wood of P. densiflora by differential transcription of the multiple PdDXS and PdHDR genes.

Molecular cloning and characterization of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (CaHDR) from Camptotheca acuminata and its functional identification in Escherichia coli

Camptothecin is an anti-cancer monoterpene indole alkaloid. The gene encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (designated as CaHDR), the last catalytic enzyme of the MEP pathway for terpenoid biosynthesis, was isolated from camptothecin-producing Camptotheca acuminata. The full-length cDNA of CaHDR was 1686 bp encoding 459 amino acids. Comparison of the cDNA and genomic DNA of CaHDR revealed that there was no intron in genomic CaHDR. Southern blot analysis indicated that CaHDR belonged to a low-copy gene family. RT-PCR analysis revealed that CaHDR expressed constitutively in all tested plant organs with the highest expression level in flowers, and the expression of CaHDR could be induced by 100 microM methyl-jasmonate (MeJA), but not by 100 mg/L salicylic acid (SA) in the callus of C. acuminata. The complementation of CaHDR in Escherichia coli ispH mutant MG1655 demonstrated its function.

1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS) is encoded by multicopy genes in gymnosperms Ginkgo biloba and Pinus taeda

Isoprenoids are synthesized through the condensation of five-carbon intermediates, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), derived from two distinct biosynthetic routes: cytosolic mevalonate (MVA) and plastidial 2-C-methyl-D: -erythritol 4-phosphate (MEP) pathways. 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (IDS; EC 1.17.1.2), which catalyzes the last step of MEP pathway, was cloned as a multicopy gene from gymnosperms Ginkgo biloba (GbIDS1, GbIDS2, and GbIDS2-1) and Pinus taeda (PtIDS1 and PtIDS2), and characterized. Phylogenetic tree constructed with other plant IDSs demonstrated gymnosperm IDSs were distinctively different from angiosperm IDSs. The gymnosperm IDS clade contained two subclades, one composed of GbIDS1 and PtIDS1, and the other composed of GbIDS2, GbIDS2-1, and PtIDS2. G. biloba IDSs, except GbIDS2-1, successfully complemented Escherichia coli DLYT1, a lytB disruptant, confirming the in vivo competency of isozymes. During the 4 weeks study period, although transcript levels of GbIDS1s were similar both in roots and leaves of cultured G. biloba embryo, the transcripts of GbIDS2 predominantly occurred in the embryo roots, where diterpene ginkgolides are biosynthesized. Levels of PtIDS2 transcripts in the diterpenoid resin-producing wood were 4-5 times higher than those in other tissues. Higher levels of GbIDS1 transcripts were induced by light, whereas those of GbIDS2 were increased by methyl jasmonate treatment. These results strongly imply GbIDS2 and PtIDS2 have high correlation with secondary metabolism. In Arabidopsis transient expression system, N-terminal 100 amino acid residues of GbIDS1 delivered fused GFP protein into chloroplast as well as cytosol and nucleus, whereas those of GbIDS2, GbIDS2-1, and two PtIDSs delivered GFP only into chloroplast.