Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Deenen, Nicole van; Bachmann, Anne-Lena; Schmidt, Thomas; Schaller, Hubert; Sand, Jennifer; Prüfer, Dirk; Gronover, Christian Schulze

doi:10.1007/s11033-011-1221-4

Cited by 30 publications

(23 citation statements)

References 35 publications

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“…Another mechanism is the feedback inhibition of HMGR, the key rate-limiting enzyme of the MVA pathway (Stermer et al, 1994). Three HMGRs were recently identified in T. brevicorniculatum (TbHMGR1 to -3), and expression analysis indicated an important role for TbHMGR1 in the provision of precursors for rubber biosynthesis in the latex (van Deenen et al, 2011). We showed that HMGR enzyme activity was inhibited in the latex of the CPTRNAi plants, presumably either by the accumulation of sterols or by IPP itself.…”

Section: Discussionmentioning

confidence: 76%

Laticifer-Specific cis-Prenyltransferase Silencing Affects the Rubber, Triterpene, and Inulin Content of Taraxacum brevicorniculatum

et al. 2012

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Certain Taraxacum species, such as Taraxacum koksaghyz and Taraxacum brevicorniculatum, produce large amounts of high-quality natural rubber in their latex, the milky cytoplasm of specialized cells known as laticifers. This high-molecular mass biopolymer consists mainly of poly(cis-1,4-isoprene) and is deposited in rubber particles by particle-bound enzymes that carry out the stereospecific condensation of isopentenyl diphosphate units. The polymer configuration suggests that the chain-elongating enzyme (rubber transferase; EC 2.5.1.20) is a cis-prenyltransferase (CPT). Here, we present a comprehensive analysis of transgenic T. brevicorniculatum plants in which the expression of three recently isolated CPTs known to be associated with rubber particles (TbCPT1 to -3) was heavily depleted by laticifer-specific RNA interference (RNAi). Analysis of the CPT-RNAi plants by nuclear magnetic resonance, size-exclusion chromatography, and gas chromatography-mass spectrometry indicated a significant reduction in rubber biosynthesis and a corresponding 50% increase in the levels of triterpenes and the main storage carbohydrate, inulin. Transmission electron microscopy revealed that the laticifers in CPT-RNAi plants contained fewer and smaller rubber particles than wild-type laticifers. We also observed lower activity of hydroxymethylglutaryl-coenzyme A reductase, the key enzyme in the mevalonate pathway, reflecting homeostatic control of the isopentenyl diphosphate pool. To our knowledge, this is the first in planta demonstration of latex-specific CPT activity in rubber biosynthesis.

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Section: Discussionmentioning

confidence: 76%

Laticifer-Specific cis-Prenyltransferase Silencing Affects the Rubber, Triterpene, and Inulin Content of Taraxacum brevicorniculatum

et al. 2012

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“…The AtHMGR1(S408A) catalytic domain with a mutated phosphorylation site at position 408 was amplified from A. thaliana leaf cDNA using primers Athmgrc1ATG-PciI-fwd and Athmgrc1(S408A)-XbaI-rev, digested with PciI/XbaI and inserted at the NcoI/XbaI sites of pLab12.1pREF [5] resulting in final vector pLab12.1pREF-Athmgrc1(S408A). The same fragment was inserted into the pCambia1305.1 vector as previously described [19]. Expression of heterologous AtHMGR variants pCambia1305.1-Athmgrc1 and pCambia1305.1-Athmgrc1(S408A) in Nicotiana benthamiana was carried out according to van Deenen et al [19].…”

Section: Methodsmentioning

confidence: 99%

“…by phosphorylation and inhibition by their own N-terminus [15–18]. T. brevicorniculatum produces three HMGRs encoded by the genes TbHMGR1 , TbHMGR2 and TbHMGR3 , with TbHMGR1 predominantly expressed in the latex [19]. MVA pathway engineering in plants is challenging [12, 20].…”

Section: Introductionmentioning

confidence: 99%

Isoprenoid biosynthesis in dandelion latex is enhanced by the overexpression of three key enzymes involved in the mevalonate pathway

et al. 2017

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BackgroundLatex from the dandelion species Taraxacum brevicorniculatum contains many high-value isoprenoid end products, e.g. triterpenes and polyisoprenes such as natural rubber. The isopentenyl pyrophosphate units required as precursors for these isoprenoids are provided by the mevalonate (MVA) pathway. The key enzyme in this pathway is 3-hydroxy-methyl-glutaryl-CoA reductase (HMGR) and its activity has been thoroughly characterized in many plant species including dandelion. However, two enzymes acting upstream of HMGR have not been characterized in dandelion latex: ATP citrate lyase (ACL), which provides the acetyl-CoA utilized in the MVA pathway, and acetoacetyl-CoA thiolase (AACT), which catalyzes the first step in the pathway to produce acetoacetyl-CoA. Here we isolated ACL and AACT genes from T. brevicorniculatum latex and characterized their expression profiles. We also overexpressed the well-characterized HMGR, ACL and AACT genes from Arabidopsis thaliana in T. brevicorniculatum to determine their impact on isoprenoid end products in the latex.ResultsThe spatial and temporal expression profiles of T. brevicorniculatum ACL and AACT revealed their pivotal role in the synthesis of precursors necessary for isoprenoid biosynthesis in latex. The overexpression of A. thaliana ACL and AACT and HMGR in T. brevicorniculatum latex resulted in the accumulation of all three enzymes, increased the corresponding enzymatic activities and ultimately increased sterol levels by ~5-fold and pentacyclic triterpene and cis-1,4-isoprene levels by ~2-fold. Remarkably high levels of the triterpene precursor squalene were also detected in the triple-transgenic lines (up to 32 mg/g root dry weight) leading to the formation of numerous lipid droplets which were observed in root cross-sections.ConclusionsWe could show the effective expression of up to three transgenes in T. brevicorniculatum latex which led to increased enzymatic activity and resulted in high level squalene accumulation in the dandelion roots up to an industrially relevant amount. Our data provide insight into the regulation of the MVA pathway in dandelion latex and can be used as a basis for metabolic engineering to enhance the production of isoprenoid end products in this specialized tissue.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-017-1036-0) contains supplementary material, which is available to authorized users.

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“…The basic building block of NR is isopentenyl pyrophosphate (IPP), which is thought to be produced mainly via the cytosolic mevalonic acid (MVA) pathway (van Deenen et al ., 2012). The same pathway also provides IPP for the synthesis of various isoprenoid end products, including sterols and pentacyclic triterpenes (Figure 1) that fulfil important roles in membrane fluidity, development and resistance against herbivores (Huber et al ., 2015; Schaller, 2003).…”

Section: Introductionmentioning

confidence: 99%

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

Stolze¹,

Wanke²,

Deenen³

et al. 2017

Plant Biotechnology Journal

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SummaryNatural rubber (NR) is an important raw material for a large number of industrial products. The primary source of NR is the rubber tree Hevea brasiliensis, but increased worldwide demand means that alternative sustainable sources are urgently required. The Russian dandelion (Taraxacum koksaghyz Rodin) is such an alternative because large amounts of NR are produced in its root system. However, rubber biosynthesis must be improved to develop T. koksaghyz into a commercially feasible crop. In addition to NR, T. koksaghyz also produces large amounts of the reserve carbohydrate inulin, which is stored in parenchymal root cell vacuoles near the phloem, adjacent to apoplastically separated laticifers. In contrast to NR, which accumulates throughout the year even during dormancy, inulin is synthesized during the summer and is degraded from the autumn onwards when root tissues undergo a sink‐to‐source transition. We carried out a comprehensive analysis of inulin and NR metabolism in T. koksaghyz and its close relative T. brevicorniculatum and functionally characterized the key enzyme fructan 1‐exohydrolase (1‐FEH), which catalyses the degradation of inulin to fructose and sucrose. The constitutive overexpression of Tk1‐FEH almost doubled the rubber content in the roots of two dandelion species without any trade‐offs in terms of plant fitness. To our knowledge, this is the first study showing that energy supplied by the reserve carbohydrate inulin can be used to promote the synthesis of NR in dandelions, providing a basis for the breeding of rubber‐enriched varieties for industrial rubber production.

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Molecular cloning of mevalonate pathway genes from Taraxacum brevicorniculatum and functional characterisation of the key enzyme 3-hydroxy-3-methylglutaryl-coenzyme A reductase

Cited by 30 publications

References 35 publications

Laticifer-Specific cis-Prenyltransferase Silencing Affects the Rubber, Triterpene, and Inulin Content of Taraxacum brevicorniculatum

Laticifer-Specific cis-Prenyltransferase Silencing Affects the Rubber, Triterpene, and Inulin Content of Taraxacum brevicorniculatum

Isoprenoid biosynthesis in dandelion latex is enhanced by the overexpression of three key enzymes involved in the mevalonate pathway

Development of rubber‐enriched dandelion varieties by metabolic engineering of the inulin pathway

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