Overexpression of a pseudo-etiolated-in-light-like protein in Taraxacum koksaghyz leads to a pale green phenotype and enables transcriptome-based network analysis of photomorphogenesis and isoprenoid biosynthesis.

Introduction: Plant growth and greening in response to light require the synthesis of photosynthetic pigments such as chlorophylls and carotenoids, which are derived from isoprenoid precursors. In Arabidopsis, the pseudo-etiolated-in-light phenotype is caused by the overexpression of repressor of photosynthetic genes 2 (RPGE2), which regulates chlorophyll synthesis and photosynthetic genes. Methods: We investigated a homologous protein in the Russian dandelion (Taraxacum koksaghyz) to determine its influence on the rich isoprenoid network in this species, using a combination of in silico analysis, gene overexpression, transcriptomics and metabolic profiling. Results: Homology-based screening revealed a gene designated pseudo-etiolated-in-light-like (TkPEL-like), and in silico analysis identified a light-responsive G-box element in its promoter. TkPEL-like overexpression in dandelion plants and other systems reduced the levels of chlorophylls and carotenoids, but this was ameliorated by the mutation of one or both conserved cysteine residues. Comparative transcriptomics in dandelions overexpressing TkPEL-like showed that genes responsible for the synthesis of isoprenoid precursors and chlorophyll were downregulated, probably explaining the observed pale green leaf phenotype. In contrast, genes responsible for carotenoid synthesis were upregulated, possibly in response to feedback signaling. The evaluation of additional differentially expressed genes revealed interactions between pathways. Discussion: We propose that TkPEL-like negatively regulates chlorophyll- and photosynthesis-related genes in a light-dependent manner, which appears to be conserved across species. Our data will inform future studies addressing the regulation of leaf isoprenoid biosynthesis and photomorphogenesis and could be used in future breeding strategies to optimize selected plant isoprenoid profiles and generate suitable plant-based production platforms.

Natural rubber reduces herbivory and alters the microbiome below ground.

Laticifers are hypothesized to mediate both plant-herbivore and plant-microbe interactions. However, there is little evidence for this dual function. We investigated whether the major constituent of natural rubber, cis-1,4-polyisoprene, a phylogenetically widespread and economically important latex polymer, alters plant resistance and the root microbiome of the Russian dandelion (Taraxacum koksaghyz) under attack of a root herbivore, the larva of the May cockchafer (Melolontha melolontha). Rubber-depleted transgenic plants lost more shoot and root biomass upon herbivory than normal rubber content near-isogenic lines. Melolontha melolontha preferred to feed on artificial diet supplemented with rubber-depleted rather than normal rubber content latex. Likewise, adding purified cis-1,4-polyisoprene in ecologically relevant concentrations to diet deterred larval feeding and reduced larval weight gain. Metagenomics and metabarcoding revealed that abolishing biosynthesis of natural rubber alters the structure but not the diversity of the rhizosphere and root microbiota (ecto- and endophytes) and that these changes depended on M. melolontha damage. However, the assumption that rubber reduces microbial colonization or pathogen load is contradicted by four lines of evidence. Taken together, our data demonstrate that natural rubber biosynthesis reduces herbivory and alters the plant microbiota, which highlights the role of plant-specialized metabolites and secretory structures in shaping multitrophic interactions.

Microscopic and Transcriptomic Analysis of Pollination Processes in Self-Incompatible Taraxacum koksaghyz.

The transition of the Russian dandelion Taraxacum koksaghyz (Asteraceae) to a profitable, alternative crop producing natural rubber and inulin requires the optimization of several agronomic traits, cultivation conditions and harvesting procedures to improve the yield. However, efficient breeding is hindered by the obligatory sexual outcrossing of this species. Several other asters have been investigated to determine the mechanism of self-incompatibility, but the underlying molecular basis remains unclear. We therefore investigated the self-pollination and cross-pollination of two compatible T. koksaghyz varieties (TkMS2 and TkMS3) by microscopy and transcriptomic analysis to shed light on the pollination process. Self-pollination showed typical sporophytic self-incompatibility characteristics, with the rare pollen swelling at the pollen tube apex. In contrast, cross-pollination was characterized by pollen germination and penetration of the stigma by the growing pollen tubes. RNA-Seq was used to profile gene expression in the floret tissue during self-pollination and cross-pollination, and the differentially expressed genes were identified. This revealed three candidates for the early regulation of pollination in T. koksaghyz, which can be used to examine self-incompatibility mechanisms in more detail and to facilitate breeding programs.

Comparative proteome and metabolome analyses of latex-exuding and non-exuding Taraxacum koksaghyz roots provide insights into laticifer biology.

Taraxacum koksaghyz has been identified as one of the most promising alternative rubber crops. Its high-quality rubber is produced in the latex of laticifers, a specialized cell type that is organized in a network of elongated tubules throughout the entire plant body. In order to gain insights into the physiological role(s) of latex and hence laticifer biology, we examine the effects of barnase-induced latex RNA degradation on the metabolite and protein compositions in the roots. We established high-quality datasets that enabled precise discrimination between cellular and physiological processes in laticifers and non-laticifer cell types of roots at different vegetative stages. We identified numerous latex-specific proteins, including a perilipin-like protein that has not been studied in plants yet. The barnase-expressing plants revealed a phenotype that did not exude latex, which may provide a valuable genetic basis for future studies of plant-environment interactions concerning latex and also help to clarify the evolution and arbitrary distribution of latex throughout the plant kingdom. The overview of temporal changes in composition and protein abundance provided by our data opens the way for a deeper understanding of the molecular interactions, reactions, and network relationships that underlie the different metabolic pathways in the roots of this potential rubber crop.

Uncovering mechanisms of rubber biosynthesis in Taraxacum koksaghyz - role of cis-prenyltransferase-like 1 protein.

The Russian dandelion Taraxacum koksaghyz synthesizes considerable amounts of high-molecular-weight rubber in its roots. The characterization of factors that participate in natural rubber biosynthesis is fundamental for the establishment of T. koksaghyz as a rubber crop. The cis-1,4-isoprene polymers are stored in rubber particles. Located at the particle surface, the rubber transferase complex, member of the cis-prenyltransferase (cisPT) enzyme family, catalyzes the elongation of the rubber chains. An active rubber transferase heteromer requires a cisPT subunit (CPT) as well as a CPT-like subunit (CPTL), of which T. koksaghyz has two homologous forms: TkCPTL1 and TkCPTL2, which potentially associate with the rubber transferase complex. Knockdown of TkCPTL1, which is predominantly expressed in latex, led to abolished poly(cis-1,4-isoprene) synthesis but unaffected dolichol content, whereas levels of triterpenes and inulin were elevated in roots. Analyses of latex from these TkCPTL1-RNAi plants revealed particles that were similar to native rubber particles regarding their particle size, phospholipid composition, and presence of small rubber particle proteins (SRPPs). We found that the particles encapsulated triterpenes in a phospholipid shell stabilized by SRPPs. Conversely, downregulating the low-expressed TkCPTL2 showed no altered phenotype, suggesting its protein function is redundant in T. koksaghyz. MS-based comparison of latex proteomes from TkCPTL1-RNAi plants and T. koksaghyz wild-types discovered putative factors that convert metabolites in biosynthetic pathways connected to isoprenoids or that synthesize components of the rubber particle shell.

Oxidosqualene Cyclase Knock-Down in Latex of Taraxacum koksaghyz Reduces Triterpenes in Roots and Separated Natural Rubber.

In addition to natural rubber (NR), several triterpenes are synthesized in laticifers of the Russian dandelion (Taraxacum koksaghyz). Detailed analysis of NR and resin contents revealed different concentrations of various pentacyclic triterpenes such as α-, ß-amyrin and taraxasterol, which strongly affect the mechanical properties of the resulting rubber material. Therefore, the reduction of triterpene content would certainly improve the industrial applications of dandelion NR. We developed T. koksaghyz plants with reduced triterpene contents by tissue-specific downregulation of major laticifer-specific oxidosqualene cyclases (OSCs) by RNA interference, resulting in an almost 67% reduction in the triterpene content of NR. Plants of the T1 generation showed no obvious phenotypic changes and the rubber yield also remained unaffected. Hence, this study will provide a solid basis for subsequent modern breeding programs to develop Russian dandelion plants with low and stable triterpene levels.

The enzymes OSC1 and CYP716A263 produce a high variety of triterpenoids in the latex of Taraxacum koksaghyz.

Only very little is known about the resin composition of natural rubber from the dandelion species Taraxacum koksaghyz, thus its full characterization could provide new insights into how the isoprenoid end-products influence the physical properties of natural rubber, and this resin might be a good source of highly diverse triterpenoids. Here, we present a comprehensive analysis of the triterpenoid composition in an acetone extract and identified 13 triterpenes and triterpenoids also including the so far unknown pentacyclic compounds lup-19(21)-en-3-ol (1) and its ketone lup-19(21)-en-3-one (2). We purified single triterpenes from the acetone extract by developing a two-step HPLC system that is adapted to the structural differences of the described triterpenoids. Furthermore, we isolated six different oxidosqualene cyclases (OSCs) and two P450 enzymes, and we functionally characterized TkOSC1 and CYP716A263 in Nicotiana benthamiana and Saccharomyces cerevisiae in detail. TkOSC1 is a multifunctional OSC that was capable of synthesizing at least four of the latex-predominant pentacyclic triterpenes (taraxasterol, α-, ß-amyrin and lup-19(21)-en-3-ol) while CYP716A263 oxidized pentacyclic triterpenes at the C-3 position. The identified enzymes responsible for biosynthesis and modification of pentacyclic triterpenes in T. koksaghyz latex may represent excellent tools for bioengineering approaches to produce pentacyclic triterpenes heterologously.

Upregulating the mevalonate pathway and repressing sterol synthesis in Saccharomyces cerevisiae enhances the production of triterpenes.

Pentacyclic triterpenes are diverse plant secondary metabolites derived from the mevalonate (MVA) pathway. Many of these molecules are potentially valuable, particularly as pharmaceuticals, and research has focused on their production in simpler and more amenable heterologous systems such as the yeast Saccharomyces cerevisiae. We have developed a new heterologous platform for the production of pentacyclic triterpenes in S. cerevisiae based on a combinatorial engineering strategy involving the overexpression of MVA pathway genes, the knockout of negative regulators, and the suppression of a competing pathway. Accordingly, we overexpressed S. cerevisiae ERG13, encoding 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase, and a truncated and deregulated variant of the rate-limiting enzyme HMG-CoA reductase 1 (tHMGR). In the same engineering step, we deleted the ROX1 gene, encoding a negative regulator of the MVA pathway and sterol biosynthesis, resulting in a push-and-pull strategy to enhance metabolic flux through the system. In a second step, we redirected this enhanced metabolic flux from late sterol biosynthesis to the production of 2,3-oxidosqualene, the direct precursor of pentacyclic triterpenes. In yeast cells transformed with a newly isolated sequence encoding lupeol synthase from the Russian dandelion (Taraxacum koksaghyz), we increased the yield of pentacyclic triterpenes by 127-fold and detected not only high levels of lupeol but also a second valuable pentacyclic triterpene product, ß-amyrin.

Functional characterization of squalene synthase and squalene epoxidase in Taraxacum koksaghyz.

The Russian dandelion Taraxacum koksaghyz produces high-value isoprenoids such as pentacyclic triterpenes and natural rubber in the latex of specialized cells known as laticifers. Squalene synthase (SQS) and squalene epoxidase (SQE) catalyze key steps in the biosynthesis of cyclic terpenoids, but neither enzyme has yet been characterized in T. koksaghyz. Genomic analysis revealed the presence of two genes (TkSQS1 and TkSQS2) encoding isoforms of SQS, and four genes (TkSQE1-4) encoding isoforms of SQE. Spatial expression analysis in different T. koksaghyz tissues confirmed that TkSQS1 and TkSQE1 are the latex-predominant isoforms, with highly similar mRNA expression profiles. The TkSQS1 and TkSQE1 proteins colocalized in the endoplasmic reticulum membrane and their enzymatic functions were confirmed by in vitro activity assays and yeast complementation studies, respectively. The functions of TkSQS1 and TkSQE1 were further characterized in the latex of T. koksaghyz plants with depleted TkSQS1 or TkSQE1 mRNA levels, produced by RNA interference. Comprehensive expression analysis revealed the coregulation of TkSQS1 and TkSQE1, along with a downstream gene in the triterpene biosynthesis pathway encoding the oxidosqualene cyclase TkOSC1. This indicates that the coregulation of TkSQS1, TkSQE1, and TkOSC1 could be used to optimize the flux toward specific terpenoids during development.

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

BACKGROUND: Latex 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. RESULTS: The 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. CONCLUSIONS: We 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.

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

Natural 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.

ATP citrate lyase activity is post-translationally regulated by sink strength and impacts the wax, cutin and rubber biosynthetic pathways.

Cytosolic acetyl-CoA is involved in the synthesis of a variety of compounds, including waxes, sterols and rubber, and is generated by the ATP citrate lyase (ACL). Plants over-expressing ACL were generated in an effort to understand the contribution of ACL activity to the carbon flux of acetyl-CoA to metabolic pathways occurring in the cytosol. Transgenic Arabidopsis plants synthesizing the polyester polyhydroxybutyrate (PHB) from cytosolic acetyl-CoA have reduced growth and wax content, consistent with a reduction in the availability of cytosolic acetyl-CoA to endogenous pathways. Increasing the ACL activity via the over-expression of the ACLA and ACLB subunits reversed the phenotypes associated with PHB synthesis while maintaining polymer synthesis. PHB production by itself was associated with an increase in ACL activity that occurred in the absence of changes in steady-state mRNA or protein level, indicating a post-translational regulation of ACL activity in response to sink strength. Over-expression of ACL in Arabidopsis was associated with a 30% increase in wax on stems, while over-expression of a chimeric homomeric ACL in the laticifer of roots of dandelion led to a four- and two-fold increase in rubber and triterpene content, respectively. Synthesis of PHB and over-expression of ACL also changed the amount of the cutin monomer octadecadien-1,18-dioic acid, revealing an unsuspected link between cytosolic acetyl-CoA and cutin biosynthesis. Together, these results reveal the complexity of ACL regulation and its central role in influencing the carbon flux to metabolic pathways using cytosolic acetyl-CoA, including wax and polyisoprenoids.

Laticifer-specific cis-prenyltransferase silencing affects the rubber, triterpene, and inulin content of Taraxacum brevicorniculatum.

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.

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

Taraxacum brevicorniculatum is known to produce high quality rubber. The biosynthesis of rubber is dependent on isopentenyl pyrophosphate (IPP) precursors derived from the mevalonate (MVA) pathway. The cDNA sequences of seven MVA pathway genes from latex of T. brevicorniculatum were isolated, including three cDNA sequences encoding for 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases (TbHMGR1-3). Expression analyses indicate an important role of TbHMGR1 as well as for the HMG-CoA synthase (TbHMGS), the diphosphomevalonate decarboxylase and the mevalonate kinase in the provision of precursors for rubber biosynthesis. The amino acid sequences of the TbHMGRs show the typical motifs described for plant HMGRs such as two transmembrane domains and a catalytic domain containing two HMG-CoA and two NADP(H) binding sites. The functionality of the HMGRs was demonstrated by complementation assay using an IPP auxotroph mutant of Escherichia coli. Furthermore, the transient expression of the catalytic domains of TbHMGR1 and TbHMGR2 in Nicotiana benthamiana resulted in a strong accumulation of sterol precursors, one of the major groups of pathway end-products.

Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz.

BACKGROUND: Natural rubber is a biopolymer with exceptional qualities that cannot be completely replaced using synthetic alternatives. Although several key enzymes in the rubber biosynthetic pathway have been isolated, mainly from plants such as Hevea brasiliensis, Ficus spec. and the desert shrub Parthenium argentatum, there have been no in planta functional studies, e.g. by RNA interference, due to the absence of efficient and reproducible protocols for genetic engineering. In contrast, the Russian dandelion Taraxacum koksaghyz, which has long been considered as a potential alternative source of low-cost natural rubber, has a rapid life cycle and can be genetically transformed using a simple and reliable procedure. However, there is very little molecular data available for either the rubber polymer itself or its biosynthesis in T. koksaghyz. RESULTS: We established a method for the purification of rubber particles--the active sites of rubber biosynthesis--from T. koksaghyz latex. Photon correlation spectroscopy and transmission electron microscopy revealed an average particle size of 320 nm, and 13C nuclear magnetic resonance (NMR) spectroscopy confirmed that isolated rubber particles contain poly(cis-1,4-isoprene) with a purity > 95%. Size exclusion chromatography indicated that the weight average molecular mass (Mw) of T. koksaghyz natural rubber is 4,000-5,000 kDa. Rubber particles showed rubber transferase activity of 0.2 pmol min(-1) mg(-1). Ex vivo rubber biosynthesis experiments resulted in a skewed unimodal distribution of [1-14C]isopentenyl pyrophosphate (IPP) incorporation at a M of 2,500 kDa. Characterization of recently isolated cis-prenyltransferases (CPTs) from T. koksaghyz revealed that these enzymes are associated with rubber particles and are able to produce long-chain polyprenols in yeast. CONCLUSIONS: T. koksaghyz rubber particles are similar to those described for H. brasiliensis. They contain very pure, high molecular mass poly(cis-1,4-isoprene) and the chain elongation process can be studied ex vivo. Because of their localization on rubber particles and their activity in yeast, we propose that the recently described T. koksaghyz CPTs are the major rubber chain elongating enzymes in this species. T. koksaghyz is amenable to genetic analysis and modification, and therefore could be used as a model species for the investigation and comparison of rubber biosynthesis.