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

IntroductionPlant 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.MethodsWe 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.ResultsHomology-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.DiscussionWe 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

Characterization of rubber particles and rubber chain elongation in Taraxacum koksaghyz

<p>Abstract</p> <p>Background</p> <p>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 <it>Hevea brasiliensis</it>, <it>Ficus spec. </it>and the desert shrub <it>Parthenium argentatum</it>, there have been no <it>in planta </it>functional studies, e.g. by RNA interference, due to the absence of efficient and reproducible protocols for genetic engineering. In contrast, the Russian dandelion <it>Taraxacum koksaghyz</it>, 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 <it>T. koksaghyz</it>.</p> <p>Results</p> <p>We established a method for the purification of rubber particles - the active sites of rubber biosynthesis - from <it>T. koksaghyz </it>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(<it>cis</it>-1,4-isoprene) with a purity >95%. Size exclusion chromatography indicated that the weight average molecular mass (<inline-formula><graphic file="1471-2091-11-11-i1.gif"/></inline-formula>w) of <it>T. koksaghyz </it>natural rubber is 4,000-5,000 kDa. Rubber particles showed rubber transferase activity of 0.2 pmol min^-1mg^-1. <it>Ex vivo </it>rubber biosynthesis experiments resulted in a skewed unimodal distribution of [1-¹⁴C]isopentenyl pyrophosphate (IPP) incorporation at a <inline-formula><graphic file="1471-2091-11-11-i1.gif"/></inline-formula>w of 2,500 kDa. Characterization of recently isolated <it>cis</it>-prenyltransferases (CPTs) from <it>T. koksaghyz </it>revealed that these enzymes are associated with rubber particles and are able to produce long-chain polyprenols in yeast.</p> <p>Conclusions</p> <p><it>T. koksaghyz </it>rubber particles are similar to those described for <it>H. brasiliensis</it>. They contain very pure, high molecular mass poly(<it>cis</it>-1,4-isoprene) and the chain elongation process can be studied <it>ex vivo</it>. Because of their localization on rubber particles and their activity in yeast, we propose that the recently described <it>T. koksaghyz </it>CPTs are the major rubber chain elongating enzymes in this species. <it>T. koksaghyz </it>is amenable to genetic analysis and modification, and therefore could be used as a model species for the investigation and comparison of rubber biosynthesis.</p

Molecular and phylogenetic characterization of the sieve element occlusion gene family in Fabaceae and non-Fabaceae plants

<p>Abstract</p> <p>Background</p> <p>The phloem of dicotyledonous plants contains specialized P-proteins (phloem proteins) that accumulate during sieve element differentiation and remain parietally associated with the cisternae of the endoplasmic reticulum in mature sieve elements. Wounding causes P-protein filaments to accumulate at the sieve plates and block the translocation of photosynthate. Specialized, spindle-shaped P-proteins known as forisomes that undergo reversible calcium-dependent conformational changes have evolved exclusively in the <it>Fabaceae</it>. Recently, the molecular characterization of three genes encoding forisome components in the model legume <it>Medicago truncatula </it>(<it>MtSEO1</it>, <it>MtSEO2 </it>and <it>MtSEO3</it>; SEO = sieve element occlusion) was reported, but little is known about the molecular characteristics of P-proteins in non-<it>Fabaceae</it>.</p> <p>Results</p> <p>We performed a comprehensive genome-wide comparative analysis by screening the <it>M. truncatula</it>, <it>Glycine max</it>, <it>Arabidopsis thaliana</it>, <it>Vitis vinifera </it>and <it>Solanum phureja </it>genomes, and a <it>Malus domestica </it>EST library for homologs of <it>MtSEO1</it>, <it>MtSEO2 </it>and <it>MtSEO3 </it>and identified numerous novel <it>SEO </it>genes in <it>Fabaceae </it>and even non-<it>Fabaceae </it>plants, which do not possess forisomes. Even in <it>Fabaceae </it>some <it>SEO </it>genes appear to not encode forisome components. All <it>SEO </it>genes have a similar exon-intron structure and are expressed predominantly in the phloem. Phylogenetic analysis revealed the presence of several subgroups with <it>Fabaceae</it>-specific subgroups containing all of the known as well as newly identified forisome component proteins. We constructed Hidden Markov Models that identified three conserved protein domains, which characterize SEO proteins when present in combination. In addition, one common and three subgroup specific protein motifs were found in the amino acid sequences of SEO proteins. <it>SEO </it>genes are organized in genomic clusters and the conserved synteny allowed us to identify several <it>M. truncatula </it>vs <it>G. max </it>orthologs as well as paralogs within the <it>G. max </it>genome.</p> <p>Conclusions</p> <p>The unexpected occurrence of forisome-like genes in non-<it>Fabaceae </it>plants may indicate that these proteins encode species-specific P-proteins, which is backed up by the phloem-specific expression profiles. The conservation of gene structure, the presence of specific motifs and domains and the genomic synteny argue for a common phylogenetic origin of forisomes and other P-proteins.</p

Zuchtmaterialerstellung durch Erschließung und Selektion bisher nicht erfasster Anbau - und Verwertungsmerkmale in bisher nicht genutzten genetischen Ressourcen der Schmalblättrigen Lupine (Lupinus angustifolius L.)

An züchterisch bisher nicht genutzten, alkaloidreichen Genbankakzessionen der Schmalblättrigen Lupine des Vavilov Institutes der Pflanzenindustrie, St. Petersburg, wurden neben agronomischen Merkmalen die Merkmalskomplexe Rhizodeposition und Methioningehalt evaluiert. Für diese Merkmale wird im aktuellen, bitterstoffarmen Zuchtmaterial keine ausreichende, züchterisch nutzbare Variabilität mehr gefunden. Für den Merkmalskomplex Rhizodeposite konnten mittels Pyrolyse-Feldionisation Massenspektrometrie (Py-FIMS) lyophilisierter Rhizodeposite sowohl Genotypen mit erhöhter mikrobieller Abbaubarkeit zur Förderung der Rhizosphärenaktivität als auch mit erhöhten Konzentrationen antimikrobieller Komponenten identifiziert werden. Für den Merkmalskomplex Methioningehalt konnten die Schlüsselgene des Methioninstoffwechsels identifiziert und sequenziert werden. Für zwei der vier Schlüsselgene liegen allerdings so viele Kopien im Genom vor, dass sie züchterisch nur sehr schwer nutzbar sind. Es konnten mittels Transkript-Profiling keine hoch aktiven Allele der Schlüsselgene der Methioninbiosynthese identifiziert werden. Demgegenüber konnten in jungen Samen jedoch hochaktive Allele von Genen gefunden werden, die für methioninreiche Speicherproteine codieren. Für die folgenden agronomisch wichtigen Merkmale konnte im untersuchten Schmalblättrigen Lupinenmaterial eine erhebliche Variabilität gefunden werden, die weit über die in Blauen Süßlupinen noch gefundene Streuung hinaus geht: Kornertrag, Kornform, Grünmasseleistung, Rohproteingehalt, Boden-pH-Toleranz, sichere und synchrone Abreife der Hülsen, synchrone Laub- und Strohreife, Standfestigkeit, Platzfestigkeit, Resistenz gegenüber Anthraknose und Verticillium, Kornform. Die gefundene Variabilität gilt es jetzt züchterisch zu nutzen und die positiven Merkmalseigenschaften in neuen Sorten zu kombinieren

Combinatorial Metabolic Engineering in Saccharomyces cerevisiae for the Enhanced Production of the FPP-Derived Sesquiterpene Germacrene

Farnesyl diphosphate (FPP)-derived isoprenoids represent a diverse group of plant secondary metabolites with great economic potential. To enable their efficient production in the heterologous host Saccharomyces cerevisiae, we refined a metabolic engineering strategy using the CRISPR/Cas9 system with the aim of increasing the availability of FPP for downstream reactions. The strategy included the overexpression of mevalonate pathway (MVA) genes, the redirection of metabolic flux towards desired product formation and the knockout of genes responsible for competitive reactions. Following the optimisation of culture conditions, the availability of the improved FPP biosynthesis for downstream reactions was demonstrated by the expression of a germacrene synthase from dandelion. Subsequently, biosynthesis of significant amounts of germacrene-A was observed in the most productive strain compared to the wild type. Thus, the presented strategy is an excellent tool to increase FPP-derived isoprenoid biosynthesis in yeast

Taraxacum brevicorniculatum rubber elongation factor TbREF associates with lipid droplets and affects lipid turn-over in yeast

A protein named TbREF that is localized on rubber particles of the rubber producing dandelion species Taraxacum brevicorniculatum was expressed in tobacco leaves and in yeast. TbREF fused to fluorescence proteins colocalized on globular, hydrophobic structures, most likely lipid droplets. Furthermore, triacylglycerol, sterol and total lipid content of TbREF expressing yeast was determined by photometric analyses of nile red stainings and GC–MS analyses. Therefore, yeast exposed an enhanced nile red fluorescence as well as an increased TAG and sterol content compared to wildtype and vector control. Altogether, these findings gave new insights into the putative function of TbREF that might be pushing rubber particle production due to its cytotoxic nature and/or shielding and preventing degradation of lipid droplets. Furthermore, these results highlight possible biotechnological applications regarding the accumulation of hydrophobic compounds in lipid droplet like structures. Keywords: Lipid droplets, Taraxacum brevicorniculatum, Rubber elongation factor, Latex, Saccharomyces cerevisa

Epigenetic variation in early and late flowering plants of the rubber-producing Russian dandelion Taraxacum koksaghyz provides insights into the regulation of flowering time

Abstract The Russian dandelion (Taraxacum koksaghyz) grows in temperate zones and produces large amounts of poly(cis-1,4-isoprene) in its roots, making it an attractive alternative source of natural rubber. Most T. koksaghyz plants require vernalization to trigger flower development, whereas early flowering varieties that have lost their vernalization dependence are more suitable for breeding and domestication. To provide insight into the regulation of flowering time in T. koksaghyz, we induced epigenetic variation by in vitro cultivation and applied epigenomic and transcriptomic analysis to the resulting early flowering plants and late flowering controls, allowing us to identify differences in methylation patterns and gene expression that correlated with flowering. This led to the identification of candidate genes homologous to vernalization and photoperiodism response genes in other plants, as well as epigenetic modifications that may contribute to the control of flower development. Some of the candidate genes were homologous to known floral regulators, including those that directly or indirectly regulate the major flowering control gene FT. Our atlas of genes can be used as a starting point to investigate mechanisms that control flowering time in T. koksaghyz in greater detail and to develop new breeding varieties that are more suited to domestication

Native and artificial forisomes : functions and applications

Forisomes are remarkable protein bodies found exclusively in the phloem of the Fabaceae. When the phloem is wounded, forisomes are converted from a condensed to a dispersed state in an ATP-independent reaction triggered by Ca(2+), thereby plugging the sieve tubes and preventing the loss of photoassimilates. Potentially, forisomes are ideal biomaterials for technical devices because the conformational changes can be replicated in vitro and are fully reversible over a large number of cycles. However, the development of technical devices based on forisomes has been hampered by the laborious and time-consuming process of purifying native forisomes from plants. More recently, the problem has been overcome by the production of recombinant artificial forisomes. This is a milestone in the development of forisome-based devices, not only because large quantities of homogeneous forisomes can be produced on demand, but also because their properties can be tailored for particular applications. In this review, we discuss the physical and molecular properties of native and artificial forisomes, focusing on their current applications in technical devices and potential developments in the future