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The Russian dandelion ( Taraxacum koksaghyz ) is a promising source of natural rubber (NR). The synthesis of NR takes place on the surface of organelles known as rubber particles, which are found in latex - the cytoplasm of specialized cells known as laticifers. As well as the enzymes directly responsible for NR synthesis, the rubber particles also contain small rubber particle proteins (SRPPs), the most abundant of which are SRPP3, 4 and 5. These three proteins support NR synthesis by maintaining rubber particle stability. We used homology-based searches to identify the whole TkSRPP gene family and qPCR to create their spatial expression profiles. Affinity enrichment-mass spectrometry was applied to identify TkSRPP3/4/5 protein interaction partners in T. koksaghyz latex and selected interaction partners were analyzed using qPCR, confocal laser scanning microscopy and heterologous expression in yeast. We identified 17 SRPP-like sequences in the T. koksaghyz genome, including three apparent pseudogenes, 10 paralogs arranged as an inverted repeat in a cluster with TkSRPP3/4/5 , and one separate gene ( TkSRPP6 ). Their sequence diversity and different expression profiles indicated distinct functions and the latex interactomes obtained for TkSRPP3/4/5 suggested that TkSRPP4 is a promiscuous hub protein that binds many partners from different compartments, whereas TkSRPP3 and 5 have more focused interactomes. Two interactors shared by TkSRPP3/4/5 (TkSRPP6 and TkUGT80B1) were chosen for independent validation and detailed characterization. TkUGT80B1 triterpenoid glycosylating activity provided first evidence for triterpenoid saponin synthesis in T. koksaghyz latex. Based on its identified interaction partners, TkSRPP4 appears to play a special role in the endoplasmic reticulum, interacting with lipidmodifying enzymes that may facilitate rubber particle formation. TkSRPP5 appears to be involved in GTPase-dependent signaling and TkSRPP3 may act as part of a kinase signaling cascade, with roles in stress tolerance. TkSRPP interaction with TkUGT80B1 draws a new connection between TkSRPPs and triterpenoid saponin synthesis in T. koksaghyz latex. Our data contribute to the functional differentiation between TkSRPP paralogs and demonstrate unexpected interactions that will help to further elucidate the network of proteins linking TkSRPPs, stress responses and NR biosynthesis within the cellular complexity of latex., Competing Interests: Author RMT was employed by the company TRM Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wolters, Laibach, Riekötter, Roelfs, Müller, Eirich, Twyman, Finkemeier, Prüfer and Schulze Gronover.)

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., (© 2024. The Author(s).)

Introduction: Plants are sessile organisms that maximize reproductive success by adapting to their environment. One of the key steps in the reproductive phase of angiosperms is flower development, requiring the perception of multiple endogenous and exogenous signals integrated via a complex regulatory network. Key floral regulators, including the main transcription factor of the photoperiodic pathway (CONSTANS, CO) and the central floral pathway integrator (FLOWERING LOCUS T, FT), are known in many species., Methods and Results: We identified several CO-like (COL) proteins in tobacco ( Nicotiana tabacum ). The NtCOL2a/b proteins in the day-neutral plant N. tabacum were most closely related to Arabidopsis CO. We characterized the diurnal expression profiles of corresponding genes in leaves under short-day (SD) and long-day (LD) conditions and confirmed their expression in phloem companion cells. Furthermore, we analyzed the orthologs of NtCOL2a/b in the maternal LD ancestor ( N. sylvestris ) and paternal, facultative SD ancestor ( N. tomentosiformis ) of N. tabacum and found that they were expressed in the same diurnal manner. NtCOL2a/b overexpression or knock-out using the CRISPR/Cas9 system did not support a substantial role for the CO homologs in the control of floral transition in N. tabacum . However, NsCOL2 overexpression induced flowering in N. sylvestris under typically non-inductive SD conditions, correlating with the upregulation of the endogenous NsFTd gene., Discussion: Our results suggest that NsFTd is transcriptionally regulated by NsCOL2 and that this COL2-dependent photoperiodic floral induction seems to be lost in N. tabacum , providing insight into the diverse genetics of photoperiod-dependent flowering in different Nicotiana species., Competing Interests: Author RT was employed by the company TRM Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Schmidt, Grundmann, Lahme, Seidemann, Schwarze, Lichtenauer, Twyman, Prüfer and Noll.)

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., Competing Interests: RMT was employed by TRM Ltd during the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Wolters, Benninghaus, Roelfs, van Deenen, Twyman, Prüfer and Schulze Gronover.)

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., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

The phloem is a highly specialized vascular tissue that forms a fundamentally important transport and signaling pathway in plants. It is therefore a system worth protecting. The main function of the phloem is to transport the products of photosynthesis throughout the whole plant, but it also transports soluble signaling molecules and propagates electrophysiological signals. The phloem is constantly threatened by mechanical injuries, phloem-sucking pests and parasites, and the spread of pathogens, which has led to the evolution of efficient defense mechanisms. One such mechanism involves structural phloem proteins, which are thought to facilitate sieve element occlusion following injury and to defend the plant against pathogens. In leguminous plants, specialized structural phloem proteins known as forisomes form unique mechanoproteins via sophisticated molecular interaction and assembly mechanisms, thus enabling reversible sieve element occlusion. By understanding the structure and function of forisomes and other structural phloem proteins, we can develop a toolbox for biotechnological applications in material science and medicine. Furthermore, understanding the involvement of structural phloem proteins in plant defense mechanisms will allow phloem engineering as a new strategy for the development of crop varieties that are resistant to pests, pathogens and parasites., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

Immunity cell-surface receptors Ve1 and Ve2 protect against fungi of the genus Verticillium causing early dying, a worldwide disease in many crops. Characterization of microbe-associated molecular pattern immunity receptors has advanced our understanding of disease resistance but signal amplification remains elusive. Here, we report that transgenic plants expressing Ve1 and Ve2 together, reduced pathogen titres by a further 90% compared to plants expressing only Ve1 or Ve2. Confocal and immunoprecipitation confirm that the two receptors associate to form heteromeric complexes in the absence of the ligand and positively regulate signaling. Bioassays show that the Ve1Ve2 complex activates race-specific amplified immunity to the pathogen through a rapid burst of reactive oxygen species (ROS). These results indicate a mechanism by which the composition of a cell-surface receptor heterocomplex may be optimized to increase immunity against devastating plant diseases., (© 2022. Crown.)

The ongoing SARS-CoV-2 pandemic demonstrates that the capacity of centralized clinical diagnosis laboratories represents a significant limiting factor in the global fight against the newly emerged virus. Scaling up these capacities also requires simple and robust methods for virus diagnosis to be easily driven by untrained personnel in a point-of-care (POC) environment. The use of impedance sensors reduces the complexity and costs of diagnostic instruments and increases automation of diagnosis processes. We present an impedance point-of-care system (IMP-POCS) that uses interdigitated electrodes surrounded by an integrated heating meander to monitor loop-mediated isothermal amplification (LAMP) and melt curve analysis (MCA) consecutively in a short time. MCA permits distinguishing false- from true-positive results and significantly raises the validity of pathogen detection. Conclusively, the herein-developed miniaturized total analysis system (µTAS) represents a powerful and promising tool for providing reliable, low-cost alternatives to standard clinical diagnosis.

Proteostasis reflects the well-balanced synthesis, trafficking and degradation of cellular proteins. This is a fundamental aspect of the dynamic cellular proteome, which integrates multiple signaling pathways, but it becomes increasingly error-prone during aging. Phosphatidylethanolamine-binding proteins (PEBPs) are highly conserved regulators of signaling networks and could therefore affect aging-related processes. To test this hypothesis, we expressed PEPBs in a heterologous context to determine their ectopic activity. We found that heterologous expression of the tobacco ( Nicotiana tabacum ) PEBP NtFT4 in Drosophila melanogaster significantly increased the lifespan of adult flies and reduced age-related locomotor decline. Similarly, overexpression of the Drosophila ortholog CG7054 increased longevity, whereas its suppression by RNA interference had the opposite effect. In tobacco, NtFT4 acts as a floral regulator by integrating environmental and intrinsic stimuli to promote the transition to reproductive growth. In Drosophila, NtFT4 engaged distinct targets related to proteostasis, such as HSP26. In older flies, it also prolonged Hsp26 gene expression, which promotes longevity by maintaining protein integrity. In NtFT4-transgenic flies, we identified deregulated genes encoding proteases that may contribute to proteome stability at equilibrium. Our results demonstrate that the expression of NtFT4 influences multiple aspects of the proteome maintenance system via both physical interactions and transcriptional regulation, potentially explaining the aging-related phenotypes we observed.

Forisomes are giant self-assembling mechanoproteins that undergo reversible structural changes in response to Ca 2+ and various other stimuli. Artificial forisomes assembled from the monomer MtSEO-F1 can be used as smart biomaterials, but the molecular basis of their functionality is not understood. To determine the role of protein polymerization in forisome activity, we tested the Ca 2+ association of MtSEO-F1 dimers (the basic polymerization unit) by circular dichroism spectroscopy and microscale thermophoresis. We found that soluble MtSEO-F1 dimers neither associate with Ca 2+ nor undergo structural changes. However, polarization modulation infrared reflection absorption spectroscopy revealed that aggregated MtSEO-F1 dimers and fully-assembled forisomes associate with Ca 2+ , allowing the hydration of poorly-hydrated protein areas. A change in the signal profile of complete forisomes indicated that Ca 2+ interacts with negatively-charged regions in the protein complexes that only become available during aggregation. We conclude that aggregation is required to establish the Ca 2+ response of forisome polymers., (© 2021 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Chronic thromboembolic pulmonary hypertension (CTEPH) remains a rare and underdiagnosed disease. After one or several episodes of acute pulmonary embolism, around 3% of patients develop CTEPH and two-thirds of these patients are potential surgical candidates. Besides surgery, additional treatment modalities are pulmonary arterial hypertension medication and balloon pulmonary angioplasty. Patients should be evaluated in CTEPH expert centers to ensure the most appropriate therapy. Pulmonary endarterectomy (PEA) is a complex, but standardized surgical procedure aiming to clear the obstructed pulmonary arteries completely. For optimal visualization, deep hypothermic circulatory arrest is a prerequisite. This article will give an overview of the evaluation, indication and surgical management of patients with CTEPH., Competing Interests: Conflicts of Interest: The authors have no conflicts of interest to declare., (2022 Annals of Cardiothoracic Surgery. All rights reserved.)

The Russian dandelion ( Taraxacum koksaghyz , family Asteraceae) produces large amounts of natural rubber in the laticifers of its roots. This species has been proposed as an alternative source of natural rubber to augment or partly replace the rubber tree ( Hevea brasiliensis ) but domestication would require genetic improvement to increase rubber yields and agronomic optimization to facilitate harvesting and processing. Optimization has focused thus far on the size and shape of the roots, the primary storage organ for natural rubber and inulin. However, the corresponding genetic factors are poorly understood. Here we describe the comparative transcriptomic analysis of root tissues from T. koksaghyz plant sets featuring different root sizes and shapes, aiming to identify differentially expressed genes correlating with root length or root diameter in the upper root and root tip. The resulting datasets revealed multiple candidate genes for each trait and root part, including a glucan endo-1,3-β-d-glucosidase, an allene oxide synthase 3, and a TIFY10A/JAZ1 homolog. These three genes were tested by qRT-PCR in outdoor-grown plants with diverse root morphology, and the expression of two genes correlated with the appropriate root morphotype, confirming the effectiveness of our method. We evaluated the candidate genes to gain insight into their potential functions in root development. Such candidate genes could be suitable for marker-assisted breeding programs in the future., Competing Interests: Author RT was employed by the company TRM Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wieghaus, Roelfs, Twyman, Prüfer and Schulze Gronover.)

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.

Potato is an important staple food crop in both developed and developing countries. However, potato plants are susceptible to several economically important viruses that reduce yields by up to 50% and affect tuber quality. One of the major threats is corky ringspot, which is a tuber necrosis caused by tobacco rattle virus (TRV). The appearance of corky ringspot symptoms on tubers prior to commercialization results in ≈ 45% of the tubers being downgraded in quality and value, while ≈ 55% are declared unsaleable. To improve current disease management practices, we have developed simple diagnostic methods for the reliable detection of TRV without RNA purification, involving minimalized sample handling (mini), subsequent improved colorimetric loop-mediated isothermal amplification (LAMP), and final verification by lateral-flow dipstick (LFD) analysis. Having optimized the mini-LAMP-LFD approach for the sensitive and specific detection of TRV, we confirmed the reliability and robustness of this approach by the simultaneous detection of TRV and other harmful viruses in duplex LAMP reactions. Therefore, our new approach offers breeders, producers, and farmers an inexpensive and efficient new platform for disease management in potato breeding and cultivation.

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 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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The FLOWERING LOCUS T ( FT )-like gene family encodes key regulators of flower induction that affect the timing of reproduction in many angiosperm species. Agricultural research has therefore focused on such genes to improve the success of breeding programs and enhance agronomic traits. We recently identified a novel FT -like gene ( NtFT5 ) that encodes a day-neutral floral activator in the model tobacco crop Nicotiana tabacum . However, further characterization is necessary to determine its value as a target for breeding programs. We therefore investigated the function of NtFT5 by expression analysis and mutagenesis. Expression analysis revealed that NtFT5 is transcribed in phloem companion cells, as is typical for FT -like genes. However, high levels of NtFT5 mRNA accumulated not only in the leaves but also in the stem. Loss-of-function mutants (generated using CRISPR/Cas9) were unable to switch to reproductive growth under long-day conditions, indicating that NtFT5 is an indispensable major floral activator during long-days. Backcrossing was achieved by grafting the mutant scions onto wild-type rootstock, allowing the restoration of flowering and pollination by a wild-type donor. The resulting heterozygous Ntft5 - / NtFT5 + plants flowered with a mean delay of only ~2 days, demonstrating that one functional allele is sufficient for near-normal reproductive timing. However, this minor extension of the vegetative growth phase also conferred beneficial agronomic traits, including a >10% increase in vegetative leaf biomass on the main shoot and the production of more seeds. The agronomic benefits of the heterozygous plants persisted under various abiotic stress conditions, confirming that NtFT5 is a promising target for crop improvement to address the effects of climate change., (Copyright © 2020 Schmidt, Zimmermann, Wiedmann, Lichtenauer, Grundmann, Muth, Twyman, Prüfer and Noll.)

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., (© 2019 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

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-, β-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 Russian dandelion (Taraxacum koksaghyz) is a promising source of inulin and natural rubber because large amounts of both feedstocks can be extracted from its roots. However, the domestication of T. koksaghyz requires the development of stable agronomic traits such as higher yields of inulin and natural rubber, a higher root biomass, and an agronomically preferable root morphology which is more suitable for cultivation and harvesting. Arabidopsis thaliana Rapid Alkalinisation Factor 1 (RALF1) has been shown to suppress root growth. We identified the T. koksaghyz orthologue TkRALF-like 1 and knocked out the corresponding gene (TkRALFL1) using the CRISPR/Cas9 system to determine its impact on root morphology, biomass, and inulin and natural rubber yields. The TkRALFL1 knockout lines more frequently developed a taproot phenotype which is easier to cultivate and harvest, as well as a higher root biomass and greater yields of both inulin and natural rubber. The TkRALFL1 gene could therefore be suitable as a genetic marker to support the breeding of profitable new dandelion varieties with improved agronomic traits. To our knowledge, this is the first study addressing the root system of T. koksaghyz to enhance the agronomic performance., Competing Interests: The authors have declared that no competing interests exist.

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-, β-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.

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.

Photoperiod is an important external stimulus governing the precise timing of the floral transition in plants. Members of the FLOWERING LOCUS T (FT)-like clade of phosphatidylethanolamine-binding proteins induce this developmental process in numerous species by forming regulatory protein complexes with FD-like bZIP transcription factors. We identified several thus far unknown FT-like and FD-like genes in the genus Nicotiana and found that, even in the day-neutral species Nicotiana tabacum, floral initiation requires the photoperiod-dependent expression of several FT-like genes. Furthermore, floral promotion under long-day (LD) and short-day (SD) conditions is mediated by an FT-like protein (NtFT5) that originates from the genome of the paternal, facultative SD ancestor Nicotiana tomentosiformis. In contrast, its ortholog of the maternal LD ancestor Nicotiana sylvestris is not present in the genome of N. tabacum cv. SR1. Expression profiling in N. tabacum and its ancestors confirmed the relevance of these FT and FD orthologs in the context of polyploidization. We also found that floral inhibition by tobacco FT-like proteins is not restricted to SD conditions, highlighting the coincident expression of tobacco FT-like genes encoding floral activators and floral inhibitors. Multicolor bimolecular fluorescence complementation analysis revealed the preferential formation of FT/FD complexes that promote rather than inhibit flowering, which in concert with the regulation of NtFT and NtFD expression could explain how floral promotion overcomes floral repression during the floral transition in tobacco., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Symptomatic patients with chronic thromboembolic disease (CTED) without pulmonary hypertension often show an excessive increase in mean pulmonary arterial pressure (MPAP) during exercise.We report on the impact of pulmonary endarterectomy (PEA) on pulmonary haemodynamics in a prospective series of 32 consecutive CTED patients who underwent PEA. All patients had a comprehensive diagnostic work-up including right heart catheterisation at baseline and 12 months after PEA. Furthermore, in 12 patients exercise right heart catheterisation was performed before and after PEA.After PEA, MPAP was lower at rest (20±3 versus 17±3 mmHg; p=0.008) and during maximal exercise (39±8 versus 31±6 mmHg; p=0.016). The mean total pulmonary resistance (TPR) decreased from 3.6±0.8 Wood Units (WU) pre-operatively to 2.7±0.7 WU 1 year after PEA (p=0.004) and the mean slope of the MPAP/cardiac output (CO) relationship decreased from 3.6±1.0 to 2.3±0.8 WU (p=0.002). Peak oxygen uptake increased from 1.2±0.4 to 1.5±0.3 L·min -1 (p=0.014) and ventilatory equivalents of carbon dioxide decreased from 39±2 to 30±2 (p=0.002). There was a significant improvement in quality of life assessed by the Cambridge Pulmonary Hypertension Outcome Review questionnaire.In CTED patients, PEA resulted in haemodynamic and clinical improvements. The means of TPR and MPAP/CO slopes decreased to <3.0 WU., Competing Interests: Conflict of interest: S. Guth reports nonfinancial support from the German Center for Lung Research, during the conduct of the study; personal fees from Actelion Pharmaceuticals, Bayer, Pfizer and GSK, outside the submitted work. Conflict of interest: C.B. Wiedenroth reports nonfinancial support from the German Center for Lung Research, during the conduct of the study; personal fees from Actelion, Bayer AG, BTG, MSD and Pfizer, outside the submitted work. Conflict of interest: A. Rieth reports nonfinancial support from the German Center for Lung Research, during the conduct of the study; personal fees from Actelion Pharmaceuticals, Novartis, Servier and St Jude Medical, and grants from Pfizer, outside the submitted work. Conflict of interest: M.J. Richter reports nonfinancial support from the German Center for Lung Research, during the conduct of the study; personal fees and nonfinancial support from Actelion Pharmaceuticals, personal fees from Bayer, Mundipharma, OMT and Roche, outside the submitted work. Conflict of interest: H.A. Ghofrani reports nonfinancial support from the German Center for Lung Research, during the conduct of the study; grants and personal fees from Actelion Pharmaceuticals, Bayer, Pfizer and Novartis, personal fees from Bellerophon Pulse Technologies, Medscape, OMT, UCB Celltech, WebMD Global, Gilead Sciences, GSK and Merck, outside the submitted work. Conflict of interest: C. Liebetrau reports nonfinancial support from the German Center for Lung Research, during the conduct of the study; personal fees from Abbott, Bayer, Pfizer, AstraZeneca, Berlin-Chemie, Boehringer Ingelheim, Daiichi Sankyo and Elixir Medical, outside the submitted work. Conflict of interest: A. Rolf reports personal fees from AstraZeneca, Boehringer Ingelheim and Pfizer-Bristol-Myers Squibb, outside the submitted work. Conflict of interest: C.W. Hamm reports nonfinancial support from the German Center for Lung Research, during the conduct of the study. Conflict of interest: E. Mayer reports nonfinancial support from the German Center for Lung Research, during the conduct of the study; personal fees from Actelion Pharmaceuticals, Bayer, Pfizer, GSK and MSD, outside the submitted work. Conflict of interest: E. Gruenig has received fees for lectures and/or consultations from Actelion Pharmaceuticals, Bayer/MSD, GSK, United Therapeutics and Pfizer., (Copyright ©ERS 2018.)

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, β-amyrin.

Natural rubber biosynthesis occurs on rubber particles, i.e. organelles resembling small lipid droplets localized in the laticifers of latex-containing plant species, such as Hevea brasiliensis and Taraxacum brevicorniculatum. The latter expresses five small rubber particle protein (SRPP) isoforms named TbSRPP1-5, the most abundant proteins in rubber particles. These proteins maintain particle stability and are therefore necessary for rubber biosynthesis. TbSRPP1-5 were transiently expressed in Nicotiana benthamiana protoplasts and the proteins were found to be localized on lipid droplets and in the endoplasmic reticulum, with TbSRPP1 and TbSRPP3 also present in the cytosol. Bimolecular fluorescence complementation confirmed pairwise interactions between all proteins except TbSRPP2. The corresponding genes showed diverse expression profiles in young T. brevicorniculatum plants exposed to abiotic stress, and all except TbSRPP4 and TbSRPP5 were upregulated. Young Arabidopsis thaliana plants that overexpressed TbSRPP2 and TbSRPP3 tolerated drought stress better than wild-type plants. Furthermore, we used rubber particle extracts and standards to investigate the affinity of the TbSRPPs for different phospholipids, revealing a preference for negatively charged head groups and 18:2/16:0 fatty acid chains. This finding may explain the effect of TbSRPP3-5 on the dispersity of artificial poly(cis-1,4-isoprene) bodies and on the lipid droplet distribution we observed in N. benthamiana leaves. Our data provide insight into the assembly of TbSRPPs on rubber particles, their role in rubber particle structure, and the link between rubber biosynthesis and lipid droplet-associated stress responses, suggesting that SRPPs form the basis of evolutionarily conserved intracellular complexes in plants., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)