Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes

<p>Abstract</p> <p>Background</p> <p>Sesquiterpene lactones are characteristic metabolites of Asteraceae (or Compositae) which often display potent bioactivities and are sequestered in specialized organs such as laticifers, resin ducts, and trichomes. For characterization of sunflower sesquiterpene synthases we employed a simple method to isolate pure trichomes from anther appendages which facilitated the identification of these genes and investigation of their enzymatic functions and expression patterns during trichome development.</p> <p>Results</p> <p>Glandular trichomes of sunflower (<it>Helianthus annuus </it>L.) were isolated, and their RNA was extracted to investigate the initial steps of sesquiterpene lactone biosynthesis. Reverse transcription-PCR experiments led to the identification of three sesquiterpene synthases. By combination of <it>in vitro </it>and <it>in vivo </it>characterization of sesquiterpene synthase gene products in <it>Escherichia coli </it>and <it>Saccharomyces cerevisiae</it>, respectively, two enzymes were identified as germacrene A synthases, the key enzymes of sesquiterpene lactone biosynthesis. Due to the very low <it>in vitro </it>activity, the third enzyme was expressed <it>in vivo </it>in yeast as a thioredoxin-fusion protein for functional characterization. In <it>in vivo </it>assays, it was identified as a multiproduct enzyme with the volatile sesquiterpene hydrocarbon δ-cadinene as one of the two main products with α-muuorlene, β-caryophyllene, α-humulene and α-copaene as minor products. The second main compound remained unidentified. For expression studies, glandular trichomes from the anther appendages of sunflower florets were isolated in particular developmental stages from the pre- to the post-secretory phase. All three sesquiterpene synthases were solely upregulated during the biosynthetically active stages of the trichomes. Expression in different aerial plant parts coincided with occurrence and maturity of trichomes. Young roots with root hairs showed expression of the sesquiterpene synthase genes as well.</p> <p>Conclusion</p> <p>This study functionally identified sesquiterpene synthase genes predominantly expressed in sunflower trichomes. Evidence for the transcriptional regulation of sesquiterpene synthase genes in trichome cells suggest a potential use for these specialized cells for the identification of further genes involved in the biosynthesis, transport, and regulation of sesquiterpene lactones.</p

Results from the DELCODE study

Previous studies have demonstrated increased tau plasma levels in patients with Alzheimer’s disease (AD) and mild cognitive impairment (MCI) due to AD. Much less is known whether increased tau plasma levels can already be detected in the pre-MCI stage of subjective cognitive decline (SCD). In the present study we measured tau plasma levels in 111 SCD patients and 134 age- and gender-matched cognitively healthy controls participating in the DZNE (German Center for Neurodegenerative Diseases) longitudinal study on cognition and dementia (DELCODE). Tau plasma levels were measured using ultra-sensitive, single-molecule array (Simoa) technology. We found no significant different tau plasma levels in SCD (3.4 pg/ml) compared with healthy controls (3.6 pg/ml) after controlling for age, gender, and education (p = 0.137). In addition, tau plasma levels did not correlate with Aβ42 (r = 0.073; p = 0.634), tau (r = −0.179; p = 0.240), and p-tau181 (r = −0.208; p = 0.171) cerebrospinal fluid (CSF) levels in a subgroup of 45 SCD patients with available CSF. In conclusion, plasma tau is not increased in SCD patients. In addition, the lack of correlation between tau in plasma and CSF in the examined cohort suggests that tau levels are affected by different factors in both biofluids

Results from the DELCODE study

Previous studies have demonstrated increased tau plasma levels in patients with Alzheimer’s disease (AD) and mild cognitive impairment (MCI) due to AD. Much less is known whether increased tau plasma levels can already be detected in the pre-MCI stage of subjective cognitive decline (SCD). In the present study we measured tau plasma levels in 111 SCD patients and 134 age- and gender-matched cognitively healthy controls participating in the DZNE (German Center for Neurodegenerative Diseases) longitudinal study on cognition and dementia (DELCODE). Tau plasma levels were measured using ultra-sensitive, single-molecule array (Simoa) technology. We found no significant different tau plasma levels in SCD (3.4 pg/ml) compared with healthy controls (3.6 pg/ml) after controlling for age, gender, and education (p = 0.137). In addition, tau plasma levels did not correlate with Aβ42 (r = 0.073; p = 0.634), tau (r = −0.179; p = 0.240), and p-tau181 (r = −0.208; p = 0.171) cerebrospinal fluid (CSF) levels in a subgroup of 45 SCD patients with available CSF. In conclusion, plasma tau is not increased in SCD patients. In addition, the lack of correlation between tau in plasma and CSF in the examined cohort suggests that tau levels are affected by different factors in both biofluids

Molecular characterization and developmental expression of key enzymes of natural product biosynthesis in sunflower glandular trichomes

In der vorliegenden Arbeit konnten durch Sequenzvergleiche mit bereits bekannten pflanzlichen Sesquiterpensynthasen drei in den Trichomen der Sonnenblume exprimierte Sesquiterpensynthasen identifiziert werden. Die Nuklein- und Aminosäuresequenzen wiesen für Terpensynthasen charakteristische Merkmale auf. Zur funktionellen Charakterisierung wurden alle identifizieren Terpensynthasen heterolog in E. coli exprimiert. Nach Aufreinigung und in vitro Umsetzungsreaktionen mit dem natürlichen Substrat Farnesylpyrophosphat wurden die Reaktionsprodukte durch GC-MS Messungen analysiert und die gebildeten Verbindungen anschließend anhand von Referenzproben bestimmt. Zwei der identifizierten Sesquiterpensynthasen (HaGAS1, HaGAS2) erwiesen sich als Germacren A-Synthasen und somit als die Schlüsselenzyme der Biosynthese der Sesquiterpenlactone der Sonnenblume. Die heterologe Expression der Germacren A-Synthasen in vivo in S. cerevisiae führte ebenfalls nur zur Bildung von Germacren A und war damit übereinstimmend mit den in vitro Daten. Die funktionelle Charakterisierung der dritten Terpensynthase erwies ich aufgrund schwacher in vitro Aktivität als schwierig. Eine heterologe Expression in vivo in S. cerevisiae führte zu einer deutlichen Steigerung der erhaltenen Produktmengen. Doch erst die Expression als Thioredoxin-Fusionsprotein in Hefen ermöglichte die Bildung genügend hoher Produktmengen zur Identifizierung der gebildeten Verbindungen. Bei dieser Sesquiterpen-synthase (HaCS) handelt es sich um ein Multiproduktenzym, welches als eine von zwei Hauptkomponenten gamma-Cadinen bildet und als weitere Produkte alpha-Copaen, alpha-Muurolen und beta-Caryophyllen produziert. In einer H. annuus Trichom EST-Bank wurde ein Cytochrom P450-Enzym identifiziert, das eine hohe Sequenzähnlichkeit zu einem bereits charakterisierten Gen aus Artemisia annua aufwies, welches an der Bildung von Artemisinin-Säure beteiligt ist. Die heterologe Expression dieses Enzyms und eines weiteren Proteins mit hoher Sequenzähnlichkeit aus Lactuca sativa zusammen mit der Germacren A-Synthase HaGAS2 in S. cerevisiae führte zur Bildung von Germacren A-Carboxylsäure und damit zur Aufklärung eines bisher nicht auf enzymatischer Ebene identifizierten Biosyntheseschrittes der Sesquiterpenlactone. Bei den identifizierten Enzymen aus H. annuus beziehungsweise L. sativa handelt es sich um multifunktionelle Germacren A-Monooxygenasen, die drei aufeinanderfolgende oxidative Schritte der Bildung von Germacren A-Carboxylsäure aus Germacren A katalysieren. Semiquantitative RT-PCR Experimente konnten die Expression aller drei identifizierten Sesquiterpensynthasen, sowie der Monooxygenase der Sonnenblume, direkt in den Trichomen zeigen. Die Expression der Gene fand nur während der biosynthetisch aktiven Phase der Trichome statt und konnte in dieser Feinheit zum ersten Mal überhaupt gezeigt werden. Daneben wurden Transkripte der Sesquiterpensynthasen in trichomtragenden Blättern und im Wurzelbereich nachgewiesen. Neben Sesquiterpenlactonen wurden von den Drüsenhaaren auch Flavonoide gebildet, deren Biosynthese ebenfalls in den Trichomen angesiedelt ist. Mit 5-Deoxynevadensin konnte ein bisher unbekanntes und trichomspezifisches 5-deoxy-Flavon identifiziert werden.Glandular trichomes from anther appendages of sunflower were collected and their RNA was isolated. Sequence comparison with known plant sesquiterpene synthases was used to identify sunflower synthases in RT-PCR reactions. Three enzymes, HaGAS1, HaGAS2 and HaCS with high similarities to already characterized sesquiterpene synthases were identified. Their nucleotide sequences were completely established on the genomic level and as RNA transcripts. The nucleotide sequences as well as the deduced amino acid sequences showed typical characteristics of terpene synthases. In order to characterize the enzymes, the sesquiterpene synthase genes were cloned and expressed in E. coli. In vitro assays with the recombinant enzymes were carried out using the native substrate farnesyldiphosphate. The resulting products were extracted and analysed by GC-MS. They were identified by comparison of data base MS-data and using reference samples under identical analytical conditions. Two expressed enzymes, HaGAS1 and HaGAS2, synthesized germacrene A as a single product. Heterologous in vivo expression of both germacrene A-synthases in S. cerevisiae confirmed the in vitro result, since the analysis of the synthesized product showed a single germacrene A peak. Due to a very low in vitro activity of HaCS, the products of the third synthase could not be directly determined by MS-analysis. Therefore, the enzyme was expressed as a thioredoxin-fusion protein in vivo in transgenic yeast. This attempt resulted in a much higher rate of product yield. Two main and at least six minor products were traced in GC-analysis. They were confirmed as sesquiterpene hydrocarbons by GC-MS analysis. One of the two main products was identified as gamma-cadinene, whereas the second main peak could not be determined conclusively. Among the minor compounds alpha-copaene, alpha-muurolene und beta-caryophyllene were identified. Screening of a H. annuus EST library (established at the Berkeley Center for Synthetic Biology, University of California, Berkeley, USA) from mRNA of trichomes revealed the presence of a cytochrome P450 protein which showed high similarity to an Artemisia annua enzyme involved in artemisinic acid biosynthesis. This enzyme and another similar protein from Lactuca sativa were cloned and coexpressed with the germacrene A-synthase HaGAS2 in yeast. The resulting product was indirectly determined as germacrene A carboxylic acid using GC-MS analysis. These novel cytochrome P450 enzymes from sunflower and lettuce can be characterized as multifunctional germacrene A-monooxygenases. They catalyse a three-step oxidation leading from germacrene A to germacrene A carboxylic acid. This oxidation process represents an essential step towards the biosynthesis of sesquiterpene lactones. Semiquantitative RT-PCR analysis demonstrated that the expression of all three sesquiterpene synthases and the sunflower P450 monooxygenase occurred directly within trichome cells. The expression was highly upregulated during the secretory stage of the capitate glandular trichomes. This developmentally regulated expression was shown for the first time in trichomes. Additionally to sesquiterpene synthase activity in trichomes of anthers and leaves, it also was detected in sunflower roots. In addition, 5-deoxynevadensin was identified as a new constituent of the glandular trichomes of sunflower. This 5-deoxy-flavone is responsible for the bright blue fluorescence of sunflower trichomes detected by fluorescence microscopy. The newly identified component may act as protectant for the STL against UV-degradation

The nucleotide sequence and genome organization of <it>Plasmopara halstedii </it>virus

Abstract Background Only very few viruses of Oomycetes have been studied in detail. Isometric virions were found in different isolates of the oomycete Plasmopara halstedii, the downy mildew pathogen of sunflower. However, complete nucleotide sequences and data on the genome organization were lacking. Methods Viral RNA of different P. halstedii isolates was subjected to nucleotide sequencing and analysis of the viral genome. The N-terminal sequence of the viral coat protein was determined using Top-Down MALDI-TOF analysis. Results The complete nucleotide sequences of both single-stranded RNA segments (RNA1 and RNA2) were established. RNA1 consisted of 2793 nucleotides (nt) exclusive its 3' poly(A) tract and a single open-reading frame (ORF1) of 2745 nt. ORF1 was framed by a 5' untranslated region (5' UTR) of 18 nt and a 3' untranslated region (3' UTR) of 30 nt. ORF1 contained motifs of RNA-dependent RNA polymerases (RdRp) and showed similarities to RdRp of Scleropthora macrospora virus A (SmV A) and viruses within the Nodaviridae family. RNA2 consisted of 1526 nt exclusive its 3' poly(A) tract and a second ORF (ORF2) of 1128 nt. ORF2 coded for the single viral coat protein (CP) and was framed by a 5' UTR of 164 nt and a 3' UTR of 234 nt. The deduced amino acid sequence of ORF2 was verified by nano-LC-ESI-MS/MS experiments. Top-Down MALDI-TOF analysis revealed the N-terminal sequence of the CP. The N-terminal sequence represented a region within ORF2 suggesting a proteolytic processing of the CP in vivo. The CP showed similarities to CP of SmV A and viruses within the Tombusviridae family. Fragments of RNA1 (ca. 1.9 kb) and RNA2 (ca. 1.4 kb) were used to analyze the nucleotide sequence variation of virions in different P. halstedii isolates. Viral sequence variation was 0.3% or less regardless of their host's pathotypes, the geographical origin and the sensitivity towards the fungicide metalaxyl. Conclusions The results showed the presence of a single and new virus type in different P. halstedii isolates. Insignificant viral sequence variation indicated that the virus did not account for differences in pathogenicity of the oomycete P. halstedii.</p

Development and Validation of an Ultrasensitive Procalcitonin Sandwich Immunoassay

Procalcitonin (PCT) is well established as a highly specific biomarker for the detection of bacterial infections and sepsis. However, the currently available diagnostic tests are not able to detect very low or very early increases of PCT or even baseline levels in healthy individuals or patients with non-bacterial infections. In order to be able to detect these very low concentrations of PCT, a sandwich immunoassay was developed using high sensitivity Single Molecule Array technology (Simoa). The assay was thoroughly validated and applied to analyze human cerebrospinal fluid (CSF) and serum samples from patients with bacterial or viral meningitis as well as CSF, serum, and K2 EDTA plasma from healthy control subjects. A 50-fold increase in sensitivity compared to the current gold standard assays was achieved, which was sensitive enough for the detection of baseline PCT levels. Both serum and CSF showed significantly elevated PCT levels in patients with bacterial meningitis compared to patients with viral meningitis and the healthy control group. Procalcitonin concentration levels for patients with viral meningitis and the control group could be measured, but were not significantly different. The determination of PCT in the low pg·mL−1 range could help to improve the monitoring of bacterial infectious diseases, as PCT level changes could be detected earlier

Study of the Humoral Immune Response towards HCV Genotype 4 Using a Bead-Based Multiplex Serological Assay

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