No systematic effects of sampling direction on climate-growth relationships in a large-scale, multi-species tree-ring data set
Gut, Urs ; Árvai, Mátyás ; Bijak, Szymon ; Camarero, J.J. ; Cedro, Anna ; Cruz-García, Roberto ; Garamszegi, Balázs ; Hacket-Pain, Andrew ; Hevia, Andrea ; Huang, Weiwei ; Isaac-Renton, Miriam ; Kaczka, Ryszard J. ; Kazimirović, Marko ; Kędziora, Wojciech ; Kern, Zoltán ; Klisz, Marcin ; Kolář, Tomáš ; Körner, Michael ; Kuznetsova, Veronica ; Montwé, David ; Petritan, Any Mary ; Petritan, Ion Catalin ; Plavcová, Lenka ; Rehschuh, Romy ; Rocha, Eva ; Rybníček, Michal ; Sánchez-Salguero, Raúl ; Schröder, Jens ; Schwab, Niels ; Stajić, Branko ; Tomusiak, Robert ; Wilmking, Martin ; Sass-Klaassen, Ute ; Buras, Allan - \ 2019
Dendrochronologia 57 (2019). - ISSN 1125-7865
Climate signal - Correlation analysis - Dendro-provenancing - Directional growth - Principal Component Gradient Analysis - Tree-rings
Ring-width series are important for diverse fields of research such as the study of past climate, forest ecology, forest genetics, and the determination of origin (dendro-provenancing) or dating of archaeological objects. Recent research suggests diverging climate-growth relationships in tree-rings due to the cardinal direction of extracting the tree cores (i.e. direction-specific effect). This presents an understudied source of bias that potentially affects many data sets in tree-ring research. In this study, we investigated possible direction-specific growth variability based on an international (10 countries), multi-species (8 species) tree-ring width network encompassing 22 sites. To estimate the effect of direction-specific growth variability on climate-growth relationships, we applied a combination of three methods: An analysis of signal strength differences, a Principal Component Gradient Analysis and a test on the direction-specific differences in correlations between indexed ring-widths series and climate variables. We found no evidence for systematic direction-specific effects on tree radial growth variability in high-pass filtered ring-width series. In addition, direction-specific growth showed only marginal effects on climate-growth correlations. These findings therefore indicate that there is no consistent bias caused by coring direction in data sets used for diverse dendrochronological applications on relatively mesic sites within forests in flat terrain, as were studied here. However, in extremely dry, warm or cold environments, or on steep slopes, and for different life-forms such as shrubs, further research is advisable.
Biomarkers of food intake for nuts and vegetable oils : an extensive literature search
Garcia-Aloy, Mar ; Hulshof, Paul J.M. ; Estruel-Amades, Sheila ; Osté, Maryse C.J. ; Lankinen, Maria ; Geleijnse, Johanna M. ; Goede, Janette De; Ulaszewska, Marynka ; Mattivi, Fulvio ; Bakker, Stephan J.L. ; Schwab, Ursula ; Andres-Lacueva, Cristina - \ 2019
Genes & Nutrition 14 (2019)1. - ISSN 1555-8932
Biomarker - Intake - Metabolomics - Nuts - Oils
Nuts and vegetable oils are important sources of fat and of a wide variety of micronutrients and phytochemicals. Following their intake, several of their constituents, as well as their derived metabolites, are found in blood circulation and in urine. As a consequence, these could be used to assess the compliance to a dietary intervention or to determine habitual intake of nuts and vegetable oils. However, before these metabolites can be widely used as biomarkers of food intake (BFIs), several characteristics have to be considered, including specificity, dose response, time response, stability, and analytical performance. We have, therefore, conducted an extensive literature search to evaluate current knowledge about potential BFIs of nuts and vegetable oils. Once identified, the strengths and weaknesses of the most promising candidate BFIs have been summarized. Results from selected studies have provided a variety of compounds mainly derived from the fatty fraction of these foods, but also other components and derived metabolites related to their nutritional composition. In particular, α-linolenic acid, urolithins, and 5-hydroxyindole-3-acetic acid seem to be the most plausible candidate BFIs for walnuts, whereas for almonds they could be α-tocopherol and some catechin-derived metabolites. Similarly, several studies have reported a strong association between selenium levels and consumption of Brazil nuts. Intake of vegetable oils has been mainly assessed through the measurement of specific fatty acids in different blood fractions, such as oleic acid for olive oil, α-linolenic acid for flaxseed (linseed) and rapeseed (canola) oils, and linoleic acid for sunflower oil. Additionally, hydroxytyrosol and its metabolites were the most promising distinctive BFIs for (extra) virgin olive oil. However, most of these components lack sufficient specificity to serve as BFIs. Therefore, additional studies are necessary to discover new candidate BFIs, as well as to further evaluate the specificity, sensitivity, dose-response relationships, and reproducibility of these candidate biomarkers and to eventually validate them in other populations. For the discovery of new candidate BFIs, an untargeted metabolomics approach may be the most effective strategy, whereas for increasing the specificity of the evaluation of food consumption, this could be a combination of different metabolites.
Insights into the ecology and evolution of the mucus-dwelling gut bacterium Mucispirillum schaedleri
Berry, David ; Pfann, Carina ; Steinberger, Michaela ; Hanson, Buck ; Herp, Simone ; Brugiroux, Sandrine ; Gomes Neto, João Carlos ; Boekschoten, M.V. ; Schwab, Clarissa ; Urich, Tim ; Ramer-Tait, Amanda E. ; Rattei, Thomas ; Stecher, Bärbel ; Loy, Alexander - \ 2017
Mus musculus - GSE83625 - PRJNA326520
Mucispirillum schaedleri is an abundant inhabitant of the intestinal mucus layer of rodents and other animals. To gain insights into its lifestyle, we analyzed the genome and transcriptome of M. schaedleri ASF 457 and tested for traits predicted by the genome using physiological experiments. Although thought to be a mucus degrader, its genome surprisingly predicts that M. schaedleri has limited capacity for degrading host-derived mucosal glycans or other complex polysaccharides. Rather, it may utilize small compounds such as peptides, amino acids, glycerol, and short chain fatty acids. Additionally, it can reduce nitrate and has systems for scavenging oxygen and reactive oxygen species, which accounts for its presence close to the mucosal tissue and during inflammation. Also of note, M. schaedleri harbors a type VI secretion system (T6SS) and several putative effector proteins containing eukaryotic domains, which suggest intimate interactions with the host and a role in inflammation. Examination of the individual phylogenies of all genes in the M. schaedleri genome revealed extensive horizontal gene transfer, primarily from intestinal Epsilon- and Deltaproteobacteria. Though M. schaedleri utilizes non-horizontally-transferred pathways (e.g. nitrate reduction), horizontally-acquired pathways from gut organisms (e.g. T6SS and glycerol-P utilization) are also likely also important for its survival in the intestine, suggesting that lateral gene transfer may have played a key role in facilitating its establishment in the gut ecosystem.
Lifestyle and Horizontal Gene Transfer-Mediated Evolution of Mucispirillum schaedleri, a Core Member of the Murine Gut Microbiota
Loy, Alexander ; Pfann, Carina ; Steinberger, Michaela ; Hanson, Buck ; Herp, Simone ; Brugiroux, Sandrine ; Gomes Neto, João Carlos ; Boekschoten, Mark V. ; Schwab, Clarissa ; Urich, Tim ; Ramer-Tait, Amanda E. ; Rattei, Thomas ; Stecher, Bärbel ; Berry, David ; Lozupone, Catherine - \ 2017
mSystems 2 (2017)1. - ISSN 2379-5077 - 15 p.
Mucispirillum schaedleri is an abundant inhabitant of the intestinal mucus layer of rodents and other animals and has been suggested to be a pathobiont, a commensal that plays a role in disease. In order to gain insights into its lifestyle, we analyzed the genome and transcriptome of M. schaedleri ASF 457 and performed physiological experiments to test traits predicted by its genome. Although described as a mucus inhabitant, M. schaedleri has limited capacity for degrading host-derived mucosal glycans and other complex polysaccharides. Additionally, M. schaedleri reduces nitrate and expresses systems for scavenging oxygen and reactive oxygen species in vivo, which may account for its localization close to the mucosal tissue and expansion during inflammation. Also of note, M. schaedleri harbors a type VI secretion system and putative effector proteins and can modify gene expression in mucosal tissue, suggesting intimate interactions with its host and a possible role in inflammation. The M. schaedleri genome has been shaped by extensive horizontal gene transfer, primarily from intestinal Epsilon- and Deltaproteobacteria, indicating that horizontal gene transfer has played a key role in defining its niche in the gut ecosystem.
Genome and transcriptome of Mucispirillum schaedleri ASF457 (MCS)
Loy, Alexander ; Pfann, Carina ; Steinberger, Michaela ; Hanson, Buck ; Herp, Simone ; Brugiroux, Sandrine ; Gomes Neto, João Carlos ; Boekschoten, M.V. ; Schwab, Clarissa ; Urich, Tim ; Ramer-Tait, Amanda E. ; Rattei, Thomas ; Stecher, Bärbel ; Berry, David ; Lozupone, Catherine - \ 2016
University of Vienna
PRJEB13534 - ERP015095 - Mucispirillum schaedleri
Mucispirillum schaedleri is an abundant inhabitant of the intestinal mucus layer of rodents and other animals. We analyzed the genome and transcriptome of M. schaedleri ASF 457 to gain insights into its lifestyle and tested traits predicted by the genome with physiological experiments. Surprisingly, though thought to be a mucus degrader its genome predicts that M. schaedleri has limited capacity for degrading host-derived mucosal glycans or other complex polysaccharides. It may rather utilize small compounds such as peptides, amino acids, glycerol, and short chain fatty acids. Additionally, it can reduce nitrate and has systems for scavenging oxygen and reactive oxygen species, which accounts for its survival close to the mucosal tissue blooms during inflammation. Interestingly, M. schaedleri harbors a type VI secretion system (T6SS) and several putative effector proteins containing eukaryotic domains, which may be involved in interacting with the host and may play a role in inflammation. An examination of individual phylogenies of all genes in the M. schaedleri genome indicated extensive lateral gene transfer, primarily from intestinal Epsilon- and Deltaproteobacteria. Though M. schaedleri utilizes non-laterally-transferred pathways (e.g. nitrate reduction), laterally acquired pathways from gut organisms (e.g. T6SS and glycerol-P utilization) are likely also important for its survival in the intestine, suggesting that lateral gene transfer may have played a key role in facilitating its establishment in the gut ecosystem.
Considerations on the shuttle mechanism of FeEDDHA chelates at the soil-root interface in case of Fe deficiency
Schenkeveld, W.D.C. ; Reichwein, A.M. ; Temminghoff, E.J.M. ; Riemsdijk, W.H. van - \ 2014
Plant and Soil 379 (2014)1-2. - ISSN 0032-079X - p. 373 - 387.
strategy i plants - calcareous soil - lolium-perenne - metal uptake - iron uptake - complexes - eddha - chlorosis - isomers - montmorillonite
A mechanism of action for the performance of Fe chelates as soil-applied fertilizer has been hypothesized by Lindsay and Schwab (J Plant Nutr 5:821-840, 1982), in which the ligand participates in a cyclic process of delivering Fe at the root surface and mobilizing Fe from the soil. This "shuttle mechanism" seems appealing in view of fertilizer efficiency, but little is known about its performance. The chelate FeEDDHA is a commonly used Fe fertilizer on calcareous soils. In this study, the performance of the shuttle mechanism has been examined for FeEDDHA chelates in soil interaction and pot trial experiments. The specificity of EDDHA ligands for chelating Fe from soils of low Fe availability is limited. Experimental support for a shuttle mechanism in soil-plant systems with FeEDDHA was found: specific metal mobilization only occurred upon FeEDDHA-facilitated Fe uptake. The mobilized metals originated at least in part from the root surface instead of the soil. The results from this study support the existence of a shuttle mechanism with FeEDDHA in soil application. If the efficiency of the shuttle mechanism is however largely controlled by metal availability in the bulk soil, it is heavily compromised by complexation of competing cations: Al, Mn and particularly Cu.
F1 hybrid of cultivated apple (Malus x domestica) and European pear (Pyrus communis) with fertile F2 offspring
Fischer, T.C. ; Malnoy, M. ; Hofmann, T. ; Schwab, W. ; Palmieri, L. ; Wehrens, H.R.M.J. ; Schuch, L.A. ; Müller, M. ; Schimmelpfeng, H. ; Velasco, R. ; Martens, S. - \ 2014
Molecular Breeding 34 (2014)3. - ISSN 1380-3743 - p. 817 - 828.
nuclear-dna content - genetic-linkage maps - flow-cytometry - japanese pear - s-alleles - borkh. - diversity - rosaceae - markers - genome
The establishment of intergeneric hybrids for horticultural and agricultural crops is still a demanding task for breeding programmes. The aim of such approaches is to introduce new quality and resistance traits and to enlarge the gene pool. Recently, an F1 hybrid between Malus × domestica and Pyrus communis became available which arose from a breeding approach undertaken in the late 1980s by the breeder Max Zwintzscher (Cologne-Vogelsang). Unlike previous reports, viable and fertile F2 plants were obtained from this F1 hybrid line by author HS, providing a unique perspective not only for genomic, transcriptomic and metabolomic studies but also for advanced breeding strategies. Here, we give the first report on the confirmation and characterization of the F1 hybrid by phenotypic, genetic and biochemical means. The intergeneric hybrid shows an intermediary phenotype of leaves, flowers and fruits, and some disorder of secondary shoot growth. Nuclear DNA content is also intermediary and corresponds to a diploid state. Apple and pear type rDNA as well as SI alleles from each genus were found. At the metabolic level, parallel biosynthesis of the apple dihydrochalcone phloridzin and of arbutin, a p-hydroquinone-glucoside typical for pear, take place leading to considerable concentrations of both in leaves. The overall data allow secure confirmation of the hybrid character and give a first insight into the hybrids genetics and physiology.
Functional molecular biology research in Fragaria
Schwab, W. ; Schaart, J.G. ; Rosati, C. - \ 2009
In: Genetics and Genomics of Rosaceae / Folta, K.M., Gardiner, S.E., Springer (Plant Genetics and Genomics: Crops and Models Volume 6) - ISBN 9780387774909 - p. 457 - 486.
Redirection of flavonoid biosynthesis through the down-regulation of an anthocyanidin glucosyltransferase in ripening strawberry fruit
Griesser, M. ; Hoffmann, T. ; Bellido, M.L. ; Rosati, C. ; Fink, B. ; Kurtzer, R. ; Aharoni, A. ; Munoz-Blanco, J. ; Schwab, W. - \ 2008
Plant Physiology 146 (2008)4. - ISSN 0032-0889 - p. 1528 - 1539.
fragaria x ananassa - phenylalanine ammonia-lyase - udp-glucose - gene-expression - molecular characterization - arabidopsis-thaliana - oxidative stress - petunia-hybrida - pathway genes - cinnamic acid
Strawberry (Fragaria x ananassa) fruit contains several anthocyanins that give the ripe fruits their attractive red color. The enzyme that catalyzes the formation of the first stable intermediate in the anthocyanin pathway is anthocyanidin-3-O-glucosyltransferase. A putative glycosyltransferase sequence (FaGT1) was cloned from a strawberry fruit cDNA library and the recombinant FaGT1 transferred UDP-glucose to anthocyanidins and, to a lesser extent, flavonols, generating the respective 3-O-glucosides. Quantitative polymerase chain reaction revealed that transcripts of FaGT1 were almost undetectable in green fruits, but gene expression increased dramatically in both turning and ripe red fruit, corresponding closely to the accumulation of anthocyanins during fruit ripening. The expression of FaGT1 is fruit associated and negatively regulated by auxin. To elucidate the in planta function of FaGT1, Agrobacterium tumefaciens cells harboring an intron-hairpin construct of a partial FaGT1 sequence were injected into midsized ripening fruits. In about one-third of the injected fruits, this led to significant down-regulation of FaGT1 transcript levels that corresponded to reduced concentrations of anthocyanin pigments in ripe strawberry fruits. In contrast, significant levels of epiafzelechin-formed by anthocyanidin reductase (ANR) from pelargonidin-were identified in FaGT1-silenced fruits, indicating competition of FaGT1 and FaANR for the common anthocyanidin substrate. Thus, FaGT1 represents an important branching-point enzyme because it is channeling the flavonoid pathway to anthocyanins. These results demonstrate a method to redirect the anthocyanin biosynthesis into flavan-3-ol production to increase the levels of bioactive natural products or modify pigments in plant tissues.
Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux
Michniewicz, M. ; Zago, M.K. ; Abas, L. ; Weijers, D. ; Schweighofer, A. ; Meskiene, I. ; Heisler, M.G. ; Ohno, C. ; Zhang, J. ; Huang, F. ; Schwab, R. ; Weigel, D. ; Meyerowitz, E.M. ; Luschnig, C. ; Offringa, R. ; Friml, J. - \ 2007
Cell 130 (2007)6. - ISSN 0092-8674 - p. 1044 - 1056.
protein-kinase - arabidopsis - efflux - transport - gradients - growth - phosphoproteomics - phosphatase-2a - gravitropism - endocytosis
In plants, cell polarity and tissue patterning are connected by intercellular flow of the phytohormone auxin, whose directional signaling depends on polar subcellular localization of PIN auxin transport proteins. The mechanism of polar targeting of PINs or other cargos in plants is largely unidentified, with the PINOID kinase being the only known molecular component. Here, we identify PP2A phosphatase as an important regulator of PIN apical-basal targeting and auxin distribution. Genetic analysis, localization, and phosphorylation studies demonstrate that PP2A and PINOID both partially colocalize with PINs and act antagonistically on the phosphorylation state of their central hydrophilic loop, hence mediating PIN apical-basal polar targeting. Thus, in plants, polar sorting by the reversible phosphorylation of cargos allows for their conditional delivery to specific intracellular destinations. In the case of PIN proteins, this mechanism enables switches in the direction of intercellular auxin fluxes, which mediate differential growth, tissue patterning, and organogenesis.
Up- and downregulation of Fragaria x ananassa O-methyltransferase: Impacts on Furanone and Phenylpropanoid metabolism
Lunkenbein, S. ; Salentijn, E.M.J. ; Coiner, H. ; Boone, M.J. ; Krens, F.A. ; Schwab, W. - \ 2006
Journal of Experimental Botany 57 (2006)10. - ISSN 0022-0957 - p. 2445 - 2453.
strawberry fragaria-ananassa - flavor compounds - gene-expression - tomato fruit - molecular-biology - dna microarrays - messenger-rnas - cell-wall - identification - aroma
A complex mixture of hundreds of substances determines strawberry (Fragariaxananassa) aroma, but only 15 volatiles are considered as key flavour compounds. Of these, 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) is regarded as the most important, but it is methylated further by FaOMT (Fragariaxananassa O-methyltransferase) to 2,5-dimethyl-4-methoxy-3(2H)-furanone (DMMF) during the ripening process. It is shown here that transformation of strawberry with the FaOMT sequence in sense and antisense orientation, under the control of the cauliflower mosaic virus 35S promoter, resulted in a near total loss of DMMF, whereas the levels of the other volatiles remained unchanged. FaOMT repression also affected the ratio of feruloyl 1-O-ß-D-glucose and caffeoyl 1-O-ß-D-glucose, indicating a dual function of the enzyme in planta. Thus, FaOMT is involved in at least two different biochemical pathways in ripe strawberry fruit
Metabolic engineering of terpenoid biosynthesis in plants
Aharoni, A. ; Jongsma, M.A. ; Kim, T.Y. ; Ri, M.B. ; Giri, A.P. ; Verstappen, F.W.A. ; Schwab, W. ; Bouwmeester, H.J. - \ 2006
Phytochemistry Reviews 5 (2006)1. - ISSN 1568-7767 - p. 49 - 58.
Metabolic engineering of terpenoids in plants is a fascinating research topic from two main perspectives. On the one hand, the various biological activities of these compounds make their engineering a new tool for improving a considerable number of traits in crops. These include for example enhanced disease resistance, weed control by producing allelopathic compounds, better pest management, production of medicinal compounds, increased value of ornamentals and fruit and improved pollination. On the other hand, the same plants altered in the profile of terpenoids and their precursor pools make a most important contribution to fundamental studies on terpenoid biosynthesis and its regulation. In this review we describe our recent results with terpenoid engineering, focusing on two terpenoid classes the monoterpenoids and sesquiterpenoids. The emerging picture is that engineering of these compounds and their derivatives in plant cells is feasible, although with some requirements and limitations. For example, in terpenoid engineering experiments crucial factors are the subcellular localisation of both the precursor pool and the introduced enzymes, the activity of endogenous plant enzymes which modify the introduced terpenoid skeleton, the costs of engineering in terms of effects on other pathways sharing the same precursor pool and the phytotoxicity of the introduced terpenoids. Finally, we will show that transgenic plants altered in their terpenoid profile exert novel biological activities on their environment, for example influencing insect behaviour
Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria ananassa)
Lunkenbein, S. ; Coiner, H. ; Vos, C.H. de; Schaart, J.G. ; Boone, M.J. ; Krens, F.A. ; Schwab, W. ; Salentijn, E.M.J. - \ 2006
Journal of Agricultural and Food Chemistry 54 (2006)6. - ISSN 0021-8561 - p. 2145 - 2153.
agrobacterium-mediated transformation - plant polyketide biosynthesis - shoot regeneration - phenolic-compounds - transgenic plants - dna microarrays - flower color - ms detection - glycine-max - gene
An octaploid (Fragaria × ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria × ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens. Out of 25 transgenic lines, nine lines showed a reduction in CHS mRNA accumulation of more than 50% as compared to the untransformed cv. Calypso control. The antisense CHS construct was found to be integrated into the genome, with a copy number ranging from one to four. The pigmentation of the fruit was only affected when less than 5% of the control CHS expression level was detected. A stable antisense phenotype over a period of 4 years was obtained in the primary transgenic lines at a rate of 1:20. As a consequence of the reduced activity of CHS, the levels of anthocyanins, flavonols, and proanthocyanidins were downregulated and precursors of the flavonoid pathway were shunted to the phenylpropanoid pathway leading to highly increased levels of cinnamoyl glucose (520% of control), caffeoyl glucose (816% of control), and feruloyl glucose (1092% of control) as well as p-coumaryl alcohol (363% of control) and p-coumaryl-1-acetate (1079% of control), which occur only as trace components in untransformed control fruits. These results demonstrate that the introduction of an antisense CHS construct in strawberry results in an unpredictable biochemical phenotype, thereby confirming that CHS function is an important regulatory point of substrate flow between the flavonoid and the phenylpropanoid pathways
Cinnamate metabolism in ripening fruit. Characterization of a UDP-glucose: Cinnamate glucosyltransferase from strawberry
Lunkenbein, S. ; Bellido, M.L. ; Aharoni, A. ; Salentijn, E.M.J. ; Kaldenhoff, R. ; Coiner, H. ; Munoz-Blanco, J. ; Schwab, W. - \ 2006
Plant Physiology 140 (2006)3. - ISSN 0032-0889 - p. 1047 - 1058.
fragaria x ananassa - heterologous expression - arabidopsis-thaliana - pectate lyase - flavonoid 3-o-glucosyltransferase - cultivated strawberries - substrate-specificity - o-glucosyltransferase - molecular-cloning - dna microarrays
Strawberry (Fragaria x ananassa) fruit accumulate (hydroxy)cinnamoyl glucose (Glc) esters, which may serve as the biogenetic precursors of diverse secondary metabolites, such as the flavor constituents methyl cinnamate and ethyl cinnamate. Here, we report on the isolation of a cDNA encoding a UDP-Glc:cinnamate glucosyltransferase (Fragaria x ananassa glucosyltransferase 2 [FaGT2]) from ripe strawberry cv Elsanta that catalyzes the formation of 1-O-acyl-Glc esters of cinnamic acid, benzoic acid, and their derivatives in vitro. Quantitative real-time PCR analysis indicated that FaGT2 transcripts accumulate to high levels during strawberry fruit ripening and to lower levels in flowers. The levels in fruits positively correlated with the in planta concentration of cinnamoyl, p-coumaroyl, and caffeoyl Glc. In the leaf, high amounts of Glc esters were detected, but FaGT2 mRNA was not observed. The expression of FaGT2 is negatively regulated by auxin, induced by oxidative stress, and by hydroxycinnamic acids. Although FaGT2 glucosylates a number of aromatic acids in vitro, quantitative analysis in transgenic lines containing an antisense construct of FaGT2 under the control of the constitutive 35S cauliflower mosaic virus promoter demonstrated that the enzyme is only involved in the formation of cinnamoyl Glc and p-coumaroyl Glc during ripening.
Gain and loss of fruit flavour compounds produced by wild and cultivated strawberry species
Aharoni, A. ; Giri, A.P. ; Verstappen, F.W.A. ; Bertea, C.M. ; Sevenier, R.E. ; Sun, Z. ; Jongsma, M.A. ; Schwab, W. ; Bouwmeester, H.J. - \ 2004
The Plant Cell 16 (2004). - ISSN 1040-4651 - p. 3110 - 3131.
messenger-rna accumulation - arabidopsis-thaliana - fragaria-vesca - functional expression - alkaloid biosynthesis - gene-expression - cdna isolation - lithospermum-erythrorhizon - alcohol-dehydrogenase - secondary metabolites
The blends of flavor compounds produced by fruits serve as biological perfumes used to attract living creatures, including humans. They include hundreds of metabolites and vary in their characteristic fruit flavor composition. The molecular mechanisms by which fruit flavor and aroma compounds are gained and lost during evolution and domestication are largely unknown. Here, we report on processes that may have been responsible for the evolution of diversity in strawberry (Fragaria spp) fruit flavor components. Whereas the terpenoid profile of cultivated strawberry species is dominated by the monoterpene linalool and the sesquiterpene nerolidol, fruit of wild strawberry species emit mainly olefinic monoterpenes and myrtenyl acetate, which are not found in the cultivated species. We used cDNA microarray analysis to identify the F. ananassa Nerolidol Synthase1 (FaNES1) gene in cultivated strawberry and showed that the recombinant FaNES1 enzyme produced in Escherichia coli cells is capable of generating both linalool and nerolidol when supplied with geranyl diphosphate (GPP) or farnesyl diphosphate (FPP), respectively. Characterization of additional genes that are very similar to FaNES1 from both the wild and cultivated strawberry species (FaNES2 and F. vesca NES1) showed that only FaNES1 is exclusively present and highly expressed in the fruit of cultivated (octaploid) varieties. It encodes a protein truncated at its N terminus. Green fluorescent protein localization experiments suggest that a change in subcellular localization led to the FaNES1 enzyme encountering both GPP and FPP, allowing it to produce linalool and nerolidol. Conversely, an insertional mutation affected the expression of a terpene synthase gene that differs from that in the cultivated species (termed F. ananassa Pinene Synthase). It encodes an enzyme capable of catalyzing the biosynthesis of the typical wild species monoterpenes, such as alpha-pinene and beta-myrcene, and caused the loss of these compounds in the cultivated strawberries. The loss of alpha-pinene also further influenced the fruit flavor profile because it was no longer available as a substrate for the production of the downstream compounds myrtenol and myrtenyl acetate. This phenomenon was demonstrated by cloning and characterizing a cytochrome P450 gene (Pinene Hydroxylase) that encodes the enzyme catalyzing the C10 hydroxylation of alpha-pinene to myrtenol. The findings shed light on the molecular evolutionary mechanisms resulting in different flavor profiles that are eventually selected for in domesticated species.
Increased and altered fragrance of tobacco plants after metabolic engineering using three monoterpene synthases from lemon
Lücker, J. ; Schwab, W. ; Hautum, B. van; Blaas, J. ; Plas, L.H.W. van der; Bouwmeester, H.J. ; Verhoeven, H.A. - \ 2004
Plant Physiology 134 (2004)1. - ISSN 0032-0889 - p. 510 - 519.
s-linalool synthase - biosynthetic-pathway - volatile compounds - nicotiana-tabacum - gene-expression - floral scents - flowers - transformation - emission - clarkia
Wild-type tobacco (Nicotiana tabacum) plants emit low levels of terpenoids, particularly from the flowers. By genetic modification of tobacco cv Petit Havana SR1 using three different monoterpene synthases from lemon (Citrus limon L. Burm. f.) and the subsequent combination of these three into one plant by crossings, we show that it is possible to increase the amount and alter the composition of the blend of monoterpenoids produced in tobacco plants. The transgenic tobacco plant line with the three introduced monoterpene synthases is emitting -pinene, limonene, and -terpinene and a number of side products of the introduced monoterpene synthases, from its leaves and flowers, in addition to the terpenoids emitted by wild-type plants. The results show that there is a sufficiently high level of substrate accessible for the introduced enzymes
Metabolic engineering of monoterpende biosysnthesis: two step production of (+)-trans-Isopiperitenol by tobacco
Lücker, J. ; Schwab, W. ; Franssen, M.C.R. ; Plas, L.H.W. van der; Bouwmeester, H.J. ; Verhoeven, H.A. - \ 2004
The Plant Journal 39 (2004)1. - ISSN 0960-7412 - p. 135 - 145.
peppermint mentha-piperita - functional expression - limonene enantiomers - linalool synthase - cdna isolation - s-linalool - plants - mint - (-)-limonene - cloning
Monoterpenoid biosynthesis in tobacco was modified by introducing two subsequent enzymatic activities targeted to different cell compartments. A limonene-3-hydroxylase (lim3h) cDNA was isolated from Mentha spicata L. 'Crispa'. This cDNA was used to re-transform a transgenic Nicotiana tabacum'Petit Havana' SR1 (tobacco) line expressing three Citrus limon L. Burm. f. (lemon) monoterpene synthases producing (+)-limonene, gamma-terpinene and (-)-beta-pinene as their main products. The targeting sequences of these synthases indicate that they are probably localized in the plastids, whereas the sequence information of the P450 hydroxylase indicates targeting to the endoplasmatic reticulum. Despite the different location of the enzymes, the introduced P450 hydroxylase proved to be functional in the transgenic plants as it hydroxylated (+)-limonene, resulting in the emission of (+)-trans-isopiperitenol. Some further modifications of the (+)-trans-isopiperitenol were also detected, resulting in the additional emission of 1,3,8-p-menthatriene, 1,5,8-p-menthatriene, p-cymene and isopiperitenone.
Terpenoid Metabolism in Wild-Type and Transgenic Arabidopsis Plants
Aharoni, A. ; Giri, A.P. ; Deuerlein, S. ; Griepink, F.C. ; Kogel, W.J. de; Verstappen, F.W.A. ; Verhoeven, H.A. ; Jongsma, M.A. ; Schwab, W. ; Bouwmeester, H.J. - \ 2003
The Plant Cell 15 (2003)12. - ISSN 1040-4651 - p. 2866 - 2884.
sesquiterpene cyclase gene - linalool synthase gene - functional expression - monoterpene biosynthesis - isoprenoid biosynthesis - diphosphate synthase - cdna isolation - s-linalool - glandular trichomes - thaliana
Volatile components, such as terpenoids, are emitted from aerial parts of plants and play a major role in the interaction between plants and their environment. Analysis of the composition and emission pattern of volatiles in the model plant Arabidopsis showed that a range of volatile components are released, primarily from flowers. Most of the volatiles detected were monoterpenes and sesquiterpenes, which in contrast to other volatiles showed a diurnal emission pattern. The active terpenoid metabolism in wild-type Arabidopsis provoked us to conduct an additional set of experiments in which transgenic Arabidopsis overexpressing two different terpene synthases were generated. Leaves of transgenic plants constitutively expressing a dual linalool/nerolidol synthase in the plastids (FaNES1) produced linalool and its glycosylated and hydroxylated derivatives. The sum of glycosylated components was in some of the transgenic lines up to 40- to 60-fold higher than the sum of the corresponding free alcohols. Surprisingly, we also detected the production and emission of nerolidol, albeit at a low level, suggesting that a small pool of its precursor farnesyl diphosphate is present in the plastids. Transgenic lines with strong transgene expression showed growth retardation, possibly as a result of the depletion of isoprenoid precursors in the plastids. In dual-choice assays with Myzus persicae, the FaNES1-expressing lines significantly repelled the aphids. Overexpression of a typical cytosolic sesquiterpene synthase resulted in the production of only trace amounts of the expected sesquiterpene, suggesting tight control of the cytosolic pool of farnesyl diphosphate, the precursor for sesquiterpenoid biosynthesis. This study further demonstrates the value of Arabidopsis for studies of the biosynthesis and ecological role of terpenoids and provides new insights into their metabolism in wild-type and transgenic plants
Domain swapping of Citrus limon monoterpene synthases: impact on enzymatic activity and product specifity.
Tamer, M.K. el; Lucker, J. ; Bosch, D. ; Verhoeven, H.A. ; Verstappen, F.W.A. ; Schwab, W. ; Tunen, A.J. van; Voragen, A.G.J. ; Maagd, R.A. de; Bouwmeester, H.J. - \ 2003
Archives of Biochemistry and Biophysics 411 (2003). - ISSN 0003-9861 - p. 196 - 203.
site-directed mutagenesis - 5-epi-aristolochene synthase - trichodiene synthase - linalool synthase - germacrene-a - biosynthesis - expression - biology
Monoterpene cyclases are the key enzymes in the monoterpene biosynthetic pathway, as they catalyze the cyclization of the ubiquitous geranyl diphosphate (GDP) to the specific monoterpene skeletons. From Citrus limon, four monoterpene synthase-encoding cDNAs for a P-pinene synthase named Cl(-)betaPINS, a gamma-terpinene synthase named ClgammaTS, and two limonene synthases named Cl(+)LIMS1 and Cl(+)LIMS2 were recently isolated [J. Lucker et al., Eur. J. Biochem. 269 (2002) 3160]. The aim of our work in this study was to identify domains within these monoterpene synthase enzymes determining the product specificity. Domain swapping experiments between Cl(-)betaPINS and ClgammaTS and between Cl(+)LIMS2 and ClyTS were conducted. We found that within the C-terminal domain of these monoterpene synthases, a region comprising 200 amino acids, of which 41 are different between Cl(-)betaPINS and ClgammaTS, determines the specificity for the formation of P-pinene or gamma-terpinene, respectively, while another region localized further downstream is required for a chimeric enzyme to yield products in the same ratio as in the wild-type ClgammaTS. For Cl(+)LIMS2, the two domains together appear to be sufficient for its enzyme specificity, but many chimeras were inactive probably due to the low homology with ClyTS. Molecular modeling was used to further pinpoint the amino acids responsible for the differences in product specificity of ClyTS and Cl(-)betaPINS. (C) 2003 Elsevier Science (USA). All rights reserved.
Monoterpene biosynthesis in lemon (Citrus limon) cDNA isolation and functional analysis of four monoterpene synthases
Lücker, J. ; Tamer, M.K. El; Schwab, W. ; Verstappen, F.W.A. ; Plas, L.H.W. van der; Bouwmeester, H.J. ; Verhoeven, H.A. - \ 2002
European Journal of Biochemistry 269 (2002). - ISSN 0014-2956 - p. 3160 - 3161.
Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit, four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis, these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were ( )-limonene (two cDNAs) (-)--pinene and -terpinene. All enzymes exhibited a pH optimum around 7; addition of Mn2 as bivalent metal ion cofactor resulted in higher activity than Mg2 , with an optimum concentration of 0.6 mm. Km values ranged from 0.7 to 3.1 ?m. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil, corresponding to more than 90␘f the main components present