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Publications - Cell and Metabolic Biology

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Publications

Hettwer, K.; Böttcher, C.; Frolov, A.; Mittasch, J.; Albert, A.; von Roepenack-Lahaye, E.; Strack, D.; Milkowski, C.; Dynamic metabolic changes in seeds and seedlings of Brassica napus (oilseed rape) suppressing UGT84A9 reveal plasticity and molecular regulation of the phenylpropanoid pathway Phytochemistry 124, 46-57, (2016) DOI: 10.1016/j.phytochem.2016.01.014

In Brassica napus, suppression of the key biosynthetic enzyme UDP-glucose:sinapic acid glucosyltransferase (UGT84A9) inhibits the biosynthesis of sinapine (sinapoylcholine), the major phenolic component of seeds. Based on the accumulation kinetics of a total of 158 compounds (110 secondary and 48 primary metabolites), we investigated how suppression of the major sink pathway of sinapic acid impacts the metabolome of developing seeds and seedlings. In UGT84A9-suppressing (UGT84A9i) lines massive alterations became evident in late stages of seed development affecting the accumulation levels of 58 secondary and 7 primary metabolites. UGT84A9i seeds were characterized by decreased amounts of various hydroxycinnamic acid (HCA) esters, and increased formation of sinapic and syringic acid glycosides. This indicates glycosylation and β-oxidation as metabolic detoxification strategies to bypass intracellular accumulation of sinapic acid. In addition, a net loss of sinapic acid upon UGT84A9 suppression may point to a feedback regulation of HCA biosynthesis. Surprisingly, suppression of UGT84A9 under control of the seed-specific NAPINC promoter was maintained in cotyledons during the first two weeks of seedling development and associated with a reduced and delayed transformation of sinapine into sinapoylmalate. The lack of sinapoylmalate did not interfere with plant fitness under UV-B stress. Increased UV-B radiation triggered the accumulation of quercetin conjugates whereas the sinapoylmalate level was not affected.
Publications

Frolov, A.; Henning, A.; Böttcher, C.; Tissier, A.; Strack, D.; An UPLC-MS/MS Method for the Simultaneous Identification and Quantitation of Cell Wall Phenolics in Brassica napus Seeds J. Agr. Food Chem. 61, 1219-1227, (2013) DOI: 10.1021/jf3042648

The seed residues left after pressing of rapeseed oil are rich in proteins and could be used for human nutrition and animal feeding. These press cakes contain, however, antinutritives, with fiber being the most abundant one. The analysis of fiber phenolic component (localized to seed coat cell walls) is, therefore, important in breeding and food quality control. However, correct structure and content assignments of cell wall-bound phenolics are challenging due to their low stability during sample preparation. Here, a novel LC-MS/MS-based method for the simultaneous identification and quantitation of 66 cell wall-bound phenolics and their derivatives is described. The method was internally standardized, corrected for degradation effects during sample preparation, and cross-validated with a well-established UV-based procedure. This approach was successfully applied to the analysis of cell wall phenolic patterns in different B. napus cultivars and proved to be suitable for marker compound search as well as assay development.
Books and chapters

Walter, M. H.; Floss, D. S.; Paetzold, H.; Manke, K.; Vollrath, J.; Brandt, W.; Strack, D.; Control of Plastidial Isoprenoid Precursor Supply: Divergent 1-Deoxy-D-Xylulose 5-Phosphate Synthase (DXS) Isogenes Regulate the Allocation to Primary or Secondary Metabolism (Bach, T. J. & Rohmer, M., eds.). 251-270, (2012) ISBN: 978-1-4614-4063-5 DOI: 10.1007/978-1-4614-4063-5_17

Following the description of two separate pathways for isoprenoid precursor biosynthesis in plants, a new level of complexity has been introduced by the discovery of two divergent gene classes encoding the first enzyme of the plastidial methylerythritol phosphate (MEP) pathway. These nonredundant 1-deoxy-d-xylulose 5-phosphate synthase (DXS) isogenes are differentially expressed in such a way that DXS1 appears to serve housekeeping functions, whereas DXS2 is associated with the production of specialized (secondary) isoprenoids involved in ecological functions. Examples of the latter are apocarotenoid formation in roots colonized by arbuscular mycorrhizal fungi and mono- or diterpenoid biosynthesis in trichomes. Knockdown of DXS2 genes can specifically suppress secondary isoprenoid formation without affecting basic plant functions. Analyzing DXS isogenes along the progression of land plant evolution shows separation in structure and complementary expression already at the level of gymnosperms, which is maintained in all angiosperms except Arabidopsis.
Publications

Walter, M. H.; Strack, D.; Carotenoids and their cleavage products: Biosynthesis and functions Nat. Prod. Rep. 28, 663-692, (2011) DOI: 10.1039/C0NP00036A

Covering: up to mid-2010This review focuses on plant carotenoids, but it also includes progress made on microbial and animal carotenoid metabolism to better understand the functions and the evolution of these structurally diverse compounds with a common backbone. Plants have evolved isogenes for specific key steps of carotenoid biosynthesis with differential expression profiles, whose characteristic features will be compared. Perhaps the most exciting progress has been made in studies of carotenoid cleavage products (apocarotenoids) with an ever-expanding variety of novel functions being discovered. This review therefore covers structural, molecular genetic and functional aspects of carotenoids and apocarotenoids alike. Apocarotenoids are specifically tailored from carotenoids by a family of oxidative cleavage enzymes, but whether there are contributions to their generation from chemical oxidation, photooxidation or other mechanisms is largely unknown. Control of carotenoid homeostasis is discussed in the context of biosynthetic and degradative reactions but also in the context of subcellular environments for deposition and sequestration within and outside of plastids. Other aspects of carotenoid research, including metabolic engineering and synthetic biology approaches, will only be covered briefly.
Publications

Wolfram, K.; Schmidt, J.; Wray, V.; Milkowski, C.; Schliemann, W.; Strack, D.; Profiling of phenylpropanoids in transgenic low-sinapine oilseed rape (Brassica napus) Phytochemistry 71, 1076-1084, (2010) DOI: 10.1016/j.phytochem.2010.04.007

A dsRNAi approach silencing a key enzyme of sinapate ester biosynthesis (UDP-glucose:sinapate glucosyltransferase, encoded by the UGT84A9 gene) in oilseed rape (Brassica napus) seeds was performed to reduce the anti-nutritive properties of the seeds by lowering the content of the major seed component sinapine (sinapoylcholine) and various minor sinapate esters. The transgenic seeds have been produced so far to the T6 generation and revealed a steady suppression of sinapate ester accumulation. HPLC analysis of the wild-type and transgenic seeds revealed, as in the previous generations, marked alterations of the sinapate ester pattern of the transformed seeds. Besides strong reduction of the amount of the known sinapate esters, HPLC analysis revealed unexpectedly the appearance of several minor hitherto unknown rapeseed constituents. These compounds were isolated and identified by mass spectrometric and NMR spectroscopic analyses. Structures of 11 components were elucidated to be 4-O-glucosides of syringate, caffeyl alcohol and its 7,8-dihydro derivative as well as of sinapate and sinapine, along with sinapoylated kaempferol glycosides, a hexoside of a cyclic spermidine alkaloid and a sinapine derivative with an ether-bridge to a C6–C3-unit. These results indicate a strong impact of the transgenic approach on the metabolic network of phenylpropanoids in B. napus seeds. Silencing of UGT84A9 gene expression disrupt the metabolic flow through sinapoylglucose and alters the amounts and nature of the phenylpropanoid endproducts.
Publications

Walter, M. H.; Floss, D. S.; Strack, D.; Apocarotenoids: hormones, mycorrhizal metabolites and aroma volatiles Planta 232, 1-17, (2010) DOI: 10.1007/s00425-010-1156-3

Apocarotenoids are tailored from carotenoids by oxidative enzymes [carotenoid cleavage oxygenases (CCOs)], cleaving specific double bonds of the polyene chain. The cleavage products can act as hormones, signaling compounds, chromophores and scent/aroma constituents. Recent advances were the identification of strigolactones as apocarotenoids and the description of their novel role as shoot branching inhibitor hormones. Strigolactones are also involved in plant signaling to both harmful (parasitic weeds) and beneficial [arbuscular mycorrhizal (AM) fungi] rhizosphere residents. This review describes the progress in the characterization of CCOs, termed CCDs and NCEDs, in plants. It highlights the importance of sequential cleavage reactions of C40 carotenoid precursors, the apocarotenoid cleavage oxygenase (ACO) nature of several CCOs and the topic of compartmentation. Work on the biosynthesis of abundant C13 cyclohexenone and C14 mycorradicin apocarotenoids in mycorrhizal roots has revealed a new role of CCD1 as an ACO of C27 apocarotenoid intermediates, following their predicted export from plastid to cytosol. Manipulation of the AM-induced apocarotenoid pathway further suggests novel roles of C13 apocarotenoids in controlling arbuscule turnover in the AM symbiosis. CCD7 has been established as a biosynthetic crosspoint, controlling both strigolactone and AM-induced C13 apocarotenoid biosynthesis. Interdependence of the two apocarotenoid pathways may thus play a role in AM-mediated reduction of parasitic weed infestations. Potential scenarios of C13 scent/aroma volatile biogenesis are discussed, including the novel mechanism revealed from mycorrhizal roots. The recent progress in apocarotenoid research opens up new perspectives for fundamental work, but has also great application potential for the horticulture, food and fragrance industries.
Publications

Vogel, J. T.; Walter, M. H.; Giavalisco, P.; Lytovchenko, A.; Kohlen, W.; Charnikhova, T.; Simkin, A. J.; Goulet, C.; Strack, D.; Bouwmeester, H. J.; Fernie, A. R.; Klee, H. J.; SlCCD7 controls strigolactone biosynthesis, shoot branching and mycorrhiza-induced apocarotenoid formation in tomato Plant J. 61, 300-311, (2010) DOI: 10.1111/j.1365-313X.2009.04056.x

The regulation of shoot branching is an essential determinant of plant architecture, integrating multiple external and internal signals. One of the signaling pathways regulating branching involves the MAX (more axillary branches) genes. Two of the genes within this pathway, MAX3/CCD7 and MAX4/CCD8, encode carotenoid cleavage enzymes involved in generating a branch‐inhibiting hormone, recently identified as strigolactone. Here, we report the cloning of SlCCD7 from tomato. As in other species, SlCCD7 encodes an enzyme capable of cleaving cyclic and acyclic carotenoids. However, the SlCCD7 protein has 30 additional amino acids of unknown function at its C terminus. Tomato plants expressing a SlCCD7 antisense construct display greatly increased branching. To reveal the underlying changes of this strong physiological phenotype, a metabolomic screen was conducted. With the exception of a reduction of stem amino acid content in the transgenic lines, no major changes were observed. In contrast, targeted analysis of the same plants revealed significantly decreased levels of strigolactone. There were no significant changes in root carotenoids, indicating that relatively little substrate is required to produce the bioactive strigolactones. The germination rate of Orobanche ramosa seeds was reduced by up to 90% on application of extract from the SlCCD7 antisense lines, compared with the wild type. Additionally, upon mycorrhizal colonization, C13 cyclohexenone and C14 mycorradicin apocarotenoid levels were greatly reduced in the roots of the antisense lines, implicating SlCCD7 in their biosynthesis. This work demonstrates the diverse roles of MAX3/CCD7 in strigolactone production, shoot branching, source–sink interactions and production of arbuscular mycorrhiza‐induced apocarotenoids.
Publications

Teutschbein, J.; Gross, W.; Nimtz, M.; Milkowski, C.; Hause, B.; Strack, D.; Identification and Localization of a Lipase-like Acyltransferase in Phenylpropanoid Metabolism of Tomato (Solanum lycopersicum) J. Biol. Chem. 285, 38374-38381, (2010) DOI: 10.1074/jbc.M110.171637

We have isolated an enzyme classified as chlorogenate: glucarate caffeoyltransferase (CGT) from seedlings of tomato (Solanum lycopersicum) that catalyzes the formation of caffeoylglucarate and caffeoylgalactarate using chlorogenate (5-O-caffeoylquinate) as acyl donor. Peptide sequences obtained by trypsin digestion and spectrometric sequencing were used to isolate the SlCGT cDNA encoding a protein of 380 amino acids with a putative targeting signal of 24 amino acids indicating an entry of the SlCGT into the secretory pathway. Immunogold electron microscopy revealed the localization of the enzyme in the apoplastic space of tomato leaves. Southern blot analysis of genomic cDNA suggests that SlCGT is encoded by a single-copy gene. The SlCGT cDNA was functionally expressed in Nicotiana benthamiana leaves and proved to confer chlorogenate-dependent caffeoyltransferase activity in the presence of glucarate. Sequence comparison of the deduced amino acid sequence identified the protein unexpectedly as a GDSL lipase-like protein, representing a new member of the SGNH protein superfamily. Lipases of this family employ a catalytic triad of Ser-Asp-His with Ser as nucleophile of the GDSL motif. Site-directed mutagenesis of each residue of the assumed respective SlCGT catalytic triad, however, indicated that the catalytic triad of the GDSL lipase is not essential for SlCGT enzymatic activity. SlCGT is therefore the first example of a GDSL lipase-like protein that lost hydrolytic activity and has acquired a completely new function in plant metabolism, functioning in secondary metabolism as acyltransferase in synthesis of hydroxycinnamate esters by employing amino acid residues different from the lipase catalytic triad.
Publications

Paetzold, H.; Garms, S.; Bartram, S.; Wieczorek, J.; Urós-Gracia, E.-M.; Rodríguez-Concepción, M.; Boland, W.; Strack, D.; Hause, B.; Walter, M. H.; The Isogene 1-Deoxy-D-Xylulose 5-Phosphate Synthase 2 Controls Isoprenoid Profiles, Precursor Pathway Allocation, and Density of Tomato Trichomes Mol. Plant 3, 904-916, (2010) DOI: 10.1093/mp/ssq032

Plant isoprenoids are formed from precursors synthesized by the mevalonate (MVA) pathway in the cytosol or by the methyl-D-erythritol 4-phosphate (MEP) pathway in plastids. Although some exchange of precursors occurs, cytosolic sesquiterpenes are assumed to derive mainly from MVA, while plastidial monoterpenes are produced preferentially from MEP precursors. Additional complexity arises in the first step of the MEP pathway, which is typically catalyzed by two divergent 1-deoxy-D-xylulose 5-phosphate synthase isoforms (DXS1, DXS2). In tomato (Solanum lycopersicum), the SlDXS1 gene is ubiquitously expressed with highest levels during fruit ripening, whereas SlDXS2 transcripts are abundant in only few tissues, including young leaves, petals, and isolated trichomes. Specific down-regulation of SlDXS2 expression was performed by RNA interference in transgenic plants to investigate feedback mechanisms. SlDXS2 down-regulation led to a decrease in the monoterpene β-phellandrene and an increase in two sesquiterpenes in trichomes. Moreover, incorporation of MVA-derived precursors into residual monoterpenes and into sesquiterpenes was elevated as determined by comparison of 13C to 12C natural isotope ratios. A compensatory up-regulation of SlDXS1 was not observed. Down-regulated lines also exhibited increased trichome density and showed less damage by leaf-feeding Spodoptera littoralis caterpillars. The results reveal novel, non-redundant roles of DXS2 in modulating isoprenoid metabolism and a pronounced plasticity in isoprenoid precursor allocation.
Publications

Mittasch, J.; Mikolajewski, S.; Breuer, F.; Strack, D.; Milkowski, C.; Genomic microstructure and differential expression of the genes encoding UDP-glucose:sinapate glucosyltransferase (UGT84A9) in oilseed rape (Brassica napus) Theor. Appl. Genet. 120, 1485-1500, (2010) DOI: 10.1007/s00122-010-1270-4

In oilseed rape (Brassica napus), the glucosyltransferase UGT84A9 catalyzes the formation of 1-O-sinapoyl-β-glucose, which feeds as acyl donor into a broad range of accumulating sinapate esters, including the major antinutritive seed component sinapoylcholine (sinapine). Since down-regulation of UGT84A9 was highly efficient in decreasing the sinapate ester content, the genes encoding this enzyme were considered as potential targets for molecular breeding of low sinapine oilseed rape. B. napus harbors two distinguishable sequence types of the UGT84A9 gene designated as UGT84A9-1 and UGT84A9-2. UGT84A9-1 is the predominantly expressed variant, which is significantly up-regulated during the seed filling phase, when sinapate ester biosynthesis exhibits strongest activity. In the allotetraploid genome of B. napus, UGT84A9-1 is represented by two loci, one derived from the Brassica C-genome (UGT84A9a) and one from the Brassica A-genome (UGT84A9b). Likewise, for UGT84A9-2 two loci were identified in B. napus originating from both diploid ancestor genomes (UGT84A9c, Brassica C-genome; UGT84A9d, Brassica A-genome). The distinct UGT84A9 loci were genetically mapped to linkage groups N15 (UGT84A9a), N05 (UGT84A9b), N11 (UGT84A9c) and N01 (UGT84A9d). All four UGT84A9 genomic loci from B. napus display a remarkably low micro-collinearity with the homologous genomic region of Arabidopsis thaliana chromosome III, but exhibit a high density of transposon-derived sequence elements. Expression patterns indicate that the orthologous genes UGT84A9a and UGT84A9b should be considered for mutagenesis inactivation to introduce the low sinapine trait into oilseed rape.
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