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Publikationen - Stoffwechsel- und Zellbiologie

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Publikation

Milkowski, C.; Baumert, A.; Strack, D.; Identification of four Arabidopsis genes encoding hydroxycinnamate glucosyltransferases FEBS Lett. 486, 183-184, (2000) DOI: 10.1016/S0014-5793(00)02270-5

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Publikation

Milkowski, C.; Baumert, A.; Strack, D.; Cloning and heterologous expression of a rape cDNA encoding UDP-glucose:sinapate glucosyltransferase Planta 211, 883-886, (2000) DOI: 10.1007/s004250000411

A cDNA encoding a UDP-glucose:sinapate glucosyltransferase (SGT) that catalyzes the formation of 1-O-sinapoylglucose, was isolated from cDNA libraries constructed from immature seeds and young seedlings of rape (Brassica napus L.). The open reading frame encoded a protein of 497 amino acids with a calculated molecular mass of 55,970 Da and an isoelectric point of 6.36. The enzyme, functionally expressed in Escherichia coli, exhibited broad substrate specificity, glucosylating sinapate, cinnamate, ferulate, 4-coumarate and caffeate. Indole-3-acetate, 4-hydroxybenzoate and salicylate were not conjugated. The amino acid sequence of the SGT exhibited a distinct sequence identity to putative indole-3-acetate glucosyltransferases from Arabidopsis thaliana and a limonoid glucosyltransferase from Citrus unshiu, indicating that SGT belongs to a distinct subgroup of glucosyltransferases that catalyze the formation of 1-O-acylglucosides (β-acetal esters).
Publikation

Mikkat, S.; Milkowski, C.; Hagemann, M.; The gene sll 0273 of the cyanobacterium Synechocystis sp. strain PCC6803 encodes a protein essential for growth at low Na+/K+ ratios Plant Cell Environ. 23, 549-559, (2000) DOI: 10.1046/j.1365-3040.2000.00565.x

A mutant of Synechocystis sp. strain PCC6803 was obtained by random cartridge mutagenesis, which could not grow at low sodium concentrations. Genetic analyses revealed that partial deletion of the sll 0273 gene, encoding a putative Na+ /H+ exchanger, was responsible for this defect. Physiological characterization indicated that the sll 0273 mutant exhibited an increased sensitivity towards K+ , even at low concentrations, which was compensated for by enhanced concentrations of Na+ . This enhanced Na+ demand could also be met by Li+ . Furthermore, addition of monensin, an ionophore mediating electroneutral Na+ /H+ exchange, supported growth of the mutant at unfavourable Na+ /K+ ratios. Measurement of internal Na+ and K+ contents of wild‐type and mutant cells revealed a decreased Na+ /K+ ratio in mutant cells pre‐incubated at a low external Na+ /K+ ratio, while it remained at the level of the wild type after pre‐incubation at a high external Na+ /K+ ratio. We conclude that the Sll0273 protein is required for Na+ influx, especially at low external Na+ concentrations or low Na+ /K+ ratios. This system may be part of a sodium cycle and may permit re‐entry of Na+ into the cells, if nutrient/Na+ symporters are not functional or operating.
Publikation

Maucher, H.; Hause, B.; Feussner, I.; Ziegler, J.; Wasternack, C.; Allene oxide synthases of barley (Hordeum vulgare cv. Salome): tissue specific regulation in seedling development Plant J. 21, 199-213, (2000) DOI: 10.1046/j.1365-313x.2000.00669.x

Allene oxide synthase (AOS) is the first enzyme in the lipoxygenase (LOX) pathway which leads to formation of jasmonic acid (JA). Two full‐length cDNAs of AOS designated as AOS1 and AOS2, respectively, were isolated from barley (H. vulgare cv. Salome) leaves, which represent the first AOS clones from a monocotyledonous species. For AOS1, the open reading frame encompasses 1461 bp encoding a polypeptide of 487 amino acids with calculated molecular mass of 53.4 kDa and an isoelectric point of 9.3, whereas the corresponding data of AOS2 are 1443 bp, 480 amino acids, 52.7 kDa and 7.9. Southern blot analysis revealed at least two genes. Despite the lack of a putative chloroplast signal peptide in both sequences, the protein co‐purified with chloroplasts and was localized within chloroplasts by immunocytochemical analysis. The barley AOSs, expressed in bacteria as active enzymes, catalyze the dehydration of LOX‐derived 9‐ as well as 13‐hydroperoxides of polyenoic fatty acids to the unstable allene oxides. In leaves, AOS mRNA accumulated upon treatment with jasmonates, octadecanoids and metabolizable carbohydrates, but not upon floating on abscisic acid, NaCl, Na‐salicylate or infection with powdery mildew. In developing seedlings, AOS mRNA strongly accumulated in the scutellar nodule, but less in the leaf base. Both tissues exhibited elevated JA levels. In situ hybridizations revealed the preferential occurrence of AOS mRNA in parenchymatic cells surrounding the vascular bundles of the scutellar nodule and in the young convoluted leaves as well as within the first internode. The properties of both barley AOSs, their up‐regulation of their mRNAs and their tissue specific expression suggest a role during seedling development and jasmonate biosynthesis.
Publikation

Maier, W.; Schmidt, J.; Nimtz, M.; Wray, V.; Strack, D.; Secondary products in mycorrhizal roots of tobacco and tomato Phytochemistry 54, 473-479, (2000) DOI: 10.1016/S0031-9422(00)00047-9

Colonization of the roots of various tobacco species and cultivars (Nicotiana glauca Grah., N. longiflora Cav., N. rustica L., N. tabacum L., N. tabacum L. cv. Samsun NN, N. sanderae hort. Sander ex Wats.) as well as tomato plants (Lycopersicon esculentum L. cv. Moneymaker) by the arbuscular mycorrhizal fungus Glomus intraradices Schenck and Smith resulted in the accumulation of several glycosylated C13 cyclohexenone derivatives. Eight derivatives were isolated from the mycorrhizal roots by preparative high performance liquid chromatography (HPLC) and spectroscopically identified (MS and NMR) as mono-, di- and triglucosides of 6-(9-hydroxybutyl)-1,1,5-trimethyl-4-cyclohexen-3-one and monoglucosides of 6-(9-hydroxybutyl)-1,5-dimethyl-4-cyclohexen-3-one-1-carboxylic acid and 6-(9-hydroxybutyl)-1,1-dimethyl-4-cyclohexen-3-one-5-carboxylic acid. In contrast to the induced cyclohexenone derivatives, accumulation of the coumarins scopoletin and its glucoside (scopolin) in roots of N. glauca Grah. and N. tabacum L. cv. Samsun NN, was markedly suppressed.
Publikation

Lehfeldt, C.; Shirley, A. M.; Meyer, K.; Ruegger, M. O.; Cusumano, J. C.; Viitanen, P. V.; Strack, D.; Chapple, C.; Cloning of the SNG1 Gene of Arabidopsis Reveals a Role for a Serine Carboxypeptidase-like Protein as an Acyltransferase in Secondary Metabolism Plant Cell 12, 1295-1306, (2000) DOI: 10.1105/tpc.12.8.1295

Serine carboxypeptidases contain a conserved catalytic triad of serine, histidine, and aspartic acid active-site residues. These enzymes cleave the peptide bond between the penultimate and C-terminal amino acid residues of their protein or peptide substrates. The Arabidopsis Genome Initiative has revealed that the Arabidopsis genome encodes numerous proteins with homology to serine carboxypeptidases. Although many of these proteins may be involved in protein turnover or processing, the role of virtually all of these serine carboxypeptidase-like (SCPL) proteins in plant metabolism is unknown. We previously identified an Arabidopsis mutant, sng1 (sinapoylglucose accumulator 1), that is defective in synthesis of sinapoylmalate, one of the major phenylpropanoid secondary metabolites accumulated by Arabidopsis and some other members of the Brassicaceae. We have cloned the gene that is defective in sng1 and have found that it encodes a SCPL protein. Expression of SNG1 in Escherichia coli demonstrates that it encodes sinapoylglucose:malate sinapoyltransferase, an enzyme that catalyzes a transesterification instead of functioning like a hydrolase, as do the other carboxypeptidases. This finding suggests that SCPL proteins have acquired novel functions in plant metabolism and provides an insight into the evolution of secondary metabolic pathways in plants.
Publikation

Lee, Y. K.; Hippe-Sanwald, S.; Jung, H. W.; Hong, J. K.; Hause, B.; Hwang, B. K.; In situ localization of chitinase mRNA and protein in compatible and incompatible interactions of pepper stems with Phytophthora capsici Physiol. Mol. Plant Pathol. 57, 111-121, (2000) DOI: 10.1006/pmpp.2000.0290

The induction of chitinase (CAChi2) mRNA started as early as 6 h after inoculation and gradually increased in the incompatible interaction of pepper stems with Phytophthora capsici. In the compatible interaction, the induction of the chitinase transcripts was detected later than that in the incompatible interaction. The CAChi2 mRNA was usually localized in the vascular tissues and their expression was constricted in the phloem-related cells. These results showed that the spatial pattern of CAChi2 mRNA expression was similar in both compatible and incompatible interactions but the temporal patterns were different from each other. In addition, the early induction ofCAChi2 mRNA was quite distinct in the incompatible interaction. Immunogold labelling data showed specific labelling of chitinase on the cell wall of the oomycete in both compatible and incompatible interactions at 24 h after inoculation. In particular, numerous gold particles were deposited on the cell wall of P. capsici with a predominant accumulation over areas showing signs of degradation in the incompatible interaction. Chitinase labelling was also detected in the intercellular space and the host cytoplasm. However, healthy pepper stem tissue was nearly free of labelling.
Publikation

Kobayashi, N.; Schmidt, J.; Nimtz, M.; Wray, V.; Schliemann, W.; Betalains from Christmas cactus Phytochemistry 54, 419-426, (2000) DOI: 10.1016/S0031-9422(00)00129-1

The presence of 14 betalain pigments have been detected by their characteristic spectral properties in flower petals of Christmas cactus (Schlumbergera x buckleyi). Along with the known vulgaxanthin I, betalamic acid, betanin and phyllocactin (6′-O-malonylbetanin), the structure of a new phyllocactin-derived betacyanin was elucidated by various spectroscopic techniques and carbohydrate analyses as betanidin 5-O-(2′-O-β-D-apiofuranosyl-6′-O-malonyl)-β-D-glucopyranoside. Among the more complex betacyanins occurring in trace amounts, the presence of a new diacylated betacyanin {betanidin 5-O-[(5″-O-E-feruloyl)-2′-O-β-D-apiofuranosyl-6′-O-malonyl)]-β-D-glucopyranoside} has been ascertained. Furthermore, the accumulation of betalains during flower development and their pattern in different organs of the flower has been examined.
Publikation

Irmler, S.; Schröder, G.; St-Pierre, B.; Crouch, N. P.; Hotze, M.; Schmidt, J.; Strack, D.; Matern, U.; Schröder, J.; Indole alkaloid biosynthesis in Catharanthus roseus: new enzyme activities and identification of cytochrome P450 CYP72A1 as secologanin synthase Plant J. 24, 797-804, (2000) DOI: 10.1111/j.1365-313X.2000.00922.x

The molecular characterization of CYP72A1 from Catharanthus roseus (Madagascar periwinkle) was described nearly a decade ago, but the enzyme function remained unknown. We now show by in situ hybridization and immunohistochemistry that the expression in immature leaves is epidermis‐specific. It thus follows the pattern previously established for early enzymes in the pathway to indole alkaloids, suggesting that CYP72A1 may be involved in their biosynthesis. The early reactions in that pathway, i.e. from geraniol to strictosidine, contain several candidates for P450 activities. We investigated in this work two reactions, the conversion of 7‐deoxyloganin to loganin (deoxyloganin 7‐hydroxylase, DL7H) and the oxidative ring cleavage converting loganin into secologanin (secologanin synthase, SLS). The action of DL7H has not been demonstrated in vitro previously, and SLS has only recently been identified as P450 activity in one other plant. We show for the first time that both enzyme activities are present in microsomes from C . roseus cell cultures. We then tested whether CYP72A1 expressed in E. coli as a translational fusion with the C . roseus P450 reductase (P450Red) has one or both of these activities. The results show that CYP72A1 converts loganin into secologanin.
Publikation

Hause, B.; Stenzel, I.; Miersch, O.; Maucher, H.; Kramell, R.; Ziegler, J.; Wasternack, C.; Tissue-specific oxylipin signature of tomato flowers: allene oxide cyclase is highly expressed in distinct flower organs and vascular bundles Plant J. 24, 113-126, (2000) DOI: 10.1046/j.1365-313x.2000.00861.x

A crucial step in the biosynthesis of jasmonic acid (JA) is the formation of its correct stereoisomeric precursor, cis (+)12‐oxophytodienoic acid (OPDA). This step is catalysed by allene oxide cyclase (AOC), which has been recently cloned from tomato . In stems, young leaves and young flowers, AOC mRNA accumulates to a low level , contrasting with a high accumulation in flower buds, flower stalks and roots. The high levels of AOC mRNA and AOC protein in distinct flower organs correlate with high AOC activity, and with elevated levels of JA, OPDA and JA isoleucine conjugate. These compounds accumulate in flowers to levels of about 20 nmol g−1 fresh weight, which is two orders of magnitude higher than in leaves. In pistils, the level of OPDA is much higher than that of JA, whereas in flower stalks, the level of JA exceeds that of OPDA. In other flower tissues, the ratios among JA, OPDA and JA isoleucine conjugate differ remarkably, suggesting a tissue‐specific oxylipin signature. Immunocytochemical analysis revealed the specific occurrence of the AOC protein in ovules, the transmission tissue of the style and in vascular bundles of receptacles, flower stalks, stems, petioles and roots. Based on the tissue‐specific AOC expression and formation of JA, OPDA and JA amino acid conjugates, a possible role for these compounds in flower development is discussed in terms of their effect on sink–source relationships and plant defence reactions. Furthermore, the AOC expression in vascular bundles might play a role in the systemin‐mediated wound response of tomato.
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  • Martin-Luther Universität Halle
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