Alfred Baumert

MOLECULAR CLONING AND HETEROLOGOUS EXPRESSION OF ACRIDONE SYNTHASE FROM ELICITED RUTA GRAVEOLENS L. CELL SUSPENSION CULTURES

Cell suspension cultures of Ruta graveolens L. produce a variety of acridone alkaloids, and the accumulation can be stimulated by the addition of fungal elicitors. Acridone synthase, the enzyme catalyzing the synthesis of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA, had been isolated from these cells, and the partial enzyme polypeptide sequence, elucidated from six tryptic fragments, revealed homology to heterologous chalcone synthases. Poly(A)+ RNA was isolated from Ruta cells that had been treated for 6 h with a crude cell wall elicitor from Phytophthora megasperma f. sp. glycinea, and a cDNA library was constructed in λ2AP. Clones harboring acridone synthase cDNA were isolated from the library by screening with a synthetic oligonucleotide probe complementary to a short stretch of sequence of the enzyme peptide with negligible homology to chalcone synthases. The identity of the clones was substantiated by DNA sequencing and by recognition of five additional peptides, determined previously from tryptic acridone synthase digests, in the translated sequence. An insert of roughly 1.4 kb encoded the complete acridone synthase, and alignments at both DNA and protein levels corroborated the high degree of homology to chalcone synthases. Expression of the enzyme in vector pET-11c in the Escherichia coli pLysS host strain proved the identity of the cloned cDNA. The heterologous enzyme in the crude E. coli extract exhibited high acridone but no chalcone synthase activity. The results were fully supported by northern blot hybridizations which revealed that the specific transcript abundance did not increase but rather decreased upon white light irradiation of cultured Ruta graveolens L. cells, a condition that commonly induces the abundance of chalcone synthase transcripts.

Reduction of sinapate ester content in transgenic oilseed rape (Brassica napus) by dsRNAi-based suppression of BnSGT1 gene expression

Seeds of oilseed rape (Brassica napus) accumulate high amounts of antinutritive sinapate esters (SE) with sinapoylcholine (sinapine) as major component, accompanied by sinapoylglucose. These phenolic compounds compromise the use of the protein-rich valuable seed meal. Hence, a substantial reduction of the SE content is considered essential for establishing rape as a protein crop. The present work focuses on the suppression of sinapine synthesis in rape. Therefore, rape (spring cultivar Drakkar) was transformed with a dsRNAi construct designed to silence seed-specifically the BnSGT1 gene encoding UDP-glucose:sinapate glucosyltransferase (SGT1). This resulted in a substantial decrease of SE content in T2 seeds with a reduction reaching 61%. In T2 seeds a high and significant correlation between the contents of sinapoylglucose and all other sinapate esters has been observed. Among transgenic plants, no significant difference in other important agronomic traits, such as oil, protein, fatty acid and glucosinolate content in comparison to the control plants was observed. Maximal reduction of total SE content by 76% was observed in seeds of one homozygous T2 plant (T3 seeds) carrying the BnSGT1 suppression cassette as a single copy insert. In conclusion, this study is an initial proof of principle that suppression of sinapoylglucose formation leads to a strong reduction of SE in rape seeds and is thus a promising approach in establishing rape, currently an important oil crop, as a protein crop as well.

Cloning and heterologous expression of a rape cDNA encoding UDP-glucose:sinapate glucosyltransferase

Abstract. 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).

Identification of four Arabidopsis genes encoding hydroxycinnamate glucosyltransferases

Members of the Brassicaceae accumulate sinapate esters as major phenylpropanoid secondary metabolites, which are £uorescent UV-protective compounds, as shown with Arabidopsis thaliana [1]. A pivotal enzyme in sinapate ester biosynthesis is UDP-glucose:sinapate glucosyltransferase (SGT, EC 2.4.1.120) which catalyzes the transfer of glucose from UDPglucose to sinapate and some other hydroxycinnamates (HCAs), including 4-coumarate, ca¡eate and ferulate [2]. 1O-Sinapoylglucose is the immediate acyl donor in reactions leading to sinapine (sinapoylcholine) in seeds [3] and sinapoylmalate plants [4,5]. The ¢rst gene in sinapate ester biosynthesis has been cloned from Arabidopsis [6] and encodes 1-O-sinapoylglucose:malate sinapoyltransferase (SMT, EC 2.3.1.92). The second gene involved in this metabolism has very recently been cloned from rape (Brassica napus) [7]. It encodes UDP-glucose:SGT. Here we report the cloning and identi¢cation of four Arabidopsis glucosyltransferase (GT) genes encoding cinnamate and HCA GTs. These genes have previously been putatively assumed to encode indole-3-acetate (IAA) GTs ([8] ; Y. Nakamura, NCBI protein database accession number BAB00006). We unambiguously demonstrate, however, that the heterologously expressed Arabidopsis enzymes do not catalyze the synthesis of the IAA glucose ester, but rather speci¢cally cinnamate and HCA glucose esters. This is a prominent example of misidenti¢cation of new genes. It shows again that the assignment of genes without functional expression carries a high risk of error. As the corresponding nucleotide sequences retrieved from the databases reveal no introns, the Arabidopsis genes encoding the proteins BAB00006, D71419, E71419 and F71419 were ampli¢ed by polymerase chain reaction (PCR) with genomic Arabidopsis DNA as template. Genomic DNA was isolated according to Brandsta«dter et al. [9] from rosette leaves of 5-week-old Arabidopsis plants (Arabidopsis thaliana Heynh. ecotype Columbia) grown in the green house. The following speci¢c primers were used: BAB00006: 5P-ATG GAG CTA GAA TCT TCT CC-3P (forward) and 5P-TTA AAA GCT TTT GAT TGA TCC-3P (reverse); E71419: 5P-ATG GAC CCG TCT CGT CAT ACT CAT G-3P (forward) and 5PCTA GTG TTC TCC GTT GTC TTC TCT CG-3P (reverse) ; F71419: 5P-ATG GAG ATG GAA TCG TCG TTA CCT C3P (forward) and 5P-TTA CAC GAC ATT ATT AAT GTT TGT C-3P (reverse); D71419: 5P-ATG GTG TTC GAA ACT TGT CCA TCT CC-3P (forward) and 5P-CTA GTA TCC ATT ATC TTT AGT CTT CG-3P (reverse). The PCR products were inserted into the SmaI-linearized vector pQE-32 (Qiagen). Sense and antisense constructs were used to transform the Escherichia coli strain M15 (pREP4). E. coli M15 (pREP4) cells harboring the Arabidopsis genes on the pQE-32 vector were grown to logarithmic phase. Expression of the recombinant Arabidopsis proteins was then induced by incubating the cells for another 20 h at 303C in the presence of 1 mM isopropylthiogalactoside (IPTG). The enzyme assays were performed as described [7]. In short : IPTG-induced E. coli cells harboring the Arabidopsis GT genes were collected, suspended in lysis bu¡er (0.1 M Mes bu¡er, pH 6.0), disrupted by soni¢cation and centrifuged. Aliquots of the supernatants were incubated for 30 min at 303C with 4 mM UDP-glucose and various acceptor molecules (cinnamate, HCAs, benzoate, hydroxybenzoates or IAA, 2 mM each) in the presence of 0.1 M Mes bu¡er (pH 6.0). The reactions were terminated by adding tri£uoroacetic acid. Enzymatic products were identi¢ed by liquid chromatographic-mass spectrometry (LC-MS) and photodiode arrayhigh performance liquid chromatography (PDA-HPLC) analyses. Quanti¢cation was achieved by external standardization with cinnamate and HCAs purchased from Sigma. Further details on PDA-HPLC and LC-MS are described elsewhere [7]. In a recent paper [7] we presented results on cloning and heterologous expression of a rape cDNA encoding UDP-glucose:sinapate GT (SGT1). The rape SGT1 sequence showed 64, 63 and 61% sequence identities to putative Arabidopsis IAA GTs [7]. In addition, a recent data base search showed the highest identity of rape SGT1 (81% on amino acid level) to a new entry assigned also to an Arabidopsis IAA GT-like protein (Y. Nakamura, NCBI protein database accession number BAB00006). These results prompted us to clone and functionally express the four Arabidopsis genes in E. coli, expecting to ¢nd rape SGT1-like enzymes. We ampli¢ed the four respective genes by PCR with genomic DNA isolated from rosette leaves of 5-week-old Arabidopsis plants. These genes were introduced into E. coli cells as sense and antisense constructs and their expression induced by the addition of IPTG. Crude E. coli protein extracts were assayed for GT activities. Testing potential acceptors, it was found that none of the recombinant enzymes was active towards IAA (Table 1). Thus, the earlier putative assignments of these genes are incorrect. Instead, the recombinant enzymes catalyzed speci¢cally the formation of cinnamate glucose ester (1-O-cinnamoylglucose) and HCA glucose esters (1-O-4-coumaroyl-, -ca¡eoyl-, -feruloyland -sinapoylglucose) with markedly di¡erent acceptor speci¢cities. Possible glucose esteri¢cation of benzoate, 4-hydroxybenzoate and salicylate was not observed. Identi¢cation of the products, cinnamoyland HCA-glucose esters, was achieved by chromatographic comparison (PDA-HPLC) with standard compounds available from a previous study [7] and by LC-MS. The latter gave the expected molecular mass for cinnamoylglucose of 310, for 4-coumaroylglucose of 326, for ca¡eoylglucose of 342; for feruloylglucose of 356, and for sinapoylglucose of 386. Neither heatdenatured proteins (5 min at 953C) nor that from E. coli

Patterns of phenylpropanoids in non-inoculated and potato virus Y-inoculated leaves of transgenic tobacco plants expressing yeast-derived invertase.

The patterns of secondary metabolites in leaves of yeast invertase-transgenic tobacco plants (Nicotiana tabacum L. cv. Samsun NN) were analyzed. Plants expressing cytosolic yeast-derived invertase (cytInv) or apoplastic (cell wall associated) yeast invertase (cwInv) showed a characteristic phytochemical phenotype compared to untransformed controls (wild-type plants). The level of phenylpropanoids decreased in the cytInv plants but increased in the cwInv plants, which showed an induced de novo synthesis of a caffeic acid amide, i.e. N-caffeoylputrescine. In addition, the level of the coumarin glucoside scopolin was markedly enhanced. Increased accumulation of scopolin in the cwInv plants is possibly correlated with the induction of defense reactions and the appearance of necrotic lesions similar to the hypersensitive response caused by avirulent pathogens. This is consistent with results from potato virus Y-infected plants. Whereas there was no additional increase in the coumarins in leaves following infection in cwInv plants, wild-type plants showed a slight increase and cytInc a marked increase.

Accumulation of tyrosol glucoside in transgenic potato plants expressing a parsley tyrosine decarboxylase

As part of the response to pathogen infection, potato plants accumulate soluble and cell wall-bound phenolics such as hydroxycinnamic acid tyramine amides. Since incorporation of these compounds into the cell wall leads to a fortified barrier against pathogens, raising the amounts of hydroxycinnamic acid tyramine amides might positively affect the resistance response. To this end, we set out to increase the amount of tyramine, one of the substrates of the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction, by placing a cDNA encoding a pathogen-induced tyrosine decarboxylase from parsley under the control of the 35S promoter and introducing the construct into potato plants via Agrobacterium tumefaciens-mediated transformation. While no alterations were observed in the pattern and quantity of cell wall-bound phenolic compounds in transgenic plants, the soluble fraction contained several new compounds. The major one was isolated and identified as tyrosol glucoside by liquid chromatography-electrospray ionization-high resolution mass spectrometry and NMR analyses. Our results indicate that expression of a tyrosine decarboxylase in potato does not channel tyramine into the hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)-transferase reaction but rather unexpectedly, into a different pathway leading to the formation of a potential storage compound.

Synthesis of 1,3-dihydroxy-N-methylacridone and its conversion to rutacridone by cell-free extracts of Ruta graveolens cell cultures

Abstract Acridone synthase was isolated from cell suspension cultures of Ruta graveolens which catalysed the formation of 1,3-dihydroxy-N- methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA. No cofactors were required for this enzyme reaction. Potassium phosphate buffer was superior compared to Tris-HCl. Sodium ascorbate instead of mercaptoethanol as oxidation protectant showed an advantageous effect on acridone synthase activity. The enzyme was strongly inhibited by 1,3-dihydroxy-N-methylacridone and by the antibiotic cerulenin. Microsomal preparations from Ruta graveolens cell suspension cultures catalysed an NADPH- and oxygen-dependent condensation of 1,3-dihydroxy-N- methylacridone and isopentenyl pyrophosphate. The reaction product was identified as rutacridone. Mg2+ or Mn2+ ions were necessary for optimal rutacridone synthase activity. The enzyme was inhibited by a number of inhibitors of cytochrome P-450 enzymes. A prenylated acridone, viz. glycocitrine-II was identified as an essential intermediate. Under in vivo conditions glycocitrine-II is incorporated into rutacridone, but a clear-cut conversion of glycocitrine-II by microsomal preparations (cyclase) was not observed. Microsomes converted rutacridone into furofoline-I. A number of detergents was used for solubilization of membrane-bound proteins of Ruta microsomes. Highest specific glycocitrine -II synthase (prenyltransferase) activity was obtained after solubilization with dodecylmaltoside.

Purification and properties of acridone synthase from cell suspension cultures of Ruta graveolens L.

Acridone synthase has been purified from cell suspension cultures of Ruta graveolens using a combination of gel filtration and ion exchange chromatography. The purified enzyme has an apparent molecular weight of 69 kDa on gel filtration and a subunit structure on SDS-PAGE of 40 kDa. The apparent Km-values are 10.64 μM and 32.8 μM for N-methylanthraniloyl-CoA and malonyl-CoA, respectively. Tryptic digestion of the homogeneous acridone synthase was performed. Seven of the peptides were chosen for microsequencing. The homology of the amino acid sequences from this particular polypeptide and corresponding peptides from chalcone synthase 3 from garden pea amounted to 76%.

Triterpenoids from Pisolithus tinctorius isolates and ectomycorrhizas

Abstract Two new triterpenoids have been identified by spectroscopic methods from mycelia of Pisolithus tinctorius as 24-ethyllanosta-8,24(24 1 )-diene-3β,22ξ-diol and (22 S )-24,25-dimethyllanosta-8-en-22,24 1 -epoxy-3β-ol-24 1 -one (25-methylpisolactone) along with the two known triterpenoids 24-methyllanosta-8,24(24 1 )-diene-3β,22ξ-diol and (22 S )-24-methyllanosta-8-en-22,24 1 -epoxy-3β-ol-24 1 -one (pisolactone). Quantification of these compounds in fungal isolates (surface and suspension cultures) and Pinus sylvestris ectomycorrhizas showed that the amount of the new triterpenoids was markedly higher in the mycorrhizas as in the isolates.

Formation of 1,3-Dihydroxy-N-methylacridone from N-Methylanthraniloyl-CoA and Malonyl-CoA by Cell-Free Extracts of Ruta graveolens

N-Methylanthraniloyl-CoA was synthesized via N-succinimidyl N-methylanthranilate and subsequent transesterification with CoA-SH . This compound was characterized by LSIMS and NMR data. An enzyme preparation from cell suspension cultures of Ruta graveolens catalyzed the formation of 1,3-dihydroxy-N-methylacridone from N-methylanthraniloyl-CoA and malonyl-CoA with a pH optimum of 7.5.