Metabolization of the Artificial Secondary Metabolite 4 ...

Plant Cell Physiol. 38(7): 844-850 (1997) JSPP © 1997

Metabolization of the Artificial Secondary Metabolite 4-Hydroxybenzoate in wft/C-Transformed Tobacco Shu-Ming Li, Zhao-Xin Wang, Emmanuel Wemakor and Lutz Heide Pharmazeutisches Institut, Eberhard-Karls-Universitdt Tubingen, Auf der Morgenstelle 8, D-72076 Tubingen, Federal Republic of Germany

Transgenic Nicotiana tabacum cell cultures formed a new, artificial secondary metabolite, i.e. 4-hydroxybenzoate (4HB), by expression of the bacterial gene ubiC, which encodes chorismate pyruvate-lyase. 4HB was converted in the cells to two different glucosides, i.e. 4-O-(l-fiD-gIucosyl)benzoic acid (4HBOG) and 4HB l-/7-D-glucosyl ester (4HBCOOG). The same metabolization was also observed with exogenously fed 4HB. This glucosylation is catalyzed by constitutively expressed glucosyltransferases which could be detected in cell-free extracts of transgenic and untransformed cells in the same activity. The enzymes forming the two respective glucosides differ in their pH optima, their affinity for 4HB and their regulation. Both in vivo and in cell-free extracts, 4HBOG is the main product at low 4HB concentration, and 4HBCOOG at high 4HB concentration. Whereas 4HBOG is accumulated as an apparently stable secondary metabolite, 4HBCOOG presents a metabolically active form of 4HB. After feeding of [U"C]4HB, a transient accumulation of 4HBCOOG was followed by incorporation of radioactive 4HB into the cell wall, suggesting that 4HBCOOG, but not 4HBOG serves as precursor of cell wall bound 4HB. Key words: Cell wall — Glucosyltransferase — 4-Hydroxybenzoate — Nicotiana tabacum — Transgenic plants — ubiC gene.

Molecular biology opens the possibility to genetically engineer the secondary metabolism of plants. This can be utilized for the production of useful secondary products, e.g. Pharmaceuticals, or for an improvement of plant disease resistance (Hain et al. 1993, Yun et al. 1992). For such purposes, it is of importance that the transformed plant is able to tolerate the formation of new, potentially toxic secondary metabolites. We therefore wanted to study the reaction of plant cells to a genetically altered secondary metabolism, and to examine the detoxification mechanisms Abbreviations: CPL, chorismate pyruvate-lyase; DHBA, 3,5dihydroxybenzoic acid; DTT, dithiothreitol; 4HB, 4-hydroxybenzoate; 4HBCOOG, 4HB 1-yS-D-glucosyl ester; 4HB0G, 4-O(l-/?-D-glucosyl)benzoic acid; KPi, potassium phosphate; PMSF, phenylmethylsulfonyl fluoride; PVPP, polyvinylpolypyrrolidone; TCA, trichloroacetic acid. 844

which may be employed in such a case. The expression of the bacterial ubiC gene in tobacco, leading to a more than 1,000-fold increase of the content of 4-hydroxybenzoate (4HB) derivatives in the plant (Siebert et al. 1996), presents a suitable object for such studies. The ubiC gene encodes chorismate pyruvate-lyase, an enzyme which converts chorismate to 4HB and which is not normally present in plants (Heide et al. 1989, Loscher and Heide 1994). As reported earlier (Siebert et al. 1996), 4HB formed in transgenic tobacco is converted into two glucosides, i.e. 4HBOG and 4HBCOOG, a reaction which may represent a detoxification mechanism. We have now studied in transgenic and wild-type tobacco cells the metabolism of 4HB, i.e. the enzymes involved in the 4HB glucosylation as well as the further metabolic fate of the 4HB glucosides. Materials and Methods Radiochemicals— [U-14C]4HB was purchased from Biotrend (Cologne, Germany). [U-I4C]4HBOG and [U-I4C]4HBCOOG were prepared as described below. Cell cultures—Cell cultures were derived from sterile-grown seedlings of wild-type tobacco (Nicotiana tabaccum cv. Petite havana SRI) or ubiC transformed tobacco, line II, as described before (Siebert et al. 1996). Cell suspension cultures were maintained in the dark at 25°C in Murashige and Skoog medium (Murashige and Skoog 1962), supplemented with 5 nM 1-naphthalenacetic acid and 1 fiM 6-benzylaminopurine. For transgenic cells, 150/^M kanamycin was added. Unless otherwise stated, cells were cultured on a rotary shaker at 80 rpm in 300 ml flasks containing 100 ml liquid medium, and subcultured at two week intervals. HPLC conditions—4HB and its glucosides were analyzed on a Waters HPLC system with a Multospher 120 RP 18-5 column at a flow rate of 1 ml min" 1 and detected at 254 nm, using as solvent system H 2 O-HCOOH (99 : 1; solvent A) and CH 3 OH-H 2 OHCOOH (50 : 49 : 1; solvent B) and employing a non-linear gradient from 10 to 50% B in A. For UV quantification, 3,5-dihydroxybenzoic acid (DHBA) was used as internal standard. An HPLC radioactivity monitor (Berthold, Bad Wildbad, Germany) was used for determination of radioactive peaks. Retention times of 4HB, 4HBOG, 4HBCOOG and DHBA were 10.9, 7.2, 9.3 and 9.8 min, respectively. Enzyme extraction—All manipulations were carried out at 04°C. Cells (7.5 g) were harvested, washed and mixed with 10 ml 0.1 M KPi buffer, pH 6.5, containing 20 mM DTT, 40 fiM PMSF and PVPP (0.5 g) and ground with a mortar and pestle to a fine slurry. The homogenate was centrifuged at 17,000 x g for 15 min. The supernatant was passed over Sephadex G-25 (Pharmacia,

4HB metabolization in tobacco Freiburg, Germany) using an unbuffered solution of 0.15 M NaCl as eluent. Determination of protein content—The protein concentration was determined according to the method of Bradford (1976) using bovine serum albumin as a standard. Glucosyltransferase assay—Each assay (100 ft\) contained 250 nmol 4HB, 500 nmol UDP-glucose and 80/A enzyme extract. For determination of 4HBOG or 4HBCOOG formation, the assay mixture contained 4/imol Tris-HCl, pH 8.0 or 4/miol KPi, pH 5.5, respectively. For examination of the pH dependence, however, these buffers were replaced by sodium citrate/potassium phosphate (2 ^mol each) or by glycine-NaOH (4 /mid). For radioactive assays, unlabeled 4HB was replaced by [U-14C]4HB. A total radioactivity of 0.185 kBq was used for assays with a 4HB concentration of 8A