of 4-hydroxybenzoic acid which is covalently bound to wall polysaccharides of the carrot cells. The possible function of phenylalanine ammonia-lyase in ...
Planta (1991) 184:362 367
Pl
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9 Springer-Verlag1991
Metabolic changes in carrot cells in response to simultaneous treatment with ultraviolet light and a fungal elicitor Johannes Gleitz, J6rg-Peter Schnitzler, Dietrich Steimle, and Hanns Ulrich Seitz* Botanisches Institut der Universitfit,AllgemeineBotanik und Pflanzenphysiologie,Auf der Morgenstelle 1, W 7400 Tiibingen, Federal Republic of Germany Received 6 October 1990; accepted 21 February 1991
Abstract. Ultraviolet light induces anthocyanin biosynthesis in cell cultures of an Afghan cultivar of Daucus carota (Daucus carota L. ssp. sativus). Simultaneous treatment with a fungal elicitor from Pythium aphanidermatum results in an inhibition of the catalytic activity of chalcone synthase (CHS), which in turn correlates with an inhibition of anthocyanin biosynthesis. On immunoblots, one isoenzyme (40 kDa) of CHS disappears upon elicitor treatment. On an mRNA level, only the m R N A for the 40-kDa-CHS is active after treatment with ultraviolet light. After inhibition of anthocyanin biosynthesis by the elicitor the enzyme protein disappears and the CHS mRNA is strongly diminished. This inhibition depends on the concentration of the elicitor. In addition, elicitor treatment leads to an induction of the general phenylpropanoid pathway as well as to the accumulation of 4-hydroxybenzoic acid which is covalently bound to wall polysaccharides of the carrot cells. The possible function of phenylalanine ammonia-lyase in providing precursors for 4-hydroxybenzoic acid is discussed. Key words: Anthocyanin - Cell culture - Chalcone synthase - Daucus - Elicitor ( Pythium ) - 4-Hydroxybenzoic acid - Phenylalanine ammonia-lyase
Introduction Plant cells respond actively to different exogeneously applied stimuli by synthesizing secondary metabolites. In cell cultures of parsley the accumulation of flavonoids is mediated by ultraviolet (UV) light. Their rate of synthesis depends on the de novo synthesis of enzymes which are part of the biosynthetic pathway and is regulated by temporal gene activation leading to transient maxima in * To whom correspondence should be addressed Abbreviations: CHI=chalcone isomerase, CHS=chalcone synthase, PAL = phenylalanineammonia-lyase,SDS-PAGE= sodium dodecyl sulfate-polyacrylamidegel electrophoresis
their catalytic activity (for a review see Hahlbrock and Scheel 1989). These flavonoids are thought to be filter substances that protect tissues from damage by excessive light irradiation (M6hle et al. 1985). On the other hand, microbial attack leads to the synthesis of phytoalexins belonging to different classes of compounds (for a review see Ebel 1986). In parsley, furanocoumarins are synthesized upon fungal infection (Jahnen and Hahlbrock 1988). The responses of parsley are also demonstrated in model systems such as cell cultures and protoplasts, and in intact plants as well (Dangl et al. 1987). In legumes, isoflavonoid-derived phytoalexins are synthesized in response to infection. The differential activation of a gene family encoding for chalcone synthase as a key enzyme in flavonoid biosynthesis has been described in Phaseolus vulgaris (Ryder et al. 1987). In soybean, glyceollin biosynthesis is also regulated by differential transcription of one of the chalconesynthase (CHS) genes (Wingender et al. 1989). Cell cultures of an anthocyanin-containing carrot (Daucus carota L. ssp. sativus) are also well suited for observing rapid responses to UV-light (Seitz and Gleitz 1988) and to a fungal elicitor from the oomycete Pythium aphanidermatum (Schnitzler and Seitz 1989). After UV irradiation the synthesis of anthocyanins is induced. Chalcone synthase is thought to be a key element in anthocyanin biosynthesis. In carrot this homodimeric enzyme is present in two isoforms exhibiting different subunit molecular weights (40 kDa and 43 kDa). The synthesis of anthocyanins is linked to a transient induction of phenylalanine ammonia-lyase (PAL) and the 40-kDa CHS (Seitz and Gleitz 1988). After treatment with an elicitor from Pythium aphanidermatum, PAL is rapidly synthesized de novo whereas the catalytic activity of CHS is inhibited, as is anthocyanin biosynthesis. On Western blots the 40-kDa CHS disappears but the second isoform with a molecular weight of 43 kDa per subunit is still present. In addition to the activation of PAL, elicitor treatment leads to metabolic changes resulting in an accumulation of 4-hydroxybenzoic acid in a wall-bound form (Schnitzler and Seitz 1989).
J. Gleitz et al. : Metabolic changes in carrot cells
OH
UNTREATED CELLS OR UV-LIGHT H0
363 Chalcone-synthase activity was determined according to Gleitz and Seitz (1989).
Sodium dodecyl sulfate-polyacrylamide 9el electrophoresis (SDS-PAGE), Western blotting, and immunostainino. All details
0
are as described earlier by Gleitz and Seitz (1989). The antisera to PAL and CHS were gifts from K. Hahlbrock.
Isolation of total RNA. Total R N A was isolated from cell cultures ANTHOCYANINS ~f !
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TREATMENT H-Hydroxybenzoic acid
of carrot. The cells were frozen in liquid nitrogen and ground with pestle and mortar. The powder was allowed to thaw in one volume of freshly prepared phenol (Maniatis 1982) and stirred for 10 min at room temperature. After the addition of equal volumes of extraction buffer (50 mM Tris-HC1 pH 7.6, 5 m M MgC12) and chloroform/isoamylalcohol (24: 1,v/v) the mixture was stirred for another 10 min. After a centrifugation at 2 0 0 0 0 ' 0 the aqueous phase was extracted twice with an equal volume of chloroform/ isoamylalcohol. Nucleic acids were precipitated overnight at - 20 ~ C in two volumes of ethanol containing 0.1 volume of 3 M sodium acetate at pH 5.5. The pellet of a 20000 - 9 centrifugation was dissolved in 0.5 ml DNase buffer (40 m M Tris-HC1 pH 7.5, 6 m M MgCI2) and incubated with 20 units of DNase I (Pharmacia LKB, Freiburg i.Br., F R G ) for 10 min at 37 ~ C. Subsequent to a chloroform/isoamylalcohol extraction and ethanol precipitation (see above) the resulting pellet was washed twice with 70% ethanol and then dissolved in sterile water. The purity of R N A was checked by non-denaturing agarose gel electrophoresis (Slater 1986). The R N A concentration was determined photometrically at 260 nm. The A260/A28o ratio varied from 1.79 to 1.9.
In-vitro translation. An mRNA-dependent rabbit reticulocyte lysate Fig. 1. Scheme illustrating the role of 4-coumaroyl-CoA in metabolic changes in carrot cells after UV irradiation and treatment with an elicitor from the oomycete Pythium aphanidermatum
In the present report we compare the responses of c u l t u r e d a n t h o c y a n i n - c o n t a i n i n g c a r r o t cells t o U V l i g h t a n d a f u n g a l elicitor. T h e m e t a b o l i c a l t e r n a t i v e is dep i c t e d in F i g . 1. T h e h i e r a r c h y in t h e r e g u l a t i o n is d i s c u s s ed.
Materials
and methods
Cell cultures. The experiments were carried out with an anthocyanin-containing cell line of Daucus carota L. ssp. sativus. Callus cultures (Seitz and Richter 1970) and cell cultures (No6 et al. 1980) were propagated as previously described. Irradiation conditions. Dark-grown cell cultures were irradiated with UV light (315-420 nm) 10d after the onset of cultivation, as described earlier (Gleitz and Seitz 1989).
Elicitor preparation. The elicitor from Pythium aphanidermatum was prepared as described previously (Schnitzler and Seitz 1989).
Protein preparation. Four grams of cells (fresh weight) and 1 g Dowex 1 x 2, equilibrated in 100 mM potassium phosphate buffer (KPi) pH 8, were resuspended in 2 ml of 200 mM KPi, 50 m M sodium hydrosulfite, pH 7. The cells were homogenized by sonification (30 s, 70 W) with a micro-tip sonifier (Branson, Danbury, Conn., USA). The supernatant of a 27000-0 centrifugation (10 min) was frozen in liquid nitrogen and stored at - 20 ~ C prior to enzyme-activity measurements.
(Boehringer, Mannheim, FRG) was used according to the suppliers instructions. The lysate was diluted in the reaction mixture to final concentrations of 0.25 mg 9m l - x of total RNA, 1.5 m M magnesium acetate, 50 m M potassium acetate and 18.5 kBq 9 pl -~ of L-[35S] methionine. The incorporation of methionine into total protein was measured as described by Lawton et al. (1983). For immunoprecipitation of the translation products, 20 HI of the sample were diluted (1 : 10) by addition of the precipitation buffer (0.1 M NaC1; 1 m M EDTA; 1% ethylphenylpolyethyleneglycol (Nonidet P-40, Fluka, Neu-Ulm, F R G ) ; 10 m M Tris-HC1 pH 7.5). The anti-CHS serum (1.5 gl) was added and then the mixture was incubated at room temperature for 3h. Then 25 pl of a suspension (100 mg - ml-~) of protein A-Sepharose were added and incubated at room temperature for 1 h. The resulting immunocomplex was collected by centrifugation and washed with precipitation buffer. The procedure was repeated twice. During the last washing step, Nonidet P-40 was omitted. The resulting pellet was heated to 100 ~ C in the presence of 10 gl sample buffer (80 mM Tris-HC1 pH 6.8; 5% mercaptoethanol (v/v); 20% glycerol (w/v); 0.01% bromphenol blue; 2% sodium dodecyl sulphate (w/v). The proteins were separated on 10% (w/v) polyacrylamide gels with a 5% stacking gel according to Laemmmli (1970). The gels were dried on a gel dryer and autoradiographed with Kodak X A R - S at room temperature. The relative m R N A activity was determined by scanning and integration of the autoradiograms with a scanner (Elscript 400; Hirschmann, Unterhaching, FRG).
Determination of 4-hydroxybenzoic acid. The 4-hydroxybenzoic acid content was determined by slightly modifying a procedure already described by Schnitzler and Seitz (1989). The butanolic extract from cell walls was dried in a stream of filtered air and the resulting residue was redissolved in methanol/H20 (1 : 1) and then used for high-performance liquid chromatography analysis by (HPLC). The chromatographic conditions were the same as already described, the sole exception being that a steeper gradient was applied ranging from 5 to 30% methanol.
Protein determination. The protein concentration was determined Enzyme assays. Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity was determined according to Koukol and Conn (1961).
by Bradford's method using bovine serum albumin (BSA) as a standard (Bradford 1976).
364
J. Gleitz et al. : Metabolic changes in carrot cells
Anthocyanin content. The anthocyanin concentration was determined photometrically in methanolic extracts prepared as previously described (Gleitz and Seitz 1989).
Results
Inhibition of UV-induced anthocyanin accumulation by a fungal elicitor. Cells of the carrot suspension culture responded to UV light by accumulating anthocyanins (Fig. 2c). Parallel to this increase CHS and chalcone isomerase (CHI), which are part of the biosynthetic pathway, were induced to a similar extent (Fig. 2a, b). Maximum activity of CHS was observed about 40 h after the onset of irradiation whereas CHI reached its maximum activity after 60 h. Simultaneous treatment of the cells with UV light and the elicitor led to a partial decrease in the extractable activities of both enzymes (Fig. 2a, b). Parallel to the inhibition of the enzymes by the elicitor the accumulation of anthocyanin decreased (Fig. 2c). The addition of the elicitor 24 h prior to UV irradiation led to nearly total inhibition of the activities of CHS and CH1 as well as of anthocyanin biosynthesis. These results indicate that the elicitor is a much stronger stimulus than UV light.
Concentration-dependent inhibition of anthocyanin biosynthesis by the elicitor. D a r k - g r o w n cells c o n t a i n e d a basal level o f C H S activity, sufficient to a c c u m u l a t e slight a m o u n t s o f a n t h o c y a n i n . U p o n light t r e a t m e n t , C H S a n d C H I activities as well as a n t h o c y a n i n a c c u m u l a t i o n were d r a m a t i c a l l y e n h a n c e d . In co n t r ast , elicitation led to a c o n c e n t r a t i o n - d e p e n d e n t i n h i b i t i o n o f these e n z y m e activities a n d o f a n t h o c y a n i n a c c u m u l a t i o n . Th e s e d a t a are s u m m a r i z e d in Fig. 3.
Effects of UV light and the elicitor on PAL and CHS proteins. Figure 4 shows a double immunoblot with antisera to PAL and CHS. Dark-grown cells had a low level of PAL activity which was hardly detectable under these conditions. The amount of immunodetectable PAL protein with an apparent molecular weight of 79 kDa was enhanced upon elicitation and UV irradiation. This observation is in good agreement with the time course of the catalytic activity of PAL (not shown). There was a second immunodetectable protein on the nitrocellulose sheet, exhibiting an apparent molecular weight of 77 kDa. It is not yet known whether it is an is 9 or a degradation product bearing the epitope (Jones 1986). As already shown in a previous report (Gleitz and
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