Planta (1989) 177:1 8

Planta

9 Springer-Verlag 1989

The induction of phenylpropanoid biosynthetic enzymes by ultraviolet light or fungal elicitor in cultured parsley cells is overriden by a heat-shock treatment Michael H. Walter* [nstitut ftir Biologic II der Universit/it, Sch/inztestrasse 1, D-7800 Freiburg i.Br., Federal Republic of Germany

Abstract. The normal (25 ~ C) response of parsley (Petroselinum crispum Mill.) cell suspension cultures to ultraviolet (UV) light was suppressed by a simultaneous 37~ C heat-shock treatment, as indicated by the loss of the inducibility of two enzymes of flavonoid biosynthesis, phenylalanine ammonia-lyase (EC4.3.1.5) and chalcone synthase. The effects on enzyme activity and on enzyme synthesis in vitro and in vivo were similar, indicating that regulatory control is at an early step of gene expression, presumably transcription. When heat shock was given during the course of an ongoing UV induction, both enzyme synthesis and enzyme activities ceased rapidly. Likewise, the induction of phenylalanine ammonia-lyase by an elicitor from Phytophthora megasperma f. sp. glycinea was terminated upon transfer from 25 ~ C to 37~ C. Based on these and previously published data, it is concluded that stress responses in this system are preferentially expressed in the order of heat shock, fungal elicitor and UV light. Key words: Chalcone synthase - Heat shock protein Petroselinum - Phenylalanine ammonia-lyase Temperature regulation

Introduction

Irradiation of dark-grown cell suspension cultures of parsley with UV light under standard temperature conditions (25 ~ C) leads to the accumulation of flavonoid glycosides, putative UV-protective Present address: Institut ffir Pflanzenphysiologie -260-, Universit/it Hohenheim, Emil Wolff-Strasse 25, D-7000 Stuttgart 70, Federal Republic of Germany Abbreviations: CHS=chalcone synthase; HSP=heat-shock protein; PAL = phenylalanine ammonia-lyase; UV ~ ultraviolet

compounds (Hahlbrock and Grisebach 1979; Hahlbrock 1981 ; Kreuzaler et al. 1983 ; Kuhn et al. 1984; Chappell and Hahlbrock 1984). Similarly, treatment with elicitor, e.g. cell-wall fragments of the fungus Phytophthora megasperrna f. sp. glycinca, causes production of furanocoumarins, the phytoalexins of parsley (Tietjen et al. 1983; Hahlbrock and Scheel 1987). Biosynthesis of these two groups of plant defense compounds proceeds via the pathway of general phenylpropanoid metabolism, starting off with the key enzyme phenylalanine ammonia-lyase (PAL). Branch pathways then consist of approximately 13 flavonoid biosynthetic enzymes including chalcone synthase (CHS) and a different, partly hypothetical group of enzymes leading to the formation of furanocoumarins (Hahlbrock et al. 1985; Hauffe et al. 1986). While both treatments stimulate the general pathway, a maximal rate of enzyme synthesis is typically reached earlier with elicitor (3-4 h after onset of treatment) than with UV irradiation (6-7 h) under the conditions used in these studies. Peaks in extractable enzyme activities occur several hours later (8 10 h and 12-15 h, respectively; Hahlbrock et al. 1981). Ultraviolet light and fungal pathogens are but two of many challenges for plants in a natural habitat. High temperature is another particularly important environmental stress. The discovery of heat-shock proteins (HSPs) has shed new light on the mechanism of temperature adaptation (see Schlesinger et al. 1982; Nover 1984; Kimpel and Key 1985; Lindquist 1986 for reviews). In cells of virtually any kind there is a rapid induction of new groups of proteins (HSPs) upon a mild heat shock (e.g. 10-15~ C above ambient growth temperature); this is often accompanied by ;suppression of the synthesis of other previously synthesized proteins. The precise function of most HSPs

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is still unknown, but their contribution to acquired heat tolerance has been demonstrated in both animal (Li and Werb 1982) and plant cells (Lin et al. 1984). The molecular mechanisms underlying the appearance of HSPs have been extensively studied in Drosophila melanogaster (Ashburner and Bonner 1979; Nover 1984). Regulatory control is exerted both at transcriptional and translational levels. Concomitant with the activation of heat-shock gene transcription and repression of other genes (Findly and Pederson 198/), m R N A s coding for HSPs are preferentially translated, while normal m R N A s are temporarily excluded from translation (Storti et al. 1980; Kr/iger and Benecke 1981; Klemenz et al. 1985; Hultmark et al. 1986). Resistance to a particular biotic or abiotic stress like the ones mentioned above is usually not an invariable trait. Changes in environmental or physiological conditions can alter the expression of a resistance response (e.g. Ward and Lazarovits 1982; Hadwiger and Wagoner /983; Wolffe et al. 1984; Classen and Ward 1985). Hahlbrock et al. (1985) have reported preliminary results on the dependence of enzyme induction on the culture conditions of parsley cells. In this paper ! describe initially some characteristic features of the heatshock response of parsley cell suspension cultures. I then present evidence that the response of cultured parsley cells to UV light and fungal elicitor, characterized by induction of PAL and CHS or PAL, respectively, can be overriden by heat shock, given before, or at various times during, UV light or elicitor treatment.

M.H Walter: Defense responses overriden by heat shock

Labelling in vivo. Protein was labelled by adding 1.11 MBq of L-[35S]methion]ne (26,39 27,77 G B q . m m o l - i , Amersham Buchler, Braunschweig, FRG) to the suspension-culture aliquots, 30 or 60 min prior to harvest as indicated. Isolation and translation of RNA. Polyribosomal R N A was isolated and translated in an mRNA-dependent retieulocyte lysate as described by Walter and Hahlbrock (1985).

Enzyme assays. The activity of PAL was determined from crude cell extracts. These were prepared by stirring 1 g of frozen cells in 2 ml of 0.2 M 2-amino-2-(hydroxymethyl)-l,3-propanediol (Tris)-HC1, pH 7.8, containing 0.1 g Dowex 1 x 2 and 14 m M mercaptoethanol, followed by centrifugation for 10 rain at 20000 g. The supernatant was used to measure PAL activity by the standard spectrophotometrie assay (Schr6der et al. 1976). Protein was determined according to Bensadoun and Weinstein (1976). The activity of CHS was assayed according to Schr6der et al. (1979) from crude extracts prepared with 0.5 M potassium phosphate, pH 8.0. All data are expressed in lakat- k g - 1 protein. Irnmunoprecipitations. Using similar procedures, PAL and CHS were immunoprecipitated from proteins labelled in vivo or those synthesized in vitro. Supernatant (200 gl) from crude cell extracts of labelled cells in Tris-HC1 buffer (see above) were incubated with 10 ~tl of PAL antiserum and 5 gl CHS antiserum at room temperature for 1 h and then overnight at 4 ~ C. Subsequent steps of the purification of the immunorecipitate, including centrifugation through a sucrose cushion, have been described (Schr6der et al. 1976). Gel electrophoresis as the final step was performed on slab gels (see below). For translation in vitro, 20 gl of the translation mixture (Walter and Hahlbrock 1985) was diluted 1 : 10 with 10 mM Tris-HC1, pH 7.5, 100 mM NaC1, 1 m M ethylenediaminetetraacetic acid (EDTA). Carrier protein (1.25 gg), consisting of a crude mixture of unlabelled parsley proteins, was then added. Smaller volumes of PAL (5 lal) and CHS (4 gl) antisera were used for immunoprecipitation and this mixture was subjected to the treatments described above.

Gel eleetrophoresis of proteins: One-dimensional slab-gel etec-

Material and methods Cell cultures. Suspension cultures of parsley (PetroseIinum crispum Mill.) were grown in the dark at the standard temperature of 2 5 ~ in slightly modified B 5 medium (Walter and Hahlbrock 1985).

Treatments. For each experiment, aliquots (about 30 ml) of a single 400-ml culture from the linear growth stage (conductivity between 1.8 and 2.5 mS) were aseptically transferred to sterile flasks and exposed to the various treatment programs with continuous shaking. Cells were irradiated under white light from fluorescent tubes (40 W-18; Philips, Eindhoven, The Netherlands) emitting high portions of UV-B at a distance of about 30 cm. Elicitor, heat-released from the cell wall of the fungus Phytophthora megasperma f. sp. glycinea by the method of Ayers et al. (1976), was a gift from J. Ebel (Institute of Biology II, University of Freiburg). Elicitor was added at a concentration of 40 lag- ml- 1 culture medium and incubation was in the dark. Rapid adjustments to the various temperatures were achieved by keeping flasks on rotary shakers in waterbaths set at the particular temperatures. Harvest and storage of cells have been described (Ragg et al. 1981).

trophoresis was carried out on gradient gels (9-15% acrylamide), containing 0.1% sodium dodecylsulfate. Labelled proteins from crude cell extracts (10-30 gg) or in vitro translations (12 p.1) were dissolved in sample buffer (Ragg et al. 1981), heated to 95 ~ C for 5 min and subjected to electrophoresis. Pellets from immunoprecipitations were treated the same way. Analysis of proteins on two-dimensional gels followed the method of O'Farrell (1975) with a few modifications described by Walter and Hahlbrock (1985). Gels were treated for fluorography (Bonner and Laskey 1974), dried onto Whatman (Maidstone, Kent, UK) 3 M M paper and exposed at - 70 ~ C. A commercial mixture of standard proteins (Amersham Buchler) was used for estimations of molecular weights. Protein staining was with Coomassie Brillant Blue R-250.

Results

Brief analysis of protein synthesis during heat shock. Since the response of parsley cell suspension cultures to heat-shock as a single stress has not previously been investigated, a 4-h, 37 ~ C heat-shock treatment was chosen for a brief characterization

M.H Walter : Defense responses overriden by heat shock

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Fig. 1A-D. Labelling patterns of parsley proteins synthesized in vitro in a reticulocyte lysate (A, B) or in vivo (C, D) in cells grown at 25~ C (A, C) or exposed to a 4-h 37~ C heat shock (B, D). Labelling in vivo was during the last hour of incubation. Proteins were separated by isoelectric focusing in the first dimension (acidic end to the left) and by sodium dodecylsulfatepolyacrylamide gel electrophoresis in the second dimension. Numbers to the right refer to molecular weights of marker proteins. To facilitate orientation, two spots (approx. Mr 70000, pI 4.8; M r 7 8 0 0 0 , pI 4.8) which are present under all conditions, are marked by arrows

o f the h e a t - s h o c k proteins o f parsley. Suspension cultures f r o m the linear g r o w t h period, a stage routinely used to measure stress responses in the parsley system, r e s p o n d e d to the elevated t e m p e r a t u r e by a drastic change in the p a t t e r n o f protein synthesis. Two-dimensional gel analysis o f proteins synthesized either in vitro in a reticulocyte lysate or in vivo (Fig. 1) revealed several m a j o r g r o u p s o f newly f o r m e d H S P s with relative m o l e c u l a r masses (Mr) o f a b o u t 15 000-18 000, 70 000, 78 000, 92000 and 95000. The M r - 7 0 0 0 0 g r o u p comprises at least four members, whereas the low-Mr H S P s are m u c h m o r e numerous. It is especially n o t e w o r t h y that there is a differential induction within the M r - 70 000 and possibly also within the M r - - 7 8 0 0 0 protein family, m o s t a p p a r e n t for the 70 000-Da class f r o m a c o m p a r i s o n o f Fig. 1 C and D. The proteins with the lowest isoelectric p o i n t (pI) in each g r o u p seem to be constitutively and

a b u n d a n t l y expressed in control as well as in heattreated cells. With these exceptions, the synthesis o f m o s t other n o n - h e a t - s h o c k proteins is terminated during heat shock. Effects o f temperature on the response to U V irradiation. In a first set o f experiments investigating

heat stress c o m b i n e d with U V light or elicitor treatment, parsley cells were p r e i n c u b a t e d at different t e m p e r a t u r e s ranging f r o m 15 ~ C to 37 ~ C for 1 h and then, while maintaining these temperatures, irradiated with U V light for a n o t h e r 6 h. As a control, an identical t r e a t m e n t was p e r f o r m e d w i t h o u t U V light. The total a r r a y o f proteins synthesized in vitro or in vivo is shown in Fig. 2. Positions o f P A L and C H S deduced f r o m a p p a r e n t molecular weights o f i m m u n o p r e c i p i t a t e d enzymes are indicated. B o t h in vitro and in vivo, the synthesis o f

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M.H Walter: Defense responses overriden by heat shock

Fig. 3A, B. Effect of temperature on inducibility of PAL and CHS. PAL and CHS were immunoprecipitated from in-vitrotranslation assays (A) or from total protein extracts of parsley cells labelled in vivo (B) and subjected to electrophoresis. Samples were identical to those used for separation of total protein (Fig. 2; lanes L ). An overexposed film is shown to also reveal faint radioactive signals of immunoprecipitated PAL and CHS; additional protein bands therefore appear which are the result of incomplete washing of the immunoprecipitate

Fig. 2A, B. Effect of temperature on the UV-irradiation response of parsley proteins. Patterns of labelled proteins are compared, representing m R N A translations in vitro (A) or labelling in vivo of corresponding samples during the last hour before harvest (B). The cultures were incubated at the indicated temperatures for 1 h in the dark followed by 6 h in the dark (lanes D) or in UV light (lanes L). Molecular weights of marker proteins are indicated (right margin) as are positions of PAL and CHS, the two most abundant UV-light-induced proteins (left margin). Approximately equal amounts of radioactivity were loaded on each lane

a protein presumably identical to PAL was induced in the UV-irradiated samples at all temperatures except at 37 ~ C, where U V light had no appreciable effect on the pattern of protein synthesis. Because of the poor separation of protein bands in the 45000-Da region, synthesis of CHS could not be analyzed (but see Fig. 3). Synthesis of HSPs was strong and selective at 37 ~ C with apparently much weaker induction at 33 ~ C. In contrast, the cold treatment (15 ~ C) alone did not cause appreciable changes in the synthesis pattern. The induction of P A L and CHS was followed more closely by performing immunoprecipitations (Fig. 3). High levels of induction by UV light were detected at 25 ~ C and 33 ~ C, but at 37 ~ C UV irradiation failed to induce the synthesis of either enzyme (Fig. 3). At 15 ~ C, induction was reduced, but CHS seemed to be more affected than PAL. Measurements at the level of m R N A activity by protein synthesis in vitro (Fig. 3 A) or enzyme synthesis in vivo (Fig. 3 B) gave similar results. Moreover, in agreement with these results, extractable enzyme activities were equally high at 2 5 ~ and 33~ (PAL 3 0 g k a t . k g -1, CHS 3 g k a t . k g - 1 ) , lower at 15~ (PAL 12 ~tkat.kg -1, CHS below

M.H Walter: Defense responses overriden by heat shock

5

detectable level), and barely detectable at 37 ~ C. In controls without UV light, higher temperatures did not affect base levels of the two enzymes (data not shown). However, at 15~ a slight induction of PAL was observed. This cold-related induction of PAL was also found at the level of enzyme activity (8 gkat- kg- 1 up from 5 gkat- k g - 1 base level at 25 ~ C and 33 ~ C). An interesting observation from the translation assay in vitro was that translational activity of total R N A was highest with R N A from cells incubated at 15 ~ C and decreased gradually with increasing temperature of cells to about 40% of maximum regardless of UV treatment.

Rapid interruption by heat shock of ongoing enzyme induction during UV light or elicitor :treatment. Parsley cells were initially given UV irradiation or elicitor treatment under normal temperature conditions (25 ~ C). At various times during the induction period of PAL and CHS, flasks were quickly transferred to 37 ~ C and maintained there for the remainder of the experiment. Ultraviolet irradiation was again given for 6 h (Fig. 4). In a similar experiment with elicitor a shorter time:of experimentation was chosen (3 h, see Fig. 5) because maximum PAL synthesis is usually observed earlier with elicitor than with UV light (Halhlbrock et al. 1981). The rapid shift to selective synthes~s of HSPs after transfer to 37~ is obvious from Figs. 4A and 5 A. While this is complete within 30 min (labelling with [35S]methionine was done during the last 30 rain of total incubation time) or earlier in the case of UV irradiation (Fig. 4A), several elicitor-specific proteins apparently continue to be synthesized at least during the initial phase of heat treatment (Fig. 5A, proteins marked by arrows). The analysis of imrnunoprecipitates (Figs. 4B, 5B) demonstrated that cells kept at a consitant 25~ showed a pronounced synthesis of PAE and CHS during UV irradiation or PAL synthesis during elicitor treatment, respectively. Incubation at 37 ~ C only during the last hour already led to a sharp decrease or even loss of enzyme synthesis. Transfer to heat-shock conditions earlier resulted in the complete absence of detectable enzyme synthesis.

Fig. 4A, B. Transfer of parsley cells to heat-shock conditions (37 ~ C) during the course of an UV irradiation initially started at 25 ~ C. All samples received 6 h of UV light and were labelled with [35-SJmethionine during the last 30 min of incubation. Numbers between the two panels indicate times at the particular temperature. Fluorographs of electrophoretic seParations of total protein (A) or immunoprecipitations of PAL and CHS (B) are shown. Extractable PAL activity (gkat.kg 1) is given in the inset of B

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M.H Walter: Defense responses overriden by heat shock

However, PAL enzyme activity was extractable from heat-shocked samples, although the amount decreased with increasing length of heat treatment. It appears that the development of new enzyme activity ceases rapidly upon a 37~ heat shock but active enzyme already accumulated at the time of transfer is not degraded to an appreciable extent. Discussion

The heat-shock response of parsley (P. crispum) cell cultures is in good agreement with the overall picture that has emerged from numerous studies in all kinds of species. That is, there is a very conserved general reaction of cells to heat stress with subtleties for individual systems (Nover 1984; Kimpel and Key 1985; Lindquist 1986). Parsley HSPs have similar molecular weights to those reported from other systems. The most ubiquitously found HSP, HSP 70, exhibits in the parsley system many features known from other organisms including multiplicity of proteins and occurrence of constitutive isoforms, the latter being reminiscent of heat-shock cognate proteins in Drosophila (Ingolia and Craig 1982; Patter et aI. i986) and mouse (Lowe and Moran 1984). Small HSPs, apparently not as conserved, are found in parsley (Fig. 1) and in other plants at slightly lower molecular weights and with a higher complexity than in animal systems (Nover and Scharf 1984; Kimpel and Key 1985). The drastic decline in the synthesis of most non-heat-shock proteins, observed here (Fig. l) is another widespread, but not universal heat-shock characteristic (Nover 1984). Plant proteins with known identity have been found to be only partly reduced (ribulose-l,5-bisphosphate carboxylase; Vierling and Key 1985) or even stimulated (embryonic storage proteins; Altschuler and Mascarenhas 1982) in their synthesis by heat shock. Stresses frequently occur in combination in natural environments. Temperature effects on other stress resistances of plants are widely known, but in most cases studied only at the level of disease symptoms and survival rates (reviews by Colhoun 1973; Ayres 1984). It has been shown in the present study that the response of parsley cell cultures to UV light is also dependent on temperature. High temperatures (37 ~ C) as well as low temperatures (15 ~ C) decreased the inducibility by UV light of Fig. 5A, B. Transfer of parsley cells to heat-shock conditions (37 ~ C) during the course of an elicitor treatment initially started at 25 ~ C. All samples received 3 h of elicitor treatment. See Fig. 4 for other conditions. The positions of PAL and of a few elicitor-specific proteins are marked in A. PAL was immunoprecipitated (B) and assayed for extractable enzyme activity (gkat. k g - 1), given in inset of B

M.H Walter: Defense responses overriden by heat shock

two characteristic enzymes of flavonoid biosynthesis, PAL and CHS. No appreciable difference was observed between the normal growth temperature (25 ~ C) and 33 ~ C. The complete loss of inducibility during a 37 ~ C heat shock was investigated further to include elicitor treatment. Elicitor provides a stimulus which appears to be a stronger stress than UV light since it has been shown to override the response to UV light, namely to suppress CHS induction, when both treatments are given under saturating conditions. No additive induction effects on PAL were found, but the two treatments rather seemed to impair each other (Hahlbrock et al. 1981). In the present combination experiments of either one of these two stimuli with heat shock, PAL induction ceased rapidly when cells were transferred to 37 ~ C during the primary treatment. This interruption of an ongoing enzyme induction and the concomitant rapid synthesis of HSPs indicate that a 37 ~ C heat shock is the "strongest" of the three stresses. While in both cases the switch to selective synthesis of HSPs is extensive (Figs. 4A, 5A), a few elicitorspecific proteins (but not PAL) are apparently exempt from the immediate shut-off of non-HSP synthesis, at least in the initial phase of the heat treatment (Fig. 5 A). I have previously used other stress combinations (Walter and Hahlbrock 1985) and conclude now that there is a preferential expression of stress responses or stress hierarchy in cultured parsley cells having the order: heat shock, nutrient depletion, fungal elicitor and UV irradiation. Clearly, this order has been established for my particular experimental conditions, notably saturating conditions for each treatment. Changing severity (e.g. elicitor concentration or purity) or duration of a particular treatment may alter this order, but in all cases studied heat shock has been found to override other stress responses. The availability of antibodies to PAL and CHS allowed different steps of enzyme formation to be monitored. When cells were preincubated at 37 ~ C and then irradiated at this temperature, neither enzyme activity, enzyme synthesis in vivo nor translatable m R N A were detectable upon UV irradiation. It therefore seems likely that the block in the development of enzyme activity occurs at an early step of gene expression, presumably transcription, possibly in a manner similar to that in Drosophila (Findly and Pederson 1981). The finding that the amount of CHS m R N A undergoes changes similar to those of its translational activity supports this view (Walter/984). Apart from transcriptional regulation (reviewed by Pelham /985) additional translational control has been reported

7

for the Drosophila system (Krfiger and Benecke 1981; Klemenz et al. 1985) and also for plants (Nover and Scharf 1984). This involves ,changes in the translation apparatus to discriminate against 25 ~ C mRNAs, leading to selective translation of HSP mRNAs. Differences occurring between protein synthesis in vivo and in vitro are indicative of this latter type of control (Kriiger and Benecke t981). Unfortunately, an attempt to study translational discrimination against PAL m R N A during heat shock in the parsley cells did not allow a clear answer, since the immunoprecipitate of. PAL from proteins translated in vitro after a brief heat shock was contamined with an abundant protein of about 70000 Da, possibly HSP(s) of this size (unpublished). My studies with parsley cells clearly show the dependency of a given response on environmental conditions, and it can be assumed that similar mechanisms are operative in whole plant-live pathogen interactions. In summarizing my findings, I conclude that the plant response to several competing stresses is not organized on a first come, first served basis as has been suggested by Hadwiger and Wagoner (1983). It rather appears that a plant, while limited in its adaptation potential, is able to preferentially respond to a new and stronger stress at the expense of defense against the', weaker stress. The author gratefully acknowledges the provision of laboratory facilities, biological materials and extensive advice from Professor K. Hahlbrock. I am indebted to Profs K. Hahlbrock, A.M. Boudet and Dr. L. Nover for critical reading of the manuscript. I thank J.-C. Molinier for photographic work and D. Lefebvre for typing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.

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M.H Walter: Defense responses overriden by heat shock

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