Plant Cell Reports
Plant Cell Reports (1989) 8:399-402
© Springer-Verlag 1989
A microtest system for the serial assay of phytotoxic compounds using photoautotrophic cell suspension cultures of Chenopodium rubrum Jutta Thiemann, Arnold Nieswandt, and Wolfgang Barz Lehrstuhl ffir Biochemie der Pflanzen, Westf/ilische Wilhelms-Universit/it, D-4400 Mtinster, Federal Republic of Germany Received August 11, 1989 - Communicated by E. W. Weiler
ABSTRACT Chenopodium rubrum photoautotrophic c e l l suspensions were grown In ~ c tissue culture dishes under photoautotrophic conditions. Growth was monitored by measuring cell number, packed c e l l volume, chlorophyll content and oxygen production. Such m i c r o t i t e r dishes are s u i t a b l e systems f o r the s e r i a l assay of growth i n h i b i t i o n and various physiological effects ( i . e . chlorophyll fluorescence, cell v i a b i l i t y , oxygen production) of photoautotrophic c e l l s as caused by herbicides and fungal phytotoxins. The a p p l i c a b i l i t y of the t e s t system is discussed. Abbreviations pcv = packed cell volume; f r . w . = fresh weight; rpm = r e v o l , per minute; DMSO = dimethyl s u l f o x i d e ; PMS = phenazine methosulfate; NBT = n i t r o - b l u e t e t r a zolium c h l o r i d e ; INTRODUCTION Heterotrophic plant c e l l cultures are s u i t a b l e systems f o r t e s t i n g the b i o l o g i c a l a c t i v i t y and the cell u l a r metabolism of n u t r i e n t s , t o x i n s , or environmental and agrochemical compounds (Gressel 1984; Grossmann et a l . 1982; Mumma and Davidonis 1983; Sandermann et a l . 1984). Several assay systems have been devised allowing the s e r i a l monitoring of numerous compounds or d i f f e r e n t concentrations in small c e l l c u l t u r e volumes in a short period of time (Potrykus et ai.1979; Grossmann 1988). Photoautotrophic plant c e l l suspension cultures which g r e a t l y resemble mesophyll c e l l s (HUsemann 1985) can well be used f o r the assay of photosynthesis-inhib i t i n g herbicides (Sato et a l . 1987). Such c e l l s are r o u t i n e l y grown in Erlenmeyer or d o u b l e - t i e r flasks (HUsemann and Barz !977) so that s e r i a l assays require considerable e f f o r t in time, labou~and shaker space. To f u r t h e r promote the a p p l i c a b i l i t y of these green c e l l s f o r the s e r i a l determination of phytotoxic compounds we have now established a microtest system using Chenopodium rubrum photoautotrophic c e l l suspensions kept in transparent m i c r o t i t e r dishes. Our i n v e s t i g a t i o n s were designed I ) to demonstrate the e f f i c i e n t growth of the photoautotrophic c e l l s in small volumes as monitored by cell number, pcv, chlorophyll content, and oxygen production, and 2) to measure the i n h i b i t o r y effects of various herbicides and a fungal phytotoxin on c e l l u l a r growth and several physiological parameters of the green c e l l s . Offprint requests to." W. Barz
F i g . l : Photograph of transparent m i c r o t i t e r dishes used as a microtest system for photoautotrophic growth of Chenopodium rubrum cell suspensions; dishes are shown withouTTTd MATERIAL AND METHODS Cell c u l t u r e . Photoautotrophic Chenopodium rubrum cell suspension cultures (HUsemann and Barz 1977) were used at the early stage of s t a t i o n a r y growth. Cells (100 mg f r . w . x 1.5 ml - I ) were suspended in fresh c u l t u r e medium. Loading of m i c r o t i t e r dishes. Plastic m i c r o t i t e r dishes (8.5 x 12.5 cm, Greiner, NUrtingen, FRG) cont a i n i n g 24 wells with 3 ml volume each were loaded under aseptic conditions with 1.5 ml c e l l suspension per w e l l . Prior to the a p p l i c a t i o n of c e l l s to the dishes the cell suspensions were mixed with the herbicides and f u s a r i c acid which were applied in s t e r i l e f i l t e r e d methanol solutions so that the organic solvent in the c e l l culture medium was set at 0.25 % ( v / v ) . The dishes f u r t h e r contain 15 additional compartments (volume I ml) which were each f i l l e d with 750 pl K2CO:/KHCO~-buffer generating a 2% CO9 p a r t i a l pressure (H~seman~ and Barz 1977) in the gas"sphere of the dishes. Four such m i c r o t i t e r dishes t i g h t l y sealed with parafilm were incubated on a MTS4 shaker (Jahnke und Kunkel, Staufen, FRG) at 350 rpm under continuous white l i g h t (80pE.m-2.s - I ) at 25oc.
400 Determination of growth parameters. For the determination of c e l l u l a r growth in the m i c r o t i t e r dishes the cell suspensions from an appropriate number of wells were removed under s t e r i l e conditions a f t e r various time i n t e r v a l s . For each value the c e l l s of four wells were separately measured and the standard deviation calculated. PCV was determined by centrifugation (1400 rpm/10 minT-Sigma 2KD-Centrifuge) of c e l l s in calibrated, conical special tubes (volume 2 ml, Fa. Schlee, Witten, FRG). Cell number was determined by resuspending the c e l l s from the pcv assays and a 200 pl aliquot was withdrawn. The c e l l s were mixed with 4.8 ml chromic acid (10% v / v ) , incubated at 70°C for 10 min and counted in a Fuchs-Rosenthal chamber under a microscope. Chlorophyll content was measured photometrically ~ i e g l e r and Egle 1965) using acetone extracts of the residual cell suspensions from the pcv assays. Production of oxygen of the cells was assayed with a Hansatech (Bachofer, Reutlingen, FRG) oxygen meter equipped with a Clark electrode. After a short preincubation period and determination of the dark respiration the net oxygen production was measured using the cells from one well. Light i n t e n s i t y was set at 1050 pE x m"2 x sec - I . Assay of herbicide tolerance. The i n h i b i t o r y e f f e c t of herbicides on the photoautotrophic c e l l s was measured a f t e r 8 days of incubation. For each herbicide concent r a t i o n one dish was used; the c e l l s from 12 wells were assayed f o r measuring pcv and chlorophyll content. The cells from the other 12 wells were separately taken for recording oxygen production, chlorophyll fluorescence and cell v i a b i l i t y ; the standard deviations were calculated. Chlorophyll fluorescence was recorded using the same cell batch as used for the measurements of oxygen production. White l i g h t was f i l t e r e d through a Corning 4-96 f i l t e r to give blue a c t i n i c l i g h t . The f i l t e r (Wratten 88A) used in the detector probe transmits red l i g h t (X>700 nm). The c e l l s were kept in darkness for 5 min, illuminated for I min and during this time period fluorescence i n t e n s i t y was recorded. The i n i t i a l maximum signal (Fp) and the terminal signal (FT) of fluorescence were used to calculate the Rfd-Values according to the equation Rfd =(Fp-FT~FT. Cell v i a b i l i t y is represented by the a b i l i t y of c e l l s to generate reducing equivalents as measured by the reduction of NBT. I ml cell suspension from the determination of the oxygen production was thoroughly mixed with 1500 pl ethylacetate, 10 pl DMSO and 200 pl test reagent (I mM NAD+, I mM PMS, I mM NBT, 50 mM TRIS-HCl, pH 7.5) on a vibromixer for 30 sec. After I hour at 20°C the v i o l e t ethylacetate phase was phot o m e t r i c a l l y assayed at 520 nm against an ethylacetate blank. This procedure had previously been checked for l i n e a r i t y of pigment production with d i f f e r e n t concentrations of NADH from a standard solution (data not shown). Chemicals. Herbicides were obtained from Bayer AG (Leverkusen), fusaric acid came from Sigma Corp., and NBT and PMS were purchased from Serva. RESULTS Growth of photoautotrophic cells in m i c r o t i t e r dishes The growth of photoautotrophic Chenopodium rubrum cell suspensions in the wells of transparent mcrot i t e r dishes ( F i g . l ) under photoautotrophic conditions proceeds e s s e n t i a l l y with the same rate as previously observed using double-tier Erlenmeyer flasks (HUsemann 1985). As documented by the data f o r cell number, chlorophyll content and oxygen production (Fig.2)~a
short lag-phase of 2-3 days is followed by a l i n e a r growth rate up to day 10. During the subsequent stationary phase cell expansion occurs as shown by a substantial increase in pcv (Fig.2) up to day 18. Beyond this time gradual c e l l u l a r decay is observed as shown by the decrease in chlorophyll content, oxygen formation and cell number (Fig.2). Increase and decrease of t o t a l chlorophyll content per well are sign i f i c a n t l y correlated with the course of total oxygen production per well showing maximum photosynthetic a c t i v i t y of the c e l l s around days 8-12. Beyond days 16-18 the c e l l s increasingly suffer from evaporation of water from the culture medium thus leading to rather viscous suspensions. In general, the satisfactory growth of photoautotrophic Ch. rubrum c e l l s in the m i c r o t i t e r dishes leads to the assumption that t h i s set up w i l l provide a v e r s a t i l e system for the serial assay of biologicall y active compounds.
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Fig.2: Growth of photoautotrophic Chenopodium rubrum cell suspensions in p l a s t i c m i c r o t i t e r ~ . Effect of herbicides on photoautotrophic c e l l s in m i c r o t i t e r dishes. To demonstrate the application of the m i c r o t i t e r system the herbicides metribuzin (an asymmetric triazinone), diuron (an urea d e r i v a t i v e ) propanil (a carboxyanilide) and dinoseb (a nitrophenol d e r i v a t i v e ) were incubated with photoautotro~hic Ch. rubrum cells in a concentration range of I0 - I 10-5M. The mode of action of these herbicides on photosynthetic electron transport (relevant for a l l herbicides tested), uncoupling of ATP-production in photosynthesis and respiration (dinoseb) are described in the l i t e r a t u r e (Fedtke 1982). The herbicide assayS were evaluated f o r pcv, chlorophyll content, oxygen production and cell v i a b i l i t y a f t e r 8 days of incubation. Furthermore, slow f l u o r e ~ cence kinetics during a dark to l i g h t t r a n s i t i o n were recorded. The i n i t i a l and the terminal values for chlorophyll fluorescence during binding of herbicides to thylakoids are supposed to be indicative of the photosynthetic capacity of photosystem I I (Havaux and Lannoye i985; Klosson and Krause 1981; Kitajima and Butler 1975). Therefore, the r e l a t i v e fluorescence Fp and the Rfd-Values allow an estimation of the photo-" synthetic capacity of herbicide-affected green c e l l s . For the determination of cell v i a b i l i t y an extrac" tion procedure f o r the water insoluble dye was developed which in comparison to e a r l i e r reports (Dixon 1985; Withers 1980) allowed the analyses of numerous assays in a much shorter time. Efficacy of herbicide action is normally recorded in terms of the pl50-values (Fedtke 1982) which are appr. 6.5-6.7 f o r metribuzin. The data measured for this herbicide in the microtest system with Ch.rubrum c e l l s show (Fig.3)that 10-7M metribuzin does not a l t e r pcv and chlorophyll content during the experimental period. A small increase in chlorophyll fluores-
401 cence, reduced values for oxygen production and the Rfd value indicate that t h i s herbicide concentration moderately reduces photosynthetic electron transport. I0-6M metribuzin represents a lethal concentration best demonstrated by the data for oxygen product i o n , cell v i a b i l i t y and the R#d-Value. The decrease in chlorophyll content is refl~cted by a s i m i l a r l y reduced value for i t s fluorescence. The residual v a l ues f o r pcv, chlorophyll content and fluorescence (Fiq.3) even found at I0-5M herbicide a f t e r 8 days f u r ther decrease upon longer incubation periods; photobleaching of chlorophyll then becomes more important. 80
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Fig.3: Concentration dependent e f f e c t of the herbicide metribuzin on growth, photosynthetic capacity and cell v i a b i l i t y of photoautotrophic Chenopodium rubrum cell suspensions The data in Fig.3 indicate that the lethal concent r a t i o n of metribuzin f o r Ch rubrum photoautotrophic cells appears to be between i0-7 and I0-6M. Additional experiments with the microtest system and metribuzin concentrations stepwise increased from I0-7M to I0-6M allowed the lethal concentration to be set at 2x10-7M (data not shown). An identical value f o r the lethal metribuzin concentration was l a t e r confirmed using our photoautotrophic Ch.rubrum c e l l s grown in double-tier flasks (HUsemann and Barz 1977) and the same methods for evaluating herbicide action(data not shown). The herbicide diuron (p150:6.7-7.5; Fedtke 1982) s i g n i f i c a n t l y affects Ch. rubrum c e l l s at I0-7M (Fig. 4) as shown by the values f o r pcv, chlorophyll content, cell v i a b i l i t y and oxygen production. Electron transport (decrease in the Rfd-Value) also appears to be reduced though a s l i g h t increase in chlorophyll f l u o rescence (Fp-value) indicates that the binding of diuron to the QB-protein of the thylakoid membranes (Fedtke 1982) has not yet i r r e v e r s i b l y damaged the membranes. Diuron at IO-DM and higher concentrations (Fig.4) led to cell death e s s e n t i a l l y as described f o r metribuzin. Propanil ~p150:5.6-6.8; Fedtke 1982) also tested at 10-7 to 10-bM showed moderate herbicide effects only at I0-6M as demonstrated by an increased Fp-value for
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Fig.4: Concentration dependent e f f e c t of the herbicide diuron on growth, photosynthetic capacity and cell v i a b i l i t y of photoautotrophic Chenopodium rubrum cell suspensions Effect of fusaric acid on photoautotrophic c e l l s in m i c r o t i t e r dishes. Fusaric acid (5-~-butyl-2-carboxypyridine) a powerful membrane-active phytotoxin produced by several Fusarium species (Mutert et alo1981) strongly i n h i b i t s photosynthesis of Ch.rubrum cells and leads to substantial photobleaching of chlorophyll (Barz 1981). When tested in the m i c r o t i t e r dishes with our c e l l s either under white l i g h t or in darkness (Fig. 5) 50 pM fusaric acid completely blocked photosynthet i c oxygen production accompanied by s i g n i f i c a n t chlorophyll destruction. This decay of the photosynthesis pigments is much less obvious in the cells cultured in darkness~though c e l l u l a r growth (pcv) w i l l p r a c t i c a l l y not occur under these conditions. DISCUSSION Exact measurements of easy-to-determine parameters for the accumulation of biomass and photosynthetic a c t i v i ty of photoautotrophic c e l l s have demonstrated that such cell suspensions r e a d i l y grow in small volumes (appr. I-1.5 ml) under photoautotrophic conditions. The growth curves shown in Fig. 2 are e s s e n t i a l l y idem t i c a l to the data obtained with Ch,rubrum c e l l s kept
402
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One shaker-load of four m i c r o t i t e r dishes contains 96 wells so that numerous d i f f e r e n t concentrations Or a substantial number of p a r a l l e l s can be measured in one experiment with a minimum amount of time, biomass and labour. I t is herewith recommended that our microtest system using photoautotrophic cells allows the serial screening of compounds such as phytohormones, growth retardants, macro- or micronutrients, herbicides, phytotoxins or any other material with a biological a c t i v i t y affecting green plant c e l l s . Furthermore, the microtest system w i l l be useful for either the analyses of e l i c i t o r - and sucrose-induced secondary compounds (HUsemann et ai.1989) or biotransformation reactions of xenobiochemicals in photoautotrophic cells i f rapid and sensitive analyses ( i . e . GC, HPLC) are available for the subsequent serial determination of compounds in small volumes of nutrient medium or cell extracts.
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Acknowledgement
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Financial support byBundesministerfUr Forschung und Technologie, Bonn, and Fonds der Chemischen Industrie is g r a t e f u l l y acknowledged. REFERENCES 50
100
f u s a r i c acid E ~ M ]
Fig.5: Concentration dependent effect of the phytotoxin fusaric acid on growth and photosynthetic capacity of photoautotrophic Chenopodium rubrum cell suspensions kept in l i g h t (open barsT-a-~-darkness (shaded bars)
in Erlenmeyer or double-tier flasks (HUsemann and Barz 1977). The only noteworthy difference between the two culture methods concerns the much shorter stationary growth phase with subsequent rapid c e l l u l a r decay (Fig.2) in the p l a s t i c dishes due to evaporation of water from the growth medium. In general, however, the m i c r o t i t e r dishes can well be applied for screening purposes with photoautotrophic c e l l s . The comparatively small values f o r the standard dev i a t i o n in Fig.2-5 indicate that c e l l s from a few wells only w i l l be s u f f i c i e n t for each quantitative assay. This, however, requires that the loading of the m i c r o t i t e r dishes with the c e l l s and the various measurements are precisely and c a r e f u l l y conducted under well established standard conditions. In addition to the parameters determined in our experiment for cell growth and physiological a c t i v i t y other, non-destructive methods could also be applied. Using special micro-electrodes determinations of I) changes in the conductivity of the nutrient medium (Hahlbrock und Kuhlen,1972), 2) nutrient uptake ( i . e . NH~+, NO3-) or 3) pH value of the growth medium are possible and such data w i l l also provide important information on the performance of a cell suspension (Grossmann 1988). The data obtained with the herbicides (Fig.3 and 4) indicate that the procedure described represents a r e l i a b l e technique to measure i n h i b i t o r y or lethal concentrations which are e s s e n t i a l l y identical to l i terature reports (Fedtke 1982) which had been obtained with d i f f e r e n t methods.
Barz W (1981) Chemische Rundschau 3 4 : 3 - 6 Dixon RA (1985) in: Dixon RA (ed) Plant Cell Culture, a practical approach. Practical Approach Services, IRL Press,Oxford Washington DC, pp 18-19. Fedtke C (1982) Biochemistry and Physiology of Herbicide Action. Springer, Berlin Heidelberg New York, pp23-85. Gressel J (1984) In: Maramorosch K (ed) Advances in Cell Culture Vol 3, Academic Press, London New York San Francisco, pp 93-181. Grossmann K, Rademacher W, Jung J (1982) Plant Cell Reports 1:281-284. Grossmann K (1988) In: Maramorosch K (ed) Advances in Cell Culture Vol 6. Academic Press, Orlando San Francisco, pp 89-136. Hahlbrock K, Kuhlen E (1972) Planta 108:271-278. Havaux M, Lannoye R (1985) Z. PflanzenzUch. 95:1-13. HUsemann W, Barz W (1977) Physiol. Plant.40:77-81. HUsemann W (1985) in: Vasil IK (ed) Cell culture and Somatic Cell Genetics of Plants,Vol 2, Academic Press, Orlando San Diego New York, pp 213-252. HUsemann W, Fischer K, Mittelbach I , Hfibner S, Richter G, Barz W (1989) In: Kurz WGW (ed) Primary and Secondary Metabolism of Plant Cell Cultures I I , Springer, Heidelberg, pp 35-46. Kitajima M, Butler WL (1975) Biochim.Biophys. Acta 376: 105-115. Klosson RJ, Krause GH (1981) Planta 151:347-352. Mumma RO, Davidonis GH (1983) Prog. Pestic, Biochem.3: 255-278. Mutert WU, LUtfring H, Barz W, Strack U (1981) Z. Naturforsch. 36c:338-339. Potrykus I , Harms CT, L~rz H (1979) Plant Sci. Lett. 14: 231-235. Sandermann H, Scheel D, Trenck Tvd (1984) Ecotoxicol. Environm. Safety 8:176-182. Sato F, Takeda S, Yamada Y (1987) Plant Cell Reports 6:401-404. Withers LA (1980) in: Ingram DS, Helgeson JP (eds) Tissue Culture Methods for Plant P a t h o l o g i s t s , pp 63-70. Ziegler R, Egle K (1965) Beit~ Biol. Planz 41:11-63.
Plant Cell Reports (1989) 8:399-402
© Springer-Verlag 1989
A microtest system for the serial assay of phytotoxic compounds using photoautotrophic cell suspension cultures of Chenopodium rubrum Jutta Thiemann, Arnold Nieswandt, and Wolfgang Barz Lehrstuhl ffir Biochemie der Pflanzen, Westf/ilische Wilhelms-Universit/it, D-4400 Mtinster, Federal Republic of Germany Received August 11, 1989 - Communicated by E. W. Weiler
ABSTRACT Chenopodium rubrum photoautotrophic c e l l suspensions were grown In ~ c tissue culture dishes under photoautotrophic conditions. Growth was monitored by measuring cell number, packed c e l l volume, chlorophyll content and oxygen production. Such m i c r o t i t e r dishes are s u i t a b l e systems f o r the s e r i a l assay of growth i n h i b i t i o n and various physiological effects ( i . e . chlorophyll fluorescence, cell v i a b i l i t y , oxygen production) of photoautotrophic c e l l s as caused by herbicides and fungal phytotoxins. The a p p l i c a b i l i t y of the t e s t system is discussed. Abbreviations pcv = packed cell volume; f r . w . = fresh weight; rpm = r e v o l , per minute; DMSO = dimethyl s u l f o x i d e ; PMS = phenazine methosulfate; NBT = n i t r o - b l u e t e t r a zolium c h l o r i d e ; INTRODUCTION Heterotrophic plant c e l l cultures are s u i t a b l e systems f o r t e s t i n g the b i o l o g i c a l a c t i v i t y and the cell u l a r metabolism of n u t r i e n t s , t o x i n s , or environmental and agrochemical compounds (Gressel 1984; Grossmann et a l . 1982; Mumma and Davidonis 1983; Sandermann et a l . 1984). Several assay systems have been devised allowing the s e r i a l monitoring of numerous compounds or d i f f e r e n t concentrations in small c e l l c u l t u r e volumes in a short period of time (Potrykus et ai.1979; Grossmann 1988). Photoautotrophic plant c e l l suspension cultures which g r e a t l y resemble mesophyll c e l l s (HUsemann 1985) can well be used f o r the assay of photosynthesis-inhib i t i n g herbicides (Sato et a l . 1987). Such c e l l s are r o u t i n e l y grown in Erlenmeyer or d o u b l e - t i e r flasks (HUsemann and Barz !977) so that s e r i a l assays require considerable e f f o r t in time, labou~and shaker space. To f u r t h e r promote the a p p l i c a b i l i t y of these green c e l l s f o r the s e r i a l determination of phytotoxic compounds we have now established a microtest system using Chenopodium rubrum photoautotrophic c e l l suspensions kept in transparent m i c r o t i t e r dishes. Our i n v e s t i g a t i o n s were designed I ) to demonstrate the e f f i c i e n t growth of the photoautotrophic c e l l s in small volumes as monitored by cell number, pcv, chlorophyll content, and oxygen production, and 2) to measure the i n h i b i t o r y effects of various herbicides and a fungal phytotoxin on c e l l u l a r growth and several physiological parameters of the green c e l l s . Offprint requests to." W. Barz
F i g . l : Photograph of transparent m i c r o t i t e r dishes used as a microtest system for photoautotrophic growth of Chenopodium rubrum cell suspensions; dishes are shown withouTTTd MATERIAL AND METHODS Cell c u l t u r e . Photoautotrophic Chenopodium rubrum cell suspension cultures (HUsemann and Barz 1977) were used at the early stage of s t a t i o n a r y growth. Cells (100 mg f r . w . x 1.5 ml - I ) were suspended in fresh c u l t u r e medium. Loading of m i c r o t i t e r dishes. Plastic m i c r o t i t e r dishes (8.5 x 12.5 cm, Greiner, NUrtingen, FRG) cont a i n i n g 24 wells with 3 ml volume each were loaded under aseptic conditions with 1.5 ml c e l l suspension per w e l l . Prior to the a p p l i c a t i o n of c e l l s to the dishes the cell suspensions were mixed with the herbicides and f u s a r i c acid which were applied in s t e r i l e f i l t e r e d methanol solutions so that the organic solvent in the c e l l culture medium was set at 0.25 % ( v / v ) . The dishes f u r t h e r contain 15 additional compartments (volume I ml) which were each f i l l e d with 750 pl K2CO:/KHCO~-buffer generating a 2% CO9 p a r t i a l pressure (H~seman~ and Barz 1977) in the gas"sphere of the dishes. Four such m i c r o t i t e r dishes t i g h t l y sealed with parafilm were incubated on a MTS4 shaker (Jahnke und Kunkel, Staufen, FRG) at 350 rpm under continuous white l i g h t (80pE.m-2.s - I ) at 25oc.
400 Determination of growth parameters. For the determination of c e l l u l a r growth in the m i c r o t i t e r dishes the cell suspensions from an appropriate number of wells were removed under s t e r i l e conditions a f t e r various time i n t e r v a l s . For each value the c e l l s of four wells were separately measured and the standard deviation calculated. PCV was determined by centrifugation (1400 rpm/10 minT-Sigma 2KD-Centrifuge) of c e l l s in calibrated, conical special tubes (volume 2 ml, Fa. Schlee, Witten, FRG). Cell number was determined by resuspending the c e l l s from the pcv assays and a 200 pl aliquot was withdrawn. The c e l l s were mixed with 4.8 ml chromic acid (10% v / v ) , incubated at 70°C for 10 min and counted in a Fuchs-Rosenthal chamber under a microscope. Chlorophyll content was measured photometrically ~ i e g l e r and Egle 1965) using acetone extracts of the residual cell suspensions from the pcv assays. Production of oxygen of the cells was assayed with a Hansatech (Bachofer, Reutlingen, FRG) oxygen meter equipped with a Clark electrode. After a short preincubation period and determination of the dark respiration the net oxygen production was measured using the cells from one well. Light i n t e n s i t y was set at 1050 pE x m"2 x sec - I . Assay of herbicide tolerance. The i n h i b i t o r y e f f e c t of herbicides on the photoautotrophic c e l l s was measured a f t e r 8 days of incubation. For each herbicide concent r a t i o n one dish was used; the c e l l s from 12 wells were assayed f o r measuring pcv and chlorophyll content. The cells from the other 12 wells were separately taken for recording oxygen production, chlorophyll fluorescence and cell v i a b i l i t y ; the standard deviations were calculated. Chlorophyll fluorescence was recorded using the same cell batch as used for the measurements of oxygen production. White l i g h t was f i l t e r e d through a Corning 4-96 f i l t e r to give blue a c t i n i c l i g h t . The f i l t e r (Wratten 88A) used in the detector probe transmits red l i g h t (X>700 nm). The c e l l s were kept in darkness for 5 min, illuminated for I min and during this time period fluorescence i n t e n s i t y was recorded. The i n i t i a l maximum signal (Fp) and the terminal signal (FT) of fluorescence were used to calculate the Rfd-Values according to the equation Rfd =(Fp-FT~FT. Cell v i a b i l i t y is represented by the a b i l i t y of c e l l s to generate reducing equivalents as measured by the reduction of NBT. I ml cell suspension from the determination of the oxygen production was thoroughly mixed with 1500 pl ethylacetate, 10 pl DMSO and 200 pl test reagent (I mM NAD+, I mM PMS, I mM NBT, 50 mM TRIS-HCl, pH 7.5) on a vibromixer for 30 sec. After I hour at 20°C the v i o l e t ethylacetate phase was phot o m e t r i c a l l y assayed at 520 nm against an ethylacetate blank. This procedure had previously been checked for l i n e a r i t y of pigment production with d i f f e r e n t concentrations of NADH from a standard solution (data not shown). Chemicals. Herbicides were obtained from Bayer AG (Leverkusen), fusaric acid came from Sigma Corp., and NBT and PMS were purchased from Serva. RESULTS Growth of photoautotrophic cells in m i c r o t i t e r dishes The growth of photoautotrophic Chenopodium rubrum cell suspensions in the wells of transparent mcrot i t e r dishes ( F i g . l ) under photoautotrophic conditions proceeds e s s e n t i a l l y with the same rate as previously observed using double-tier Erlenmeyer flasks (HUsemann 1985). As documented by the data f o r cell number, chlorophyll content and oxygen production (Fig.2)~a
short lag-phase of 2-3 days is followed by a l i n e a r growth rate up to day 10. During the subsequent stationary phase cell expansion occurs as shown by a substantial increase in pcv (Fig.2) up to day 18. Beyond this time gradual c e l l u l a r decay is observed as shown by the decrease in chlorophyll content, oxygen formation and cell number (Fig.2). Increase and decrease of t o t a l chlorophyll content per well are sign i f i c a n t l y correlated with the course of total oxygen production per well showing maximum photosynthetic a c t i v i t y of the c e l l s around days 8-12. Beyond days 16-18 the c e l l s increasingly suffer from evaporation of water from the culture medium thus leading to rather viscous suspensions. In general, the satisfactory growth of photoautotrophic Ch. rubrum c e l l s in the m i c r o t i t e r dishes leads to the assumption that t h i s set up w i l l provide a v e r s a t i l e system for the serial assay of biologicall y active compounds.
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Fig.2: Growth of photoautotrophic Chenopodium rubrum cell suspensions in p l a s t i c m i c r o t i t e r ~ . Effect of herbicides on photoautotrophic c e l l s in m i c r o t i t e r dishes. To demonstrate the application of the m i c r o t i t e r system the herbicides metribuzin (an asymmetric triazinone), diuron (an urea d e r i v a t i v e ) propanil (a carboxyanilide) and dinoseb (a nitrophenol d e r i v a t i v e ) were incubated with photoautotro~hic Ch. rubrum cells in a concentration range of I0 - I 10-5M. The mode of action of these herbicides on photosynthetic electron transport (relevant for a l l herbicides tested), uncoupling of ATP-production in photosynthesis and respiration (dinoseb) are described in the l i t e r a t u r e (Fedtke 1982). The herbicide assayS were evaluated f o r pcv, chlorophyll content, oxygen production and cell v i a b i l i t y a f t e r 8 days of incubation. Furthermore, slow f l u o r e ~ cence kinetics during a dark to l i g h t t r a n s i t i o n were recorded. The i n i t i a l and the terminal values for chlorophyll fluorescence during binding of herbicides to thylakoids are supposed to be indicative of the photosynthetic capacity of photosystem I I (Havaux and Lannoye i985; Klosson and Krause 1981; Kitajima and Butler 1975). Therefore, the r e l a t i v e fluorescence Fp and the Rfd-Values allow an estimation of the photo-" synthetic capacity of herbicide-affected green c e l l s . For the determination of cell v i a b i l i t y an extrac" tion procedure f o r the water insoluble dye was developed which in comparison to e a r l i e r reports (Dixon 1985; Withers 1980) allowed the analyses of numerous assays in a much shorter time. Efficacy of herbicide action is normally recorded in terms of the pl50-values (Fedtke 1982) which are appr. 6.5-6.7 f o r metribuzin. The data measured for this herbicide in the microtest system with Ch.rubrum c e l l s show (Fig.3)that 10-7M metribuzin does not a l t e r pcv and chlorophyll content during the experimental period. A small increase in chlorophyll fluores-
401 cence, reduced values for oxygen production and the Rfd value indicate that t h i s herbicide concentration moderately reduces photosynthetic electron transport. I0-6M metribuzin represents a lethal concentration best demonstrated by the data for oxygen product i o n , cell v i a b i l i t y and the R#d-Value. The decrease in chlorophyll content is refl~cted by a s i m i l a r l y reduced value for i t s fluorescence. The residual v a l ues f o r pcv, chlorophyll content and fluorescence (Fiq.3) even found at I0-5M herbicide a f t e r 8 days f u r ther decrease upon longer incubation periods; photobleaching of chlorophyll then becomes more important. 80
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Fig.3: Concentration dependent e f f e c t of the herbicide metribuzin on growth, photosynthetic capacity and cell v i a b i l i t y of photoautotrophic Chenopodium rubrum cell suspensions The data in Fig.3 indicate that the lethal concent r a t i o n of metribuzin f o r Ch rubrum photoautotrophic cells appears to be between i0-7 and I0-6M. Additional experiments with the microtest system and metribuzin concentrations stepwise increased from I0-7M to I0-6M allowed the lethal concentration to be set at 2x10-7M (data not shown). An identical value f o r the lethal metribuzin concentration was l a t e r confirmed using our photoautotrophic Ch.rubrum c e l l s grown in double-tier flasks (HUsemann and Barz 1977) and the same methods for evaluating herbicide action(data not shown). The herbicide diuron (p150:6.7-7.5; Fedtke 1982) s i g n i f i c a n t l y affects Ch. rubrum c e l l s at I0-7M (Fig. 4) as shown by the values f o r pcv, chlorophyll content, cell v i a b i l i t y and oxygen production. Electron transport (decrease in the Rfd-Value) also appears to be reduced though a s l i g h t increase in chlorophyll f l u o rescence (Fp-value) indicates that the binding of diuron to the QB-protein of the thylakoid membranes (Fedtke 1982) has not yet i r r e v e r s i b l y damaged the membranes. Diuron at IO-DM and higher concentrations (Fig.4) led to cell death e s s e n t i a l l y as described f o r metribuzin. Propanil ~p150:5.6-6.8; Fedtke 1982) also tested at 10-7 to 10-bM showed moderate herbicide effects only at I0-6M as demonstrated by an increased Fp-value for
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Fig.4: Concentration dependent e f f e c t of the herbicide diuron on growth, photosynthetic capacity and cell v i a b i l i t y of photoautotrophic Chenopodium rubrum cell suspensions Effect of fusaric acid on photoautotrophic c e l l s in m i c r o t i t e r dishes. Fusaric acid (5-~-butyl-2-carboxypyridine) a powerful membrane-active phytotoxin produced by several Fusarium species (Mutert et alo1981) strongly i n h i b i t s photosynthesis of Ch.rubrum cells and leads to substantial photobleaching of chlorophyll (Barz 1981). When tested in the m i c r o t i t e r dishes with our c e l l s either under white l i g h t or in darkness (Fig. 5) 50 pM fusaric acid completely blocked photosynthet i c oxygen production accompanied by s i g n i f i c a n t chlorophyll destruction. This decay of the photosynthesis pigments is much less obvious in the cells cultured in darkness~though c e l l u l a r growth (pcv) w i l l p r a c t i c a l l y not occur under these conditions. DISCUSSION Exact measurements of easy-to-determine parameters for the accumulation of biomass and photosynthetic a c t i v i ty of photoautotrophic c e l l s have demonstrated that such cell suspensions r e a d i l y grow in small volumes (appr. I-1.5 ml) under photoautotrophic conditions. The growth curves shown in Fig. 2 are e s s e n t i a l l y idem t i c a l to the data obtained with Ch,rubrum c e l l s kept
402
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One shaker-load of four m i c r o t i t e r dishes contains 96 wells so that numerous d i f f e r e n t concentrations Or a substantial number of p a r a l l e l s can be measured in one experiment with a minimum amount of time, biomass and labour. I t is herewith recommended that our microtest system using photoautotrophic cells allows the serial screening of compounds such as phytohormones, growth retardants, macro- or micronutrients, herbicides, phytotoxins or any other material with a biological a c t i v i t y affecting green plant c e l l s . Furthermore, the microtest system w i l l be useful for either the analyses of e l i c i t o r - and sucrose-induced secondary compounds (HUsemann et ai.1989) or biotransformation reactions of xenobiochemicals in photoautotrophic cells i f rapid and sensitive analyses ( i . e . GC, HPLC) are available for the subsequent serial determination of compounds in small volumes of nutrient medium or cell extracts.
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Acknowledgement
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Financial support byBundesministerfUr Forschung und Technologie, Bonn, and Fonds der Chemischen Industrie is g r a t e f u l l y acknowledged. REFERENCES 50
100
f u s a r i c acid E ~ M ]
Fig.5: Concentration dependent effect of the phytotoxin fusaric acid on growth and photosynthetic capacity of photoautotrophic Chenopodium rubrum cell suspensions kept in l i g h t (open barsT-a-~-darkness (shaded bars)
in Erlenmeyer or double-tier flasks (HUsemann and Barz 1977). The only noteworthy difference between the two culture methods concerns the much shorter stationary growth phase with subsequent rapid c e l l u l a r decay (Fig.2) in the p l a s t i c dishes due to evaporation of water from the growth medium. In general, however, the m i c r o t i t e r dishes can well be applied for screening purposes with photoautotrophic c e l l s . The comparatively small values f o r the standard dev i a t i o n in Fig.2-5 indicate that c e l l s from a few wells only w i l l be s u f f i c i e n t for each quantitative assay. This, however, requires that the loading of the m i c r o t i t e r dishes with the c e l l s and the various measurements are precisely and c a r e f u l l y conducted under well established standard conditions. In addition to the parameters determined in our experiment for cell growth and physiological a c t i v i t y other, non-destructive methods could also be applied. Using special micro-electrodes determinations of I) changes in the conductivity of the nutrient medium (Hahlbrock und Kuhlen,1972), 2) nutrient uptake ( i . e . NH~+, NO3-) or 3) pH value of the growth medium are possible and such data w i l l also provide important information on the performance of a cell suspension (Grossmann 1988). The data obtained with the herbicides (Fig.3 and 4) indicate that the procedure described represents a r e l i a b l e technique to measure i n h i b i t o r y or lethal concentrations which are e s s e n t i a l l y identical to l i terature reports (Fedtke 1982) which had been obtained with d i f f e r e n t methods.
Barz W (1981) Chemische Rundschau 3 4 : 3 - 6 Dixon RA (1985) in: Dixon RA (ed) Plant Cell Culture, a practical approach. Practical Approach Services, IRL Press,Oxford Washington DC, pp 18-19. Fedtke C (1982) Biochemistry and Physiology of Herbicide Action. Springer, Berlin Heidelberg New York, pp23-85. Gressel J (1984) In: Maramorosch K (ed) Advances in Cell Culture Vol 3, Academic Press, London New York San Francisco, pp 93-181. Grossmann K, Rademacher W, Jung J (1982) Plant Cell Reports 1:281-284. Grossmann K (1988) In: Maramorosch K (ed) Advances in Cell Culture Vol 6. Academic Press, Orlando San Francisco, pp 89-136. Hahlbrock K, Kuhlen E (1972) Planta 108:271-278. Havaux M, Lannoye R (1985) Z. PflanzenzUch. 95:1-13. HUsemann W, Barz W (1977) Physiol. Plant.40:77-81. HUsemann W (1985) in: Vasil IK (ed) Cell culture and Somatic Cell Genetics of Plants,Vol 2, Academic Press, Orlando San Diego New York, pp 213-252. HUsemann W, Fischer K, Mittelbach I , Hfibner S, Richter G, Barz W (1989) In: Kurz WGW (ed) Primary and Secondary Metabolism of Plant Cell Cultures I I , Springer, Heidelberg, pp 35-46. Kitajima M, Butler WL (1975) Biochim.Biophys. Acta 376: 105-115. Klosson RJ, Krause GH (1981) Planta 151:347-352. Mumma RO, Davidonis GH (1983) Prog. Pestic, Biochem.3: 255-278. Mutert WU, LUtfring H, Barz W, Strack U (1981) Z. Naturforsch. 36c:338-339. Potrykus I , Harms CT, L~rz H (1979) Plant Sci. Lett. 14: 231-235. Sandermann H, Scheel D, Trenck Tvd (1984) Ecotoxicol. Environm. Safety 8:176-182. Sato F, Takeda S, Yamada Y (1987) Plant Cell Reports 6:401-404. Withers LA (1980) in: Ingram DS, Helgeson JP (eds) Tissue Culture Methods for Plant P a t h o l o g i s t s , pp 63-70. Ziegler R, Egle K (1965) Beit~ Biol. Planz 41:11-63.
