Planta
Planta (1984) 162:215-219
9 Springer-Verlag 1984
Incorporation of phytol precursors into chlorophylls of tobacco cell cultures* Jfirgen Benz, Ulrika Lempert and Wolfhart Rfidiger Botanisches Institut der Universitfit, Menzinger Strasse 67, D-8000 Mtinchen 19, Federal Republic of Germany
Abstract. The incorporation of [1-3H] geranylgeranyl diphosphate (GGPP), [1-3H] geranylgeranyl monophosphate (GGMP) and [U-14C] phytyl diphosphate (PhPP) into chlorophylls a and b in growing tobacco cell cultures was investigated. The substrates were effectively incorporated into chlorophylls a and b, 3.2% of the total activity of applied GGPP or G G M P and 12.4% of the total activity of applied PhPP being found in chlorophylls a and b after 24 h incubation. The radioactivity was found in phytyl chlorophyllide throughout which means effective hydrogenation of the alcohol moiety in the case of GGPP and GGMP. With increasing substrate concentration, the specific radioactivity of chlorophyll increased up to a saturation level which was reached either at 20 40 gM PhPP or at 60 gM GGPP and GGMP. The specific radioactivity of the chlorophyll formed during the 24-h incubation period was the same as that of the applied substrate at saturating substrate concentration. The specific radioactivity of chlorophyll a was higher than that of chlorophyll b only in the case of PhPP. Key words: Chlorophyll biosynthesis - Nicotiana (chlorophyll biosynthesis) - Geranylgeranyl phosphate - Phytol.
Introduction The lipophilic property of chlorophylls (Chl) - a prerequisite for their incorporation into protein* Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday Abbreviations: Chlide = chlorophyllide a; Chlph = phytyl chlorophyllide; C h l ~ = geranylgeranyl chlorophyllide a; G G P P = geranylgeranyI diphosphate; G G M P = g e r a n y l g e r a n y l monophosphate; H P L C = high-performance liquid chromatography; PhPP = phytyl diphosphate
pigment complexes - is a consequence of the presence of the phytol residue. Insertion of this residue is one of the last steps in Chl biosynthesis. Investigation of greening, etiolated seedlings demonstrated that newly formed chlorophyllide a (Chlide) reacts at first with geranylgeranyl diphosphate (GGPP). The product of this reaction, geranylgeranyl chlorophyllide a (ChlG~), is then hydrogenated to the final pigment phytyl chlorophyllide (Chlph; Schoch etal. 1977; Schoch 1978; Schoch et al. 1980). This reaction sequence was indirectly supported by in-vitro investigations. Chlorophyll synthetase from oat etioplasts accepts GGPP more readily than phytyl diphosphate (PhPP; Rfidiger et al. 1980). The soluble GGPP pool of etiolated oat seedlings is depleted during the onset of chlorophyll formation (Benz et al. 1983). However, the situation in green plants is possibly different. Chlorophyll synthetase of broken spinach chloroplasts accepts PhPP more readily than GGPP (Soil et al. 1983). Phytyl diphosphate is formed from GGPP in the chloroplast envelope (Soll and Schultz 1981; Soll et al. 1983). Therefore, it would seem to be of interest to investigate the incorporation of GGPP and PhPP in an intact green plant system which synthesizes chlorophyll. It is well known that GGPP and similar compounds cannot readily penetrate intact plant tissues. Incubation of etiolated barley shoots with [14C]PhPP in the light did not lead to significant labelling of newly formed Chl (Riidiger 1960). Incorporation of PhPP and similar compounds was therefore investigated with crude plant homogenates (Watts and Kekwick 1974) which do not necessarily reflect the physiological situation of Chl biosynthesis. The penetration barrier is probably not the chloroplast envelope but the plasmalemma linked to the cell wall: GGPP readily penetrates the protoplast and plastid membranes of etiolated oat leaves (Benz et al. 1981). In the present paper we report the incorporation of GGPP, geranylger-
216
J. Benz et al. : Incorporation of phytol precursors into chlorophylls of tobacco cell cultures
anyl monophosphate (GGMP) and PhPP into Chl in growing tobacco cell suspension cultures which are capable of net synthesis of chlorophyll a and b. Material and methods The tissue culture used for the present experiments originated from a callus culture of Nieotiana tabaeum isolated by Bergmann (1960). Tobacco tissues were grown as suspension in a modified Murashige and Skoog medium (Logemann and Bergmann 1974) at 28_+ 1~ C and 60-70% humidity under continuous illumination (5 W m -2) by fluorescent lamps (L 58 W/21 Lumilux weiss and L 65 W/77 R Fluora; Osram, Mtinchen, FRG). The culture was continuously agitated at 50 rpm on a gyratory shaker (Model G-10; New Brunswick, Edison, N.J., USA). Subcultivation of the parent culture was done every 10 d by dilution with a six-fold volume of fresh medium. The experiments were started with samples of freshly transferred cells (1-2 ml packed cells per 10 ml medium, determined in aliquots of each sample). After a lag phase of 1-2 d, exponential growth continued for about two weeks. Incubation with the radioactive substrates was started on day 4. The medium was adjusted to pH 6.0 at the beginning of incubation. Substrates were dissolved either in methanol (GGPP, GGMP) or in water (PhPP). The maximum volume of methanol added to a 10-ml sample was 75 gl. The same amount of methanol was added to each controI culture. The substrates were [13H]GGPP and [1-3H]GGMP (0.85 TBq tool -1) the preparation of which was described by R/idiger et al. (1980). [U-14C] phytyl diphosphate was prepared accordingly from [U14C]phytol (0.14TBq tool 1) (NEN Chemicals, Dreieich, FRG). After 24 h, the cells (3-5 ml packed cells per sample) were collected by centrifugation at 500 g for 10 rain and washed twice with fresh medium. The cells were rapidly frozen with liquid nitrogen and, after thawing, repeatedly extracted with acetone (at first 15-20 ml, then 4-5 ml each time) until the residue was colorless. Pigments of the combined acetone extracts were transferred into diethylether which was washed with water until it was free of acetone. Aliquots Of this organic phase were taken for spectrophotometric determination of chlorophylls (French 1960) and radioactivity. Chlorophylls were transformed into pheophytins which were then purified by column chromatography on powdered sugar and by high-performance liquid chromatography (HPLC) on LiChrosorb RP-8 (Merck, Darmstadt, FRG) with methanol:water (95 : 5, v/v) as previously described (Riidiger et al. 1980). Pigments and radioactivity were determined in aliquots after column chromatography and after HPCL. Radioactivity was determined with a Liquid Scintillation Counter equipped with channels for 3H and 14C (Beckman LS 100 C, Mtinchen, FRG) in Rotiszint 22 (Roth, Karlsruhe, FRG).
Results and discussion
The aim of the present paper was to investigate the incorporation of GGPP, GGMP and PhPP into chlorophyll during the maximum of growth and chlorophyll accumulation in tobacco cell cultures. Under the experimental conditions applied here (see Material and methods), this was the case about 3 5 d after the transfer of the cell suspension
Table 1. Distribution of radioactivity in the culture medium
and extracts from tobacco cell cultures after incubation with GGPP, G G M P or PhPP. Each sample (1-2 ml packed cells per 10 ml medium) was pregrown for 4 d and then incubated for another day with [1-3H]GGPP (final concentration 10 26 btmol t- 1), [1-3H] G G M P (final concentration 60-93 gmol 1-1) or [U-14C]PhPP (final concentration 40 61 btmol 1-1). The specific radioactivity was 0.85 TBq tool 1 for GGPP and G G M P and 0.14 TBq mol-1 for PhPP. Both GGPP and G G M P were added as methanolic solution (maximum volume 75 ~tl per sample), PhPP was added as aqueous solution. All values are given as percent of total activity of the applied substrate Substrate
GGPP, G G M P PhPP
Radioactivity in Culture medium
Pellet after extraction
Lipid fraction (diethylether)
Chl a and b after purification by HPLC
63.0_+2.5 65.8_+3.2
5.1_+1.1 6.9_+0.9
26.3_+2.1 23.0_+2.9
3.2_+0.7 12.4_+1.9
to the fresh medium. In most experiments, the increase of chlorophylls a and b was between 10 and 30% within 24 h, at this age of the cultured cells. The time of incubation was always 24 h. Control experiments demonstrated that the substrates (GGPP, PhPP, GGMP) are stable in the culture medium. Hydrolysis of added GGPP or PhPP was less than 3%, and of GGMP 1-2% during the course of experiment. Therefore, hydrolysis of substrates could be neglected for the calculations. The substrates were (in part) applied in methanolic solutions. Control experiments verified that the addition of methanol to the culture medium (final concentration below 1%, v/v) did not appreciably hamper either growth or chlorophyll accumulation. The present investigation clearly demonstrated the incorporation of GGPP, GGMP, and PhPP into chlorophylls a and b in growing tobacco cell cultures (Table 1). About 60-70% of the applied radioactivity remained in the culture medium under the applied conditions and only minor amounts (5% of GGPP, 7% of PhPP) were incorporated into non-extractable compounds. Whereas incorporation into the lipid fraction was about equal for GGPP and PhPP (26.3 versus 23.0% of the applied activity), an appreciable difference was found for the incorporation into Chl a and b. The pigments (isolated as pheophytins) contain 3.2% of the applied activity (i.e. 12% of the activity of the lipid fraction) after incubation with GGPP but 12.4% of the applied activity (i.e. 54% of the activity of the lipid fraction) after incubation with PhPP. The meaning of this difference will be dis-
J. Benz et al. : Incorporation of phytol precursors into chlorophylls of tobacco cell cultures
I~176 I 7 6~ 2o-
g 10o (3
._u u CI. if)
11
(ol
5- Io
if" o
io
(o)
i'o 2'o 3'o io s'o 6'0 7'o 8'o 9'0 Substrate concentration [IJ.M] Fig. 1. Incorporation of G G P P and PhPP into Chl a and b in tobacco cell cultures. Incubation for 24 h in the light with various substrate concentrations 2.5 to 93 ~tM. The specific radioactivity of the applied substrates (100% =0.85 TBq mol 1 for [3H]GGPP and [3H]GGMP; 0 . 1 4 T B q mo1-1 for [I~C]PhPP). Open symbols are for Chl a, closed symbols are for C h l b . Incubation with o o, G G P P ; [] m, G G M P ; A A, PhPP. Only the values for Chl a from PhPP (zx--z~) form a curve separate from that of all the other values. Experiments involving both incubations with only one substrate (either G G P P or G G M P or PhPP) and simultaneous incubations with two substrates ( G G P P and PhPP) are included
cussed below. It was verified in each experiment that the radioactivity coincided with the pigment peak in chromatography and that the fractions eluting before and after the Chl were inactive. The incorporation of GGPP was similar to that in broken oat etioplasts where 1.5-4% of the applied radioactivity was found in Chl~a (Benz et al. 1983). However, HPLC analysis proved that the radioactivity incorporated in tobacco cell cultures was mainly ( ~ 99%) in Chlph whereas only a very small activity ( ~ 1%) was found in ChlGa, dihydrogeranylgeranyl chlorophyllide, and tetrahydrogeranylgeranyl chlorophyllide, if GGPP or GGMP were applied as substrates. Thus, hydrogenation of geranylgeranyl to phytyl residues is very effective in this system. We assume that this reflects the physiological situation in green plants. Other plant systems were much less effective in this respect. Broken spinach chloroplasts produced up to 90% Chlph (basis esterified Chl= 100%) from labelled GGPP under optimum conditions but much less under most conditions (Soll et al. 1983). Broken etioplasts which normally do not hydrogenate ChlGa (Rfidiger et al. 1980) produced only up to 5% Chlph from labelled GGPP in the presence of excess NADPH (Benz et al.
217
1980). The degree of hydrogenation of Chl~G to Chlph has been taken as the criterion for the intactness of etioplast or chloroplast, although formation of Chlph from labelled GGPP did not exceed 9% in mesophyll protoplasts from etiolated oat (Benz et al. 1981). The radioactivity was determined in purified Chl a and b and calculated as specific radioactivity for the Chl increase. This calculation was based on the assumption that Chl turnover was negligible under the experimental conditions, i.e. that the (inactive) Chl present at the beginning of incubation was still present at the end of the experiment. In accordance with this assumption, the specific radioactivity of the newly formed Chl was identical to that of the applied substrate at substrate saturation (Fig. 1). The specific radioactivity of Chl a and b increased with increasing concentrations of the substrates in the medium, as expected (Fig. 1). This correlation was about equal for GGPP and GGMP. The presence of a kinase which transforms GGMP into GGPP with ATP was found earlier in etioplast preparations (Riidiger et al. 1980). It can be assumed that such a kinase is also active in the tobacco cell cultures. Interestingly, incorporation of PhPP into Chl a seems to be more efficient than incorporation of GGPP or GGMP (Fig. 1) since the specific radioactivity of Chl a was higher after incubation with PhPP than with equal concentrations of GGPP. This was clearly demonstrated in experiments in which tobacco cell cultures were incubated simultaneously with [3H]GGPP and [14C]PhPP (Table 2). Saturation (=100% specific radioactivity of newly formed Chl, based on the specific radioactivity of the substrate) was reached at 20-40 gM PhPP but only at about 60 gM GGPP or GGMP. This result could possibly be explained by a higher specificity of chlorophyll synthetase for PhPP than for GGPP, as in spinach chloroplasts (Sollet al. 1983). Substrate saturation for chlorophyll synthetase of broken oat etioplasts was found at 80-100 gM GGPP or PhPP (Rfidiger et al. 1980). No difference was found between GGPP and PhPP in these experiments although the enzyme of etioplasts shows a 2:1 preference for GGPP. Substrate saturation was found in spinach chloroplasts only at about 670 gM GGPP for Chl a and at about 170 ~tM GGPP for Chl b (Soll et al. 1983); PhPP was not investigated. Another possible explanation for the preferred incorporation of PhPP in the cell culture system is that GGPP can be partly used up in several metabolic pathways (e.g. biosynthesis of carotenoids and gibberellins)
218
J. Benz et al. : Incorporation of phytol precursors into chlorophylls of tobacco cell cultures
Table 2. Analysis of Chl a and b after simultaneous incubation of tobacco cell cultures with [3H] GGPP and [14C] PhPP. Conditions as in Fig. 1. The values of the specific activities of isolated Chlph of these experiments are also included in Fig. 1 [3H] G G P P (I.tM)
[1'~C]PhPP (p.M)
Chl a (nmol)
Increase (%)"
Specific radioactivity 3H (GBq mol-1)
2.6 2.6 5.2 5.2 20.0
2.6 2.6 5.2 5.2 20.0
6.1 6.1 4.3 4.2 20.0
6.1 6.1 4.3 4.2 20.0
12.4 13.8 7.2 11.5 8.0
12.6 39.7 7.4 27.3 11.3
22.1 62.9 34.0 85.0 42.5
Chl b (nmol)
Increase %a
Specific radioactivity
2.2 3.7 2.3 3.6 2.7
6.7 42.5 7.1 26.5 11.9
(2.6) b (7.4) (4) (10) (5)
14C (GBq m o l - 1) 33.6 37.1 47.6 37.1 128.8
(24) b (26.5) (34) (26.5) (92)
3H (GBq mol 1)
1'*C (GBq mol 1)
44.2 (5.2) b 40.8 (4.8) 10.2 (1.2)
27.6 (19.7) b 18.2 (13) 51.8 (37)
a % of original Chl (at the beginning of the incubation) b Radioactivity was determined for isolated Pheph and calculated for Chlph increase. Values in brackets-specific radioactivity of Chlph expressed as % of the specific radioactivity of applied substrate=0.85 TBq mol 1 for [3H]GGPP and 0.14 TBq m o l - 1 for [14C]PhPP
Table 3. Specific radioactivity of Chl a and b after incubation with GGPP, G G M P and PhPP. Given are the ratios of specific activities Chl a/Chl b based on the increase of chlorophylls during the incubation (means of five independent determinations with various substrate concentrations) Substrate
Ratio specific activity Chl a/Chl b
GGPP GGMP PhPP
1.05 0.98 1.83
which do not use PhPP. This would result in a lower incorporation of G G P P into Chl (as compared with PhPP) even if there were no difference in substrate specificity of chlorophyll synthetase. Consequently, Chl should contain a higher percentage of metabolized PhPP than of metabolized GGPP. This can clearly be seen from Table 1 (see above). Another aspect which should be mentioned here is the specific radioactivity of Chl b in comparison with Chl a. In all experiments with G G P P and GGMP, at various substrate concentrations, both chlorophylls have about the same specific radioactivity. With PhPP, however, Chl a always has a higher specific activity than Chl b in the same experiment (see Table 3). Thus, it seems as if PhPP would be incorporated more directly into Chl a than into Chl b whereas this difference does not exist for GGPP. Incorporation of PhPP into Chl b
follows the saturation curve for incorporation of GGPP or G G M P into Chl a and Chl b (see Fig. 1). It should be recalled here that the investigated products of the tobacco cell culture system were always phytylated, i.e. Chl aph and Chl bph. Our data agree with the assumption that Chl aph can be formed by two pathways, either i) Chlide a GGPP , Chl acG --+ Chl aph, or ii) Chlide a PhPP , Chl apla. The pathway which predominates depends on the availability of the substrates G G P P and PhPP. Normally, it is pathway i) via G G P P (Benz et al. 1983; Soll et al. 1983) but it can be pathway ii) via PhPP if this substrate is supplied in sufficient amounts. Our data agree with the assumption that formation of Chl beh occurs preferentially on pathway i) via G G P P i.e. that pathway ii) does not operate for Chl b formation. Because Chlide b is a good substrate for chlorophyll synthetase (Benz and Rfidiger 1981), Chlide b could be the direct precursor of Chl ba~. However, we could not detect any Chlide b in greening etiolated plants (data not shown) contrary to the report o f Aronoff (1981). It has been suggested that the transformation of the Chl a structure to the Chl b structure takes place with newly formed (esterified) Chl a (Shlyk 1971; Virgin 1977). It is tempting to suggest from our data that Chl a c e is transformed to Chl bGc which only then is hydrogenated to Chl bph. This
J. Benz et al. : Incorporation of phytol precursors into chlorophylls of tobacco cell cultures
would also agree with the result of Oelze-Karow and Mohr (1978) who found that completion of Chl b formation requires approximately the same space of time as the esterification of Chlide a to Chl a. However, it is premature to draw a final conclusion on the pathway of Chl b biosynthesis at this time. We thank Professor L. Bergmann, K61n, for the inoculum of the tobacco cell suspension culture and initial advice. The work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.
References Aronoff, S. (1981) Chlorophyllide b. Biochem. Biophys. Res. Commun. 102, 108-112 Benz, J., Hampp, R., R/idiger, W. (1981) Chlorophyll biosynthesis by mesophyll protoplasts and plastids from etiolated oat (Arena sativa L.) leaves. Planta 152, 54-58 Benz, J., Haser, A., Rfidiger, W. (1983) Changes in the endogenous pools of tetraprenyl diphosphates in etiolated oat seedlings after irradiation. Z. Pflanzenphysiol. 111,349-356 Benz, J., Riidiger, W. (1981) Chlorophyll biosynthesis: various chlorophyllides as exogenous substrates for chlorophyll synthetase. Z. Naturforsch. Tell C 36, 51-57 Benz, J., Wolf, C., Rfidiger, W. (1980) ChIorophyll biosynthesis: hydrogenation of geranylgeraniol. Plant Sci. Lett. 19, 225-230 Bergmann, L. (1960) Growth and division of single cells of higher plants in vitro. J. Gen. Physiol. 43, 841-851 French, C.S. (1960) The chlorophylls in vivo and in vitro. In: Handbuch der Pflanzenphysiologie, Bd. 5/I, pp. 252-297, Ruhland, W., ed. Springer, Berlin G6ttingen Heidelberg Logemann, H., Bergmann, L. (1974) Einflul3 von Licht und
219
Medium auf die ,,Plating Efficiency" isolierter Zellen aus Calluskulturen von Nicotiana tabacum var. ,,Samsun". Planta 121,283 292 Oelze-Karow, H., Mohr, H. (1978) Control of chlorophyll b biosynthesis by phytochrome. Photochem. Photobiol. 27, 189-193 Rfidiger, W. (1960) fOber die Biosynthese des Phytols und den Phytolgehalt der Protochlorophylle. Dissertation, University of Wiirzburg Rfidiger, W., Benz, J., Guthoff, C. (1980) Detection and partial characterization of activity of chlorophyll synthetase in etioplast membranes. Eur. J. Biochem. 109, 193-200 Schoch, S. (1978) The esterification of chlorophyllide a in greening bean leaves. Z. Naturforsch. Teil C 33, 712-714 Schoch, S., Hehlein, C., Rfidiger, W. (1980) Influence of anaerobiosis on chlorophyll biosynthesis in greening oat seedlings (Arena saliva L.). Plant Physiol. 66, 576 579 Schoch, S., Lempert, U., Riidiger, W. (1977) Uber die letzten Stufen der Chlorophyll-Biosynthese. Zwischenprodukte zwischen Chlorophyllid und phytolhaltigem Chlorophyll. Z. Pflanzenphysiol. 83, 427 436 Shlyk, A.A. (1971) Biosynthesis of chlorophyll b. Annu. Rev. Plant Physiol. 22, 169-184 Soll, J., Schultz, G. (1981) Phytol synthesis from geranylgeraniol in spinach chloroplasts. Biochem. Biophys. Res. Commun. 99, 907-912 Soll, J., Schultz, G., Rfidiger, W., Benz, J. (1983) Hydrogenation of geranylgeraniol two pathways exist in spinach chIoroplast. Plant Physiol. 71, 849 854 Virgin, H. (1977) The spectral response of light dependent chlorophyll b formation. Physiol. Plant. 40, 45-49 Watts, R.B., Kekwick, R.G.O. (1974) Factors affecting the formation of phytol and its incorporation into chlorophyll by homogenates of the leaves of the French bean Phaseolus vulgaris. Arch. Biochem. Biophys. 160, 469-475 Received 9 February; accepted 11 May 1984
Planta (1984) 162:215-219
9 Springer-Verlag 1984
Incorporation of phytol precursors into chlorophylls of tobacco cell cultures* Jfirgen Benz, Ulrika Lempert and Wolfhart Rfidiger Botanisches Institut der Universitfit, Menzinger Strasse 67, D-8000 Mtinchen 19, Federal Republic of Germany
Abstract. The incorporation of [1-3H] geranylgeranyl diphosphate (GGPP), [1-3H] geranylgeranyl monophosphate (GGMP) and [U-14C] phytyl diphosphate (PhPP) into chlorophylls a and b in growing tobacco cell cultures was investigated. The substrates were effectively incorporated into chlorophylls a and b, 3.2% of the total activity of applied GGPP or G G M P and 12.4% of the total activity of applied PhPP being found in chlorophylls a and b after 24 h incubation. The radioactivity was found in phytyl chlorophyllide throughout which means effective hydrogenation of the alcohol moiety in the case of GGPP and GGMP. With increasing substrate concentration, the specific radioactivity of chlorophyll increased up to a saturation level which was reached either at 20 40 gM PhPP or at 60 gM GGPP and GGMP. The specific radioactivity of the chlorophyll formed during the 24-h incubation period was the same as that of the applied substrate at saturating substrate concentration. The specific radioactivity of chlorophyll a was higher than that of chlorophyll b only in the case of PhPP. Key words: Chlorophyll biosynthesis - Nicotiana (chlorophyll biosynthesis) - Geranylgeranyl phosphate - Phytol.
Introduction The lipophilic property of chlorophylls (Chl) - a prerequisite for their incorporation into protein* Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday Abbreviations: Chlide = chlorophyllide a; Chlph = phytyl chlorophyllide; C h l ~ = geranylgeranyl chlorophyllide a; G G P P = geranylgeranyI diphosphate; G G M P = g e r a n y l g e r a n y l monophosphate; H P L C = high-performance liquid chromatography; PhPP = phytyl diphosphate
pigment complexes - is a consequence of the presence of the phytol residue. Insertion of this residue is one of the last steps in Chl biosynthesis. Investigation of greening, etiolated seedlings demonstrated that newly formed chlorophyllide a (Chlide) reacts at first with geranylgeranyl diphosphate (GGPP). The product of this reaction, geranylgeranyl chlorophyllide a (ChlG~), is then hydrogenated to the final pigment phytyl chlorophyllide (Chlph; Schoch etal. 1977; Schoch 1978; Schoch et al. 1980). This reaction sequence was indirectly supported by in-vitro investigations. Chlorophyll synthetase from oat etioplasts accepts GGPP more readily than phytyl diphosphate (PhPP; Rfidiger et al. 1980). The soluble GGPP pool of etiolated oat seedlings is depleted during the onset of chlorophyll formation (Benz et al. 1983). However, the situation in green plants is possibly different. Chlorophyll synthetase of broken spinach chloroplasts accepts PhPP more readily than GGPP (Soil et al. 1983). Phytyl diphosphate is formed from GGPP in the chloroplast envelope (Soll and Schultz 1981; Soll et al. 1983). Therefore, it would seem to be of interest to investigate the incorporation of GGPP and PhPP in an intact green plant system which synthesizes chlorophyll. It is well known that GGPP and similar compounds cannot readily penetrate intact plant tissues. Incubation of etiolated barley shoots with [14C]PhPP in the light did not lead to significant labelling of newly formed Chl (Riidiger 1960). Incorporation of PhPP and similar compounds was therefore investigated with crude plant homogenates (Watts and Kekwick 1974) which do not necessarily reflect the physiological situation of Chl biosynthesis. The penetration barrier is probably not the chloroplast envelope but the plasmalemma linked to the cell wall: GGPP readily penetrates the protoplast and plastid membranes of etiolated oat leaves (Benz et al. 1981). In the present paper we report the incorporation of GGPP, geranylger-
216
J. Benz et al. : Incorporation of phytol precursors into chlorophylls of tobacco cell cultures
anyl monophosphate (GGMP) and PhPP into Chl in growing tobacco cell suspension cultures which are capable of net synthesis of chlorophyll a and b. Material and methods The tissue culture used for the present experiments originated from a callus culture of Nieotiana tabaeum isolated by Bergmann (1960). Tobacco tissues were grown as suspension in a modified Murashige and Skoog medium (Logemann and Bergmann 1974) at 28_+ 1~ C and 60-70% humidity under continuous illumination (5 W m -2) by fluorescent lamps (L 58 W/21 Lumilux weiss and L 65 W/77 R Fluora; Osram, Mtinchen, FRG). The culture was continuously agitated at 50 rpm on a gyratory shaker (Model G-10; New Brunswick, Edison, N.J., USA). Subcultivation of the parent culture was done every 10 d by dilution with a six-fold volume of fresh medium. The experiments were started with samples of freshly transferred cells (1-2 ml packed cells per 10 ml medium, determined in aliquots of each sample). After a lag phase of 1-2 d, exponential growth continued for about two weeks. Incubation with the radioactive substrates was started on day 4. The medium was adjusted to pH 6.0 at the beginning of incubation. Substrates were dissolved either in methanol (GGPP, GGMP) or in water (PhPP). The maximum volume of methanol added to a 10-ml sample was 75 gl. The same amount of methanol was added to each controI culture. The substrates were [13H]GGPP and [1-3H]GGMP (0.85 TBq tool -1) the preparation of which was described by R/idiger et al. (1980). [U-14C] phytyl diphosphate was prepared accordingly from [U14C]phytol (0.14TBq tool 1) (NEN Chemicals, Dreieich, FRG). After 24 h, the cells (3-5 ml packed cells per sample) were collected by centrifugation at 500 g for 10 rain and washed twice with fresh medium. The cells were rapidly frozen with liquid nitrogen and, after thawing, repeatedly extracted with acetone (at first 15-20 ml, then 4-5 ml each time) until the residue was colorless. Pigments of the combined acetone extracts were transferred into diethylether which was washed with water until it was free of acetone. Aliquots Of this organic phase were taken for spectrophotometric determination of chlorophylls (French 1960) and radioactivity. Chlorophylls were transformed into pheophytins which were then purified by column chromatography on powdered sugar and by high-performance liquid chromatography (HPLC) on LiChrosorb RP-8 (Merck, Darmstadt, FRG) with methanol:water (95 : 5, v/v) as previously described (Riidiger et al. 1980). Pigments and radioactivity were determined in aliquots after column chromatography and after HPCL. Radioactivity was determined with a Liquid Scintillation Counter equipped with channels for 3H and 14C (Beckman LS 100 C, Mtinchen, FRG) in Rotiszint 22 (Roth, Karlsruhe, FRG).
Results and discussion
The aim of the present paper was to investigate the incorporation of GGPP, GGMP and PhPP into chlorophyll during the maximum of growth and chlorophyll accumulation in tobacco cell cultures. Under the experimental conditions applied here (see Material and methods), this was the case about 3 5 d after the transfer of the cell suspension
Table 1. Distribution of radioactivity in the culture medium
and extracts from tobacco cell cultures after incubation with GGPP, G G M P or PhPP. Each sample (1-2 ml packed cells per 10 ml medium) was pregrown for 4 d and then incubated for another day with [1-3H]GGPP (final concentration 10 26 btmol t- 1), [1-3H] G G M P (final concentration 60-93 gmol 1-1) or [U-14C]PhPP (final concentration 40 61 btmol 1-1). The specific radioactivity was 0.85 TBq tool 1 for GGPP and G G M P and 0.14 TBq mol-1 for PhPP. Both GGPP and G G M P were added as methanolic solution (maximum volume 75 ~tl per sample), PhPP was added as aqueous solution. All values are given as percent of total activity of the applied substrate Substrate
GGPP, G G M P PhPP
Radioactivity in Culture medium
Pellet after extraction
Lipid fraction (diethylether)
Chl a and b after purification by HPLC
63.0_+2.5 65.8_+3.2
5.1_+1.1 6.9_+0.9
26.3_+2.1 23.0_+2.9
3.2_+0.7 12.4_+1.9
to the fresh medium. In most experiments, the increase of chlorophylls a and b was between 10 and 30% within 24 h, at this age of the cultured cells. The time of incubation was always 24 h. Control experiments demonstrated that the substrates (GGPP, PhPP, GGMP) are stable in the culture medium. Hydrolysis of added GGPP or PhPP was less than 3%, and of GGMP 1-2% during the course of experiment. Therefore, hydrolysis of substrates could be neglected for the calculations. The substrates were (in part) applied in methanolic solutions. Control experiments verified that the addition of methanol to the culture medium (final concentration below 1%, v/v) did not appreciably hamper either growth or chlorophyll accumulation. The present investigation clearly demonstrated the incorporation of GGPP, GGMP, and PhPP into chlorophylls a and b in growing tobacco cell cultures (Table 1). About 60-70% of the applied radioactivity remained in the culture medium under the applied conditions and only minor amounts (5% of GGPP, 7% of PhPP) were incorporated into non-extractable compounds. Whereas incorporation into the lipid fraction was about equal for GGPP and PhPP (26.3 versus 23.0% of the applied activity), an appreciable difference was found for the incorporation into Chl a and b. The pigments (isolated as pheophytins) contain 3.2% of the applied activity (i.e. 12% of the activity of the lipid fraction) after incubation with GGPP but 12.4% of the applied activity (i.e. 54% of the activity of the lipid fraction) after incubation with PhPP. The meaning of this difference will be dis-
J. Benz et al. : Incorporation of phytol precursors into chlorophylls of tobacco cell cultures
I~176 I 7 6~ 2o-
g 10o (3
._u u CI. if)
11
(ol
5- Io
if" o
io
(o)
i'o 2'o 3'o io s'o 6'0 7'o 8'o 9'0 Substrate concentration [IJ.M] Fig. 1. Incorporation of G G P P and PhPP into Chl a and b in tobacco cell cultures. Incubation for 24 h in the light with various substrate concentrations 2.5 to 93 ~tM. The specific radioactivity of the applied substrates (100% =0.85 TBq mol 1 for [3H]GGPP and [3H]GGMP; 0 . 1 4 T B q mo1-1 for [I~C]PhPP). Open symbols are for Chl a, closed symbols are for C h l b . Incubation with o o, G G P P ; [] m, G G M P ; A A, PhPP. Only the values for Chl a from PhPP (zx--z~) form a curve separate from that of all the other values. Experiments involving both incubations with only one substrate (either G G P P or G G M P or PhPP) and simultaneous incubations with two substrates ( G G P P and PhPP) are included
cussed below. It was verified in each experiment that the radioactivity coincided with the pigment peak in chromatography and that the fractions eluting before and after the Chl were inactive. The incorporation of GGPP was similar to that in broken oat etioplasts where 1.5-4% of the applied radioactivity was found in Chl~a (Benz et al. 1983). However, HPLC analysis proved that the radioactivity incorporated in tobacco cell cultures was mainly ( ~ 99%) in Chlph whereas only a very small activity ( ~ 1%) was found in ChlGa, dihydrogeranylgeranyl chlorophyllide, and tetrahydrogeranylgeranyl chlorophyllide, if GGPP or GGMP were applied as substrates. Thus, hydrogenation of geranylgeranyl to phytyl residues is very effective in this system. We assume that this reflects the physiological situation in green plants. Other plant systems were much less effective in this respect. Broken spinach chloroplasts produced up to 90% Chlph (basis esterified Chl= 100%) from labelled GGPP under optimum conditions but much less under most conditions (Soll et al. 1983). Broken etioplasts which normally do not hydrogenate ChlGa (Rfidiger et al. 1980) produced only up to 5% Chlph from labelled GGPP in the presence of excess NADPH (Benz et al.
217
1980). The degree of hydrogenation of Chl~G to Chlph has been taken as the criterion for the intactness of etioplast or chloroplast, although formation of Chlph from labelled GGPP did not exceed 9% in mesophyll protoplasts from etiolated oat (Benz et al. 1981). The radioactivity was determined in purified Chl a and b and calculated as specific radioactivity for the Chl increase. This calculation was based on the assumption that Chl turnover was negligible under the experimental conditions, i.e. that the (inactive) Chl present at the beginning of incubation was still present at the end of the experiment. In accordance with this assumption, the specific radioactivity of the newly formed Chl was identical to that of the applied substrate at substrate saturation (Fig. 1). The specific radioactivity of Chl a and b increased with increasing concentrations of the substrates in the medium, as expected (Fig. 1). This correlation was about equal for GGPP and GGMP. The presence of a kinase which transforms GGMP into GGPP with ATP was found earlier in etioplast preparations (Riidiger et al. 1980). It can be assumed that such a kinase is also active in the tobacco cell cultures. Interestingly, incorporation of PhPP into Chl a seems to be more efficient than incorporation of GGPP or GGMP (Fig. 1) since the specific radioactivity of Chl a was higher after incubation with PhPP than with equal concentrations of GGPP. This was clearly demonstrated in experiments in which tobacco cell cultures were incubated simultaneously with [3H]GGPP and [14C]PhPP (Table 2). Saturation (=100% specific radioactivity of newly formed Chl, based on the specific radioactivity of the substrate) was reached at 20-40 gM PhPP but only at about 60 gM GGPP or GGMP. This result could possibly be explained by a higher specificity of chlorophyll synthetase for PhPP than for GGPP, as in spinach chloroplasts (Sollet al. 1983). Substrate saturation for chlorophyll synthetase of broken oat etioplasts was found at 80-100 gM GGPP or PhPP (Rfidiger et al. 1980). No difference was found between GGPP and PhPP in these experiments although the enzyme of etioplasts shows a 2:1 preference for GGPP. Substrate saturation was found in spinach chloroplasts only at about 670 gM GGPP for Chl a and at about 170 ~tM GGPP for Chl b (Soll et al. 1983); PhPP was not investigated. Another possible explanation for the preferred incorporation of PhPP in the cell culture system is that GGPP can be partly used up in several metabolic pathways (e.g. biosynthesis of carotenoids and gibberellins)
218
J. Benz et al. : Incorporation of phytol precursors into chlorophylls of tobacco cell cultures
Table 2. Analysis of Chl a and b after simultaneous incubation of tobacco cell cultures with [3H] GGPP and [14C] PhPP. Conditions as in Fig. 1. The values of the specific activities of isolated Chlph of these experiments are also included in Fig. 1 [3H] G G P P (I.tM)
[1'~C]PhPP (p.M)
Chl a (nmol)
Increase (%)"
Specific radioactivity 3H (GBq mol-1)
2.6 2.6 5.2 5.2 20.0
2.6 2.6 5.2 5.2 20.0
6.1 6.1 4.3 4.2 20.0
6.1 6.1 4.3 4.2 20.0
12.4 13.8 7.2 11.5 8.0
12.6 39.7 7.4 27.3 11.3
22.1 62.9 34.0 85.0 42.5
Chl b (nmol)
Increase %a
Specific radioactivity
2.2 3.7 2.3 3.6 2.7
6.7 42.5 7.1 26.5 11.9
(2.6) b (7.4) (4) (10) (5)
14C (GBq m o l - 1) 33.6 37.1 47.6 37.1 128.8
(24) b (26.5) (34) (26.5) (92)
3H (GBq mol 1)
1'*C (GBq mol 1)
44.2 (5.2) b 40.8 (4.8) 10.2 (1.2)
27.6 (19.7) b 18.2 (13) 51.8 (37)
a % of original Chl (at the beginning of the incubation) b Radioactivity was determined for isolated Pheph and calculated for Chlph increase. Values in brackets-specific radioactivity of Chlph expressed as % of the specific radioactivity of applied substrate=0.85 TBq mol 1 for [3H]GGPP and 0.14 TBq m o l - 1 for [14C]PhPP
Table 3. Specific radioactivity of Chl a and b after incubation with GGPP, G G M P and PhPP. Given are the ratios of specific activities Chl a/Chl b based on the increase of chlorophylls during the incubation (means of five independent determinations with various substrate concentrations) Substrate
Ratio specific activity Chl a/Chl b
GGPP GGMP PhPP
1.05 0.98 1.83
which do not use PhPP. This would result in a lower incorporation of G G P P into Chl (as compared with PhPP) even if there were no difference in substrate specificity of chlorophyll synthetase. Consequently, Chl should contain a higher percentage of metabolized PhPP than of metabolized GGPP. This can clearly be seen from Table 1 (see above). Another aspect which should be mentioned here is the specific radioactivity of Chl b in comparison with Chl a. In all experiments with G G P P and GGMP, at various substrate concentrations, both chlorophylls have about the same specific radioactivity. With PhPP, however, Chl a always has a higher specific activity than Chl b in the same experiment (see Table 3). Thus, it seems as if PhPP would be incorporated more directly into Chl a than into Chl b whereas this difference does not exist for GGPP. Incorporation of PhPP into Chl b
follows the saturation curve for incorporation of GGPP or G G M P into Chl a and Chl b (see Fig. 1). It should be recalled here that the investigated products of the tobacco cell culture system were always phytylated, i.e. Chl aph and Chl bph. Our data agree with the assumption that Chl aph can be formed by two pathways, either i) Chlide a GGPP , Chl acG --+ Chl aph, or ii) Chlide a PhPP , Chl apla. The pathway which predominates depends on the availability of the substrates G G P P and PhPP. Normally, it is pathway i) via G G P P (Benz et al. 1983; Soll et al. 1983) but it can be pathway ii) via PhPP if this substrate is supplied in sufficient amounts. Our data agree with the assumption that formation of Chl beh occurs preferentially on pathway i) via G G P P i.e. that pathway ii) does not operate for Chl b formation. Because Chlide b is a good substrate for chlorophyll synthetase (Benz and Rfidiger 1981), Chlide b could be the direct precursor of Chl ba~. However, we could not detect any Chlide b in greening etiolated plants (data not shown) contrary to the report o f Aronoff (1981). It has been suggested that the transformation of the Chl a structure to the Chl b structure takes place with newly formed (esterified) Chl a (Shlyk 1971; Virgin 1977). It is tempting to suggest from our data that Chl a c e is transformed to Chl bGc which only then is hydrogenated to Chl bph. This
J. Benz et al. : Incorporation of phytol precursors into chlorophylls of tobacco cell cultures
would also agree with the result of Oelze-Karow and Mohr (1978) who found that completion of Chl b formation requires approximately the same space of time as the esterification of Chlide a to Chl a. However, it is premature to draw a final conclusion on the pathway of Chl b biosynthesis at this time. We thank Professor L. Bergmann, K61n, for the inoculum of the tobacco cell suspension culture and initial advice. The work was supported by the Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg.
References Aronoff, S. (1981) Chlorophyllide b. Biochem. Biophys. Res. Commun. 102, 108-112 Benz, J., Hampp, R., R/idiger, W. (1981) Chlorophyll biosynthesis by mesophyll protoplasts and plastids from etiolated oat (Arena sativa L.) leaves. Planta 152, 54-58 Benz, J., Haser, A., Rfidiger, W. (1983) Changes in the endogenous pools of tetraprenyl diphosphates in etiolated oat seedlings after irradiation. Z. Pflanzenphysiol. 111,349-356 Benz, J., Riidiger, W. (1981) Chlorophyll biosynthesis: various chlorophyllides as exogenous substrates for chlorophyll synthetase. Z. Naturforsch. Tell C 36, 51-57 Benz, J., Wolf, C., Rfidiger, W. (1980) ChIorophyll biosynthesis: hydrogenation of geranylgeraniol. Plant Sci. Lett. 19, 225-230 Bergmann, L. (1960) Growth and division of single cells of higher plants in vitro. J. Gen. Physiol. 43, 841-851 French, C.S. (1960) The chlorophylls in vivo and in vitro. In: Handbuch der Pflanzenphysiologie, Bd. 5/I, pp. 252-297, Ruhland, W., ed. Springer, Berlin G6ttingen Heidelberg Logemann, H., Bergmann, L. (1974) Einflul3 von Licht und
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Medium auf die ,,Plating Efficiency" isolierter Zellen aus Calluskulturen von Nicotiana tabacum var. ,,Samsun". Planta 121,283 292 Oelze-Karow, H., Mohr, H. (1978) Control of chlorophyll b biosynthesis by phytochrome. Photochem. Photobiol. 27, 189-193 Rfidiger, W. (1960) fOber die Biosynthese des Phytols und den Phytolgehalt der Protochlorophylle. Dissertation, University of Wiirzburg Rfidiger, W., Benz, J., Guthoff, C. (1980) Detection and partial characterization of activity of chlorophyll synthetase in etioplast membranes. Eur. J. Biochem. 109, 193-200 Schoch, S. (1978) The esterification of chlorophyllide a in greening bean leaves. Z. Naturforsch. Teil C 33, 712-714 Schoch, S., Hehlein, C., Rfidiger, W. (1980) Influence of anaerobiosis on chlorophyll biosynthesis in greening oat seedlings (Arena saliva L.). Plant Physiol. 66, 576 579 Schoch, S., Lempert, U., Riidiger, W. (1977) Uber die letzten Stufen der Chlorophyll-Biosynthese. Zwischenprodukte zwischen Chlorophyllid und phytolhaltigem Chlorophyll. Z. Pflanzenphysiol. 83, 427 436 Shlyk, A.A. (1971) Biosynthesis of chlorophyll b. Annu. Rev. Plant Physiol. 22, 169-184 Soll, J., Schultz, G. (1981) Phytol synthesis from geranylgeraniol in spinach chloroplasts. Biochem. Biophys. Res. Commun. 99, 907-912 Soll, J., Schultz, G., Rfidiger, W., Benz, J. (1983) Hydrogenation of geranylgeraniol two pathways exist in spinach chIoroplast. Plant Physiol. 71, 849 854 Virgin, H. (1977) The spectral response of light dependent chlorophyll b formation. Physiol. Plant. 40, 45-49 Watts, R.B., Kekwick, R.G.O. (1974) Factors affecting the formation of phytol and its incorporation into chlorophyll by homogenates of the leaves of the French bean Phaseolus vulgaris. Arch. Biochem. Biophys. 160, 469-475 Received 9 February; accepted 11 May 1984
