Planta (1984)161:55~554
P l a n t a 9 Springer-Verlag 1984
The biosynthesis of chlorophyll in greening barley (Hordeum vulgate). Is there a light-independent protochlorophyllide reductase? Klaus Apel, Michael Motzkus artd Katayoon Dehesh Botanisches Institut der Universit/it, Olshausenstrasse40, D-2300 Kiel, Federal Republic of Germany.
Abstract. Recently, some evidence for the occurrence of a light-independent protochlorophyllidereducing enzyme in greening barley plants has been presented. In the present work this problem was reinvestigated. 6-[14C] Aminolevulinic acid was fed to isolated barley shoots from plants which had been preilluminated for various lengths of time. Porphyrins which had been synthesized during the dark incubation were analyzed by high-performance liquid chromatography. There was no evidence for a light-independent synthesis of chlorphyll(ide). The 14C-labelled precursor was incorporated almost exclusively into protochlorophyllide. The reduction of labelled protochlorophyllide to chlorophyllide was strictly light-dependent. These results are not consistent with the existence of a light-independent protochlorophyllide-reductase in barley as proposed previously. Key words: Chlorophyll biosynthesis - Hordeum (chlorophyll) - Protochlorophyllide reductase.
Introduction
Angiosperms are unable to form chloroplasts in the absence of light. When seedlings are grown in the dark, the proplastids in the developing leaves are transformed into another plastid type, the etioplast. These plastids lack chlorophyll and possess no extensive thylakoid membranes. The transformation of etioplasts into chloroplasts is mediated by light (Boardman 1966; Boardman et al. 1978; Bogorad 1976; Castelfranco and Beale 1983, Kirk and Tilney-Bassett 1978; Schopfer and Apel 1983). Abbreviation:
phy
HPLC=high-performanceliquid chromatogra-
The first detectable light-dependent step toward the formation of the chloroplast is the phototransformation of protochlorophyllide to chlorophyllide. This reaction is catalyzed by the enzyme NADPH-protochlorophyllide oxidoreductase (Griffiths 1974, 1978; Apel etal. 1980). Surprisingly in barley both the concentration of the enzyme as well as the amount of the m R N A encoding the enzyme protein decrease rapidly upon illumination (Apel 1981; H/iuser et al. 1984; Mapleston and Griffiths 1980; Santel and Apel 1981). Similar light effects on the NADPH-protochlorophyllide oxidoreductase have been observed also in other plants (Ikeuchi and Murakami 1982; Kay and Griffiths 1983; Meyer et al. 1983). In barley the level of the NADPH-protochlorophyllide oxidoreductase has dropped beyond the limit of detection, when the maximum rate of chlorophyll accumulation is reached (Santel and Apel 1981). These results have led us to believe that the function of this enzyme may be restricted to the initial phase of greening and that, later on, the conversion of protochlorophyllide to chlorophyllide may be catalyzed by another, as yet unknown biosynthetic mechanism (Santel and Apel 1981). It is known that in lower plants at least one additional enzyme exists which is capable of reducing protochlorophyllide to chlorophyllide in the dark (e.g. Castelfranco and Beale 1983; Kirk and Tilney-Bassett 1978). Recently some evidence for the occurence of such a light-independent protochlorophyllide-reducing enzyme in angiosperms including barley, has been presented (Adamson 1983; Adamson et al. 1980, 1983). In the case of barley the ability to accumulate chlorophyll in the dark was reported to be confined to young expanding leaves which had been exposed to continuous light prior to the dark incubation (Adamson 1983). The light-independent reduction of protochloro-
K. Apel et al. : Is there a light-independent protochlorophyllide reductase?
phyllide to chlorophyllide could be part of an additional route by which chlorophyll is formed in green angiosperms. In the present paper the possible presence of a light-independent protochlorophyllide-reducing enzyme in barley was reinvestigated using an experimental approach which was different from the one which had been used previously (Adamson 1983).
551
!
m
11
1V
E c -4
=o C3
Material
and methods
Plant material. Barley (Hordeum vutgare L., cv. Carina) was grown for 5 d in the dark as described previously (Apel et al. 1980).
L
rr"
Incubation of plants. The synthesis of porphyrins in the dark was measured by feeding 6-[14C] aminolevulinic acid to isolated barley shoots. Dark-grown plants were illuminated for various lengths of time with incandescent white light. The shoots were excised just above the coleoptile sheath and incubated with the labelled compound (1.6-I0 ~ Bq per leaf; 2.109 Bq m M - t 6-aminolevulinic acid) for 2 h in the dark as described previously (Redlinger and Apel 1980). At the end of the incubation the leaves were directly frozen in liquid nitrogen or exposed to white light prior to the freezing step. The frozen leaves were ground with a precooled mortar and a pestle and the pigments extracted with 80% acetone.
A
Liquid chromatography. High-performance liquid chromatogra-
.>
phy (HPLC) was carried out with two Consta Metric pumps (Milton Roy) using a Ct8 reversed-phase column (Spherisorb OD 5, 5 ~tm, 25 cm long 4.6 mm diameter). Pigments were eluted according to the method of Eskins and Harris (1981) with the following modifications : A linear gradient of 20 to 60% ethylacetate in methanol/water (80:20; v/v) was applied to the column at a flow rate of 1 ml min- ~. The pigments were detected spectrophotometrically using a variable wavelength detector (spectro Monitor D ; Milton Roy). The various porphyrins were identified either by recording the elution at different wavelengths (630, 650 and 670 nm) or by coelution of isolated standard pigments. Protochlorophyllide was purified as described elsewhere (Redlinger and Apel 1980). Protochlorophyll was isolated according to Houssier and Sauer (1969). The eluate was fractionated into 250-gl portions. The radioactivity in each fraction was measured by liquid scintillation counting and quenching was corrected for by the external-standard method.
o
b• c6 u~
>,
92 ..... ,. f
/
J.......
"ff. . . . .
I. . . . . . .
L .......
5O
0
B
Fraction
h ......
k ......
L ......
100
number
• c u~
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_~2
Results
A
and discussion
Chlorophyll accumulation in redarkened plants was measured by feeding C14-1abelled 6-aminolevulinic acid to isolated barley shoots. Barley plants were illuminated for 6 h with incandescent white light before the shoots were excised and incubated for 2 h in the dark with the labelled chlorophyll precursor. At the end of the incubation the shoots were either treated for 10 min with continuous white light or were used directly for the extraction of pigments. These samples were separated by reverse-phase HPLC. Protochlorophyllide and chlorophyllide were separated from each other and were
..... ,." }'--"-.J ".....
L
~
50 C
Fraction
,
.....,~.. ' ......9 ~....... 100
number
Fig. 1 A-C. The analysis of porphyrins of barley by HPLC. Shoots were excised from 6-h preilluminated plants and incubated in the dark for 2 h with fi-[14C]aminolevulinic acid. A The absorbance changes at 436 nm during pigment separation, I = chlorophyllide, II = protochlorophyllide, I I I = chlorophyll b, I V - c h l o r o p h y l l a. These compounds were identified as described under Material and methods. B, C The distribution of radioactively labelled porphyrins extracted from leaves which were kept in the dark (B) or received a 15-min light treatment at the end of the dark period (C)
552
K. Apel et al. : Is there a light-independent protochlorophyllide reductase ?
Table 1. The distribution of labelled porphyrins (%) (incorporation of fi-[14C]aminolevulinic acid into porphyrins of greening barley leaves). Shoots were excised from preilluminated barley plants and incubated in the dark for 2 h with fi-aminolevulinic acid as described under Material and methods. The pigments were extracted directly from dark-incubated shoots or following an additional 15-rain light treatment of the shoots. The radioactivity of individual porphyrins was determined after HPLC of the various pigment samples, m E = n o t detectable; L = l i g h t ; D = darkness Light regime
0hL, 0hL, 0hL, 0hL, 24hL, 24hL, 48hL, 48hL, 72hL, 72hL,
2hD 2hD, 2hD 2hD, 2hD 2hD, 2hD 2hD, 2hD 2hD,
15' L 15' L 15' L 15' L 15' L
Protochlorophyllide
Chlorophyllide
Chlorophyll a
Expt. I
Expt. II
Expt. I
Expt. II
Expt. I
Expt. II
99.16 0.83 99.42 30.85 99.91 87.43 99.42 91.08 99.60 76.25
98.92 17.12 99.81 82.41 99.19 75.63 -
1.08 1.98 0.19 1.21 0.81 1.70 -
n.d. 88.01 n.d. 66.24 n.d. 12.05 n.d. 8.70 n.d. 23.60
n.d. 80.90 n.d. 17.59 n.d. 22.67 -
0.84 11.16 0.58 2.92 0.10 0.57 0.58 0.23 0.40 0.15
identified either by coelution of the purified pigment and-or by measuring the absorbance spectrum of the eluted pigment fractions (Fig. 1). In addition to these two pigments, chlorophylls a and b were also resolved and their radioactivity determined. The only porphyrin whose concentration increased during the dark incubation of 6-h-preilluminated plants was protochlorophyllide. The amounts of chlorophyll(ide) a and b did not change significantly during this incubation period. The distribution of radioactivity among the various pigment fractions was consistent with this observation. Only two radioactively labelled major peaks were detected when pigment samples of 6-h-preilluminated barley shoots were separated by HPLC. The first peak was shown to contain free fi-aminolevulinic acid, the second one coeluted with protochlorophyllide (Fig. 1). The identification of the labelled component as protochlorophyllide was confirmed by the result of an illumination experiment. In those shoots which were exposed for 10 min to continuous white light the labelled protochlorophyllide peak decreased by more than 70%. At the same time, new radioactively labelled peaks appeared within the elution profile, whose retention times were identical to those of chlorophyllide a and chlorophyll a (Fig. 1, Table 1). This result indicates that protochlorophyllide is the only. major freshly synthesized prophyrin compound which accumulates in shoots of 6-h-preilluminated barley plants during a subsequent dark incubation. Only traces of radioactively labelled chlorophyllide were found in these plants, far less than 1% of
the radioactivity incorporated into the total fraction of porphyrin (Table 1). Even though at present we cannot exclude the possibility that the formation of these traces of chlorophyllide might be catalyzed by a light-independent protochlorophyllide-reducing enzyme, it seems more likely that exposure of the plants to the green safelight has led to the formation of minute traces of chlorophyltide. It has been reported that the capability of barley and pea plants to synthesize chlorophyll in the dark develops only during the illumination of etiolated plants (Adamson 1983; Adamson etal. 1983). Thus, it is conceivable that the 6 h of illumination which had been used in the initial experiment described above might not be sufficient enough to allow the activation of the light-independent protochlorophyllide reduction. In the following experiments the length of the illumination period was extended to 24, 48 and 72 h before the shoots were excised and incubated with fi-[14C] aminolevulinic acid for 2 h in the dark. In all cases, the labelled precursor was incorporated almost exclusively into protochlorophyllide, Only traces of chlorophyllide were detectable which did not account for more than 0.5% of the total radioactivity of the pigment fraction. The conversion of protochlorophyllide to chlorophyllide occurred only in the light. The amount of phototransformable protochlorophyllide declined steadily with increasing lengths of the preillumination period. In etiolated leaves almost 100% of the protochlorophyllide was photoreduced during the first 10 min of illumination. In shoots which were derived from
K. Apel et al. : Is there a light-independent protochlorophyllide reductase?
6-h-preilluminated plants 70% of the labelled protochlorophyllide was photoreduced. The fraction of photoconvertible protochlorophyllide decreased to less than 30% in samples of plants which had been illuminated for more than 24 h prior to the dark incubation. It is not known yet which factor is responsible for this drastically reduced phototransformation of protochlorophyllide. It is well documented that in the primary leaf of etiolated plants different maturation stages of etioplasts are found in a linear series, with the youngest in cells near the base and the oldest in cells near the tip (Boffey et al. 1980; Robertson and Laetsch 1974). This distribution of different plastid forms is paralleled by drastic differences in the NADPH-protochlorophyllide oxidoreductase content of the plastids and their capacity to accumulate chlorophyll during illumination (Dehesh et al. 1983). Similar differences in the distribution of a light-independent protochlorophyllide-reducing enzyme have been described also for the various parts of barley leaves (Adamson 1983). The primary leaves of excised shoots which had been incubated with 6-[~4C] aminolevulinic acid in the dark were cut into three pieces of equal lengths and the pigments of each segment were extracted and measured separately. In all three segments protochlorophyllide was the only pigment which was labelled radioactively during the dark incubation. Traces of labelled chlorophyllide were present in equal amounts in all three leaf segments and did not exceed 1% of the total incorporated radioactivity (Table 2). There were, however, significant differences in the extent of protochlorophyllide reduction in the three leaf sections. While in the tip part only 40% of the protochlorophyllide was photoreduced, in the middle and the basal parts of the leaf approximately 70% of the protochlorophyllide was phototransformed into chlorophyll(ide) (Table 2). Our results clearly demonstrate that during the dark incubation of excised shoots which had been derived from preilluminated barley plants only protochlorophyllide but not chlorophyll(ide) incorporated substantial amounts of 14C-labelled g-aminolevulinic acid. These results are not consistent with earlier reports on the occurrence of a light-independent protochlorophyllide-reducing enzyme in barley leaves. In the work by Adamson and coworkers (1980, 1983) changes in chlorophyll contents were determined by extracting the pigments from leaves and measuring them spectroscopically. In the present work a different experimental approach was used. The incorporation of 6-[14C] aminolevulinic acid allows one to follow
553
Table 2. The distribution of labelled porphyrins (%) (incorporation of 3-[14C]aminolevulinic acid into porphyrins of different leaf sections of barley plants). Shoots were isolated from 19-h preilluminated barley plants and cut into three pieces of equal lengths. The basal, middle and tip sections were incubated with 6-aminolevulinic acid for 2 h in the dark and were kept in the dark or treated for 10 min with white light before the pigments were extracted. The analysis of the radioactively labelled porphyrins was done as described in Table 1. n.d. = n o t detectable; L = light; D = darkness
Sample
Protochloro- Chlorophyllide phyllide
Chlorophyll a
99.50 59.15
0.50 1.21
n.d. 39.64
Middle part of the leaf 19hL, 2hD 99.58 19hL, 2hD, 10'L 30.23
0.42 0.74
n.d. 69.03
Basal section of the leaf 19hL, 2hD 99.59 19hL, 2hD, 10'L 35.46
0.41 0.13
n.d. 64.41
Tip of the leaf 19hL, 2hD 19hL, 2hD, 10'L
directly the de-novo synthesis of porphyrins. At the moment we cannot explain the apparent discrepancy between the results of these two approaches. One could argue that perhaps an additional pool of cLaminolevulinic acid exists which leads to chlorophyll formation via a dark reduction of protochlorophyllide and which does not exchange with the pool of radioactively labelled precursor. However, as far as we know no evidence exists which supports such an assumption. The breakdown of chlorophyll, which might affect the steady-state levels of pigments (Bennett 1981) did not appear to interfere with the accumulation of radioactively labelled protochlorophyllide. Even after an overnight incubation of excised shoots the amount of accumulated protochlorophyllide did not decrease and, at the same time, no new radioactively labelled porphyrin derivatives could be observed during the HPLC separation of pigment samples (data not shown). The transformation of radioactively labelled protochlorophyllide to chlorophyllide took place only in the light. The almost complete absence of chlorophyll(ide) in dark-incubated shoots provides good evidence that light had been excluded from the incubation assay and that it did not influence the porphyrin synthesis during the dark incubation. A light-independent protochlorophyllide-reducing enzyme might be involved in an alternative route for the synthesis of chlorophyll such as has been proposed previously (Santel and Apel 1981).
554
K. Apel et al. : Is there a light-independent protochlorophyllide reductase?
However, we have failed to detect such an enzyme in barley. The important question of whether or not the NADPH-protochlorophyllide oxidoreductase is responsible for chlorophyll(ide) formation throughout the greening process remains unsolved and will have to await the development of a new experimental approach. This investigation was supported by Deutsche Forschungsgemeinschaft (Ap 16/5-1).
References Adamson, H. (1983) Evidence for a light-independent protochlorophyllide reductase in green barley leaves. In: Cell function and differentiation 2, pp. 33-41, Akoyunoglou, G., Evangelopoilos, A.E., Georgtsos, J., Palaiologos, G., Trakatellis, A., Tsiganos, C.P., eds. Proc. Spec. FEBS Meet., Athens, 1982. Liss, New York Adamson, H., Hiller, R.G., Vesk, M. (1980) Chloroplast development and the synthesis of chlorophyll a and b and chlorophyll protein complexes I and II in the dark in Tradescantia albiflora (Knuth). Planta 150, 269-274 Adamson, H., Packer, N., Sanders, N. (1983) Chlorophyll synthesis in the dark in peas. 6th Int. Congr. Photosynthesis, Brussels, Book of Abstracts, vol. 2, p. 242 Apel, K. (1981) The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Phytochrome-induced decrease of translatable mRNA coding for the NADPH-protochlorophyllide oxidoreductase. Eur. J. Biochem. 120, 89-93 Apel, K., Santel, H.-J., Redlinger, T.E., Falk, H. (1980) The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Isolation and characterization of the NADPH protochlorophyllide oxidoreductase. Eur. J. Biochem. 111, 251-258 Bennett, J. (1981) Biosynthesis of the light-harvesting chlorophyll a/b protein. Polypeptide turnover in darkness. Eur. J. Biochem. 118, 61-70 Boardman, N.K. (1966) Protochlorophyll. In: The chlorophylls, pp. 437-476, Vernon, L.P., Seely, G.R., eds. Academic Press, New York London Boardman, N.K., Anderson, J.M., Goodchild, D.J. (1978) Chlorophyll-protein complexes and structure of mature and developing chloroplasts. Curr. Top. Bioenerg. 8, 35-109 Boffey, S.A., Sellden, G., Leech, R.M. (1980) The influence of cell age on chlorophyll formation in light-grown and etiolated wheat seedlings. Plant Physiol. 65, 680-684 Bogorad, L. (1976) Chlorophyll biosynthesis. In: Chemistry and biochemistry of plant pigments, 2nd edn., vol. 1, pp. 64-148, Goodwin, T.W., ed. Academic Press, New York London
Castelfranco, P.A., Beale, S.T. (1983) Chlorophyll biosynthesis: recent advances and areas of current interest. Annu. Rev. Plant Physiol. 34, 241 278 Dehesh, K., Hfiuser, I., Apel, K., Kloppstech, K. (1983) The distribution of NADPH-protochlorophyllide oxidoreductase in relation to chlorophyll accumulation along the barley leaf gradient. Planta 158, 134-139 Eskins, K., Harris, L. (1981) High-performance liquid chromatography of etioplast pigments in red kidney bean leaves. Photochem. Photobiol. 33, 131-133 Griffiths, W.T. (1974) Protochlorophyll and protochlorophyllide as precursors for chlorophyll synthesis in vitro. FEBS Lett. 49, 196-200 Griffiths, W.T. (1978) Reconstitution of chlorophyllide formation by isolated etioplast membranes. Biochem. J. 174, 681-692 Hfiuser, I., Dehesh, K., Apel, K. (1984) The proteolytic degradation in vitro of the NADPH-protochlorophyllide oxidoreductase of barley (Hordeum vulgare L.). Arch. Biochem. Biophys. 228, 577-586 Houssier, C., Sauer, K. (1969) Optical properties of the pr 0tochlorophyll pigments. I. Isolation, characterization, and infrared spectra. Biochim. Biophys. Acta 172, 476-491 Ikeuchi, M., Murakami, S. (1982) Behavior of the 36000 dalton protein in the internal membranes of squash etioplasts during greening. Plant Cell Physiol. 23, 575-583 Kay, S.A., Griffiths, W.T. (1983) Light-induced breakdown of NADPH-protochlorophyllide oxidoreductase in vitro. Plant PhysioL 72, 229-236 Kirk, J.T.O., Tilney-Bassett, R.A.E. (1978) The plastids. Freeman, London San Francisco Mapleston, E.R., Griffiths, W.T. (1980) Light modulation of the activity of protochlorophyllide reductase. Biochem. J. 189, 125-133 Meyer, G., Bliedung, H., Kloppstceh, K. (1983) NADPH-protochlorophyllide oxidoreductase: reciprocal regulation in mono- and dicotyledonean plants. Plant Cell Reports 2, 26-29 Redlinger, T.E., Apel, K. (1980) The effect of light on four protochlorophyllide-binding polypeptides of barley (Hordeum vulgate). Arch. Biochem. Biophys. 200, 253-260 Robertson, D., Laetsch, M. (1974) Structure and function of developing barley plastids. Plant Physiol. 54, 148-159 Santel, H.L, Apel, K. (1981) The protochlorophyllide holtchrome of barley (Hordeum vulgate L.). The effect of light on the NADPH: protochlorophyllide oxidoreductase. Eur. J. Biochem. 120, 95-103 Schopfer, P., Apel, K. (1983) Intracellular photomorphogenesis. In: Encyclopedia of plant physiology, N.S., vol. 16: Photomorphogenesis, pp. 258-288, Shropshire, W., jr., Mohr, H., eds. Springer, Berlin Heidelberg New York Received 27 December 1983; accepted 15 March 1984
P l a n t a 9 Springer-Verlag 1984
The biosynthesis of chlorophyll in greening barley (Hordeum vulgate). Is there a light-independent protochlorophyllide reductase? Klaus Apel, Michael Motzkus artd Katayoon Dehesh Botanisches Institut der Universit/it, Olshausenstrasse40, D-2300 Kiel, Federal Republic of Germany.
Abstract. Recently, some evidence for the occurrence of a light-independent protochlorophyllidereducing enzyme in greening barley plants has been presented. In the present work this problem was reinvestigated. 6-[14C] Aminolevulinic acid was fed to isolated barley shoots from plants which had been preilluminated for various lengths of time. Porphyrins which had been synthesized during the dark incubation were analyzed by high-performance liquid chromatography. There was no evidence for a light-independent synthesis of chlorphyll(ide). The 14C-labelled precursor was incorporated almost exclusively into protochlorophyllide. The reduction of labelled protochlorophyllide to chlorophyllide was strictly light-dependent. These results are not consistent with the existence of a light-independent protochlorophyllide-reductase in barley as proposed previously. Key words: Chlorophyll biosynthesis - Hordeum (chlorophyll) - Protochlorophyllide reductase.
Introduction
Angiosperms are unable to form chloroplasts in the absence of light. When seedlings are grown in the dark, the proplastids in the developing leaves are transformed into another plastid type, the etioplast. These plastids lack chlorophyll and possess no extensive thylakoid membranes. The transformation of etioplasts into chloroplasts is mediated by light (Boardman 1966; Boardman et al. 1978; Bogorad 1976; Castelfranco and Beale 1983, Kirk and Tilney-Bassett 1978; Schopfer and Apel 1983). Abbreviation:
phy
HPLC=high-performanceliquid chromatogra-
The first detectable light-dependent step toward the formation of the chloroplast is the phototransformation of protochlorophyllide to chlorophyllide. This reaction is catalyzed by the enzyme NADPH-protochlorophyllide oxidoreductase (Griffiths 1974, 1978; Apel etal. 1980). Surprisingly in barley both the concentration of the enzyme as well as the amount of the m R N A encoding the enzyme protein decrease rapidly upon illumination (Apel 1981; H/iuser et al. 1984; Mapleston and Griffiths 1980; Santel and Apel 1981). Similar light effects on the NADPH-protochlorophyllide oxidoreductase have been observed also in other plants (Ikeuchi and Murakami 1982; Kay and Griffiths 1983; Meyer et al. 1983). In barley the level of the NADPH-protochlorophyllide oxidoreductase has dropped beyond the limit of detection, when the maximum rate of chlorophyll accumulation is reached (Santel and Apel 1981). These results have led us to believe that the function of this enzyme may be restricted to the initial phase of greening and that, later on, the conversion of protochlorophyllide to chlorophyllide may be catalyzed by another, as yet unknown biosynthetic mechanism (Santel and Apel 1981). It is known that in lower plants at least one additional enzyme exists which is capable of reducing protochlorophyllide to chlorophyllide in the dark (e.g. Castelfranco and Beale 1983; Kirk and Tilney-Bassett 1978). Recently some evidence for the occurence of such a light-independent protochlorophyllide-reducing enzyme in angiosperms including barley, has been presented (Adamson 1983; Adamson et al. 1980, 1983). In the case of barley the ability to accumulate chlorophyll in the dark was reported to be confined to young expanding leaves which had been exposed to continuous light prior to the dark incubation (Adamson 1983). The light-independent reduction of protochloro-
K. Apel et al. : Is there a light-independent protochlorophyllide reductase?
phyllide to chlorophyllide could be part of an additional route by which chlorophyll is formed in green angiosperms. In the present paper the possible presence of a light-independent protochlorophyllide-reducing enzyme in barley was reinvestigated using an experimental approach which was different from the one which had been used previously (Adamson 1983).
551
!
m
11
1V
E c -4
=o C3
Material
and methods
Plant material. Barley (Hordeum vutgare L., cv. Carina) was grown for 5 d in the dark as described previously (Apel et al. 1980).
L
rr"
Incubation of plants. The synthesis of porphyrins in the dark was measured by feeding 6-[14C] aminolevulinic acid to isolated barley shoots. Dark-grown plants were illuminated for various lengths of time with incandescent white light. The shoots were excised just above the coleoptile sheath and incubated with the labelled compound (1.6-I0 ~ Bq per leaf; 2.109 Bq m M - t 6-aminolevulinic acid) for 2 h in the dark as described previously (Redlinger and Apel 1980). At the end of the incubation the leaves were directly frozen in liquid nitrogen or exposed to white light prior to the freezing step. The frozen leaves were ground with a precooled mortar and a pestle and the pigments extracted with 80% acetone.
A
Liquid chromatography. High-performance liquid chromatogra-
.>
phy (HPLC) was carried out with two Consta Metric pumps (Milton Roy) using a Ct8 reversed-phase column (Spherisorb OD 5, 5 ~tm, 25 cm long 4.6 mm diameter). Pigments were eluted according to the method of Eskins and Harris (1981) with the following modifications : A linear gradient of 20 to 60% ethylacetate in methanol/water (80:20; v/v) was applied to the column at a flow rate of 1 ml min- ~. The pigments were detected spectrophotometrically using a variable wavelength detector (spectro Monitor D ; Milton Roy). The various porphyrins were identified either by recording the elution at different wavelengths (630, 650 and 670 nm) or by coelution of isolated standard pigments. Protochlorophyllide was purified as described elsewhere (Redlinger and Apel 1980). Protochlorophyll was isolated according to Houssier and Sauer (1969). The eluate was fractionated into 250-gl portions. The radioactivity in each fraction was measured by liquid scintillation counting and quenching was corrected for by the external-standard method.
o
b• c6 u~
>,
92 ..... ,. f
/
J.......
"ff. . . . .
I. . . . . . .
L .......
5O
0
B
Fraction
h ......
k ......
L ......
100
number
• c u~
o >,
_~2
Results
A
and discussion
Chlorophyll accumulation in redarkened plants was measured by feeding C14-1abelled 6-aminolevulinic acid to isolated barley shoots. Barley plants were illuminated for 6 h with incandescent white light before the shoots were excised and incubated for 2 h in the dark with the labelled chlorophyll precursor. At the end of the incubation the shoots were either treated for 10 min with continuous white light or were used directly for the extraction of pigments. These samples were separated by reverse-phase HPLC. Protochlorophyllide and chlorophyllide were separated from each other and were
..... ,." }'--"-.J ".....
L
~
50 C
Fraction
,
.....,~.. ' ......9 ~....... 100
number
Fig. 1 A-C. The analysis of porphyrins of barley by HPLC. Shoots were excised from 6-h preilluminated plants and incubated in the dark for 2 h with fi-[14C]aminolevulinic acid. A The absorbance changes at 436 nm during pigment separation, I = chlorophyllide, II = protochlorophyllide, I I I = chlorophyll b, I V - c h l o r o p h y l l a. These compounds were identified as described under Material and methods. B, C The distribution of radioactively labelled porphyrins extracted from leaves which were kept in the dark (B) or received a 15-min light treatment at the end of the dark period (C)
552
K. Apel et al. : Is there a light-independent protochlorophyllide reductase ?
Table 1. The distribution of labelled porphyrins (%) (incorporation of fi-[14C]aminolevulinic acid into porphyrins of greening barley leaves). Shoots were excised from preilluminated barley plants and incubated in the dark for 2 h with fi-aminolevulinic acid as described under Material and methods. The pigments were extracted directly from dark-incubated shoots or following an additional 15-rain light treatment of the shoots. The radioactivity of individual porphyrins was determined after HPLC of the various pigment samples, m E = n o t detectable; L = l i g h t ; D = darkness Light regime
0hL, 0hL, 0hL, 0hL, 24hL, 24hL, 48hL, 48hL, 72hL, 72hL,
2hD 2hD, 2hD 2hD, 2hD 2hD, 2hD 2hD, 2hD 2hD,
15' L 15' L 15' L 15' L 15' L
Protochlorophyllide
Chlorophyllide
Chlorophyll a
Expt. I
Expt. II
Expt. I
Expt. II
Expt. I
Expt. II
99.16 0.83 99.42 30.85 99.91 87.43 99.42 91.08 99.60 76.25
98.92 17.12 99.81 82.41 99.19 75.63 -
1.08 1.98 0.19 1.21 0.81 1.70 -
n.d. 88.01 n.d. 66.24 n.d. 12.05 n.d. 8.70 n.d. 23.60
n.d. 80.90 n.d. 17.59 n.d. 22.67 -
0.84 11.16 0.58 2.92 0.10 0.57 0.58 0.23 0.40 0.15
identified either by coelution of the purified pigment and-or by measuring the absorbance spectrum of the eluted pigment fractions (Fig. 1). In addition to these two pigments, chlorophylls a and b were also resolved and their radioactivity determined. The only porphyrin whose concentration increased during the dark incubation of 6-h-preilluminated plants was protochlorophyllide. The amounts of chlorophyll(ide) a and b did not change significantly during this incubation period. The distribution of radioactivity among the various pigment fractions was consistent with this observation. Only two radioactively labelled major peaks were detected when pigment samples of 6-h-preilluminated barley shoots were separated by HPLC. The first peak was shown to contain free fi-aminolevulinic acid, the second one coeluted with protochlorophyllide (Fig. 1). The identification of the labelled component as protochlorophyllide was confirmed by the result of an illumination experiment. In those shoots which were exposed for 10 min to continuous white light the labelled protochlorophyllide peak decreased by more than 70%. At the same time, new radioactively labelled peaks appeared within the elution profile, whose retention times were identical to those of chlorophyllide a and chlorophyll a (Fig. 1, Table 1). This result indicates that protochlorophyllide is the only. major freshly synthesized prophyrin compound which accumulates in shoots of 6-h-preilluminated barley plants during a subsequent dark incubation. Only traces of radioactively labelled chlorophyllide were found in these plants, far less than 1% of
the radioactivity incorporated into the total fraction of porphyrin (Table 1). Even though at present we cannot exclude the possibility that the formation of these traces of chlorophyllide might be catalyzed by a light-independent protochlorophyllide-reducing enzyme, it seems more likely that exposure of the plants to the green safelight has led to the formation of minute traces of chlorophyltide. It has been reported that the capability of barley and pea plants to synthesize chlorophyll in the dark develops only during the illumination of etiolated plants (Adamson 1983; Adamson etal. 1983). Thus, it is conceivable that the 6 h of illumination which had been used in the initial experiment described above might not be sufficient enough to allow the activation of the light-independent protochlorophyllide reduction. In the following experiments the length of the illumination period was extended to 24, 48 and 72 h before the shoots were excised and incubated with fi-[14C] aminolevulinic acid for 2 h in the dark. In all cases, the labelled precursor was incorporated almost exclusively into protochlorophyllide, Only traces of chlorophyllide were detectable which did not account for more than 0.5% of the total radioactivity of the pigment fraction. The conversion of protochlorophyllide to chlorophyllide occurred only in the light. The amount of phototransformable protochlorophyllide declined steadily with increasing lengths of the preillumination period. In etiolated leaves almost 100% of the protochlorophyllide was photoreduced during the first 10 min of illumination. In shoots which were derived from
K. Apel et al. : Is there a light-independent protochlorophyllide reductase?
6-h-preilluminated plants 70% of the labelled protochlorophyllide was photoreduced. The fraction of photoconvertible protochlorophyllide decreased to less than 30% in samples of plants which had been illuminated for more than 24 h prior to the dark incubation. It is not known yet which factor is responsible for this drastically reduced phototransformation of protochlorophyllide. It is well documented that in the primary leaf of etiolated plants different maturation stages of etioplasts are found in a linear series, with the youngest in cells near the base and the oldest in cells near the tip (Boffey et al. 1980; Robertson and Laetsch 1974). This distribution of different plastid forms is paralleled by drastic differences in the NADPH-protochlorophyllide oxidoreductase content of the plastids and their capacity to accumulate chlorophyll during illumination (Dehesh et al. 1983). Similar differences in the distribution of a light-independent protochlorophyllide-reducing enzyme have been described also for the various parts of barley leaves (Adamson 1983). The primary leaves of excised shoots which had been incubated with 6-[~4C] aminolevulinic acid in the dark were cut into three pieces of equal lengths and the pigments of each segment were extracted and measured separately. In all three segments protochlorophyllide was the only pigment which was labelled radioactively during the dark incubation. Traces of labelled chlorophyllide were present in equal amounts in all three leaf segments and did not exceed 1% of the total incorporated radioactivity (Table 2). There were, however, significant differences in the extent of protochlorophyllide reduction in the three leaf sections. While in the tip part only 40% of the protochlorophyllide was photoreduced, in the middle and the basal parts of the leaf approximately 70% of the protochlorophyllide was phototransformed into chlorophyll(ide) (Table 2). Our results clearly demonstrate that during the dark incubation of excised shoots which had been derived from preilluminated barley plants only protochlorophyllide but not chlorophyll(ide) incorporated substantial amounts of 14C-labelled g-aminolevulinic acid. These results are not consistent with earlier reports on the occurrence of a light-independent protochlorophyllide-reducing enzyme in barley leaves. In the work by Adamson and coworkers (1980, 1983) changes in chlorophyll contents were determined by extracting the pigments from leaves and measuring them spectroscopically. In the present work a different experimental approach was used. The incorporation of 6-[14C] aminolevulinic acid allows one to follow
553
Table 2. The distribution of labelled porphyrins (%) (incorporation of 3-[14C]aminolevulinic acid into porphyrins of different leaf sections of barley plants). Shoots were isolated from 19-h preilluminated barley plants and cut into three pieces of equal lengths. The basal, middle and tip sections were incubated with 6-aminolevulinic acid for 2 h in the dark and were kept in the dark or treated for 10 min with white light before the pigments were extracted. The analysis of the radioactively labelled porphyrins was done as described in Table 1. n.d. = n o t detectable; L = light; D = darkness
Sample
Protochloro- Chlorophyllide phyllide
Chlorophyll a
99.50 59.15
0.50 1.21
n.d. 39.64
Middle part of the leaf 19hL, 2hD 99.58 19hL, 2hD, 10'L 30.23
0.42 0.74
n.d. 69.03
Basal section of the leaf 19hL, 2hD 99.59 19hL, 2hD, 10'L 35.46
0.41 0.13
n.d. 64.41
Tip of the leaf 19hL, 2hD 19hL, 2hD, 10'L
directly the de-novo synthesis of porphyrins. At the moment we cannot explain the apparent discrepancy between the results of these two approaches. One could argue that perhaps an additional pool of cLaminolevulinic acid exists which leads to chlorophyll formation via a dark reduction of protochlorophyllide and which does not exchange with the pool of radioactively labelled precursor. However, as far as we know no evidence exists which supports such an assumption. The breakdown of chlorophyll, which might affect the steady-state levels of pigments (Bennett 1981) did not appear to interfere with the accumulation of radioactively labelled protochlorophyllide. Even after an overnight incubation of excised shoots the amount of accumulated protochlorophyllide did not decrease and, at the same time, no new radioactively labelled porphyrin derivatives could be observed during the HPLC separation of pigment samples (data not shown). The transformation of radioactively labelled protochlorophyllide to chlorophyllide took place only in the light. The almost complete absence of chlorophyll(ide) in dark-incubated shoots provides good evidence that light had been excluded from the incubation assay and that it did not influence the porphyrin synthesis during the dark incubation. A light-independent protochlorophyllide-reducing enzyme might be involved in an alternative route for the synthesis of chlorophyll such as has been proposed previously (Santel and Apel 1981).
554
K. Apel et al. : Is there a light-independent protochlorophyllide reductase?
However, we have failed to detect such an enzyme in barley. The important question of whether or not the NADPH-protochlorophyllide oxidoreductase is responsible for chlorophyll(ide) formation throughout the greening process remains unsolved and will have to await the development of a new experimental approach. This investigation was supported by Deutsche Forschungsgemeinschaft (Ap 16/5-1).
References Adamson, H. (1983) Evidence for a light-independent protochlorophyllide reductase in green barley leaves. In: Cell function and differentiation 2, pp. 33-41, Akoyunoglou, G., Evangelopoilos, A.E., Georgtsos, J., Palaiologos, G., Trakatellis, A., Tsiganos, C.P., eds. Proc. Spec. FEBS Meet., Athens, 1982. Liss, New York Adamson, H., Hiller, R.G., Vesk, M. (1980) Chloroplast development and the synthesis of chlorophyll a and b and chlorophyll protein complexes I and II in the dark in Tradescantia albiflora (Knuth). Planta 150, 269-274 Adamson, H., Packer, N., Sanders, N. (1983) Chlorophyll synthesis in the dark in peas. 6th Int. Congr. Photosynthesis, Brussels, Book of Abstracts, vol. 2, p. 242 Apel, K. (1981) The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Phytochrome-induced decrease of translatable mRNA coding for the NADPH-protochlorophyllide oxidoreductase. Eur. J. Biochem. 120, 89-93 Apel, K., Santel, H.-J., Redlinger, T.E., Falk, H. (1980) The protochlorophyllide holochrome of barley (Hordeum vulgare L.). Isolation and characterization of the NADPH protochlorophyllide oxidoreductase. Eur. J. Biochem. 111, 251-258 Bennett, J. (1981) Biosynthesis of the light-harvesting chlorophyll a/b protein. Polypeptide turnover in darkness. Eur. J. Biochem. 118, 61-70 Boardman, N.K. (1966) Protochlorophyll. In: The chlorophylls, pp. 437-476, Vernon, L.P., Seely, G.R., eds. Academic Press, New York London Boardman, N.K., Anderson, J.M., Goodchild, D.J. (1978) Chlorophyll-protein complexes and structure of mature and developing chloroplasts. Curr. Top. Bioenerg. 8, 35-109 Boffey, S.A., Sellden, G., Leech, R.M. (1980) The influence of cell age on chlorophyll formation in light-grown and etiolated wheat seedlings. Plant Physiol. 65, 680-684 Bogorad, L. (1976) Chlorophyll biosynthesis. In: Chemistry and biochemistry of plant pigments, 2nd edn., vol. 1, pp. 64-148, Goodwin, T.W., ed. Academic Press, New York London
Castelfranco, P.A., Beale, S.T. (1983) Chlorophyll biosynthesis: recent advances and areas of current interest. Annu. Rev. Plant Physiol. 34, 241 278 Dehesh, K., Hfiuser, I., Apel, K., Kloppstech, K. (1983) The distribution of NADPH-protochlorophyllide oxidoreductase in relation to chlorophyll accumulation along the barley leaf gradient. Planta 158, 134-139 Eskins, K., Harris, L. (1981) High-performance liquid chromatography of etioplast pigments in red kidney bean leaves. Photochem. Photobiol. 33, 131-133 Griffiths, W.T. (1974) Protochlorophyll and protochlorophyllide as precursors for chlorophyll synthesis in vitro. FEBS Lett. 49, 196-200 Griffiths, W.T. (1978) Reconstitution of chlorophyllide formation by isolated etioplast membranes. Biochem. J. 174, 681-692 Hfiuser, I., Dehesh, K., Apel, K. (1984) The proteolytic degradation in vitro of the NADPH-protochlorophyllide oxidoreductase of barley (Hordeum vulgare L.). Arch. Biochem. Biophys. 228, 577-586 Houssier, C., Sauer, K. (1969) Optical properties of the pr 0tochlorophyll pigments. I. Isolation, characterization, and infrared spectra. Biochim. Biophys. Acta 172, 476-491 Ikeuchi, M., Murakami, S. (1982) Behavior of the 36000 dalton protein in the internal membranes of squash etioplasts during greening. Plant Cell Physiol. 23, 575-583 Kay, S.A., Griffiths, W.T. (1983) Light-induced breakdown of NADPH-protochlorophyllide oxidoreductase in vitro. Plant PhysioL 72, 229-236 Kirk, J.T.O., Tilney-Bassett, R.A.E. (1978) The plastids. Freeman, London San Francisco Mapleston, E.R., Griffiths, W.T. (1980) Light modulation of the activity of protochlorophyllide reductase. Biochem. J. 189, 125-133 Meyer, G., Bliedung, H., Kloppstceh, K. (1983) NADPH-protochlorophyllide oxidoreductase: reciprocal regulation in mono- and dicotyledonean plants. Plant Cell Reports 2, 26-29 Redlinger, T.E., Apel, K. (1980) The effect of light on four protochlorophyllide-binding polypeptides of barley (Hordeum vulgate). Arch. Biochem. Biophys. 200, 253-260 Robertson, D., Laetsch, M. (1974) Structure and function of developing barley plastids. Plant Physiol. 54, 148-159 Santel, H.L, Apel, K. (1981) The protochlorophyllide holtchrome of barley (Hordeum vulgate L.). The effect of light on the NADPH: protochlorophyllide oxidoreductase. Eur. J. Biochem. 120, 95-103 Schopfer, P., Apel, K. (1983) Intracellular photomorphogenesis. In: Encyclopedia of plant physiology, N.S., vol. 16: Photomorphogenesis, pp. 258-288, Shropshire, W., jr., Mohr, H., eds. Springer, Berlin Heidelberg New York Received 27 December 1983; accepted 15 March 1984
