- Email: [email protected]
Formation of prolamellar bodies without correlative accumulation of protochlorophyllide or chlorophyllide in pine cotyledons
Plant Science Letters, 2 (1974) 45--54 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
FORMATION OF PROLAMELLAR BODIES WITHOUT CORRELATIVE ACCUMULATION OF PROTOCHLOROPHYLLIDE OR CHLOROPHYLLIDE IN PINE COTYLEDONS
M.R. MICHEL-WOLWERTZand R. BRONCHART Laboratory of Biochemical Systematics and Laboratory of Plant Morphology, Department of Botany, University of LiJge (Belgium) (Received June 12th, 1973) (Revision received August 24th, 1973)
SUMMARY Chloroplast formation and accumulation of chlorophyllous pigments were studied in pine cotyledons (Pinus jeffreyi) during germination in complete darkness or under continuous light. During the first days of germination (in the light and in the dark), cotyledons grew inside the seed; at this moment, no membranes were visible in proplastids and no chlorophyllous pigments could be detected. When the radicle broke the seed integuments, the cotyledons had developed green plastids. These contained crystalline prolamellar bodies as well as lamellae and grana. The same plastid structure was found in fully expanded cotyledons from dark-grown pine seedlings. Under continuous light, even when cotyledons emerged from the seed integuments, the plastids still contained prolamellar bodies. The prolamellar bodies remained for several days but gradually decreased and finally disappeared. Analysis of pigment content could not establish a correlation between the formation of prolamellar bodies and the accumulation of protochlorophyllide a or/and chlorophyllide a.
INTRODUCTION When grown in the dark, angiosperm seedlings develop etioplasts which contain prolamellar bodies bearing a few perforated double membrane sheets (for areview, see ref. 1). Protochlorophyll(-ide) a is the only chlorophyll present in these plastids. However, a few angiosperms have some ability to synAbbreviation: F.W., fresh weight. 45
thesize chlorophyll in the dark: Goodwin and Owens 2, for instance, have reported that seedlings of oats form chlorophyll a in the absence of light; R6bbelen 3 found traces of chlorophyll a in etio|ated ~eedlings of Arabidopsis thaliana. Upon illumination, prolamellar bodies usually lose their regularity and, according to many authors, are progressively transformed into "an irreguhr network of tubules". This process is correlated with the photoconversion of protochlorophyllide a into chlorophyllide a 4 or the phytylation of chlorophyllide a to chlorophyll a s. Protochl~rophyll(-ide) a has been proposed by m~ny authors 6-9 to be a constituent of prolamellar body membrane. According to Henningsen and Boynton 1o and Weier and Brown ~1, the formation of prolamellar bodies in etioplasts of dark-grown angiosperms is dependent o~ the synthesis of protochlorophyll(-ide). In etiolated bean leaves, Dujardin ~nd Sironval ~2 and Klein and Schiff 13 found three protochlorophyll(-ide) a forms which they characterized by the in vivo absorption maxima at 628 nm (P62s), 635 nm (P63s) ~nd 650 nm (P6s0), respectively. The P63s and P6s0 were phototransformable whereas the form P62s was not transformable by light. Klein and Schiff ~3 observed a correlation between the accumulation of P6s0 and the formation and enlargement of prolamellar bodies. Most gymnosperms, especially conifers, can make chlorophylls in complete darkness ~4-~6. Protochlorophyllide and chlorophyllide have been reported ~7 to be present as well as chlorophyll in dark-grown seedlings of three species of gymnos.perms: Pinus silvestris, Picea exeelsa, Larix sibirica (for a review, see ref. 18). Chloroplasts of conifer seedlings developed in the dark contain lamellae, grana and also characteristic paracrystalline prolamellar bodies ~9- 21. It was of interest to investigate whether such plastids accumulate protochlorophyll(-ide) a as do angiosperm etioplasts and also to follow the evolution of the prolamellar bodies upon illumination. We present here details about plastid structure and pigment composition of Pinus jeffreyi cotyledons grown either in the dark or in the light. MATERIALS AND METHODS
Plan t materials The seeds of Pinus jeffreyi (obtained from Vilmorin, Paris, Prance) were germinated on vermiculite and tap water in rooms, under continuous light, or in complete darkness at 20--23 °. Illuminated plants received 1000 lux; this intensity was obtained with daylight supplemented with continuous light from fluorescent tubes. Dark-grown seedlings were handled in the dark or under a dim green safelight. At different stages of germination, cotyledons were studied with respect to plastid structure and pigment composition.
46
Electron microscopy Cotyledons were prefixed during a period of 3--4 h with 4% glutaraldehyde in a Na-cacodylate buffer (pH 7.0) and post-fixed during another period of 3--4 h with 2% OsO4 in distilled water. They were then dehydrated with ethanol and embedded in Epon. After sectioning the material was doublestained in uranyl acetate and lead citrate according to Reynolds 22. Then the sections were coated with a carbon film. The observations were carried out with a Siemens Elmiskop 101 electron microscope (tension 80 kV). Pigment composition Pigments were extracted in 80% acetone by grinding the cotyledons with sand in a mortar. The extracts were then centrifuged. Chlorophyll a and chlorophyll b contents of the extracts were calculated according to the equations of Mackinney 23 . Protochlorophyll(-ide) a and chlorophyllide a were detected by fluorescence measurements (in vivo and in vitro) or by thin-layer chromatography of acetone extracts, respectively. Fluorescence measurements We measured fluorescence emission spectra of whole cotyledons and of acetone extracts of cotyledons at liquid nitrogen temperature and at room temperature, respectively. Spectra at liquid nitrogen temperature were recorded with an instrument described by Sironval et al. 24 equipped with an EMI 9558 B photomultiplier. For fluorescence measurements at room temperature the Dewar flask was replaced by a Zeiss ZFM4 sample holder. Thin-layer chromatography The pigments were transferred from 80% acetone to diethyl ether and then separated by thin-layer chromatography according to Bacon 55. RESULTS
Plastid structure: During the first days of germination (in the light and in the dark) the embryo of Pinus jeffreyi grew inside the seed, cotyledons elongated but remained yellowish; plastid structure was difficult to recognize (Fig. 1) and chlorophyllous pigments were not detectible by means of fluorescence measurements. When the radicle broke through the integuments of the seed (6--12 days after sowing) cotyledons were still entirely embedded in the endosperm but they had become pale green in colour. Plastids could easily be observed (Fig. 2). At this stage, cotyledon plastids from pine cultivated under continuous light ("light plastids") had the same structure as cotyledon plastids from dark-grown pine ("dark plastid~"). The plastids were delimited by a double membrane; they contained typical crystalline prolamellar bodies, single lamellae and also grana (Fig. 3). They were full of big starch grains. Usually more than one prolamellar body was observed per plastid. When cotyledons were 47
,
i.
i
),
".
~v
~ .
.
. J " "
.
-,
p -
°
~
,
°
.
i
~-.~,
¢c"
~,,~,
4
Fit~. 1 Portion o f a c e l l f r o a l a c o t y l e d o n o f P t u u s j e f f r e y i 3 d a y s after plantitlg in t h e dark. No m e m b r a i l e s are visible w i t h i n p r o p l a s t i d s w h i c h are o n l y ree()gnizable by t h e p r e s e n c e o f ~t,~rch ~rain,,,. tP, pr()ph~slid, S, starch grains), x 12 000. i'i~. 2. Plastid s t r u c t u r e in a dark-~rown pine c o t y l e d o n 1. 2 d a y s after planting. At this sta~e of ~erminati(~n, c o t y l e d o n s were still e m b e d d e d in e n d o s p e r m , but the radicle had br()ken t h r o u g h the seed i n t e g u m e n t s . T h e p r o l a m e l l a r b o d y as well as ~rana a n d v o l u m i n o u s stai'ch ~rains :~re ~isible. ,: 24 (100. -i ,",
•
.~..
~,
'ai
Fig. 3. Portion of a plastid from cotyledon of pine seedlings grown in darkness for 1 2 days. A large crystalline prolarnellar body and several grana with up to 5 to 8 disks are seen. × 90 000.
.19
fully expanded, "dark plastids" still showed a similar structure but the number of prolamellar bodies diminished and the number of lamellae and grana increased. Fig. 4 shows plastids from cotyledons of pine seedlings (12 days old) grown under continuous light. At this stage, cotyledons were coming out of the integuments and we observed the portions which emerged outside the teguments and were exposed to light. These plastids showed lamellae, ~'ana and also crystalline prolamellar bodies. Compared to "dark plastids" from cotyledons of the same age, "light plastids" were enriched in lamellae and grana and ~he size of the prolameUar bodies was smaller (compare Fig. 4 and Fig. 2). During a few days in the light, prolamellar bodies kept their regular, crystalline structure but progressively, their size decreased though the number of grana and lamellae increased. In cotyledon plastids of pine seedlings kept under continuous light for 3--4 weeks, no prolamellar bodies were found; plastids only contained lamellae and grana (Fig. 5).
Pigment composition: We measured the chlorophyll contents of acetone extracts from pine seedlings grown for 2 weeks either under continuous light or in complete darkness. Seedlings grown in the light contained 130 pg chl (a + b)/100 mg F.W.~ the a/b ratio varied from 1.8 to 2.3. Seedlings grown in complete darkness contained less chlorophyll: 65 pg chl ta + b)/100 mg F.W.; the a/b ratio was higher: 2.2 to 2.7. We registered fluorescence emission spectra (at --196 ° ) of cotyledons from pine seedlings cultivated for different times in the dark or in the light. As soon as fluorescence was detectible in cotyledons (grown in the dark or under continuous light), the emission spectrum showed a main band at 696 nm; a second peak was found near 730 nm. No distinct peak appeared in the 630-650 nm region where protochlorophyll(-ide) fluoresces. Similar fluorescence emission spectra (see, for instance, Fig. 6) were always obtained for pine cotyledons whatever their age and whatever the conditions of culture (continuous light or darkness). In Fig. 7, we compared the fluorescence emission spectrum (at room temperature) of an acetone extract of cotyledons grown under complete darkness (cotyledons with plastid structure as in Fig. 2), with the spectrum of an acetone extract of cotyledons grown under continuous light (cotyledons with plastid structure as in Fig. 5). Both spectra showed an intense emission band at 668 nm; a weak emission band was visible around 630--635 nm. This means that in both extracts the bulk of green pigments consisted of chlorophylls. If protochlorophyll(-ide) was present, it would be in t~.aces, as the emission band located at 630--635 nm ~,as feeble, even in the extract from dark-grown cotyledons, in which the plastids contained many prolamellar bodies. Thin-layer chromatography of concentrated extracts from dark cotyledons (plastid structure as in Fig. 2) showed in addition to pheophytin and carotenoids, two green spots: chlorophyll a and chlorophyll b. No green pigment 50
• .+*+
~++'-
, +
,.+"
.~*'+~?.~.
~ "t'~ t:.
L
.
+. ~.++
s
.~:2+: % ~ .
+
4 .
.
.
.
+.5 Fig. 4. Plastid structure in pine cotyledons cultivated during 12 days under continuous light. At this stage, cotyledons were coming out of the seed integuments and sections had been made in the cotyledon portion exposed to continuous light for 48 h. Three prolamellar bodies interlinked by grana and single thylakoids are visible in this picture. × 18 000. Fig. 5. Plastid in Pinus jeffreyi cotyledons cultivated for 3 weeks under continuous illumination. Prolamellar bodies are no longer visible, grana and single thylakoids extend throughout all the plastid volume. × 15 000. 51
FL UORE SCE NCE (RELATIVE:
FLUORESCENCE ~REL ATi vIE) °
668 p 696
I
7~0-32
/ •
lO0
750
(n m)
600
650
'
7*0o '
|
~. (rim)
Fig. 6. Fluorescence emission spectrum (at--196 ° ) of Pinusjeffreyi cotyledons 12 days after planting in darkness (excitation wavelength: 436 nm). Spectrum was not corrected for wavelength variation of photomultiplier response. Fig. 7. Fluorescence emission spectra (at room temperature) of acetone extracts from pine cotyledons 8 days after planting in complete darkness ( ~ ) and 28 days after planting under continuous light (------); excitation wavelength: 436 nm. Dark-grown seedlings
had cotyledons buried in the endosperm and radicle just coming out of the seed integuments. Cotyledons from seedlings cultivated under continuous light were fully expanded. Spectra were not corrected for wavelength variation of photomultiplier response. was visible even under UV light at the starting line of the chromatogram where u n p h y t y l a t e d pigments (protochlorophyllide a and chlorophyllide a) are usually located. This indicates that if protochlorophyll pigments were present in dark-grown pine cotyledons, they were not due to pretochlorophyllide a, b u t it does not exclude the possibility that traces of protochlorophyll a ( p h y t y l a t e d pigment) might be present. DISCUSSION The first plastids which developed during germination (in the light o: i , the dark) of pine, while cotyledons were still embedded in endosperrrJ, contained lamellae, grana and regular prolamellar bodies. Compared w~th angiosperms, this piastid structure reminds one of b o t h the structure of chloroplast and of etioplast. Prolamellar bodies from pine cotyledons showed the same regular structur~ as those of etiolated angiosperm leaves; t h e y were usually smaller b u t t h e y were more numerous per plastid. Upon illumination, angiosperm prolamellar bodies usually lose their crystalline configuration and the membranes are rearranged into lamellae. In certain cases, however, regular prolamellar bodies can persist for some time after dark-grown angiosperms are illuminated ~0. Treffry 26reported the develop52
ment of regular prolamellar bodies in etiolated peas exposed to red light. These facts are to be related to our results: in pine, the crystalline configuration of prolamellar bodies persisted for several days in the light, while the volume of prolamellar bodies progressively reduced. Many authors 6-9 proposed that in angiosperms, prolamellar bodies are the site of phototransformable protochlorophyll(-ide) a which absorbs in vivo at 650 nm (P6s0). According to Weier and Brown ~ and Henningsen and Boynton 10, protochlorophyllide would be an indispensable component for the formation of prolamellar bodies of angiosperms. Treffry 26, however, reported that prolamellar bodies can remain regular and even be formed in the absence of protochlorophyllide. He suggested that these prolamellar bodies may contain chlorophyllide and concluded that in general, it is the accumulation of an unphytylated form of chlorophyll (protochlorophyllide or chlorophyllide) which is associated with the development of prolamellar bodies. Such conclusions can hardly be drawn in the case of prolamellar bodies of cotyledons from Pinus jeffreyi: (1) Dark-grown cotyledons did not show at any stage of germination a fluorescence emission band in vivo at --196 ° in the 630--650 nm region (Fig. 6), where the different protochlorophyll(-ide) forms fluoresce, although their plastids contained numerous prolamellar bodies. (2) During germination in the dark, the first chlorophyll pigment appearing in cotyledons was chlorophyll a: as soon as fluorescence was detectible in an acetone extract from cotyledons of pine germinating in darkness, the emission band was located near 668 nm (unpublished data). (3) No traces of protochlorophyllide a or chlorophyUide a have been detected by chromatography of acetone extract from dark-grown cotyledons. If it can be assumed that the prolamellar body in gymnosperms has the same significance and the same structure as in angiosperms, the present findings could indicate that, protochlorophyllide or/and chlorophyllide a are not necessary for the formation of the prolamellar bodies. Since in our conditions, prolamellar bodies were found in plastids from dark-grown and light.grown cotyledons, it could be supposed that in gymnosperm cotyledons prolamellar bodies are an obligate transient in the development of proplastids into chloroplasts. ACKNOWLEDGEMENTS
Thanks are due to Miss F. Cogniaux for her help in ultramicrotomy. This investigation was supported in part by the Fonds National de la Recherche Scientifique, Cr6dit aux Chercheurs, and by the Fonds National de la Recherche Fondamentaie Collective, Grant No. 10.118.
53
REFERENCES J.T.O. Kirk and R.A.E. TilneyoBassett, The Piastids, Freeman, London, 1967. R.H. Goodwin and O.H. Owens, Plant Physiol., 22 (1947) 197. G. R~bbelen, P~an~a, 47 (1956) 532. K.W. Henningsen, An action spectrum for vesicle dispersal in bean plastids, in T.W. Goodwin, Biochemistry c f the Chloroplast, Vol. lI, Academic Press, New York, 1967, p. 453. 5 T. Treffry, Planta, 91 (1970) 279. 6 N.K. Boardman and S.G. Wildman, Biochim. Biophys. Acta, 59 (1962) 222. 7 L. Bogorad, Biosynthesis and morphogenesis in plastids, in T.W. Goodwin, Biochemistry of Chloroplasts, Voi. II, Academic Press, London, 1967, p. 615. 8 A. Kahn, Plant Physiol., 43 (1968) 1769. 9 D. Lafl~che, J.M. Bov~ and J. Duranton, J. Ultrastruct. Res., 40 (1972) 205. 10 K.W. Henningsen and J.E. Boynton, J. Cell Biol., 44 (1970) 290. 11 T.E. Weier and D.L. Brown, Am. J. Botany, 57 (1970) 267. 12 E. Dujardin and C. Sironval, Photosynthetica, 4 (2) (1970) 129. 13 S. Klein and J.A. Schiff, Plant Physiol., 49 (1972) 619. 14 A. Burgerstein, Bet. Deut. Bot. Ges., 18 (1900) 168. 15 V. Lubimenko, Rev. Gen. Bot., 40 (1928) 88. 16 L. Bogorad, Botanical Gazette, 111 (1950)221. 17 E.G. Sudyina, Photochem. Photobiol., 2 (1963) 181. 18 N.K. Boardman, Protochorophyll, in L.P. Vernon and G.R. Seely, The Chlorophylls, Academic Press, London, 1966, p. 455. 19 D. yon Wettstein, Brookl. Symp. BioL, 11 (1958) 138. 20 H. Camefort, Compt. Rend., 257 (1963) 2876. 21 D. Nikoli~ and M. Bogdanovi~, Protoplasma, 75 (1972) 205. 22 E.S. Reynolds, J. CellBiol., 17 (1963) 208. 23 F.G. Mackinney, J. Biol. Chem., 140 (.1941) 315.. 24 C. Sironvai, M. Brouers, J.-M. Michel and J. Kuiper, Photosynthetica, 2 (4~ (1968) 268. 25 H.F. B,con, J. Chromatog., 17 (1965) 322. 26 T. Treffry, J. Exptl. Botany, 24 (1973) -85. I 2 3 4
FORMATION OF PROLAMELLAR BODIES WITHOUT CORRELATIVE ACCUMULATION OF PROTOCHLOROPHYLLIDE OR CHLOROPHYLLIDE IN PINE COTYLEDONS
M.R. MICHEL-WOLWERTZand R. BRONCHART Laboratory of Biochemical Systematics and Laboratory of Plant Morphology, Department of Botany, University of LiJge (Belgium) (Received June 12th, 1973) (Revision received August 24th, 1973)
SUMMARY Chloroplast formation and accumulation of chlorophyllous pigments were studied in pine cotyledons (Pinus jeffreyi) during germination in complete darkness or under continuous light. During the first days of germination (in the light and in the dark), cotyledons grew inside the seed; at this moment, no membranes were visible in proplastids and no chlorophyllous pigments could be detected. When the radicle broke the seed integuments, the cotyledons had developed green plastids. These contained crystalline prolamellar bodies as well as lamellae and grana. The same plastid structure was found in fully expanded cotyledons from dark-grown pine seedlings. Under continuous light, even when cotyledons emerged from the seed integuments, the plastids still contained prolamellar bodies. The prolamellar bodies remained for several days but gradually decreased and finally disappeared. Analysis of pigment content could not establish a correlation between the formation of prolamellar bodies and the accumulation of protochlorophyllide a or/and chlorophyllide a.
INTRODUCTION When grown in the dark, angiosperm seedlings develop etioplasts which contain prolamellar bodies bearing a few perforated double membrane sheets (for areview, see ref. 1). Protochlorophyll(-ide) a is the only chlorophyll present in these plastids. However, a few angiosperms have some ability to synAbbreviation: F.W., fresh weight. 45
thesize chlorophyll in the dark: Goodwin and Owens 2, for instance, have reported that seedlings of oats form chlorophyll a in the absence of light; R6bbelen 3 found traces of chlorophyll a in etio|ated ~eedlings of Arabidopsis thaliana. Upon illumination, prolamellar bodies usually lose their regularity and, according to many authors, are progressively transformed into "an irreguhr network of tubules". This process is correlated with the photoconversion of protochlorophyllide a into chlorophyllide a 4 or the phytylation of chlorophyllide a to chlorophyll a s. Protochl~rophyll(-ide) a has been proposed by m~ny authors 6-9 to be a constituent of prolamellar body membrane. According to Henningsen and Boynton 1o and Weier and Brown ~1, the formation of prolamellar bodies in etioplasts of dark-grown angiosperms is dependent o~ the synthesis of protochlorophyll(-ide). In etiolated bean leaves, Dujardin ~nd Sironval ~2 and Klein and Schiff 13 found three protochlorophyll(-ide) a forms which they characterized by the in vivo absorption maxima at 628 nm (P62s), 635 nm (P63s) ~nd 650 nm (P6s0), respectively. The P63s and P6s0 were phototransformable whereas the form P62s was not transformable by light. Klein and Schiff ~3 observed a correlation between the accumulation of P6s0 and the formation and enlargement of prolamellar bodies. Most gymnosperms, especially conifers, can make chlorophylls in complete darkness ~4-~6. Protochlorophyllide and chlorophyllide have been reported ~7 to be present as well as chlorophyll in dark-grown seedlings of three species of gymnos.perms: Pinus silvestris, Picea exeelsa, Larix sibirica (for a review, see ref. 18). Chloroplasts of conifer seedlings developed in the dark contain lamellae, grana and also characteristic paracrystalline prolamellar bodies ~9- 21. It was of interest to investigate whether such plastids accumulate protochlorophyll(-ide) a as do angiosperm etioplasts and also to follow the evolution of the prolamellar bodies upon illumination. We present here details about plastid structure and pigment composition of Pinus jeffreyi cotyledons grown either in the dark or in the light. MATERIALS AND METHODS
Plan t materials The seeds of Pinus jeffreyi (obtained from Vilmorin, Paris, Prance) were germinated on vermiculite and tap water in rooms, under continuous light, or in complete darkness at 20--23 °. Illuminated plants received 1000 lux; this intensity was obtained with daylight supplemented with continuous light from fluorescent tubes. Dark-grown seedlings were handled in the dark or under a dim green safelight. At different stages of germination, cotyledons were studied with respect to plastid structure and pigment composition.
46
Electron microscopy Cotyledons were prefixed during a period of 3--4 h with 4% glutaraldehyde in a Na-cacodylate buffer (pH 7.0) and post-fixed during another period of 3--4 h with 2% OsO4 in distilled water. They were then dehydrated with ethanol and embedded in Epon. After sectioning the material was doublestained in uranyl acetate and lead citrate according to Reynolds 22. Then the sections were coated with a carbon film. The observations were carried out with a Siemens Elmiskop 101 electron microscope (tension 80 kV). Pigment composition Pigments were extracted in 80% acetone by grinding the cotyledons with sand in a mortar. The extracts were then centrifuged. Chlorophyll a and chlorophyll b contents of the extracts were calculated according to the equations of Mackinney 23 . Protochlorophyll(-ide) a and chlorophyllide a were detected by fluorescence measurements (in vivo and in vitro) or by thin-layer chromatography of acetone extracts, respectively. Fluorescence measurements We measured fluorescence emission spectra of whole cotyledons and of acetone extracts of cotyledons at liquid nitrogen temperature and at room temperature, respectively. Spectra at liquid nitrogen temperature were recorded with an instrument described by Sironval et al. 24 equipped with an EMI 9558 B photomultiplier. For fluorescence measurements at room temperature the Dewar flask was replaced by a Zeiss ZFM4 sample holder. Thin-layer chromatography The pigments were transferred from 80% acetone to diethyl ether and then separated by thin-layer chromatography according to Bacon 55. RESULTS
Plastid structure: During the first days of germination (in the light and in the dark) the embryo of Pinus jeffreyi grew inside the seed, cotyledons elongated but remained yellowish; plastid structure was difficult to recognize (Fig. 1) and chlorophyllous pigments were not detectible by means of fluorescence measurements. When the radicle broke through the integuments of the seed (6--12 days after sowing) cotyledons were still entirely embedded in the endosperm but they had become pale green in colour. Plastids could easily be observed (Fig. 2). At this stage, cotyledon plastids from pine cultivated under continuous light ("light plastids") had the same structure as cotyledon plastids from dark-grown pine ("dark plastid~"). The plastids were delimited by a double membrane; they contained typical crystalline prolamellar bodies, single lamellae and also grana (Fig. 3). They were full of big starch grains. Usually more than one prolamellar body was observed per plastid. When cotyledons were 47
,
i.
i
),
".
~v
~ .
.
. J " "
.
-,
p -
°
~
,
°
.
i
~-.~,
¢c"
~,,~,
4
Fit~. 1 Portion o f a c e l l f r o a l a c o t y l e d o n o f P t u u s j e f f r e y i 3 d a y s after plantitlg in t h e dark. No m e m b r a i l e s are visible w i t h i n p r o p l a s t i d s w h i c h are o n l y ree()gnizable by t h e p r e s e n c e o f ~t,~rch ~rain,,,. tP, pr()ph~slid, S, starch grains), x 12 000. i'i~. 2. Plastid s t r u c t u r e in a dark-~rown pine c o t y l e d o n 1. 2 d a y s after planting. At this sta~e of ~erminati(~n, c o t y l e d o n s were still e m b e d d e d in e n d o s p e r m , but the radicle had br()ken t h r o u g h the seed i n t e g u m e n t s . T h e p r o l a m e l l a r b o d y as well as ~rana a n d v o l u m i n o u s stai'ch ~rains :~re ~isible. ,: 24 (100. -i ,",
•
.~..
~,
'ai
Fig. 3. Portion of a plastid from cotyledon of pine seedlings grown in darkness for 1 2 days. A large crystalline prolarnellar body and several grana with up to 5 to 8 disks are seen. × 90 000.
.19
fully expanded, "dark plastids" still showed a similar structure but the number of prolamellar bodies diminished and the number of lamellae and grana increased. Fig. 4 shows plastids from cotyledons of pine seedlings (12 days old) grown under continuous light. At this stage, cotyledons were coming out of the integuments and we observed the portions which emerged outside the teguments and were exposed to light. These plastids showed lamellae, ~'ana and also crystalline prolamellar bodies. Compared to "dark plastids" from cotyledons of the same age, "light plastids" were enriched in lamellae and grana and ~he size of the prolameUar bodies was smaller (compare Fig. 4 and Fig. 2). During a few days in the light, prolamellar bodies kept their regular, crystalline structure but progressively, their size decreased though the number of grana and lamellae increased. In cotyledon plastids of pine seedlings kept under continuous light for 3--4 weeks, no prolamellar bodies were found; plastids only contained lamellae and grana (Fig. 5).
Pigment composition: We measured the chlorophyll contents of acetone extracts from pine seedlings grown for 2 weeks either under continuous light or in complete darkness. Seedlings grown in the light contained 130 pg chl (a + b)/100 mg F.W.~ the a/b ratio varied from 1.8 to 2.3. Seedlings grown in complete darkness contained less chlorophyll: 65 pg chl ta + b)/100 mg F.W.; the a/b ratio was higher: 2.2 to 2.7. We registered fluorescence emission spectra (at --196 ° ) of cotyledons from pine seedlings cultivated for different times in the dark or in the light. As soon as fluorescence was detectible in cotyledons (grown in the dark or under continuous light), the emission spectrum showed a main band at 696 nm; a second peak was found near 730 nm. No distinct peak appeared in the 630-650 nm region where protochlorophyll(-ide) fluoresces. Similar fluorescence emission spectra (see, for instance, Fig. 6) were always obtained for pine cotyledons whatever their age and whatever the conditions of culture (continuous light or darkness). In Fig. 7, we compared the fluorescence emission spectrum (at room temperature) of an acetone extract of cotyledons grown under complete darkness (cotyledons with plastid structure as in Fig. 2), with the spectrum of an acetone extract of cotyledons grown under continuous light (cotyledons with plastid structure as in Fig. 5). Both spectra showed an intense emission band at 668 nm; a weak emission band was visible around 630--635 nm. This means that in both extracts the bulk of green pigments consisted of chlorophylls. If protochlorophyll(-ide) was present, it would be in t~.aces, as the emission band located at 630--635 nm ~,as feeble, even in the extract from dark-grown cotyledons, in which the plastids contained many prolamellar bodies. Thin-layer chromatography of concentrated extracts from dark cotyledons (plastid structure as in Fig. 2) showed in addition to pheophytin and carotenoids, two green spots: chlorophyll a and chlorophyll b. No green pigment 50
• .+*+
~++'-
, +
,.+"
.~*'+~?.~.
~ "t'~ t:.
L
.
+. ~.++
s
.~:2+: % ~ .
+
4 .
.
.
.
+.5 Fig. 4. Plastid structure in pine cotyledons cultivated during 12 days under continuous light. At this stage, cotyledons were coming out of the seed integuments and sections had been made in the cotyledon portion exposed to continuous light for 48 h. Three prolamellar bodies interlinked by grana and single thylakoids are visible in this picture. × 18 000. Fig. 5. Plastid in Pinus jeffreyi cotyledons cultivated for 3 weeks under continuous illumination. Prolamellar bodies are no longer visible, grana and single thylakoids extend throughout all the plastid volume. × 15 000. 51
FL UORE SCE NCE (RELATIVE:
FLUORESCENCE ~REL ATi vIE) °
668 p 696
I
7~0-32
/ •
lO0
750
(n m)
600
650
'
7*0o '
|
~. (rim)
Fig. 6. Fluorescence emission spectrum (at--196 ° ) of Pinusjeffreyi cotyledons 12 days after planting in darkness (excitation wavelength: 436 nm). Spectrum was not corrected for wavelength variation of photomultiplier response. Fig. 7. Fluorescence emission spectra (at room temperature) of acetone extracts from pine cotyledons 8 days after planting in complete darkness ( ~ ) and 28 days after planting under continuous light (------); excitation wavelength: 436 nm. Dark-grown seedlings
had cotyledons buried in the endosperm and radicle just coming out of the seed integuments. Cotyledons from seedlings cultivated under continuous light were fully expanded. Spectra were not corrected for wavelength variation of photomultiplier response. was visible even under UV light at the starting line of the chromatogram where u n p h y t y l a t e d pigments (protochlorophyllide a and chlorophyllide a) are usually located. This indicates that if protochlorophyll pigments were present in dark-grown pine cotyledons, they were not due to pretochlorophyllide a, b u t it does not exclude the possibility that traces of protochlorophyll a ( p h y t y l a t e d pigment) might be present. DISCUSSION The first plastids which developed during germination (in the light o: i , the dark) of pine, while cotyledons were still embedded in endosperrrJ, contained lamellae, grana and regular prolamellar bodies. Compared w~th angiosperms, this piastid structure reminds one of b o t h the structure of chloroplast and of etioplast. Prolamellar bodies from pine cotyledons showed the same regular structur~ as those of etiolated angiosperm leaves; t h e y were usually smaller b u t t h e y were more numerous per plastid. Upon illumination, angiosperm prolamellar bodies usually lose their crystalline configuration and the membranes are rearranged into lamellae. In certain cases, however, regular prolamellar bodies can persist for some time after dark-grown angiosperms are illuminated ~0. Treffry 26reported the develop52
ment of regular prolamellar bodies in etiolated peas exposed to red light. These facts are to be related to our results: in pine, the crystalline configuration of prolamellar bodies persisted for several days in the light, while the volume of prolamellar bodies progressively reduced. Many authors 6-9 proposed that in angiosperms, prolamellar bodies are the site of phototransformable protochlorophyll(-ide) a which absorbs in vivo at 650 nm (P6s0). According to Weier and Brown ~ and Henningsen and Boynton 10, protochlorophyllide would be an indispensable component for the formation of prolamellar bodies of angiosperms. Treffry 26, however, reported that prolamellar bodies can remain regular and even be formed in the absence of protochlorophyllide. He suggested that these prolamellar bodies may contain chlorophyllide and concluded that in general, it is the accumulation of an unphytylated form of chlorophyll (protochlorophyllide or chlorophyllide) which is associated with the development of prolamellar bodies. Such conclusions can hardly be drawn in the case of prolamellar bodies of cotyledons from Pinus jeffreyi: (1) Dark-grown cotyledons did not show at any stage of germination a fluorescence emission band in vivo at --196 ° in the 630--650 nm region (Fig. 6), where the different protochlorophyll(-ide) forms fluoresce, although their plastids contained numerous prolamellar bodies. (2) During germination in the dark, the first chlorophyll pigment appearing in cotyledons was chlorophyll a: as soon as fluorescence was detectible in an acetone extract from cotyledons of pine germinating in darkness, the emission band was located near 668 nm (unpublished data). (3) No traces of protochlorophyllide a or chlorophyUide a have been detected by chromatography of acetone extract from dark-grown cotyledons. If it can be assumed that the prolamellar body in gymnosperms has the same significance and the same structure as in angiosperms, the present findings could indicate that, protochlorophyllide or/and chlorophyllide a are not necessary for the formation of the prolamellar bodies. Since in our conditions, prolamellar bodies were found in plastids from dark-grown and light.grown cotyledons, it could be supposed that in gymnosperm cotyledons prolamellar bodies are an obligate transient in the development of proplastids into chloroplasts. ACKNOWLEDGEMENTS
Thanks are due to Miss F. Cogniaux for her help in ultramicrotomy. This investigation was supported in part by the Fonds National de la Recherche Scientifique, Cr6dit aux Chercheurs, and by the Fonds National de la Recherche Fondamentaie Collective, Grant No. 10.118.
53
REFERENCES J.T.O. Kirk and R.A.E. TilneyoBassett, The Piastids, Freeman, London, 1967. R.H. Goodwin and O.H. Owens, Plant Physiol., 22 (1947) 197. G. R~bbelen, P~an~a, 47 (1956) 532. K.W. Henningsen, An action spectrum for vesicle dispersal in bean plastids, in T.W. Goodwin, Biochemistry c f the Chloroplast, Vol. lI, Academic Press, New York, 1967, p. 453. 5 T. Treffry, Planta, 91 (1970) 279. 6 N.K. Boardman and S.G. Wildman, Biochim. Biophys. Acta, 59 (1962) 222. 7 L. Bogorad, Biosynthesis and morphogenesis in plastids, in T.W. Goodwin, Biochemistry of Chloroplasts, Voi. II, Academic Press, London, 1967, p. 615. 8 A. Kahn, Plant Physiol., 43 (1968) 1769. 9 D. Lafl~che, J.M. Bov~ and J. Duranton, J. Ultrastruct. Res., 40 (1972) 205. 10 K.W. Henningsen and J.E. Boynton, J. Cell Biol., 44 (1970) 290. 11 T.E. Weier and D.L. Brown, Am. J. Botany, 57 (1970) 267. 12 E. Dujardin and C. Sironval, Photosynthetica, 4 (2) (1970) 129. 13 S. Klein and J.A. Schiff, Plant Physiol., 49 (1972) 619. 14 A. Burgerstein, Bet. Deut. Bot. Ges., 18 (1900) 168. 15 V. Lubimenko, Rev. Gen. Bot., 40 (1928) 88. 16 L. Bogorad, Botanical Gazette, 111 (1950)221. 17 E.G. Sudyina, Photochem. Photobiol., 2 (1963) 181. 18 N.K. Boardman, Protochorophyll, in L.P. Vernon and G.R. Seely, The Chlorophylls, Academic Press, London, 1966, p. 455. 19 D. yon Wettstein, Brookl. Symp. BioL, 11 (1958) 138. 20 H. Camefort, Compt. Rend., 257 (1963) 2876. 21 D. Nikoli~ and M. Bogdanovi~, Protoplasma, 75 (1972) 205. 22 E.S. Reynolds, J. CellBiol., 17 (1963) 208. 23 F.G. Mackinney, J. Biol. Chem., 140 (.1941) 315.. 24 C. Sironvai, M. Brouers, J.-M. Michel and J. Kuiper, Photosynthetica, 2 (4~ (1968) 268. 25 H.F. B,con, J. Chromatog., 17 (1965) 322. 26 T. Treffry, J. Exptl. Botany, 24 (1973) -85. I 2 3 4