- Email: [email protected]
Ultrastructure of Chromoplasts in Detached Cotyledons of Cucumber Treated with Growth Retardant (2-Chloroethyl-trimethylammonium Chloride)
Biochem. Physiol. Pflanzen (BPP), Bd. 164, S.
514~521
(1973)
Laboratory of Plant Cytology and Cytochemistry, University of Lodz, Poland
Ultrastructure of Chromoplasts in Detached Cotyledons of Cucumber Treated with Growth Retardant (2-Chloroethyl-trimethylammonium Chloride) By E.
MIKULSKA,
H.
ZOLNIEROWICZ
With plate
and B.
NAROLEWSKA
7~14
(Received February 26, 1973)
Summary Chromoplasts develop in the detached cotyledons of etiolated cucumber seedlings growing in the light in the presence of 0.05 M CCC (2-chloroethyl-trimethylammonium chloride) solution. In control cotyledons, however, chloroamyloplasts appear. Ultrastructure of the cucumber cotyledon chromoplasts differentiated under experimental conditions is similar to that of other plant chromoplasts developed under natural conditions. Lycopene and {i-carotene, occuring in the chromoplasts of cucumber cotyledons, are localized in inter-thylakoid spaces where they crystallize. In addition, the rigid membrane piles present in the stroma of chromoplasts under examination seem to be the site of synthesis and/or accumulation of lycopene.
Introduction
The chromoplasts develop in the course of biological process of some flowers, fruits and carrot roots ripening. They are a final stage of plastid metamorphosis and considered as ageing plastids. The results of recent investigations show that chromoplasts can also be formed under experimental conditions. For example, the detached cotyledons of Cucurbita grown in light and in the presence of 0.05 M eee solution became orange-red coloured (KNYPL 1969a). The results of chemica.l analyses point to great amounts of lycopene accumulated in them while in control material only some traces of this compound are present at a simultaneous decrease of carotenoid pigments. Under these conditions the synthesis of chlorophyll is completely inhibited (KNYPL 1970). It seemed interesting to investigate whether chromoplasts developed under these conditions have the same structure as those formed under natural conditions. The site of pigment accumulation was also studied, as well as the form in which the pigments are localized and whether the differentiation of these chromoplasts proceeds in conformity with that described in the literature.
Ultrastructure of Chromoplasts in Detached Cotyledons etc.
515
Material and methods Seeds of Cucumis sativus L., variety "Delicatess" were placed in Petri dishes on Whatritan Nr. 2 blotting paper wetted with 4-5 ml of distilled water. The seeds were germinated in the dark at 24.8 ± 0.2 DC. When seedlings were 5 day old, the seeds were decoated and cotyledons dissected with a razor blade. Separated cotyledons were placed in Petri dishes on Whatman Nr. 2 paper moistened with distilled water (control) or 0.05 M CCC solution. The dishes were incubated for 48 hours in continuous fluorescent light (1300 lux) at 26-27 DC (KNYPL 1969a). Then the cotyledons grown in water became intensively green, those however incubated in te presence of CCC orange-red. Electron microscopy. Pieces (1 mm square) of 5 day old cotyledons grown in darkness (etiolated) and those of 7 day old green and orange-red cotyledons were fixed in 5% glutaraldehyde in 0.1 11{ phosphate buffer (pH 7.2) for 3 hours at 4 DC and postosmicated in the same buffer for 4 hours. Dehydration in graded ethanol series and embedding in Epon 812 followed standard methods. Thin sections prepared on a LKB ultramicrotome and double stained with uranyl acetate followed by lead citrate were examined in a Tesla BS 513 A electron microscope. Some grids with sections before staining were incubated in 0.04 M veronal buffer (pH 7.5) + 0.0025 M MgS04 containing DNase (Serva, Heidelberg, crystalline, 1 mg/ml buffer) with no DNase in the control, at 37 DC for 24 hours.
Observations
Amyloplasts occur in the cells of dark-grown cucumber cotyledons. The sections of amyloplasts reveal their circular, oval, triangular, or comma-like shapes (pI. 7). The comma-shaped amyloplasts resemble proplastids. The dimensions of amyloplasts varied within 0.97 -2.9 ,urn. They are surrounded with a double membrane which is usually finely folded. In the finely granulated matrix electron-transparent areas are visible. They contain granular or rod-shaped bodies with radiated fine fibrilis, which could be interpreted as DNA material (pI. 7, fig. 2 -5). Each of the described plastids contains one or several starch grains surrounded with an electron-transparent envelope. Some of the starch grains are extremely electron-dense (pI. 7, fig. 3). The internal system of amyloplast membranes under examination is very poor. It is limited to single short or long tubules and vesicles dispersed irregularly in the stroma. The folded lamellae formed in the region of vesicles fusing are visible near the surrounding membrane (pI. 7, fig. 4). In the cells of green cotyledons plastids are present. In consideration of the great amount of starch grains and slightly developed lamellar system these plastids are classified into the category of chloroamyloplasts (pI. 8). Their countours are rounded, oval or elongated. Some plastids form outgrowths enclosing the fragments of cytoplasm. The diameter of the described chloroamyloplasts varied within 3.6 and 7.4 ,urn. They are surrounded with a double membrane, which is sometimes finely folded, similarly to that of amyloplasts. The presence of numerous and clearly visible of ribo- and polysomes should be noticed in a dense stroma (pI. 8, 9). Similarly as in
516
E.
MIKULSKA,
H.
ZOLNIEROWICZ
and B.
NAROLEWSKA
amyloplasts, the starch grains surrounded with electron-transparent envelope occur in most chloroamyloplasts. The fragment of chloroamyloplast shown in pI. 9, fig. 9, contains a large area more transparent than the surrounding stroma in whieh fine DNA fibrils, ribosomes and small starch grain are visible. The internal lamellar system of ehloroamyloplasts under examination is eomposed of thylakoids orientated usually parallel to the long axis of the plastid (pI. 8, fig. 7, 8). They are not always differentiated into grana and inter-grana thylakoids. Prolamellar bodies remain in the stroma of a few chloroamyloplasts (pI. 9, fig. 10). In the cells of detached cotyledons growing in the light, in the presence of 0,05 M eee chromoplasts are present, which developed from amyloplasts of etiolated cucumber cotyledons (pI. 10). These chromoplasts increased significantly in size, changing often their shape as well. Many chromoplasts form on one or both lips elongated outgrowths enclosing large fragments of cytoplasm (pI. 10, fig. 14, dotted arrow). The stroma of chromoplasts is dense, finely granulated and contains numerous ribosomes. Osmiophilic globules not observed either in amylo- or in chloroamyloplasts appear in the stroma of some chromoplasts singly or in small groups (pI. 10, fig. 13; pI. 13, fig. 21, 22). Only very few chromoplasts contain single, small starch grains surrounded, similarly as in amylo- and chloroamyloplasts, with an electron-transparent areas. DNA-containing areas clearly visible in the stroma of the chromoplasts described should be noticed (pI. 10, fig. 12, 14; pI. 11). A detailed observation of these structures can be made at higher magnifications. They are formed from a central body of various dimensions and shape, with radiating fine filaments (pI. 11, fig. 15). The smallest of them are 15-20 A in diameter. After DNase treatment the described structures disappear completely or they leave behind very fine shadelike fragments (pI. 11, fig. 16). This suggests that these structures contain DNA. Small, electron-transparent areas situated usually near the plastid membrane, where the electron-dense structures are localized, were also found in the stroma of numerous chromoplasts. These structures observed in transversal seetions (pI. 12, fig. 17, 18) appear as granular globules, in longitudinal ones, however, as straight, or bent, sometimes ramified strands orientated parallel each other (pI. 12, fig. 19, 20). An internal membrane system in the described chromoplasts is slightly developed. In numerous plastids short or long single membranes randomly orientated in the stroma were observed (pI. 10, fig. 14; pI. 14, arrows). In the sections of other chromoplasts several flattened membranes adhering to eaeh other are present. They form flat piles (pI. 13, fig. 22 -26). These piles can be long or short ones; the latter resemble grana (pI. 14). These membranes give the impression of rigid crystalline structures. Sometimes certain fragments of these membrane piles show a structure resembling
Ultrastructure of Chrornoplasts in Detached Cotyledons etc.
517
that of protein crystals (pI. 11, fig. 15). Microscopic examination at lower magnifications suggests, that the external membranes in these piles enclose a homogenous electron-dense substance (pI. 13, fig. 22, 23). Nevertheless, magnification of the order of 40000 X reveals the unit membranes laying very near to each other. Neither intrathylakoid space nor any space betweE'l1 neighbouring thylakoids can be seen (pI. 13, fig. 25, 26; pI. 14). Electron-cIear, usually S- shaped spacos occuring in the area of stroma, parallel or perpendicularly orientated to the long axis of the plastid arc a charakteristic feature of most chromoplasts observed (pI. 10, fig. 11-13; pI. 14). They can be separated from the stroma by a membrane. These spaces correspond to the areas in which the crystals of orange-red pigments, appearing in the intra-thylakoid spaces were present. These crystals were dissolved during ma,terial dehydration. The fact that the remainder of crystals as an electron-dense structure is maintained in some areas supports this opinion (pI 10, fig. 11, arrow).
Discussion
The presence of chromoplasts in the cells of detached cucumber cotyledons treated with 0.05 M eee solution confirms the results of biochemical analyses, proving that the growth retardant 2-chloroethyltrimethylammonium chloride inhibits the synthesis of chlorophyll (KNYPL and eHYLINSKA 1972a, b) and induces a considerable accumulation of lycopene together with a simultaneous reduction of p-carotene content as compared to the control (KNYPL 1969 b). These processes occuring in the cotyledons under examination result in chromoplasts development. Presumably an inhibitory effect of eeG on the process of greening is due to inhibition of the activity of some enzymes catalysing the biosynthesis of chlorophylls (KNYPL 1971). DNA-containing areas occurring in the stroma are a common feature of amylo-, chloroamyloplasts and chromoplasts of cucumber cotyledons. The morphology of these areas does not differ from the morphology of the areas described in the leaf chloroplasts of higher plants (KISLEV et a1. 1965; SALEMA and BADENHUIZEN 1969; ODINTSOVA et aI. 1970; GRAN and POSSINGHAM 1972), in those of some algae (RIS and PLAUT 1962; BISALPUTRA and BISALPUTRA 1967), in the amyloplasts of corn endosperm, amyloplasts of growing green potato tubers and also in proteinoplasts of the epidermal cells of Helleborus corsicus (SALEMA and BADENHUIZEN 1969). After fixing the material in glutaraldehyde these authors observed in the chromoplasts of Solanum and Physalis fruits the areas containing fibrillar structures comparable with DNAareas in chloroplasts. Recently Kow ALLIK and HERRMANN (1972) describe the areas with a low electron density occuring in chromopla'lts of Narcissus pseudonarcissus~ 34
BiocheIn. Physiol. Pflanzen, Bd. 164
518
E.
MIKULSKA,
H.
ZOL"IEROWICZ
and B.
NAROLEWSKA
containing fibrils of 25-30 A in diameter resembling DNA in chloroplasts and mitochondria of this species. DNA isolated from chromoplasts of Narcissus flowers (HERRMANN 1972) resembles the DNA from chloroplast preparations of the same plant. The digestion of ultrathin sections with DNase leads to a distinct decrease of the number of fibrils in the electron-transparent areas of chromoplasts of the cucumber cotyledons, and removes also the so-called core-like structures containing DNA but almost always small fragments of these fibrils remain. KOWALLIK and HERRMANN (1972) noticed that the enzyme digestion of fibrils in the transparent areas of chromoplasts of Narcissus pseudo narcissus was unsuccessful, although he succeeded in DNA isolation from these chromoplasts (HERRMANN 1972). This supports the opinion that fibrillar structures occuring in the chromoplasts can be really recognized as bodies provided with DNA. The chloroamyloplasts of control cucumber cotyledons considerably increase in their dimensions during 48 hours of incubation in the light as compared with the amy lop lasts from which they developed. A great number of ribo- and polysomes occuring in the stroma of these chloroplasts suggests an intensive synthesis of proteins in the area of chloroamyloplasts. Ribosomes of the described chloroamyloplasts occur freely in the matrix. We did not observe "rough thylakoids", described by FALK (1969), to occur in the chloroplasts of the leaves of young seedlings of Phaseolus vulgaris. The chromoplasts are considerably larger in size than the amyloplasts from which they differentiated. Their stroma, similarly to that of the chloroamyloplasts, contains a great number of free ribosomes. The ultrastructure of cucumber cotyledon chromoplasts formed under the influence of 0.05 M eee is similar to that of the chromoplasts differentiated under natural conditions and described by other authors. Based on the electron microscopic pictures it could be considered that lycopene and j1-carotene present in the cucumber cotyledon chromoplasts accumulate and crystallize in the inter-thylakoid spaces. Electron-transparent regions, adjacent to the wavy lines representing areas where membranes became dissociated from the carotene crystalloid during alcohol dehydration (which extracted some of the lycopen e), were observed in the stroma of cucumber cotyledons. These images resemble those described by MOLLENHAUER and KOGUT (1968) and KUHN (1970). The compact membrane structures can be spaces of the synthesis and/or accumulation of a part of lycopene as well. BEN-SHAUL et al. (1968) maintain this opinion. BEN-SHAUL and NAFTALI (1969) suggest that lycopene is synthesised in great amounts in Lycopersicum esculentum plastids during the later developmental stages and it forms crystals associated with rigid membranes constituting a lycopene body. At the final developmental stage the lycopene body forms a crystal. According to the authors, the pattern of the formation of lycopene bodies and lycopene crystals is similar. HARRIS and SPURR
Ultrastructure of Chromoplasts in Detached Cotyledons etc.
519
(1969a) found that fJ-carotene, occuring in high-beta mutant tomato fruits presumably largely in the globules, crystallizes into elongated or druse type forms, lycopene however forms crystaloids associated with thylakoids. In the normal red tomato, lycopene is the predominant carotene and it aggregates on thylakoid membranes (HARRIS and SPURR 1969b). It cannot be excluded, that osmiophilic globules, occuring in the stroma of some cucumber cotyledon chromoplasts, can also be the sites of the localization of a certain amount of the pigments. It is known that the chromo· plast pigments are localized in a lipid component of globular bodies occuring in the chromoplast stroma (FREy-WYSSLING and KREUTZER 1958; THOMSON 1966). The developed chromoplasts of cucumber cotyledons differentiated under the influence of CCC do not differ in t.heir structure, pigment localization, and form of carotenoid crystals from those formed under natural conditions. The only slight deviation from the model accepted in the literature was observed in the course of the transformation of amyloplasts into chromoplasts. During normal ontogenesis of the chromoplasts, which develop directly from amylpplasts, a great number of vesicle appears in the stroma. They are arranged in a prolamellar body. Part of the vesicles is transformed into short tubules. Then osmiophilic globules with accumullated pigments appear (GRILl 1965). If the process of transformation of amyloplasts into chromoplasts takes place in the light, as in the present experiments, the system of grana and intergrana thylakoids develops on a small area of the stroma. Then, the membran system desintegrates to vesicles next to which globules with oarotenoid pigments appear (GRILl 1965). In the presence of CCC, very few membranes, showing no differentiation into grana and stroma thylakoids, appear during the development of chromoplasts. Membrane piles are tightly com part. Neither vesicles nor osmiophilic globules appear in the stroma of these chromoplasts, which are characteristic for this process proceeding under natural conditions. For this reason, it should be supposed that under experimental conditions the process of transformation of amyloplasts into chromoplasts starts with the formation of an internal membrane system, but under the influence of CCC, inhibiting the synthesis of chlorophyll, morphogenesis of the membrane system is also inhibited. This results in the degeneration of these membranes and crystallization of carotenoid pigments in them. The structures which look like globules in transversal section, when observed longitudinal section, represent the strands arranged parallel to each other. There occur in the light areas of the stroma of cucumber cotyledon chromoplasts. They can be compared with the tubular structures observed in the chromoplasts of cucumber flowers by SMITH and BUTLER (1971). The authros interpret these bodies as the pigment containing tubules. 34*
520
E. MIKULSKA, H. ZOLNIEROWICZ a.nd B. NAROLEWSKA
Acknowledgements We are greatly indebted to Doc. Dr. J. S. KNYPL for providing us with experimental material.
References BEN-SHAUL, Y., and NAFTALI, Y., The development and ultrastructure of lycopene bodies in chloroplasts of Lycopersicum esculeniuln. Protoplasm a 67, 333-344 (1969). - TREFFRY, '1'., and KLEIN, S., Fine structure studies of carotene body development. J. }[irrosc. 7, 265-274 (1968). BISALPUTRA, '1'., and BISALPUTRA, A. A., The occurrence of DXA fibrils in chloroplasts of Laurencia spectabilis. J. Ultrastruct. Res. 17, 14-22 (1967). CRAN, D.O., and POSSINGHHf, J. V., Variation of plastid types in spinach. Protoplasma 74, 345-356 (1972). FALK, H., ROUGH thylakoids polysomes attached to chloroplasts membranes. J. Cell BioI. 42, 582-587 (1969). FREY- WYSSLING, A., and KREUTZER, E., Die submikroskopische Entwicklung der Chromoplasten in den Bliiten von Ranunculus repens L. Planta a1, 104-114 (1958). GRILl, l\L, Origine e sviluppo dei cromoplasti nei frutti di Zucca americana (Cucurbita pepo L. cv. Small Sugar). I. Origine dei cromoplasti da amiloplasti. Caryologia 18, 409-433 (1965). HARRIS, W. M., and SPURR, A. R., Chromoplasts of tomato fruits. 1. Ultrastructure of low-pigmet and high-beta mutant. Carotene analysis. Amer. J. Bot. 56 (4),369-379 (1969a). - - Chromoplasts of tomato fruits. II. The red tomato. Amer. J. Bot 56 (4), 380-389 (1969 b). HERRMANN, R. G., Do chromoplasts contain DNA? II. The isolation and characterisation of DNA from chromoplasts, chloroplasts, mitochondria, and nuclei of Narcissus. Protoplasma 74,7-17 (1972). KISLEV, ;\[., SWIFT, H., and BOGORAD, L., Nucleic acids of chloroplasts and mitochondria in swiss chard. J. Cell BioI. 25, 327-344 (1965). KNYPL, J. S., Inhibition of chlorophyll synthesis by growth retardants ami coumarin, and its reversal by potassium. Nature 224, 1025-1026 (1969a). -- Accumulation of lycopene in detached cotyledones of pumpkin treated with (2-chloroethyl)trimethylammonium chloride. Naturwiss. 56, 572 (1969b). - Wplyw retardantow wzrostu, kumaryny, gibereliny i cytokinin na kielkowanie nasion oraz syntez~ i rozpad chlorofilu, bialek i RNA w lisciach i liscieniach. £6dz 1970, 1-95. - Control of protein and RNA synthesis by AMO-1618 and other growth retardants in cucumber cotyledons. Bioch. Physiol. Pf!. 162, 127-141 (1971). - CIIYLINSKA, K. M., The inhibitory effect of (2-chloroethyl)-trimethylammonium chloride on chlorophyll and protein synthesis in lettuce cotyledons, and its reversal by potassium. J. Exp. Bot. 23. 525-529 (1972a). - - Chlorophyll accumulation and protein synthesis in lettuce cotyledons treated with growth retardants, gibberellin, and benzylaminopurine. Z. Pflanzenphysiol. 66, 297-306 (1972b). KOWALLIK, K. V., and HERRMANN, R. G., Do chromoplasts contain DNA? I. Electron-microscopic investigation of Narcissus chromoplast. Protoplasm a 74, 1-6 (1972). KUHN, H., Chemismus, Struktur und Entstehung der Carotinkristallchen in der Nebenkrone von N,1fcissus poeticus L. var. "La Riante". J. Ultrastruct. Res. 33, 332-355 (1970). ~J\IOLLENHAUER, H. H., and KOGUT, C., Chromoplast development in daffodil. J. Microsc. 7, 1045 bis 1050 (1968).
Ultrastructure of Chromoplasts in Detached Cotyledons etc.
521
ODINTSOVA, :VI. S., I\hKULSKA, E., and TURISCIIEVA, M. S., Electron microscopy of DNA in pea chloroplasts. Exp. Cell Res. 61, 423-432 (1970). RIS, H., and PLAUT, W., Ultrastructure of DNA-containing areas in the chloroplast of Chlamydomonas. J. Cell BioI. 13, 383-391 (1962). SALEMA, R., and BADENHUIZEN, N. P., Nucleic acids in plastids and starch formation. Acta Bot. Neerl. 18 (1),203-215 (1969). SMITH, :VI., and BUTLER, R. D., Ultrastructural aspects of petal development in Cucumis sativus with particular reference to the chromoplasts. Protoplasma 73,1-13 (1971). THOMSON, W. W., Ultrastructural development of chromoplasts in Valencia omnges. Bot. Gaz. 127, 133-139 (1966). Authors' address: Doc. Dr. E. :VhKUSKA, H. ZOLNIEROWICZ and B. NAROLEWSKA, Laboratory of Plant Cytology and Cytochemistry, Institute of Biochemistry and Physiology, University of L6di, Nowopoludniowa 12/16, L6di (Poland).
522
E. MIKULSKA, H. ZOL:'{IEROWICZ and B. NARoLEwsKA
Explanations of plates Plate 7 Fig. 1-6. Amyloplasts in cells of dark-grown cucumber cotyledons showing DNA-areas with fine fibrils (arrows) and starch grains (S). X 25700. Plate 8 Fig. 7-8. Ohloroplasts from detached green cotyledons of cucumber (control material) showing thylakoids, ribo- and polysomes (arrows) and starch grains (S). X 25700. Plate9 Fig. 9-10. Sections of chloroplasts from detached green cucumber cotyledons. 9, a portion of chloroplast showing the electron transparent DNA-containing area with starch granule (S). Ribo- and polysomes in chloroplasts stroma are also observed (arrows). x80500. 10, chloroplast with pro lamellar body (PB) and starch grains. X 25 700. Plate 10 Fig. 11-14. Ohromoplasts from detached cotyledons of cucumber treated with 0.06 l\I 000. 11-13, elongated and clear spaces in the stroma probably represent the sites of lycopene crystals, the crystals themselves being lost by dissolution in the solvents used for tissue dehydration. 11, fragment of undissolved crystal (double arrow). X 80500. 14, outline of dissolved crystal. Areas with DNA fibrils (arrows). 12-14. X 25700. Plate 11 Fig. 15. Electron transparent area in chromoplast at higher magnification (x 80500) showing the occurrence of D~A fibrils. Fragment of thylakoids pile shows crystalline structure (arrow). Fig. 16. Fragment of the chromoplast after DNase digestion showing small fragments of DNA fibrils. X 80500. Plate 12 Ohromoplast fragments with areas containing tubule-like structures in and longitudinal (fig. 19,20) sections. X 80500.
transvers~l
(fig. 17, 18)
Plate 13 Fig. 21, 22. Ohromoplasts with osmiophilic globules and membrane piles. 23, 24, fragments of membrane piles at X 26700. 25, 26, the same at X 80500. Plate 14 Section of chromoplast with grana-like membrane piles and single membranes (arrows).
X
80500.
BPP, Bd. 164
Plate 7
Mi kuls ka et al. VEB GUSTAV FISCHER VERLAG JENA
Plate 8
BPP, Bd. 164
l\Iikulska et al. VEB GUSTAV FISCHER VERLAG JENA
BPP, Ed. 164
Plate 9
Mikulska et al. VEB GUSTAV FISCHER VERLAG JENA
BI'P, Bd. 164
Plate 10
Mikulska et al. VEB GUSTAV FISCHER VERLAG JENA
BPP, Bd. 164
Plate 11
Mikulska et al. VEB GUSTAV FISCHER VERLAG JENA
Plate 12
BBP, Bd. 164
Mi kuls ka et al. VEB GUSTAV FISCHER VERLAG JENA
Plate 13
BPP, Rd. 164
Mikulska et al. VEB GUSTAV FISCHER VERLAG JENA
Plate 14
BPP, Ed. 164
Mikulska et al. VEB GUSTAV F:SCHER VERLAG JENA
514~521
(1973)
Laboratory of Plant Cytology and Cytochemistry, University of Lodz, Poland
Ultrastructure of Chromoplasts in Detached Cotyledons of Cucumber Treated with Growth Retardant (2-Chloroethyl-trimethylammonium Chloride) By E.
MIKULSKA,
H.
ZOLNIEROWICZ
With plate
and B.
NAROLEWSKA
7~14
(Received February 26, 1973)
Summary Chromoplasts develop in the detached cotyledons of etiolated cucumber seedlings growing in the light in the presence of 0.05 M CCC (2-chloroethyl-trimethylammonium chloride) solution. In control cotyledons, however, chloroamyloplasts appear. Ultrastructure of the cucumber cotyledon chromoplasts differentiated under experimental conditions is similar to that of other plant chromoplasts developed under natural conditions. Lycopene and {i-carotene, occuring in the chromoplasts of cucumber cotyledons, are localized in inter-thylakoid spaces where they crystallize. In addition, the rigid membrane piles present in the stroma of chromoplasts under examination seem to be the site of synthesis and/or accumulation of lycopene.
Introduction
The chromoplasts develop in the course of biological process of some flowers, fruits and carrot roots ripening. They are a final stage of plastid metamorphosis and considered as ageing plastids. The results of recent investigations show that chromoplasts can also be formed under experimental conditions. For example, the detached cotyledons of Cucurbita grown in light and in the presence of 0.05 M eee solution became orange-red coloured (KNYPL 1969a). The results of chemica.l analyses point to great amounts of lycopene accumulated in them while in control material only some traces of this compound are present at a simultaneous decrease of carotenoid pigments. Under these conditions the synthesis of chlorophyll is completely inhibited (KNYPL 1970). It seemed interesting to investigate whether chromoplasts developed under these conditions have the same structure as those formed under natural conditions. The site of pigment accumulation was also studied, as well as the form in which the pigments are localized and whether the differentiation of these chromoplasts proceeds in conformity with that described in the literature.
Ultrastructure of Chromoplasts in Detached Cotyledons etc.
515
Material and methods Seeds of Cucumis sativus L., variety "Delicatess" were placed in Petri dishes on Whatritan Nr. 2 blotting paper wetted with 4-5 ml of distilled water. The seeds were germinated in the dark at 24.8 ± 0.2 DC. When seedlings were 5 day old, the seeds were decoated and cotyledons dissected with a razor blade. Separated cotyledons were placed in Petri dishes on Whatman Nr. 2 paper moistened with distilled water (control) or 0.05 M CCC solution. The dishes were incubated for 48 hours in continuous fluorescent light (1300 lux) at 26-27 DC (KNYPL 1969a). Then the cotyledons grown in water became intensively green, those however incubated in te presence of CCC orange-red. Electron microscopy. Pieces (1 mm square) of 5 day old cotyledons grown in darkness (etiolated) and those of 7 day old green and orange-red cotyledons were fixed in 5% glutaraldehyde in 0.1 11{ phosphate buffer (pH 7.2) for 3 hours at 4 DC and postosmicated in the same buffer for 4 hours. Dehydration in graded ethanol series and embedding in Epon 812 followed standard methods. Thin sections prepared on a LKB ultramicrotome and double stained with uranyl acetate followed by lead citrate were examined in a Tesla BS 513 A electron microscope. Some grids with sections before staining were incubated in 0.04 M veronal buffer (pH 7.5) + 0.0025 M MgS04 containing DNase (Serva, Heidelberg, crystalline, 1 mg/ml buffer) with no DNase in the control, at 37 DC for 24 hours.
Observations
Amyloplasts occur in the cells of dark-grown cucumber cotyledons. The sections of amyloplasts reveal their circular, oval, triangular, or comma-like shapes (pI. 7). The comma-shaped amyloplasts resemble proplastids. The dimensions of amyloplasts varied within 0.97 -2.9 ,urn. They are surrounded with a double membrane which is usually finely folded. In the finely granulated matrix electron-transparent areas are visible. They contain granular or rod-shaped bodies with radiated fine fibrilis, which could be interpreted as DNA material (pI. 7, fig. 2 -5). Each of the described plastids contains one or several starch grains surrounded with an electron-transparent envelope. Some of the starch grains are extremely electron-dense (pI. 7, fig. 3). The internal system of amyloplast membranes under examination is very poor. It is limited to single short or long tubules and vesicles dispersed irregularly in the stroma. The folded lamellae formed in the region of vesicles fusing are visible near the surrounding membrane (pI. 7, fig. 4). In the cells of green cotyledons plastids are present. In consideration of the great amount of starch grains and slightly developed lamellar system these plastids are classified into the category of chloroamyloplasts (pI. 8). Their countours are rounded, oval or elongated. Some plastids form outgrowths enclosing the fragments of cytoplasm. The diameter of the described chloroamyloplasts varied within 3.6 and 7.4 ,urn. They are surrounded with a double membrane, which is sometimes finely folded, similarly to that of amyloplasts. The presence of numerous and clearly visible of ribo- and polysomes should be noticed in a dense stroma (pI. 8, 9). Similarly as in
516
E.
MIKULSKA,
H.
ZOLNIEROWICZ
and B.
NAROLEWSKA
amyloplasts, the starch grains surrounded with electron-transparent envelope occur in most chloroamyloplasts. The fragment of chloroamyloplast shown in pI. 9, fig. 9, contains a large area more transparent than the surrounding stroma in whieh fine DNA fibrils, ribosomes and small starch grain are visible. The internal lamellar system of ehloroamyloplasts under examination is eomposed of thylakoids orientated usually parallel to the long axis of the plastid (pI. 8, fig. 7, 8). They are not always differentiated into grana and inter-grana thylakoids. Prolamellar bodies remain in the stroma of a few chloroamyloplasts (pI. 9, fig. 10). In the cells of detached cotyledons growing in the light, in the presence of 0,05 M eee chromoplasts are present, which developed from amyloplasts of etiolated cucumber cotyledons (pI. 10). These chromoplasts increased significantly in size, changing often their shape as well. Many chromoplasts form on one or both lips elongated outgrowths enclosing large fragments of cytoplasm (pI. 10, fig. 14, dotted arrow). The stroma of chromoplasts is dense, finely granulated and contains numerous ribosomes. Osmiophilic globules not observed either in amylo- or in chloroamyloplasts appear in the stroma of some chromoplasts singly or in small groups (pI. 10, fig. 13; pI. 13, fig. 21, 22). Only very few chromoplasts contain single, small starch grains surrounded, similarly as in amylo- and chloroamyloplasts, with an electron-transparent areas. DNA-containing areas clearly visible in the stroma of the chromoplasts described should be noticed (pI. 10, fig. 12, 14; pI. 11). A detailed observation of these structures can be made at higher magnifications. They are formed from a central body of various dimensions and shape, with radiating fine filaments (pI. 11, fig. 15). The smallest of them are 15-20 A in diameter. After DNase treatment the described structures disappear completely or they leave behind very fine shadelike fragments (pI. 11, fig. 16). This suggests that these structures contain DNA. Small, electron-transparent areas situated usually near the plastid membrane, where the electron-dense structures are localized, were also found in the stroma of numerous chromoplasts. These structures observed in transversal seetions (pI. 12, fig. 17, 18) appear as granular globules, in longitudinal ones, however, as straight, or bent, sometimes ramified strands orientated parallel each other (pI. 12, fig. 19, 20). An internal membrane system in the described chromoplasts is slightly developed. In numerous plastids short or long single membranes randomly orientated in the stroma were observed (pI. 10, fig. 14; pI. 14, arrows). In the sections of other chromoplasts several flattened membranes adhering to eaeh other are present. They form flat piles (pI. 13, fig. 22 -26). These piles can be long or short ones; the latter resemble grana (pI. 14). These membranes give the impression of rigid crystalline structures. Sometimes certain fragments of these membrane piles show a structure resembling
Ultrastructure of Chrornoplasts in Detached Cotyledons etc.
517
that of protein crystals (pI. 11, fig. 15). Microscopic examination at lower magnifications suggests, that the external membranes in these piles enclose a homogenous electron-dense substance (pI. 13, fig. 22, 23). Nevertheless, magnification of the order of 40000 X reveals the unit membranes laying very near to each other. Neither intrathylakoid space nor any space betweE'l1 neighbouring thylakoids can be seen (pI. 13, fig. 25, 26; pI. 14). Electron-cIear, usually S- shaped spacos occuring in the area of stroma, parallel or perpendicularly orientated to the long axis of the plastid arc a charakteristic feature of most chromoplasts observed (pI. 10, fig. 11-13; pI. 14). They can be separated from the stroma by a membrane. These spaces correspond to the areas in which the crystals of orange-red pigments, appearing in the intra-thylakoid spaces were present. These crystals were dissolved during ma,terial dehydration. The fact that the remainder of crystals as an electron-dense structure is maintained in some areas supports this opinion (pI 10, fig. 11, arrow).
Discussion
The presence of chromoplasts in the cells of detached cucumber cotyledons treated with 0.05 M eee solution confirms the results of biochemical analyses, proving that the growth retardant 2-chloroethyltrimethylammonium chloride inhibits the synthesis of chlorophyll (KNYPL and eHYLINSKA 1972a, b) and induces a considerable accumulation of lycopene together with a simultaneous reduction of p-carotene content as compared to the control (KNYPL 1969 b). These processes occuring in the cotyledons under examination result in chromoplasts development. Presumably an inhibitory effect of eeG on the process of greening is due to inhibition of the activity of some enzymes catalysing the biosynthesis of chlorophylls (KNYPL 1971). DNA-containing areas occurring in the stroma are a common feature of amylo-, chloroamyloplasts and chromoplasts of cucumber cotyledons. The morphology of these areas does not differ from the morphology of the areas described in the leaf chloroplasts of higher plants (KISLEV et a1. 1965; SALEMA and BADENHUIZEN 1969; ODINTSOVA et aI. 1970; GRAN and POSSINGHAM 1972), in those of some algae (RIS and PLAUT 1962; BISALPUTRA and BISALPUTRA 1967), in the amyloplasts of corn endosperm, amyloplasts of growing green potato tubers and also in proteinoplasts of the epidermal cells of Helleborus corsicus (SALEMA and BADENHUIZEN 1969). After fixing the material in glutaraldehyde these authors observed in the chromoplasts of Solanum and Physalis fruits the areas containing fibrillar structures comparable with DNAareas in chloroplasts. Recently Kow ALLIK and HERRMANN (1972) describe the areas with a low electron density occuring in chromopla'lts of Narcissus pseudonarcissus~ 34
BiocheIn. Physiol. Pflanzen, Bd. 164
518
E.
MIKULSKA,
H.
ZOL"IEROWICZ
and B.
NAROLEWSKA
containing fibrils of 25-30 A in diameter resembling DNA in chloroplasts and mitochondria of this species. DNA isolated from chromoplasts of Narcissus flowers (HERRMANN 1972) resembles the DNA from chloroplast preparations of the same plant. The digestion of ultrathin sections with DNase leads to a distinct decrease of the number of fibrils in the electron-transparent areas of chromoplasts of the cucumber cotyledons, and removes also the so-called core-like structures containing DNA but almost always small fragments of these fibrils remain. KOWALLIK and HERRMANN (1972) noticed that the enzyme digestion of fibrils in the transparent areas of chromoplasts of Narcissus pseudo narcissus was unsuccessful, although he succeeded in DNA isolation from these chromoplasts (HERRMANN 1972). This supports the opinion that fibrillar structures occuring in the chromoplasts can be really recognized as bodies provided with DNA. The chloroamyloplasts of control cucumber cotyledons considerably increase in their dimensions during 48 hours of incubation in the light as compared with the amy lop lasts from which they developed. A great number of ribo- and polysomes occuring in the stroma of these chloroplasts suggests an intensive synthesis of proteins in the area of chloroamyloplasts. Ribosomes of the described chloroamyloplasts occur freely in the matrix. We did not observe "rough thylakoids", described by FALK (1969), to occur in the chloroplasts of the leaves of young seedlings of Phaseolus vulgaris. The chromoplasts are considerably larger in size than the amyloplasts from which they differentiated. Their stroma, similarly to that of the chloroamyloplasts, contains a great number of free ribosomes. The ultrastructure of cucumber cotyledon chromoplasts formed under the influence of 0.05 M eee is similar to that of the chromoplasts differentiated under natural conditions and described by other authors. Based on the electron microscopic pictures it could be considered that lycopene and j1-carotene present in the cucumber cotyledon chromoplasts accumulate and crystallize in the inter-thylakoid spaces. Electron-transparent regions, adjacent to the wavy lines representing areas where membranes became dissociated from the carotene crystalloid during alcohol dehydration (which extracted some of the lycopen e), were observed in the stroma of cucumber cotyledons. These images resemble those described by MOLLENHAUER and KOGUT (1968) and KUHN (1970). The compact membrane structures can be spaces of the synthesis and/or accumulation of a part of lycopene as well. BEN-SHAUL et al. (1968) maintain this opinion. BEN-SHAUL and NAFTALI (1969) suggest that lycopene is synthesised in great amounts in Lycopersicum esculentum plastids during the later developmental stages and it forms crystals associated with rigid membranes constituting a lycopene body. At the final developmental stage the lycopene body forms a crystal. According to the authors, the pattern of the formation of lycopene bodies and lycopene crystals is similar. HARRIS and SPURR
Ultrastructure of Chromoplasts in Detached Cotyledons etc.
519
(1969a) found that fJ-carotene, occuring in high-beta mutant tomato fruits presumably largely in the globules, crystallizes into elongated or druse type forms, lycopene however forms crystaloids associated with thylakoids. In the normal red tomato, lycopene is the predominant carotene and it aggregates on thylakoid membranes (HARRIS and SPURR 1969b). It cannot be excluded, that osmiophilic globules, occuring in the stroma of some cucumber cotyledon chromoplasts, can also be the sites of the localization of a certain amount of the pigments. It is known that the chromo· plast pigments are localized in a lipid component of globular bodies occuring in the chromoplast stroma (FREy-WYSSLING and KREUTZER 1958; THOMSON 1966). The developed chromoplasts of cucumber cotyledons differentiated under the influence of CCC do not differ in t.heir structure, pigment localization, and form of carotenoid crystals from those formed under natural conditions. The only slight deviation from the model accepted in the literature was observed in the course of the transformation of amyloplasts into chromoplasts. During normal ontogenesis of the chromoplasts, which develop directly from amylpplasts, a great number of vesicle appears in the stroma. They are arranged in a prolamellar body. Part of the vesicles is transformed into short tubules. Then osmiophilic globules with accumullated pigments appear (GRILl 1965). If the process of transformation of amyloplasts into chromoplasts takes place in the light, as in the present experiments, the system of grana and intergrana thylakoids develops on a small area of the stroma. Then, the membran system desintegrates to vesicles next to which globules with oarotenoid pigments appear (GRILl 1965). In the presence of CCC, very few membranes, showing no differentiation into grana and stroma thylakoids, appear during the development of chromoplasts. Membrane piles are tightly com part. Neither vesicles nor osmiophilic globules appear in the stroma of these chromoplasts, which are characteristic for this process proceeding under natural conditions. For this reason, it should be supposed that under experimental conditions the process of transformation of amyloplasts into chromoplasts starts with the formation of an internal membrane system, but under the influence of CCC, inhibiting the synthesis of chlorophyll, morphogenesis of the membrane system is also inhibited. This results in the degeneration of these membranes and crystallization of carotenoid pigments in them. The structures which look like globules in transversal section, when observed longitudinal section, represent the strands arranged parallel to each other. There occur in the light areas of the stroma of cucumber cotyledon chromoplasts. They can be compared with the tubular structures observed in the chromoplasts of cucumber flowers by SMITH and BUTLER (1971). The authros interpret these bodies as the pigment containing tubules. 34*
520
E. MIKULSKA, H. ZOLNIEROWICZ a.nd B. NAROLEWSKA
Acknowledgements We are greatly indebted to Doc. Dr. J. S. KNYPL for providing us with experimental material.
References BEN-SHAUL, Y., and NAFTALI, Y., The development and ultrastructure of lycopene bodies in chloroplasts of Lycopersicum esculeniuln. Protoplasm a 67, 333-344 (1969). - TREFFRY, '1'., and KLEIN, S., Fine structure studies of carotene body development. J. }[irrosc. 7, 265-274 (1968). BISALPUTRA, '1'., and BISALPUTRA, A. A., The occurrence of DXA fibrils in chloroplasts of Laurencia spectabilis. J. Ultrastruct. Res. 17, 14-22 (1967). CRAN, D.O., and POSSINGHHf, J. V., Variation of plastid types in spinach. Protoplasma 74, 345-356 (1972). FALK, H., ROUGH thylakoids polysomes attached to chloroplasts membranes. J. Cell BioI. 42, 582-587 (1969). FREY- WYSSLING, A., and KREUTZER, E., Die submikroskopische Entwicklung der Chromoplasten in den Bliiten von Ranunculus repens L. Planta a1, 104-114 (1958). GRILl, l\L, Origine e sviluppo dei cromoplasti nei frutti di Zucca americana (Cucurbita pepo L. cv. Small Sugar). I. Origine dei cromoplasti da amiloplasti. Caryologia 18, 409-433 (1965). HARRIS, W. M., and SPURR, A. R., Chromoplasts of tomato fruits. 1. Ultrastructure of low-pigmet and high-beta mutant. Carotene analysis. Amer. J. Bot. 56 (4),369-379 (1969a). - - Chromoplasts of tomato fruits. II. The red tomato. Amer. J. Bot 56 (4), 380-389 (1969 b). HERRMANN, R. G., Do chromoplasts contain DNA? II. The isolation and characterisation of DNA from chromoplasts, chloroplasts, mitochondria, and nuclei of Narcissus. Protoplasma 74,7-17 (1972). KISLEV, ;\[., SWIFT, H., and BOGORAD, L., Nucleic acids of chloroplasts and mitochondria in swiss chard. J. Cell BioI. 25, 327-344 (1965). KNYPL, J. S., Inhibition of chlorophyll synthesis by growth retardants ami coumarin, and its reversal by potassium. Nature 224, 1025-1026 (1969a). -- Accumulation of lycopene in detached cotyledones of pumpkin treated with (2-chloroethyl)trimethylammonium chloride. Naturwiss. 56, 572 (1969b). - Wplyw retardantow wzrostu, kumaryny, gibereliny i cytokinin na kielkowanie nasion oraz syntez~ i rozpad chlorofilu, bialek i RNA w lisciach i liscieniach. £6dz 1970, 1-95. - Control of protein and RNA synthesis by AMO-1618 and other growth retardants in cucumber cotyledons. Bioch. Physiol. Pf!. 162, 127-141 (1971). - CIIYLINSKA, K. M., The inhibitory effect of (2-chloroethyl)-trimethylammonium chloride on chlorophyll and protein synthesis in lettuce cotyledons, and its reversal by potassium. J. Exp. Bot. 23. 525-529 (1972a). - - Chlorophyll accumulation and protein synthesis in lettuce cotyledons treated with growth retardants, gibberellin, and benzylaminopurine. Z. Pflanzenphysiol. 66, 297-306 (1972b). KOWALLIK, K. V., and HERRMANN, R. G., Do chromoplasts contain DNA? I. Electron-microscopic investigation of Narcissus chromoplast. Protoplasm a 74, 1-6 (1972). KUHN, H., Chemismus, Struktur und Entstehung der Carotinkristallchen in der Nebenkrone von N,1fcissus poeticus L. var. "La Riante". J. Ultrastruct. Res. 33, 332-355 (1970). ~J\IOLLENHAUER, H. H., and KOGUT, C., Chromoplast development in daffodil. J. Microsc. 7, 1045 bis 1050 (1968).
Ultrastructure of Chromoplasts in Detached Cotyledons etc.
521
ODINTSOVA, :VI. S., I\hKULSKA, E., and TURISCIIEVA, M. S., Electron microscopy of DNA in pea chloroplasts. Exp. Cell Res. 61, 423-432 (1970). RIS, H., and PLAUT, W., Ultrastructure of DNA-containing areas in the chloroplast of Chlamydomonas. J. Cell BioI. 13, 383-391 (1962). SALEMA, R., and BADENHUIZEN, N. P., Nucleic acids in plastids and starch formation. Acta Bot. Neerl. 18 (1),203-215 (1969). SMITH, :VI., and BUTLER, R. D., Ultrastructural aspects of petal development in Cucumis sativus with particular reference to the chromoplasts. Protoplasma 73,1-13 (1971). THOMSON, W. W., Ultrastructural development of chromoplasts in Valencia omnges. Bot. Gaz. 127, 133-139 (1966). Authors' address: Doc. Dr. E. :VhKUSKA, H. ZOLNIEROWICZ and B. NAROLEWSKA, Laboratory of Plant Cytology and Cytochemistry, Institute of Biochemistry and Physiology, University of L6di, Nowopoludniowa 12/16, L6di (Poland).
522
E. MIKULSKA, H. ZOL:'{IEROWICZ and B. NARoLEwsKA
Explanations of plates Plate 7 Fig. 1-6. Amyloplasts in cells of dark-grown cucumber cotyledons showing DNA-areas with fine fibrils (arrows) and starch grains (S). X 25700. Plate 8 Fig. 7-8. Ohloroplasts from detached green cotyledons of cucumber (control material) showing thylakoids, ribo- and polysomes (arrows) and starch grains (S). X 25700. Plate9 Fig. 9-10. Sections of chloroplasts from detached green cucumber cotyledons. 9, a portion of chloroplast showing the electron transparent DNA-containing area with starch granule (S). Ribo- and polysomes in chloroplasts stroma are also observed (arrows). x80500. 10, chloroplast with pro lamellar body (PB) and starch grains. X 25 700. Plate 10 Fig. 11-14. Ohromoplasts from detached cotyledons of cucumber treated with 0.06 l\I 000. 11-13, elongated and clear spaces in the stroma probably represent the sites of lycopene crystals, the crystals themselves being lost by dissolution in the solvents used for tissue dehydration. 11, fragment of undissolved crystal (double arrow). X 80500. 14, outline of dissolved crystal. Areas with DNA fibrils (arrows). 12-14. X 25700. Plate 11 Fig. 15. Electron transparent area in chromoplast at higher magnification (x 80500) showing the occurrence of D~A fibrils. Fragment of thylakoids pile shows crystalline structure (arrow). Fig. 16. Fragment of the chromoplast after DNase digestion showing small fragments of DNA fibrils. X 80500. Plate 12 Ohromoplast fragments with areas containing tubule-like structures in and longitudinal (fig. 19,20) sections. X 80500.
transvers~l
(fig. 17, 18)
Plate 13 Fig. 21, 22. Ohromoplasts with osmiophilic globules and membrane piles. 23, 24, fragments of membrane piles at X 26700. 25, 26, the same at X 80500. Plate 14 Section of chromoplast with grana-like membrane piles and single membranes (arrows).
X
80500.
BPP, Bd. 164
Plate 7
Mi kuls ka et al. VEB GUSTAV FISCHER VERLAG JENA
Plate 8
BPP, Bd. 164
l\Iikulska et al. VEB GUSTAV FISCHER VERLAG JENA
BPP, Ed. 164
Plate 9
Mikulska et al. VEB GUSTAV FISCHER VERLAG JENA
BI'P, Bd. 164
Plate 10
Mikulska et al. VEB GUSTAV FISCHER VERLAG JENA
BPP, Bd. 164
Plate 11
Mikulska et al. VEB GUSTAV FISCHER VERLAG JENA
Plate 12
BBP, Bd. 164
Mi kuls ka et al. VEB GUSTAV FISCHER VERLAG JENA
Plate 13
BPP, Rd. 164
Mikulska et al. VEB GUSTAV FISCHER VERLAG JENA
Plate 14
BPP, Ed. 164
Mikulska et al. VEB GUSTAV F:SCHER VERLAG JENA