Microscope Studies on the Morphogenesis of Plastids V

Bot. Mag. Tokyo 84: 123-136 (March 25, 1971)

Electron Microscope Studies on the Morphogenesis of Plastids V. Concerning One-dimensional Metamorphosis of the Plastids in Cryptomeriac Leaves*

by Susumu TOYAMA** and Kazuo FUNAZAKIS**

Received November 25, 1970

Abstract

To know about the mechanism of interconversion of plastids, electron microscopic observations were made on Cryptomeria leaves which aquire a reddish brown color in winter and recover their green color in coming spring to summer. In normal green leaves, two different kinds of plastids have been observed, viz, the chloroplasts having well organized grana structure in mesophyll cells and those completely lacking grana lamellae in bundle sheath cells. General feature of plastids in reddish brown leaves, may be summarized as follows : (1) The presence of red granules of rhodoxanthin (a carotenoid), and a well developed lamellar system involving grana- and intergrana-lamellae. (2) The plastoglobules, osmiophilic granules in plastids, increase in number and size as compared with the chloroplasts in normal green leaves. (3) Shrinkage which is one of the character- istic features of senescent plastids is not observed. (4) RNA content in plastid stroma is almost unchanged throughout an entire leaf stage ranging from normal green to subsequent regreening. Basic structure of the plastids in regreened leaves is quite similar to that in winter leaves, except for some increase of thylakoid membrane and decrease of osmiophilic granules. Accordingly, it is presumed that the plastids appearing in reddish brown leaves are not mere chromoplasts but those having an incipient nature of the chloroplast, since real chromoplasts can not be converted into the chloroplasts. It seems that monotrope Plastiden-Metamorphose " is plausible in this case.

Until now, two controversial opinions have been presented as regards the metamorphosis of the plastids. According to Schimperl', three kinds of plastids, chloroplast, chromoplast and leucoplast, are capable of interconversion from one to another. However, Frey-Wyssling et al.2' are of the opinion that the metamorphosis of plastids proceeds only in a linear fashion. Recently, Toyama and Ueda3' have shown that the reversion of chromoplast into active chloroplast does not occur in autumn leaves of Ginkgo biloba. On the other hand, Thomson et al4' have demonst- rated the reversion of chromoplast into chloroplast in pericarp cells of Valencia orange (Citrus sinensis L.), whose color is orange in winter and green in the next spring to summer. Similar color change may also be observed in Cryptomeria * This work was presented at the 33 rd Annual Meeting of the Botanical Society of Japan held at Kumamoto on Nov. 1-3, 1968. ** Department of Botany , Faculty of Science, Tokyo Kyoiku University, Otsuka, Tokyo, Japan (Present address: Department of Botany, University of California, Davis, California 95616) *** Biological Laboratory , Jissen Joshi-Gakuen, Shibuya, Tokyo, Japan. 124 TOYAMA, S. and FUNAZAKI, K. Vol. 84

leaves, which become reddish brown in winter and undergo regreening in spring. Two mechanisms may be conceivable in the analysis of regreening phenomena in these plants. The one is a reversion of the chromoplasts into the chloroplasts, and the other is the de novo formation of chloroplasts from the proplastids replacing Fig. 1 Scheme of reversible meta- the collapsing chromoplasts. Using Crypto- morphosis of the plastids proposed by meria leaves, the present experiment was Schimper (1885). carried out in order to clarify the process, of plastid metamorphosis in detail and to determine the alternative of both mechanisms mentioned above.

Fig. 2. Scheme of monotrope Plastiden-Metamorphose " proposed by Frey-Wyssling et at. (1955).

Materials and Methods

Leaves of Cryptomeria japonica were collected at two different locations,, at Yunoyama in Mie Prefecture on March, 1965 and at Higashimatsuyama in Saitama Prefecture on February, 1968. Some of the fresh leaf samples were used immediately for electron microscopic observation, and the other leaves were cultured in tap water under continuous illumination (fluorescent light, 25001ux) at ca. 20°. In order to examine the membrane system within the plastids, small leaf pieces were fixedd with 1 solution of KMnO4 for 2 hours at 4° . For the observation of granules and fine structure in the stroma, samples were prefixed with 3.5°Q solution of glutaraldehyde for 1 hour and post-fixed with 2°o aqueous 0504 for 2 hours at 4° . Thenn the fixed materials were dehydrated with acetone series, embedded in epoxy-resin - and polymerized at 50-60 ° . Sections were cut with an ultramicrotome (Porter-Blum, . Type MT-P, and examined with an electron microscope (Type JEM-7). Sections were stained withh a saturated solution of uranyl acetate for 2 hours and then with Millonig's solutions' for 30 minutes. Cytochemical examination on the senescence of plastids was made by the use of light microscope. For this purpose, several sections of fresh materials were stained with azure-B for the discrimination of RNA, and with fast green at pH 2 for the evaluation of total protein (Shaw et al.)6'. The sections to be stained with azure-B were treated in advance with DNA-ase to remove DNA. The staining with fast green was carreid out after removal of both DNA and RNA by pretreatment with 5% trichloroacetic acid. March, 1971 Electron Microscope Studies on the Morphogenesis of Plastids 125

Results

1. Plastids in normal green leaves In green leaves of Cryptomeria japonica, two different kinds of chloroplast were observed. The chloroplasts in mesophyll cells such as seen in photo 1, are typical ones with respect to their size and internal structure. These chloroplasts possess not only well-developed grana stacks connected with intergrana-lamellae but also plastoglobules and other fine granules. Another sort of chloroplast is shown in photo 2. The most remarkable feature in this chloroplast is a lack of grana structure. The internal lamellae are composed in general of a series of uninter- rupted membrane running in parallel along the long axis of the chloroplasts. In common to both kinds of chloroplast, starch grains, plastoglobules and ribosome like granules are contained, irrespective of the conspicuous difference in lamellar system. All leaves of Cryptomeria do not always attain a reddish brown color in winter. The leaves in the shade remain to be green as before. In these leaves, all plastids possess well-developed membrane structure together with a small number of plastoglobules (photo 3).

2. Plastids in reddish brown leaves Typical plastids appearing in the course of leaf color change, from green to reddish brown, are shown in photo 4. These plastids are characterized by gradual increase in number and size of plastoglobules and formation of small vacuoles in a periphery of stroma region. All plastids in reddish brown leaves possess more or less complicated lamellar system involving typical grana- and intergrana-lamellae, whose size is almost same as that in the normal green leaves (Table 1). Upon fixation with a solution of KMnO4, plastids usually retain their membrane system in an intact state (photo 5), but in some cases they are devoid of fine structure in the stroma. The most distinctive feature in the plastids of reddish brown leaves consists in an existence of numerous plastoglobules in the stroma. As a rule, 40- 80 globules of 0.1-0.5, t in size may be seen in one section of a plastid (photo 6 and 15). These globules are arranged among thylakoids often in a row along a longi- tudial axis of each plastid (photo 3, 15 and 18). Upon KMnO4 fixation spherical globules are disrupted into irregular, star-shaped bodies (photo 5, 7 and 16). In reddish brown leaves obtained from Yunoyama, most of the membrane systems in plastids have been disrupted into many discrete vesicles of ca. 0.05 i in

Table 1. Some charcteristic features of the plastids in Cryptomeria leaves from summer to spring. 126 TOYAMA, S. and FUNAZAKI,K. Vol. 84

size. These minute bodies may be regarded as a constituent of the thylakoid (photo 11). The vesicles are enveloped with double membrane and seem to be formed from thylakoids one after another by successive constriction of its terminal part. Accordingly, it may be that the collapse of thylakoid membrane is a coun- terpart of the process of lamellar formation (Toyama et al .)3. Osmiophilic globules are also observed in the matrix of cytoplasm. They are larger in size (1-2~€ in diameter), smaller in number than the plastoglobules, and found often in contact with the plastids (photo 13). Reddish brown leaves, which had been stored in an ice-box for 4 months after collection, were gradually brought into regreening upon culture in the light at 20 ° . Plastids in such leaves contain a large number of plastoglobules together with a few membrane system (photo 15). Abnormal lamellar systems were also observed in the plastids, which seems to be derived from the plastids in bundle sheath cells (photo 14). In some plastids, many vacuoles have been formed seemingly due to the swelling of the thylakoid membrane (photo 16). By the examinations under the light microscope, red granules of carotenoids (mainly rhodoxanthin)7' were observed, 6-12 pieces in a single plastid of reddish brown leaves (Table 1). However, electron micrographs have shown that they were lost during the course of dehydration with acetone. The presence of grana is dif- ficult to confirm under the light microscope because of the concurrence of carotenoid granules within the plastids, while it is discernible with ease under the electron microscope. Throughout the whole process from normal green to regreening of leaves, the changes in size and number of plastoglobules are shown in Fig. 3.

3. Plastids in the leaves undergone regreening Reddish brown leaves of Cryptomeria recover their capacity of greening during the culture for 4-5 days under 2500 lux at 20° . At this time, various morphological features of the plastids may be observed according to the physiological conditions to which they are exposed. Some plastids in the leaves undergoing regreening are able to regain all the lamellar structure comparable to that in typical chloroplasts

Fig. 3. Changes in number and magnitude of plastoglobules within the plastids of Cryptomeria leaves. Diameter of plastoglobule 0 Number of plastoglobule in one section of a plastid March, 1971 Electron Microscope Studies on the Morphogenesis of Plastids 1.27

of normal green leaves (photo 17). The other chloroplasts are degraded to the loss of regreening capacity due to the collapse and vacuolation of thylakoid (photo 16). The remarkable changes occurring in the plastid structure during regreening of leaves are the decrease in size and number of plastoglobules (Fig. 3) and a significant development of thylakoid membrane. Even if the regreening proceeds to some extent, all plastids do not always develop thoroughly into the chloroplast, because they do not attain a synchronized growth even in a single cell. The reddish brown leaves kept in ice-box for 4 months can recover their capacity of greening at room temperature in a few days. In this case, conspicuous changes in the feature of plastids are almost the same as mentioned above. Here, it is noteworthy that numerous ribosomal granules are seen in the matrix of plastids after regreening (photo 18). In this connection, it is conceivable that the two processes are involved in the course of regreening, i. e., the reactivation of preexisting plastids on the one hand, and the de novo development of proplastids into chloroplasts on the other. In some cases, proplastids are also produced from mature chloroplasts by budding (photo 8), although their reproduction is due to the division of themselves in general. Photo 9 shows some proplastids, which are filled with matrix and enveloped by double limiting membrane within the cells of reddish brown leaf. Young chloroplasts having small thylakoids are also shown in photo 10, in papallel with a mature plastid.

Discussion

The origin of the chloroplasts is still in a controversy. In general, however, it is surmised that they are autonomous self-duplicating bodies (Prey-Wyssling and Muhlethaler8' ; Kirk and Tilney-Bassett9'). The plastids increase in number by the following processes: (1) the division of proplastids, (2) the transverse constriction of chloroplasts, and (3) the budding of chloroplasts. The budding such as shown in photo 8 has not been observed in general plastid ontogenesis, so that this may be regarded as an exceptional phenomenon. It has been known for many years that the chloroplasts in the bundle-sheath cells are different from the chloroplasts in the mesophyll cells. The details of these dimorphic chloroplasts were studied on maize by Hodge et aL10', on many tropical grasses by Johnsonll', and on sugar cane by Laetsch12'. The mature chloroplasts in the bundle-sheath cells of these plants are larger ones, lacking grana and having many starch grains (photo 2). On the other hand, the chloroplasts in mesophyll cells are smaller, containing grana and having a few starch grains (photo 1). Laetsch13,14) has suggested that the chloroplast structure in tropical grasses is accomodated with C02-fixation, and has presumed to be an adaptation for rapid transportation of precursors and end products of photosynthesis. Along with the senescence of leaf cells, chloroplasts are converted into chromoplasts, in which many plastoglobules are produced, being probably due to the collapse of lamellar structure. The chromoplasts gradually undergo crumbling and end their life. In general, the chromoplasts which have been derived from the chloroplasts, such as seen in fruits of Capsicum, Physalis and in normal leaf cells, are not reverted into the chloroplasts (Fret'-Wyssling and Kreutzer1J' ; Toyama and Ueda3'). However, Thomson et al4' have shown that the chromoplasts in a mature pericarp of Valencia orange are able to come back again to the chloroplasts. In their electron micrographs, the chromoplasts had a small number of incomplete 128 TOYAMA, S. and FUNAZAKI, K. Vol. 84

lamellar systems together with many plastoglobules. It is likely to us that the plastids in this orange are not mature chromoplasts but intermediate forms traps itioning from chloroplasts to chromoplasts. On the other hand, in the plastids seenn in orange-yellow pericarp of Citrus natsudaidai, small lamellar structures and a red fluorescence of porphyrin compounds have been observed by the present authours. (unpublished) ; the plastids as such retain the activity and structure sufficient for the reversion of active chloroplasts. The development of proplastids into chloroplasts would take place during 3-4 months necessary for regreening of Valencia orange, although Thomson et al.4' are positive in denying the fact. The present writers have, observed numerous proplastids in mature pericarp cells of Citrus: natsudaidai (unpublished). On the other hand, Orsenigo16' has stated from his experiment on the bleaching of green hyacinth petals that the membrane system in the chloroplasts gradually disappear during bleaching, and that limiting membrane of the plastids are broken. down in bleached petals. He said that the conversion of chloroplasts into leucoplasts does not take place in usual cases. On the contrary, Thomson et al.4' have surmisedd that the transformation of chloroplasts into leucoplasts is possible, because of the structural analogy between the leucoplasts and the plastids in bleached petals. To our mind, however, it is desired that the plastids must be classified not only on their structure but also on their functional detail. In this case, the distinction betweenn the final and transitional features should, of course, be taken into account. Cryptomeria leaves surviving for 2 or more years apparently differ in their characteristics from the leaves of many deciduous plants. The reddish browning of leaves is due not to the senescence of plastids but to a transient change in function leading to the production of a red carotenoid pigment (rhodoxanthin). Inn fact, no lethal change was observed in fine structure and function of the plastids.. Therefore, it is unlikely that the regreening of Cryptomeria plastids has occurred as a result of the conversion of chromoplasts into chloroplasts. Within the one Cryptomeria tree, branches in the shade bear green leaves even in winter, whose chloroplasts have well-developed lamellar system. Accordingly, the abnormal accumulation of carotenoid pigment may take part in the maintenance of photosynthe- tic activity and also in the protection of plastids from bleaching by bright lightt under low temperature. Some of the significant changes during senescence of plastids are the formationn of plastoglobules followed by collapse of internal thylakoid systems and a conspicuous, shrinkage of plastids. Ikeda17 and Baker (unpublished) have also described the disintegration of chloroplast lamellae leading in turn to an accumulation of dense globules in the stroma. However, in reddish brown leaves of Cryptomeria, no in- dication of senescence has been recognized even in size and shape of the plastids,, except for a slight physiological senility indicated by the appearance of some plastoglobules. According to the biochemical studies of senescence made by Shawl$',, the disintegration of chloroplasts is preceded by an appreciable loss of RNA and protein. Besides, Oota19 has reported that the degeneration of ribosomes involving the liberation of RNA and protein is a main feature of the senescence. As shown qualitatively by cytochemical test (Table 1), a slight or no decrease was observed in contents of RNA and total protein in the plastids of reddish brown leaves of Cryptomeria. Therefore, it seems that the biochemical senility has not proceeded in Cryptomeria plastids, although the number and size of plastoglobules have increased to some extent. Plastoglobules also exist in the stroma of young March, 1971 Electron Microscope Studies on the Morphogenesis of Plastids 129

plastids during the development of lamellar system, so that they are not always regarded as disintegration product of lamellar structures. It seems that the f or- mation of plastoglobules is caused by metabolic unbalance between lipid synthesis and lamellar formation, and that they play a significant part in the storage of ex- cessive lipid materials (Barton20' ; Peveling and Lichtenthaler21'). The validity of such speculation may be supported by the facts that the plastoglobules are also contained in proplastids of the seedling cultured in the dark, and that they decrease in number rapidly in the light leading in turn to the formation of lamellar structure (Sprey and Lichtenthaler22'). According to Lichtenthaler and Peveling23', the giant osmiophilicc globules in the cytoplasm are derived from plastoglobules and perform a function of lipid storage in the cells. Dynamic interrelation between plastoglobules and giant globules inn cytoplasm can not be answered by the present study, though the latter are found frequently in closer connection with the plastids.

Acknowledgement

The present authors express their sincere thanks to Prof. K. Hayashi and Assist. Prof. R. Ueda in the department of Botany, Tokyo Kyoiku University, and also to President S. Tadenuma of Jissen Joshi-Gakuen, for their kind guidance and encouragement throughout this investigation. We are also grateful to Prof. C. R. Stocking and Emer. Prof. T. E. Weier in University of California, Davis, for their valuable criticism and correcting the manuscript.

Literature cited

1) Schimper, A. F. W., Bot. Ztg. 41: 105, Texas, Austin. 112, 137, 1.53 (1883). 12) Laetsch, W. M., Z. Pflanzenphysiol. 54 2) Frey-Wyssling, A., Ruch, F. and Berger, 472 (1965). X., Protoplasma 45: 97 (1955). 13) Amer. J. Bot. 55: 875 (1968). 3) Toyama, S. and Ueda, R., Sci. Rep. 14) , P. 36-46 in Progress in Photo- Tokyo Kyoiku Digaku 12 B : 31 (1965). synthesis Research" Publication spon- 4) Thomson, W. W., Lewis, L. N. and sored by International Union of Biological Coggins, C. W., Cytoloia 32. 117 (1967). Science. (1969). 5) Millonig, G., J. Biophys. Biochem. Cytol. 1.5) Frey-Wyssling, A. and Kreutzer, E., J. 11: 746 (1961). Ultrastruct. Res. 1 : 397 (1958). 6) Shaw, M., Bhattacharya, P. K. and Quick, 16) Orsenigo, M., Giorn. Bot. Ital. 70: 467 W. A., Can. J. Bot. 43: 739 (1965). (1963). 7) Hida, M. and Ida, K., Bot. Mag. Tokyo 17) Ikeda, T. and Ueda, R., Bot. Mag. Tokya 77: 458 (1964). 77: 336 (1964). 8) Frey-Wyssling, A. and Mi hlethaler, K., 18) Shaw, M. and Manocha, M. S., Can. J. P. 230-241 in Ultrastructural Plant Bot. 43: 747 (1965). Cytology" Elsevier Publishing Company. 19) Oota, Y., Ann. Rev. Plant Physiol. 15: (1.965). 1.7 (1964). 9) Kirk, J. T. O. and Tilney-Bassett, R. A. E., 20) Barton, R., Planta 71: 31.4 (1.966). P. 402-523 in The Plastids" W. H. 21) Peveling, E. and Lichtenthaler, H. K., Z. Freeman and Company. (1967). Pflanzenphysiol. 56: 299 (1967). 10) Hodge, A. J., McLean, J. D. and Mercer, 22) Sprey, B. and Lichtenthaler, H. K., Z. F. V., J. Biophys. Biochem. Cytol. 1 . 605 Naturforschg. 21 B : 697 (1966). (1955). 23) Lichtenthaler, H. K. and Peveling, E., Z. 11) Johnson, Sr. M. C., Ph. D. Thesis, Univ. Pflanzenphysiol. 56: 1.53 (1.967). 130 TOYAMA, S. and FUNAZAKI, K. Vol. 84

Explanation of Plates

Plates I-VI are the electron micrographs of plastids in leaf cells of Crypomeria japonica, which were fixed with a solution of glutaraldehyde-osmium tetraoxide or with an aqueous solution of potassium permanganate. Millonig's method for staining was applied to all sections for giving contrast in membrane structures. Designations in the figures are as follows : CG, osmiophilic globule in the cytoplasm ; CW, cell wall ; GL, grana lamellae M, mitochondria ; PG, plastoglobule ; PP, proplastid ; R, ribosome ; S, starch ; SL, stroma lamellae; V, vacuole ; VE, vesicle ; YC, young chloroplast. Plate 1. 1) Chloroplast in a green leaf cell in summer. Inner membrane system is composed of the grana- and intergrana-lamellae similar to those of angiosperm plants. 2) Chlo- roplast in a bundle sheath cell, which is characterized by the absence of grana structure. 3) Chloroplast in green leaf cell in winter, in which lamellar system is well preserved and several plastoglobules have come into appearance. Plate II. 4) Plastid at an intermediary stage of leaves from green to begining reddish brown in winter. Enhancement of plastoglobules and formation of vacuoles in a periphery of stroma region are especially noteworthy. 5) Plastids in reddish brown leaf fixed with KMnO4, in which the well developed grana structure, many plastoglobules and a large starch grain are observed. Plate III. 6) Plastid in reddish brown leaf fixed with 0s04, in which lamellar system is still retained and the occurrence of plastoglobules is quite evident. Ribosome granules in both cytoplasm and plastids are also observed. 7) Plastoglobules in reddish brown leaf fixed with KMnO4. 8) Proplastid of a reddish brown leaf, showing its formation by budding from the mature plastid. Plate IV. 9) proplastid in reddish brown leaf in winter, which are enveloped by double limiting membrane and filled with matrix. 1.0) Young chloroplast containing small thylakoids. This seems to show the development of plastid even in a reddish brown leaf in winter. 11) and 1.2) show the disintegration process of senescent plastids. 1.1) Plastid fixed with KMnO4, in which thylakoids are degraded into many fine vesicles. Grana lamellae are preserved until intergrana lamellae have disappeared. 1.2) Plastid fixed with 0504, in which disappear- ance of thylakoids and matrix, and remarkable reduction of plastoglobules can be recognized. Plate V. 1.3) Comparison of plastoglobules with osmiophilic globules in the cytoplasm of a reddish brown leaf. Both are easily distinguishable by their size, though scarcely dis- criminate from their density and affinity to 0504. 1.4)-1.6) show the plastids kept for about 4 months in the cold and dark. 1.4) Plastid possessing abnormal lamellar system. This seems to be a plastid in bundle sheath cell. 1.5) One of the typical plastids at a maximal stage of the reddish browning. Note the size and number of plastoglobules in the plastid. Plate VI. 1.6) Vacuolation of thylakoid by fixation with KMnO4. 1.7)-1.8) Plastids of a leaf under regreening in the light (2500lux) at 20°. Reduction in size and number of plas- tolo~ales and existence of ribosome granules are clearly shown. 1.7) Plastid in mesophyll cell. 1.8) Plastid in bundle sheath cell. March, 1971 Electron Microscope Studies on the Morphogenesis of Plastids 131

Plate I 132 TOYAMA, S. and FUNAZAKI, K. Vol. $4

Plate II March, 1971 Electron Microscope Studies on the Morphogenesis of Plastids 133

Plate III 134 TO YAMA, S. and TUNAZAKI, K. Vol. 84

Plate IV 135 March, 1971 Electron Microscope Studies on the Morphogenesis of Plastids

Plate V 136 TOYAMA, S. and FUNAZAKI, K. Vol. 84

Plate VI