ULTRASTRUCTURAL CHANGES OF PLASTIDS IN FLAX EMBRYOS CULTIVATED IN VITRO

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1 New Phytol. (1981) 87, ULTRASTRUCTURAL CHANGES OF PLASTIDS IN FLAX EMBRYOS CULTIVATED IN VITRO BY MILADA CIAMPOROVA AND ANNA PRETOVA Slovak Acadetny of Sciences, Institute of Experimental Biology and Ecology, Bratislava, Czechoslovakia {Accepted 15 June 1980) SUMMARY The ultrastructure of plastids in the cells of flax cotyledons was investigated at three stages during embryogenesis and germination: (1) immature 14-day old green embryos excised from seeds, (2) 24-day old embryos which yellowed following a 10-day cultivation in vitro and (3) 34-day old embryos which grew for 20 days on the medium and after a temporary loss of pigment regreened and germinated. For comparison, the ultrastructure ot chloroplasts from the mature leaves was also investigated. The chloroplasts of 14-day old embryos are characterized by a well-developed system of both the grana and the intergranal lamellae and stroma which contains starch grains and phytoferritin. During cultivation in vitro the dimensions of plastids decrease and the internal structure of the chloroplasts undergoes disintegration. In the stroma of plastids of the 24-day old embryos, lamellae of different length, vesicles and electron-dense, membrane-bound bodies and numerous plastoglobuli can be identified and amoeboid plastids occur in the cells. During continued cultivation the dimensions of plastids again increase and in them again differentiation of the system of grana and intergranal lamellae can be observed. The structure of plastids in the 34-day old embryos is in good agreement with the structure of those in the mature, assimilating leaves of flax. The results indicate that the basic developmental pathway takes place in the same plastids of the flax embryos once during the embryogenesis and once during germination. INTRODUCTION Plants with green coloured embryos such asflaxare classified as chloroembryophyta (Yakovlev and Zhukova, 1973). Inside the seed, the flax embryos lose their green colour just prior to ripening and they become yellow (Kantorova, 1957). Similar colour changes occur also under in vitro conditions. The excised 14-day old green embryos cultivated in vitro for 7 to 10 days lose their chlorophyll pigments and become yellow, thus reaching the stage deflned as 'dormancy in vitro' (Pretova and Erdelska, 1977). After a further 7 to 10 days cultivation, the flax embryos begin to germinate and at the same time, chlorophyll again can be observed in their cotyledons (Pretova, 1977, 1978). Amyloplasts with very few lamellae occur in the flax oosphere (D'Alascio- Deschamps, 1973). The bicellular flax proemhryo contains amyloplasts with dense accumulations of phytoferritin in the stroma (Deschamps, 1970). The plastids of the embryo in its heart stage have already developed grana and stroma lamellae but they do not contain starch (Deschamps, 1969). F'urther stages of plastid development have not been investigated yet. The present study shows three further stages of the plastid structural development in the cotyledons of the flax embryos cultivated itt vitro. The stages investigated correspond to those used for the determination of the quality and quantity of pigments in the flax embryos cultivated in situ and in vitro (Pretova, 1977, 1978) X/81/ S02.U0/ The New Phytologist

2 474 M. CiAMPOROVA AND A. PRETOVA MATERIALS AND METHODS Immature 14-day old embryos of flax {Linum usitatissimum, L., cv. Viera) were excised from seeds and placed on White's (1942) medium with 5 % sucrose. The embryos were grown in test tubes at 25 C and light intervals of 3000 lx for 16 h per day. Cotyledons of immature, 14-day old green embryos immediately after excision, cotyledons of 24-day old yellow embryos which were grown for 10 days in vitro and had lost their chlorophyll pigment during this period (i.e. embryos in the stage of 'dormancy in vitro') and finally cotyledons of 34-day old embryos which were cultivated on the medium for 20 days and which again regreened atid started to germinate, were fixed for the electron microscopical investigation. For comparison, a sample from the mature leaf of fiax plants growing in the field was taken. The material was fixed in 3 % glutaraldehyde for 1-5 h, washed in phosphate buffer (0-66 M, ph 7 2) and postfixed in 1 % OsO^ in the same buffer. F'ollowing dehydration in an ethanol series and propylene oxide the specimens were embedded in Epon 812. The ultrathin sections were stained with 1 % aqueous uranyl acetate solution for 1 h and with lead citrate (Venable and Coggeshall, 1965) for 6 min. The material was examined in Philips EM 300 and Tesla BS 500 electron microscopes. RESULTS Fourteen-day old embryo (green) The degree of vacuolation is relatively low in the mesophyll cells of the cotyledons. In addition to all the basic organelles, the cytoplasm contains numerous lipid bodies and plastids randomly distributed throughout the entire cell. According to their well developed inner structure the plastids are classified as chloroplasts (Fig. 1 a). Most of them are of oval shape, their length being 3 to 4 //m and width 1 5 //m. In a cell section there are usually 6 chloroplasts with 1 or 2 starch grains embedded in the stroma of each. The chloroplasts contain 5 to 8 grana. The cisternae of both the intergranal and grana lamellae are narrow and electron transparent. The plastoglobuli, maximum six in number in a cross section of a plastid, are present in the stroma between the intergranal lamellae. In the stroma of some chloroplasts, dense accumulations of phytoferritin can be seen (Fig. 1 b). Twenty-four-day old embryo (in vitro, yellow) The largest part of the cell volume is occupied by the large central vacuole. The most numerous structural components are the lipid bodies (Fig. 1 c). In a cell section there are 1 to 6 plastids of an oval or nearly spherical shape usually 1 to 1 5 /<m long and 0 8 /im wide. No occurrence of a granal arrangement of membranes has been observed in any plastid. Vesicles and long, irregularly orientated lamellae are present in the stroma of the plastids. A large number of plastoglobuli (up to 25 in a plastid section) form aggregations in the stroma. Other structures in the stroma are the electron-dense circular or angular membrane-bound bodies. In some places bounding membrane and membrane projections are discernible (Figs 1 c and 2 a. Starch grains occur in fewer than 50 % of plastids. The shape of some plastids is amoeboid revealing enclosed portions of cytoplasm in their cross sections (Fig. 2b).

Plastids in cultivated flax embryos 475 Fig. 1. N, nucleus; M, mitochondria; L, lipid bodies; cw, cell wall; pg, plastoglobuli; S, starch grain; I, inclusion (membrane-bound body); cyt.

3 Plastids in cultivated flax embryos 475 Fig. 1. N, nucleus; M, mitochondria; L, lipid bodies; cw, cell wall; pg, plastoglobuli; S, starch grain; I, inclusion (membrane-bound body); cyt., cytoplasm, (a) Chloroplasts of a 14-day old green embryo, x (b) Portion of a chloroplast with phytoferritin (arrow) of a 14-day old green embryo, x (c) Plastids of the 24-day old yellow embryo. Electron-dense, membrane-bound bodies m the stroma (arrows). X (C)

4 476 M. ClAMPOROVA AND A. PRETOVA Fig. 2. (a), (b) Plastids of the 24-day old yellow etnbryos. (a) Starch grain atid tnembranes ptojeetitik out from tbe membratie-bound body (arrow) in tbe plastid stroma. x (b) Enclosed portion of cytoplasm in the amoeboid plastid. x (c) Cbloroplast of tbe 34-day old Krcen embryo. X 267yo. (d) Chloroplasts of a mature assimilating leaf, x 9840.

Plastids in cultivated flax embryos 477 Thirty-four-day old embryos {germinating in vitro, green) The cells of cotyledons have a large central vacuole.

5 Plastids in cultivated flax embryos 477 Thirty-four-day old embryos {germinating in vitro, green) The cells of cotyledons have a large central vacuole. The narrow layer of cytoplasm along the cell wall contains no lipid hodies. The plastids, hy their arrangement of lamellae in a system of grana and intergranal lamellae, correspond with the structure of chloroplasts (Fig. 2c). The lens-shaped chloroplasts are 4 to 5 //m in length and 15 to 2 /im wide. The chloroplasts contain 10 to 20 grana. They usually do not contain starch hut in some of them a small rounded grain of starch has heen observed. Between the intergranal lamellae about 1 to 3 plastoglobuli are present. Mature leaf The chloroplasts of the fully functional leaf mesophyll (Fig. 2d) are very similar to the chloroplasts of the green cotyledons of the germinating 34-day old embryos, six of them usually being present in cross sections of cells. Their shape is lens-like, usually 7 /ym long and 2 5 //m wide. In the stroma there is a well developed system of grana (10 to 20 in chloroplast cross section) and intergranal lamellae. The chloroplasts contain 1 to 3 large starch grains and only a few plastoglobuli. The electron microscopic investigation has suggested that, along with the loss of the green colour of the Rax embryos during their 10 day long cultivation on White's medium, the granal arrangement of chloroplast becomes destroyed. During continuation of the embryo cultivation simultaneously with the greening process, a complex system of grana and intergranal lamellae redifferentiate probably in the same plastids. DISCUSSION The degree of differentiation of chloroplast structure seems to depend, among other factors, on the light conditions to which embryos are exposed. With embryos embedded below a thick layer of fruit, like those of Citrulus nobilis (Orsenigo, 1964, cf. Yakovlev and Zhukova, 1973), the structure of chloroplasts is very simple and imperfect. The structure of chloroplasts in embryos with thinner envelope layers, e.g. Iberis umbellata (Yakovlev and Zhukova, 1973) approaches the structure of chloroplasts of the assimilation leaves. The same feature is likely to occur with the flax plant. From the heart stage of the flax embryo, i.e. 5 to 7 day after pollination (Deschamps, 1969) to the stage of a 14-day old embryos in situ, starch accumulates and the number of grana increases in the embryo chloroplasts. However, they are smaller and contain fewer grana than the chloroplasts of mature leaves. Their ultrastructure correlates with the lower amount of chlorophyll when compared to that of the assimilating leaves of flax. In the 14-day old embryos, chlorophyll a and chlorophyll b, violaxanthin, lutein and //-carotene but not neoxanthin were identified (Pretova, 1977). Following the placement of the 14-day old green flax embryos on the cultivation medium for 10 days, the emhryos lose their green colour. In this stage, neither green nor yellow pigments were present except some trace of lutein (Pretova, 1978). In the cotyledon cells, the development of a large central vacuole was observed. It was shown that the cell division declined in the mesophyll of the cotyledons of the 9 to 14-day old emhryos and that there were practically no mitoses during their further in-vitro cultivation (Frdelska, 1978). Since the plastid division (judged by the absence of constricted plastids) does not occur as well, the number of plastids in the cells appears to remain unchanged, but the dimensions of the plastids

478 M. CiAMPOROVA AND A. PRETOVA decrease significantly. Some of them are in the stage of amoeboid plastids, e.g. the Stage 3 according to Whatley (1977).

6 478 M. CiAMPOROVA AND A. PRETOVA decrease significantly. Some of them are in the stage of amoeboid plastids, e.g. the Stage 3 according to Whatley (1977). In the plastids, disintegration of both the granal and the intergranal membranes takes place and the number of plastoglobuli mcreases. At the same time, membrane-bound bodies occur in the stroma. These are more frequently in their angular form, which is the more advanced stage in the process of formation of plastid lamellae compared with the circular form (Platt-Aloia and Thomson, 1977). The dense inclusions probably represent the accumulated protein which might be utilized later in the process of the formation of the lamellar system of chloroplasts as in the leaves of some plants (Stetler and Laetsch, 1969; Cran and Possingham, 1974; Platt-Aloia and Thomson, 1977). During the continued in-vitro cultivation of the fiax embryos the yellowed embryos regreen and germination begins. The dimensions of plastids increase and they become lens-shaped. In the stroma of the plastids the protein inclusions (membrane-bound bodies) disappear and a simultaneous differentiation of both the grana and the intergranal lamellae occurs. With the 34-day old embryo, the internal structure of chloroplasts does not differ qualitatively from the structure of the functional chloroplasts in the mesophyll of mature leaves. The latter are only slightly larger and contain more grana along with 1 to 3 large starch grains. The embryos in this stage contained chlorophyll a, chlorophyll b, lutein, neoxanthin violaxanthin and /?-carotene (Pretova, 1978). ' Whatley (1977) suggested that chloroplast development follows a single basic pathway including certain consistent basic stages and certain optional stages regardless of the tissue type and the species. The processes of plastid dedifi^'erentiation and redifferentiation in the ripening and germinating seeds respectively, have already been observed and the results have recently been summarized (Whatley, 1978). Dedifferentiation of amyloplasts to undifferentiated plastids (Stage 1) and their redifferentiation to chloroplasts (Stage 5) in the same species was observed in primary leaves of Phaseolus vulgaris during seed development and subsequent germination (Whatley, 1979). It seems probable that in individual plastids of the fiax embryo cotyledons, the single basic pathway takes place twice. During fiax embryogenesis, the first chloroplast differentiation is accompanied by the accumulation of phytoferritin (Deschamps, 1970) representing the optional stage B (Whatley, 1977). During the embryo maturation in vitro, the loss of green colour is accompanied by the disintegration of the chloroplast structure. Following the further in vitro cultivation chloroplasts again differentiate. In this process the membrane-bound protein deposits occur representing the optional stage D (Whatley, 1977). ^ f K It was shown that, from the stage of the heart embryo up to the 14-day old fiax embryo, the dififerentiation of the chloroplast structure occurred in situ. However, further developmental processes in situ do not need to be the same as we have showri for the in vitro conditions. In the in situ greening cotyledons of Cucurbita maxima (Lott and Castelfranco, 1970) and of Pinus nigra (Nikolic and Bogdanovic, 1972), prolamellar bodies as optional stages were found. We failed to find these structures in the in vitro cultivated fiax embryos. The development of chloroplasts and primarily the optional stages of this process vary in accordance with the total developmental and physiological state of plants (Platt-Aloia and Thomson, 1977) and also as a result of changes in the outer environmental conditions (Whatley, 1977), which include also the factor of the in vitro cultivation. Therefore, the further stages of plastid development in both, in situ and in vitro will be investigated.

7 Plastids in cultivated flax embryos 479 REFERENCES CHAN, D. G. & POSSINGHAM, J. K. (1974). Plastid thylakoid formation. Annals of Botany, 38, 843. D'AI.ASCIO-DESCHAMPS, R. (1973). Organisation du sac embryonaire du Linum catharticum L. espece recoltee en station naturelle; etude ultrastructurale. Btdletin Societe botanique de France, 120, 189. DESCHAMPS, R. (1969). Premiers stades du developement de l'embryo et de l'albumen du lin. Revue de cytologie et de bintogie vegetates, 32, 379. DESCHAMPS, R. (1970). Sur la presence de pbytoferritine dans l'ovule du lin, Linum tisitatissimum L. Revue de cytologie et de biologie vegetates, 33, 101. ERDELSKA, O. (1978). Reaction of immature Hax embryos to excision. Biologia (Bratislava). 33, 17. KANTOROVA, T. S. (1957). On tbe embryology of cultured Hax (in Russ). Byulleten' Glavnogo botanicheskogo sada, 29, 48. LoTT, J. N. A. & CASTELFRANCO, P. (1970). Cbanges in tbe cotyledons of Cucurbita maxima during germination III. Plastids and cbloropbylls. Canadian Journal of Botany, 18, NiKOI.IC, D. & BoGDANOVlc, M. (1972). Plastid diffet-entiation and cblorophyll syntbesis in cotyledons of black pine seedlings grown in tbe dark. Protoplasma, 75, 205. PLATT-.AI.OIA, K. A. & THOMSON, W. W. (1977). Cbloroplast development in young sesame plants. Neio Phytologist, 78, 599. PREfoVA, A. (1977). Pigments in young embryos of Linum usitatissumum L. Photosynthetica, II, 217. PREfovA, A. (1978). Flax embryogenesis. Cbanges in tbe pigments and lipid content in vitro and in situ Biologia {Bratislava), 33, 29. PREfovA, A. & ERDELSKA, O. (1977). Some differences in tbe embryogenesis of excised Rax embryos. In: Use of Tissue Culture in Plant Breeding - a Symposium (Ed. by F. Novak), pp Prague STETLER, D. A. & LAETSCH, W. M. (1969). Cbloroplast development in Nicotiana tabacum 'Maryland Mammotb'. American Jotirnal of Botany, 56, 260. VENABLE, J. H. & COOGESHALL, R. (1965). A simplified lead citrate stain in electron microscopy. Journa/ of Cell Biology, 25, 407. WHATLEY, J. M. (1977). Variations in tbe basic patbway of cbloroplast development. Netv Phytologist, 78, 407. WHATLEY, J. M. (1978). A suggested eyele of plastid developmental interrelationsbips. New Phytologist, 80, 489. WHATLEV, J. M. (1979). Plastid development in tbe primary leaf of Phaseolus vulgaris. variations between different types of cell. New Phytologist, 82, 1. WHITE, P. R. (1942). Plant tissue culture. Annual Review of Biochemistry, 11, 615. YAKOVLEV, M. S. & ZHUKOVA, G. Ya. (1973). Angiosperms with Green and Colotirless Embryos (in Russ). Izd. Nauka, Leningrad.

THE BEHAVIOUR OF CHLOROPLASTS DURING CELL DIVISION OF ISOETES LACUSTRIS L.

New Phytol (1974) 73, 139-142. THE BEHAVIOUR OF CHLOROPLASTS DURING CELL DIVISION OF ISOETES LACUSTRIS L. BY JEAN M. WHATLEY Botany School, University of Oxford (Received 2 July 1973) SUMMARY Cells in