INFLUENCE OF LEAF DIFFERENTIATION ON THE DEVELOPMENTAL PATHWAY OF COLEUS CHLOROPLASTS

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New Phytol. (1982) 92, 273-278 277 INFLUENCE OF LEAF DIFFERENTIATION ON THE DEVELOPMENTAL PATHWAY OF COLEUS CHLOROPLASTS BY P. JACOB VARKEY AND MATHEW J.

1 New Phytol. (1982) 92, INFLUENCE OF LEAF DIFFERENTIATION ON THE DEVELOPMENTAL PATHWAY OF COLEUS CHLOROPLASTS BY P. JACOB VARKEY AND MATHEW J. NADAKAVUKAREN Biological Sciences Department, Illinois State University, Normal, Illinois 61761, U.S.A. (Accepted 22 April 1982) SUMMARY Ultrastructural observations of young Coleus leaves revealed two pathways of chloroplast development within the same leaf. Developing plastids in the mesophyll cells from the basal regions of a leaf contained dense-staining inclusion bodies associated v^^ith newly forming thylakoids while these inclusion bodies were absent from the plastids in the cells of the leaf tip. Studies of the chemical nature of these inclusion bodies suggest that they are made up of proteins and possibly lipids, with no carbohydrates. The material present in the newly-forming thylakoids is similar in electron density to the inclusion bodies. The difference in chloroplast ontogeny appears to be related to the difference in cell development between the leaf tip and base. INTRODUCTION Proplastids of meristematic tissue are considered to be the precursors of chloroplasts. However, the developmental pathway of proplastids to mature chloroplasts can vary (Whatley, 1977). Many studies have focussed on etioplasts of dark-grown plants and their transformation to mature chloroplasts when exposed to light (Kirk and Tilney-Bassett, 1978). There are also a number of reports on the development of proplastids to chloroplasts in plants that are grown under normal light-dark conditions. However, there is a lack of consensus as to the exact developmental sequence. Membrane-bounded inclusions have been seen in proplastids of many normally grown plants (Srivastava, 1966; Badenhuizen and Salema, 1967; Israel and Steward, 1967; Ames and Pivorum, 1974; Cran and Possingham, 1974; Esau, 1975; Platt-Aloia and Thomson, 1977; Mares, Coote and Possingham, 1979). Usually these bodies are granular in nature and stain densely with osmium tetroxide. Although their chemical composition and function are speculative, investigators have suggested that these bodies are storage sites for carbohydrates (Badenhuizen and Salema, 1967) or protein (Marinos, 1967). Srivastava (1966) speculated that these inclusion bodies may have a role in the formation of thylakoid membranes. Israel and Steward (1967) called them prethylakoid bodies and suggested that they are the sites of accumulation of thylakoid membrane precursors. Close association, and in many cases, continuity between the darkly stained inclusion bodies and the thylakoid membranes have been shown (Platt-Aloia and Thomson, 1977; Casadoro and Rascio, 1978; Mares et al, 1979). Studies using successively older leaves have shown that these inclusions are more frequent in young leaves and that they are absent in fully developed leaves (Platt-Aloia and Thomson, 1977; Casadoro and Rascio, 1978, 1979). In this paper we present our observations ofthe membrane-bound inclusions of Coleus leaf proplastids and their X/82/ $03-00/ The New Phytologist

274 P- J- VARKEY AND M. J. NADAKAVUKAREN fate during leaf development. The results of enzyme digestion and histochemical studies are also presented.

2 274 P- J- VARKEY AND M. J. NADAKAVUKAREN fate during leaf development. The results of enzyme digestion and histochemical studies are also presented. MATERIALS AND METHODS Coleus plants (Coleus blumii Benth.) grown in the greenhouse under natural summer light-dark conditions were used. Shoot apices, young leaves of different ages measuring 1-5 to 18 mm, and mature leaves were taken for electron microscopy. Pieces of tissue close to the margin and near the midrib from both the tip and base of these leaves were fixed in 5 % phosphate buffered (ph 7-3) glutaraldehyde and post-fixed in buffered 1 % osmium tetroxide. They were dehydrated in a graded series of ethyl alcohol and in propylene oxide. Block staining with uranyl acetate was carried out during dehydration in 70 % alcohol. Tissue pieces were embedded in Epon 812 and sections were made using a diamond knife on a Reichert Om-U2 ultramicrotome. The sections were post-stained with uranyl acetate and lead citrate. Thin sections picked up on gold grids were stained for carbohydrates using the method of Thiery (1.967). For protease and pepsin digestions, thin sections picked up on gold grids were floated for 14h on 1 % solution of phosphate buffered protease (Sigma) adjusted to ph 7 or on 1 % solution of buffered pepsin (Worthington Biochemical Corporation) adjusted to ph 2. Appropriate controls using phosphate buffer adjusted to ph 7 and ph 2 without protease or pepsin were run for the same length of time. The sections were washed for 2 h by floating them on distilled water and then stained with lead citrate. These procedures were carried out at room temperature. The sections were examined with a Hitachi HU-llA electron microscope operating at 50 kv. RESULTS The plastids in the shoot apex and primordial leaves were not fully developed; many of them showed densely staining membrane-bound inclusion bodies in the stroma [Fig. 1 (a)]. The size of the inclusions varied from 0-2 to 0-9 /*m across the long axis. Although the inner membrane of the plastid envelope showed invaginations into the stroma, no connection to the inclusion was noticed. Plastids at all stages of development were found in young leaves measuring 3 to 18 mm. Mesophyll cells from the tip of leaves as small as 3 mm contained fully developed chloroplasts. However, the plastids in the mesophyll cells from the basal regions of these leaves were less developed than those from the tip. Fully developed chloroplasts were not seen in the basal cells. Plastid inclusions associated with grana at different developmental stages were frequently seen in these cells [Fig. l(b)]. Grana made up of several thylakoids and attached to the inclusion can be recognized in these plastids after digestion with pepsin [Fig. l(d)]. In general, a decrease in the density of the inclusion bodies was noticed in successively older leaves. In addition we found fewer plastids with inclusions in older leaves. The newly formed thylakoids of these plastids contained lightly stained membranes bordering electron dense material [Fig. l(c)]. We did not find any inclusions in the mesophyll cell plastids from the tip of the smallest leaf we observed. Plastids of the bundle sheath cells in all parts of the leaf showed slower rate of development than the mesophyll cell plastids. Prolamellar bodies were not seen in any of the plastids examined. Carbohydrate specific staining of the inclusion bodies yielded negative results.

3 Differentiation and chloroplast development - 'S. J ' '. i...*.- '.?, ' * : '. " '; V 'I H;. (c) Fig. 1. (a) Plastid in a mesophyll cell from the bast- of a youiik lt-al" showing a di-nscly st inclusion body, (b) Grana formation from an inclusion body in a plastid from a basal CL-11 of a ycunk leaf, (c) Plastid from the basal cell of a youn^ leaf.showing lightly stained th.ylakoid membranes, (d) Plastids from a basal cell of a young leaf after digestion with pepsin. Note decreased stain densits ofthe inclusion body and grana compared to those in (b). Arrow indicates thylakoid membranes attached to the inclusion body, (b) and (d) are serial seciions of the same plastid. All markers represent ()-5 //m.

4 276 p. J. VARKEY AND M. J, NADAKAVUKAREN After protease digestion, there was a slight reduction in the density of the inclusions. Pepsin digestion led to a considerable reciuction in the density of the inclusions and the mlaterial in the thylakoids [Fig. l(d)]. No reduction in density of the inclusions was noticed in the sections floated on phosphate buffer without protease or pepsin. DISCUSSION The complete absence of inclusion bodies in the developing plastids of the mesophyll cells at the tip of young leaves and the predominance of these bodies in the developing plastids of the mesophyll cells at the leaf base suggest two pathways of chloroplast development in Coleus. It has long been known that in leaves there is a differentiation gradient along the long axis (Avery, 1933; Saurer and Possingham, 1970; Steer, 1971). Cells at the leaf tip develop faster than cells in other parts of the leaf. Our observation that plastid development within a given cell is highly uniform suggests that it is regulated by the rate of cell development. Our results also suggest that the state of development of the cell seems to determine the pathway of plastid development in Coleus. Plastid development is retarded in less developed mesophyll cells at the base of young leaves. Because of the slow organization of thylakoid membranes in these cells, the precursors accumulate in the form of inclusion bodies and as the cell develops, a gradual organization of thylakoid membranes from these inclusions takes place. The complete absence of such inclusion bodies in the plastids of the leaf tip mesophyll cells shows that inclusion bodies are not prerequisites for the formation of thylakoid membranes. Except for the absence of prolamellar bodies, our observations are consistent with those of Casadoro and Rascio (1977) on tobacco (their fig. 14, patterns A and B), thus providing further support for the hypothesis that deviation from the standard developmental sequence is due to a change in the equilibrium between the rate of synthesis of the thylakoid precursors and the organization of the thylakoid membranes. Studies on the chemical nature of the inclusions show that they are mainly made up of porteins, but since the inclusions were not completely digested by pepsin or protease, protein is not the only component present. Salema and Abreu (1972) suggested that the inclusions in developing chloroplasts of Phyllanthus were lipoproteinaceous in nature. Since we did not detect any carbohydrates in the inclusions using Thiery's (1967) method, we suggest that the inclusions are most likely to have lipids in addition to proteins. The decreased electron density of both inclusion bodies and the material present in the newly formed thylakoids [Fig. l(d)] following enzyme digestion clearly suggests a similarity in their chemical composition and provides further evidence that thylakoids can be formed from these inclusions. A gradual differentiation of thylakoid membranes from plastid inclusions resulting in grana formation has been described previously by others (Cran and Possingham, 1974; Platt-Aloia and Thomson, 1977; Hurkman and Kennedy, 1977; Casadoro and Rascio, 1978). We conclude that in Coleus the developmental pathway of proplastids to chloroplasts is influenced by the state of leaf development. A model for chloroplast development in Coleus leaves based on our ultrastructural observations is presented in Figure 2.

5 Differentiation and chloroplast development 277 Fig. 2. Schematic representation of two different pathways of chloroplast development in one leaf. Path A represents development in cells from the tip and path B represents development in cells from the base of the same leaf. ACKNOWLEDGEMENT We thank Professor Derek McCracken for his valuable comments during the preparation of this manuscript. REFERENCES AMES, I. H. & PIVORUM, J. P. (1974). A cytochemical investigation of chloroplast inclusion. American Journal of Botany, 61, ^ AvEav, G. S. JR (1933). Structure and development of tobacco leaf. American Journal of Botany, 20, BADENHUIZEN, N. P. & SALEMA, R. (1967). Observations on the development of chloroamyloplast. Revista de Biologia, (1-2), CASADORO, G. & RASCIO, N. (1978). Chloroplast ontogenesis in Helianthus annus L. Protoplasma, 97, CASADORO, G. & RASCIO, N. (1979). Patterns of thylakoid system formation. Journal of Ultrastructure Research, 69, CRAN, D. G. & PossiNGHAM, J. V. (1974). Plastid thylakoid formation. Annals of Botany, 38, ESAU, K. (1975). Crystalline inclusion in the thylakoid of spinach chloroplast. Journal of Ultrastructure Research, 52,225-2^%. F ^ y HuRKMAN, W. J. & KENNEDY, G. (1977). Thylakoid body in tobacco chloroplast. Americanjournal of Botany, 64, ISRAEL, H. W. & STEWARD, F. C. (1967). The fine structure and development of plastids in cultured cells of Dancus carota. Annals of Botany, 31, KIRK, J. T. D. & TILNEY-BASSETT, R. A. E. (1978). The Plastids, pp Elsevier North-Holland Biomedical Press, Amsterdam. MARES, D. J., COOTE, M. A. & POSSINGHAM, J. V. (1979). Membrane bound plastid inclusions and chloroplast thylakoid formation in sunflower (Helianthus annus L.) Annals of Botany, 43, MARINOS, N. G. (1967). Multifunctional plastids in the meristematic region of potato tuber buds. Journal of Ultrastructure Research, 17, PLATT-ALOIA, K. A. & THOMSON, W. W. (1977). Chloroplast development in young sesame plants. Netv Phytologist, 78, SALEMA, R. & ABREU, I. (1972). Development of photosynthetic lamella in Phyllanthus nivosus. Broteria, 41, SAURER, W. & POSSINGHAM, J. V. (1970). Studies on the growth of spinach leaves (Spinacea oleracea). Journal of Experimental Botany, 21,

6 278 p. J. VARKEY AND M. J. NADAKAVUKAREN SRIVASTAVA, L. M. (1966). On the fine structure of the cambium of Fraxinus americana L. Journal of Cell Biology, 31, STEER, B. T. (1971). The dynamics of leaf growth and photosynthetic capacity in Capsicum fruitiscens L. Annals of Botany, 35, THIERY, J. P. (1967). Mise en evidence des polysaccharides sur coupes fines en microscopie electronique. Journal de Microscopie, 6, WHATLEY, J. M. (1977). Variations in the basic pathway of chloroplast development. New Phytologist, 78,

Ontogeny of Chloroplast in Satsuma Mandarin Young Leaves Sprayed with Urea

Pakistan Journal of Biological Sciences, 2 (2): 571-574, 1999 Research Article Ontogeny of Chloroplast in Satsuma Mandarin Young Leaves Sprayed with Urea S.E. Aguja, P. Mohammad, M. Shiraishi* and T. Saga*