Planta
Planta 143, 21-27 (1978)
9 by Springer-Verlag 1978
Endoplasmic Reticulum and Crystalline Fibrils in the Root Protophloem of Nymphoidespeltata Karl J. Oparka and Richard P.C. Johnson Department of Botany, University of Aberdeen, St. Machar Drive, Aberdeen AB9 2UD, U.K.
Abstract. Endoplasmic reticulum in the root protophloem ofNymphoidespeltata (S.G. Gruel.) O. Kuntze changes form as sieve elements differentiate. In immature sieve elements the individual endoplasmic reticulure (ER) cisternae form large irregular aggregates in the cytoplasm. In older immature sieve elements the ER aggregates are more ordered and membranes in them are convoluted. Although convoluted ER predominates in immature sieve elements the ER of the mature sieve elements consists mainly of flattened stacks of ER cisternae. Some of these stacks of ER may be derived from the existing convoluted ER. "Crystalline fibrils" first appear in the cytoplasm of the sieve element when the ER starts to aggregate. The crystalline fibrils move to the parietal layer of the sieve element along with the aggregates of ER. A possible ontogenetic relationship between ER and crystalline fibrils is discussed. Key words: Crystalline fibril - Endoplasmic reticulure - Nyrnphoides - Protophloem.
Introduction Much work with the electron microscope has now established that the endoplasmic reticulum becomes considerably modified in sieve elements as they differentiate. In mature sieve elements the ER cisternae commonly appear aligned parallel to one another in stacks in the parietal layer (Esau 1969 and references therein). The ER becomes stacked by an apparent re-organisation of the rough ER present in immature sieve elements. As the cisternae of the rough ER become
Abbreviation." ER =endoplasmic reticulum
aligned, a densely staining material accumulates between adjacent cisternae while ribosomes may disappear from all but the outermost cisternaein a stack (Esau and Gill, 1971, 1972; Dute and Evert, 1977; Melaragno and Walsh, 1976). Zee (1969a) referred to the stacking process as a "zipping u p " of ER cisternae. As well as stacked ER, aggregates of convoluted ER membranes have also been found in sieve tubes (Wooding, 1967; Evert and Deshpande, 1969; Esau and Gill, 1971; Behnke, 1968, 1973). The stages by which the convoluted ER develops have received less attention and are the subject of the present paper. The conspicuous ER of the sieve element was given the name "sieve tube reticulum" by Bouck and Cronshaw (1965), a term later altered to "sieve element reticulum" by Srivastava and O'Brien (1966). Esau and Gill (1971), however, have since proposed that these specific terms be replaced by more descriptive expressions referring to aggregated, stacked or convoluted ER. In this paper we examine how the ER changes as the root protophloem of Nyrnphoides peltata differentiates. The advantages of using root protophloem, in particular, to study how cells differentiate, have been pointed out by Esau and Gill (1971). Johnson (1969) described complexes of ER membranes which occur in the parietal layer in mature sieve elements of Nymphoides. In contact with these membranes were structures which appeared in cross section in the electron microscope as hexagonal lattices and, in longitudinal section under the light microscope, probably as fibrils. He referred to these structures as "crystalline fibrils". Structures which appear similar to crystalline fibrils have been seen in other plants by other authors (Behnke, 1968 ; Weintraub et al., 1975). Here we consider a possible ontogenetic relationship between crystalline fibrils and ER.
0032-0935/78/0143/0021/$01.40
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K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem
Fig. 1. Two adjoining sieve elements. A number of convoluted ER aggregates are present in the upper sieve element (darts) x 11,000. SP, sieve plate; P plastid; Pp P-protein; V vacuole
Materials and Methods Plants of Nymphoides peltata (S.G. Gruel.) O. Kuntze were grown in plastic cisterns under constant illumination in the laboratory. Main roots were removed gently from the surrounding soil, cut into 1 cm portions and immediately immersed in 4% glutaraldehyde bufferedto pH 6.8 with NaOH-PIPES buffer (Salema and Brand~,o, 1973). After 1 h the roots were shortened to 1 mm and left for a further 2 h under fixative. They were then postfixed in buffered 2% osmium tetroxide for 1 h at room temperature, dehydrated through a graded acetone series and embedded in Epon 812 resin. Thick sections (0.5-t lain) for light rnicroscopy were dried on to glass slides and stained with toluidene blue. Thin
sections for electron microscopy were cut on glass knives, stained with uranyl acetate followed by lead citrate, and viewed with an AEI EM6B electron microscope operating at 60 KV.
Results I n t h e r o o t o f N. peltata five p r o t o p h l o e m p o l e s develop to the inside of the root pericycle with a single protophloem sieve-element forming at each pole. The d i f f e r e n t i a t i n g s i e v e e l e m e n t s , as s e e n in l o n g i t u d i n a l section, elongate and increase their wall thickness w h i l e s u r r o u n d i n g cells r e m a i n t h i n w a l l e d .
K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem
23
Fig. 2. Part of a longitudinal section through an immature sieve element to show accumulation of ER cisternae in the cytoplasm, x 24,000. ER endoplasmic reticulum; Ar nucleus; D dictyosome; SP sieve plate Fig. 3. An older sieve element than the one shown in Figure 2. The ER cisternae arc convoluted and the intcrcisternal spaces are densely stained, x 51,000. W cell wall D u r i n g the early stages o f their d e v e l o p m e n t the sieve elements have dense p r o t o p l a s t s and c o n t a i n a full c o m p l e m e n t o f cell organelles i n c l u d i n g nuclei, plastids, e n d o p l a s m i c r e t i c u l n m (ER), m i t o c h o n d r i a a n d d i c t y o s o m e s . P - p r o t e i n b o d i e s also occur in the sieve elements (Fig. 1). One o f the first signs to distinguish sieve elements f r o m s u r r o u n d i n g cells is a c h a n g e in a p p e a r a n c e of their ER. Initially the sieve-element E R is present as i n d i v i d u a l profiles o f r o u g h E R which a p p e a r scattered t h r o u g h o u t the dense p r o t o p l a s m o f the cell. As d i f f e r e n t i a t i o n p r o c e e d s the E R cisternae c o m e
t o g e t h e r in the c y t o p l a s m o f the sieve e l e m e n t to f o r m large irregular aggregates. W i t h i n each a g g r e g a t e the cisternae are n o t a l i g n e d p a r a l l e l b u t are w o u n d sinu o u s l y t h r o u g h o u t the aggregate. I n F i g u r e 2 such an a g g r e g a t e is seen lying close t o a d e v e l o p i n g sieve plate. The cisternae a p p e a r d i l a t e d a n d lack r i b o s o m e s a n d are s e p a r a t e d f r o m one a n o t h e r by a n a r r o w intercisternal space which later b e c o m e s filled with densely staining material. In later stages o f d i f f e r e n t i a t i o n the a g g r e g a t e s o f E R t a k e on a m o r e c o n v o l u t e d a p p e a r a n c e . T h e cisternae r e m a i n i r r e g u l a r in w i d t h a l t h o u g h the
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K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem
Fig. 4. Higher magnification view of the large ER aggregate shown in Figure 1. The cisternae and intercisternal spaces show a repeating pattern within the aggregate x 29,500 Fig. 5. ER aggregate showing transition from flat to convoluted forms. The cisternae are continuous from the convoluted to the stacked region of the aggregate, x 51,000
densely staining intercisternal spaces remain remarkably u n i f o r m with a width o f a b o u t 25 n m (Fig. 3). Several aggregates of convoluted E R m a y f o r m in a single sieve element (Fig. 1). In some aggregates the cisternae and intercisternal spaces b e c o m e regularly folded in a repeating pattern (Fig. 4). Eventually all the convoluted E R aggregates are f o u n d only in the parietal layer of the sieve element. Flat stacks of ER, which consist of cisternae lying closely appressed and parallel to one another, also occur in i m m a t u r e sieve elements, a l t h o u g h less fre-
quently than the convoluted forms. However, stacked E R predominates in the parietal layer of those sieve elements which are mature e n o u g h to lack tonoplasts (Fig. 9). Our observations o f the developing protop h l o e m sieve elements suggest that some of the convoluted E R o f the i m m a t u r e sieve element gives rise to the stacked forms of E R f o u n d in the mature sieve element. Some of the sieve elements a p p r o a c h i n g maturity a m o n g s t others in a file can be seen to contain E R aggregates which appear intermediate between flat and convoluted forms. A n aggregate of intermedi-
K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem
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Fig. 6. Two crystalline fibrils in transverse section (darts). The upper crystalline fibril is between two ER cisternae, x 107,000 Fig. 7. Longitudinal section through the parietal layer of a mature sieve element, showing crystalline fibril in longitudinal section (dart). x i 11,000 Fig. 8. Crystalline fibril in Iongiludinal sectioninanimrnaturesieveelement~ • 60,500 Fig. 9. Parietal layer of mature sieve element showing stacked ER. • 66,000. F P-protein filament
ate f o r m is shown in F i g u r e 5. A c o n t i n u i t y exists between the cisternae o f the c o n v o l u t e d region a n d the cisternae o f the s t a c k e d region. As c o n v o l u t e d E R c o n v e r t s to s t a c k e d E R the width o f the cisternae b e c o m e s m u c h r e d u c e d a n d they b e c o m e as n a r r o w , or n a r r o w e r , t h a n the intercisternal spaces. The w i d t h o f the i n t e r c i s t e r n a l spaces, however, r e m a i n s as before. S t a c k e d aggregates b e c o m e m o s t c o n s p i c u o u s following the d i s a p p e a r a n c e o f the sieve e l e m e n t nucleus.
Crystalline fibrils were f o u n d in c o n t a c t with the d e v e l o p i n g E R aggregates in the sieve tubes o f the p r o t o p h l o e m . T h e y first a p p e a r e d in the c y t o p l a s m after the E R cisternae h a d lost their r i b o s o m e s a n d h a d aggregated. Crystalline fibrils were n o t f o u n d in y o u n g cells in which the EP. was present as i n d i v i d u a l r o u g h profiles. In transverse section the cystalline fibrils a p p e a r e d as h e x a g o n a l lattices with a r e p e a t p e r i o d o f a b o u t 10 nm. F i g u r e 6 shows two crystalline fibrils in t r a n s v e r s e section. The u p p e r o f the two
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K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem
fibrils is in the intercisternal space between two dilated E R profiles. In longitudinal section each crystalline fibril appears as a series of electron-dense parallel lines, about 10 nm apart, separated by clear regions. Usually only short lengths of crystalline fibrils appeared in the thickness of a section but longer lengths of fibril were encountered occasionally (Fig. 8). In older sieve elements the crystalline fibrils were frequently found in contact with the stacked ER in the parietal layer of mature sieve elements (Fig. 7). Discussion
The convoluted aggregates of ER found in the petiolar sieve tubes of N. peltata (Johnson, 1969), and shown here in the root, are similar to those reported in the developing protophloem of Phaseolus by Esau and Gill (1971). These authors also found that flat and convoluted forms of ER may occur in the same aggregate and suggested that different arrangements of ER, as well as different orientations of stacked cisternae within the cell, " a r e secondary variations in the basic phenomenon of aggregation of the ER system ". Our observations on the protophloem in N. peltata indicate that convoluted ER is gradually converted to stacked ER during ontogeny. It might be converted when the nucleus breaks down since intermediate aggregates of ER become conspicuous while the nucleus breaks down in the sieve element. A similar change in ER occurs in developing sieve elements of Pisum, where, before and after the nucleus breaks down, dilated aggregates of ER give way to flattened layers of cisternae parallel to the wall (Bouck and Cronshaw, 1965). The ordered convoluted ER aggregates found in the present study resemble those reported by Wooding (1967) to occur in immature sieve elements of Acer. Wooding found mainly stacks of flat cisternae in the mature sieve element and thought that the ordered aggregates degenerated before the cell matures. Perhaps some of the convoluted ER aggregates reported by Wooding were also converted into stacked ER. Behnke (1968) also depicted ordered ER aggregates in sieve elements. In Dioscorea he found that some of the ER membranes in the sieve element became arranged "like sinoidal curves" while the tonoplast degenerated. He was able to construct a 3-dimensional representation of the ordered ER aggregates, The arrangement of cisternae found in the present study also appears to conform to Behnke's model. Interestingly, in mature petiolar sieve elements of Nymphoides a close contact occurs also between ER in the parietal layer and crystalline fibrils (Johnson, 1969). Structures similar to the crystalline fibril are
the hexagonal tubules reported in sieve tubes of apple leaves by Weintraub, Stace-Smith and Schroeder (1975). However, these authors did not report an association between their hexagonal tubules and the ER of the sieve element although they suggested that some of the tubules may have broken away from larger groupings close to the walls. In the present study the crystalline fibrils were always found close to the sieve element ER. Behnke (1968) reported a lattice-like structure in sieve elements of Dioscorea and found the cisternae and intercisternal spaces of the ER to be continuous with the respective electron dense and electron lucent regions of the lattice. In Nymphoides, however, we have been unable to demonstrate continuity between the ER membranes a n d the crystalline fibrils although they occur very close together. It is possible that the crystalline fibrils are independent structures formed as the ER reorganises, either from material produced by the ER or from the actual material of the ER membranes. Parthasarathy (1974a) found tubules varying between 15-20 nm in diameter in contact with the ER in sieve tubes of a number of Palm species and suggested that the tubules might be derived from a conversion of the densely staining material in the intercisternal spaces. Our own micrographs (e.g. Fig. 6) support the idea that this is how the crystalline fibrils originate. Johnson (1969) thought that crystalline fibrils might be precursors of banded P-protein filaments since the two occur close to one another in sieve elements in N. peltata. Thus, in N. peltata, at least, some P-protein filaments might be derived from ER via crystalline fibrils. Conversion of membranes to filaments and vice versa in sieve tubes has been discussed by other authors (Franke, 1971; Melaragno and Walsh, 1976; Parthasarathy, 1974b; Kleinig, D6rr and Kollmann, 1971 ; Weber, Franke and Kartenbeck, 1974; Sauter, 1977). However, the function of the ER retained in the mature sieve element is still obscure. Wooding (1967) suggested that the aggregated ER is in an inactive form. On the other hand Zee (1969b) and Esau and Gill (1972) suggested that it is the site of autolytic enzymes. The possibility that the parietal membranes of sieve elements are able to reorganise into filamentous structures requires further careful scrutiny; the demonstration of such a process might show whether mature sieve elements are able to produce P-protein. References
Behnke, H.-D.: Zum Aufbau gitterartiger Membranstrukturen im Siebelementplasma von Dioscorea. Protoplasma 66, 287-310 (1968) Behnke, H.-D.: Struktur~inderungen des endoplasmatischen Reticulums und Auftreten yon Proteinfilamenten wS_hrend tier
K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem Siebr6hrendifferenzierung bei Smilax excelsa. Protoplasma 77, 279 289 (1973) Bouck, G.B., Cronshaw, J.: The fine structure of differentiating sieve tube elements. J. Cell Biol. 25, 79 96 (1965) Dute, R.R., Evert, R.F. : Sieve-element ontogeny in the root of Equisetum hyemale. Amer. J. Bot. 64, 421-438 (1977) Esau, K. : The Phloem. In: Handbuch der Pflanzenanatomie Histologie, vol. 5, pt. 2. Zimmermann, W., Ozenda, P., Wulff, H.D., eds. Berlin, Stuttgart: Borntraeger 1969 Esau, K., Gill, R.H.: Aggregation of endoplasmic reticulum and its relation to the nucleus in a differentiating sieve element. J. Ultrastruct. Res. 34, 144-158 (1971) Esau, K., Gill, R.H. : Nucleus and endoplasmic reticulum in differentiating root protophloem of Nicotiana tabacum. J. Ultrastruct. Res. 41, 160-175 (1972) Evert, R.F., Deshpande, B.F.: Electron microscope investigation of sieve-element ontogeny and structure in Ulmus americana. Protoplasma 68, 403 432 (1969) Franke, W.W. : Relationship of nuclear membranes with filaments and microtubules. Protoplasma 73,,263-292 (1971) Johnson, R.P.C. : Crystalline fibrils and complexes of membranes in the parietal layer in sieve elements. Planta 84, 68-80 (1969) Kleinig, H., D6rr, I., Kollmann, R. : Vinblastine-induced precipitation of phloem proteins in vitro. Protoplasma 73, 293 302 (1971) Melaragno, J.E., Walsh, M.A. : Ultrastructural features of developing sieve elements in Lemna minor L. the protoplast. Amer. J. Bpt. 63, 1145-1157 (1976) Parthasarathy, M.V. : Ultrastructure of phloem in palms. II Struc-
27
tural changes and fate of the organdies in differentiating sieve elements. Protoplasma 79, 93-125 (1974a) Parthasarathy, M.V.: Ultrastructure of phleom in palms, lII. Mature phloem. Protoplasma 79, 265-315 (1974b) Salema, R., Brandao, I.: The use of PIPES buffer in the fixation of plant cells for electron microscopy. J. Submicr. Cytol. 5, 79-96 (1973) Sauter, J.J.: Electron microscopial localisation of adenosine triphosphatase in sieve cells of Pinus nigra var. austriaca (HOESS) BADOUX. Z. Pflanzenphysiol. 81,438 458 (1977) Srivastava, L.M., O'Brien, T.P. : Secondary phloem ofPinus strobas L. Protoplasma 61, 277 293 (1966) Weber, C., Franke, W.W., Kartenbeck, J. : Structure and biochemistry of phloem-proteins isolated from Cucurbita maxima. Exp. Ceil Res. 87, 79-106 (1974) Weintraub, M., Stace-Smith, R., Schroeder, B. : Hexagonal tubular structures in sieve tubes of apple leaves. Phytopath. 65, 660 663 (I975) Wooding, F.B.P.: Endoplasmic reticulum aggregates of ordered structure Planta 76, 205-208-(1967) Zee, S.-Y.: The developmental fate of endoplasmic reticulum in the sieve element of Pisum. Aust. J. Biol. Sci. 22, 257-259 (1969a) Zee, S.-Y. : The localisation of acid phosphatase in the sieve element of Pisum. Aust. J. Biol. Sci. 22, 1051 1054 (1969b)
Received 28 April; accepted 26 June 1978
Planta 143, 21-27 (1978)
9 by Springer-Verlag 1978
Endoplasmic Reticulum and Crystalline Fibrils in the Root Protophloem of Nymphoidespeltata Karl J. Oparka and Richard P.C. Johnson Department of Botany, University of Aberdeen, St. Machar Drive, Aberdeen AB9 2UD, U.K.
Abstract. Endoplasmic reticulum in the root protophloem ofNymphoidespeltata (S.G. Gruel.) O. Kuntze changes form as sieve elements differentiate. In immature sieve elements the individual endoplasmic reticulure (ER) cisternae form large irregular aggregates in the cytoplasm. In older immature sieve elements the ER aggregates are more ordered and membranes in them are convoluted. Although convoluted ER predominates in immature sieve elements the ER of the mature sieve elements consists mainly of flattened stacks of ER cisternae. Some of these stacks of ER may be derived from the existing convoluted ER. "Crystalline fibrils" first appear in the cytoplasm of the sieve element when the ER starts to aggregate. The crystalline fibrils move to the parietal layer of the sieve element along with the aggregates of ER. A possible ontogenetic relationship between ER and crystalline fibrils is discussed. Key words: Crystalline fibril - Endoplasmic reticulure - Nyrnphoides - Protophloem.
Introduction Much work with the electron microscope has now established that the endoplasmic reticulum becomes considerably modified in sieve elements as they differentiate. In mature sieve elements the ER cisternae commonly appear aligned parallel to one another in stacks in the parietal layer (Esau 1969 and references therein). The ER becomes stacked by an apparent re-organisation of the rough ER present in immature sieve elements. As the cisternae of the rough ER become
Abbreviation." ER =endoplasmic reticulum
aligned, a densely staining material accumulates between adjacent cisternae while ribosomes may disappear from all but the outermost cisternaein a stack (Esau and Gill, 1971, 1972; Dute and Evert, 1977; Melaragno and Walsh, 1976). Zee (1969a) referred to the stacking process as a "zipping u p " of ER cisternae. As well as stacked ER, aggregates of convoluted ER membranes have also been found in sieve tubes (Wooding, 1967; Evert and Deshpande, 1969; Esau and Gill, 1971; Behnke, 1968, 1973). The stages by which the convoluted ER develops have received less attention and are the subject of the present paper. The conspicuous ER of the sieve element was given the name "sieve tube reticulum" by Bouck and Cronshaw (1965), a term later altered to "sieve element reticulum" by Srivastava and O'Brien (1966). Esau and Gill (1971), however, have since proposed that these specific terms be replaced by more descriptive expressions referring to aggregated, stacked or convoluted ER. In this paper we examine how the ER changes as the root protophloem of Nyrnphoides peltata differentiates. The advantages of using root protophloem, in particular, to study how cells differentiate, have been pointed out by Esau and Gill (1971). Johnson (1969) described complexes of ER membranes which occur in the parietal layer in mature sieve elements of Nymphoides. In contact with these membranes were structures which appeared in cross section in the electron microscope as hexagonal lattices and, in longitudinal section under the light microscope, probably as fibrils. He referred to these structures as "crystalline fibrils". Structures which appear similar to crystalline fibrils have been seen in other plants by other authors (Behnke, 1968 ; Weintraub et al., 1975). Here we consider a possible ontogenetic relationship between crystalline fibrils and ER.
0032-0935/78/0143/0021/$01.40
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K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem
Fig. 1. Two adjoining sieve elements. A number of convoluted ER aggregates are present in the upper sieve element (darts) x 11,000. SP, sieve plate; P plastid; Pp P-protein; V vacuole
Materials and Methods Plants of Nymphoides peltata (S.G. Gruel.) O. Kuntze were grown in plastic cisterns under constant illumination in the laboratory. Main roots were removed gently from the surrounding soil, cut into 1 cm portions and immediately immersed in 4% glutaraldehyde bufferedto pH 6.8 with NaOH-PIPES buffer (Salema and Brand~,o, 1973). After 1 h the roots were shortened to 1 mm and left for a further 2 h under fixative. They were then postfixed in buffered 2% osmium tetroxide for 1 h at room temperature, dehydrated through a graded acetone series and embedded in Epon 812 resin. Thick sections (0.5-t lain) for light rnicroscopy were dried on to glass slides and stained with toluidene blue. Thin
sections for electron microscopy were cut on glass knives, stained with uranyl acetate followed by lead citrate, and viewed with an AEI EM6B electron microscope operating at 60 KV.
Results I n t h e r o o t o f N. peltata five p r o t o p h l o e m p o l e s develop to the inside of the root pericycle with a single protophloem sieve-element forming at each pole. The d i f f e r e n t i a t i n g s i e v e e l e m e n t s , as s e e n in l o n g i t u d i n a l section, elongate and increase their wall thickness w h i l e s u r r o u n d i n g cells r e m a i n t h i n w a l l e d .
K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem
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Fig. 2. Part of a longitudinal section through an immature sieve element to show accumulation of ER cisternae in the cytoplasm, x 24,000. ER endoplasmic reticulum; Ar nucleus; D dictyosome; SP sieve plate Fig. 3. An older sieve element than the one shown in Figure 2. The ER cisternae arc convoluted and the intcrcisternal spaces are densely stained, x 51,000. W cell wall D u r i n g the early stages o f their d e v e l o p m e n t the sieve elements have dense p r o t o p l a s t s and c o n t a i n a full c o m p l e m e n t o f cell organelles i n c l u d i n g nuclei, plastids, e n d o p l a s m i c r e t i c u l n m (ER), m i t o c h o n d r i a a n d d i c t y o s o m e s . P - p r o t e i n b o d i e s also occur in the sieve elements (Fig. 1). One o f the first signs to distinguish sieve elements f r o m s u r r o u n d i n g cells is a c h a n g e in a p p e a r a n c e of their ER. Initially the sieve-element E R is present as i n d i v i d u a l profiles o f r o u g h E R which a p p e a r scattered t h r o u g h o u t the dense p r o t o p l a s m o f the cell. As d i f f e r e n t i a t i o n p r o c e e d s the E R cisternae c o m e
t o g e t h e r in the c y t o p l a s m o f the sieve e l e m e n t to f o r m large irregular aggregates. W i t h i n each a g g r e g a t e the cisternae are n o t a l i g n e d p a r a l l e l b u t are w o u n d sinu o u s l y t h r o u g h o u t the aggregate. I n F i g u r e 2 such an a g g r e g a t e is seen lying close t o a d e v e l o p i n g sieve plate. The cisternae a p p e a r d i l a t e d a n d lack r i b o s o m e s a n d are s e p a r a t e d f r o m one a n o t h e r by a n a r r o w intercisternal space which later b e c o m e s filled with densely staining material. In later stages o f d i f f e r e n t i a t i o n the a g g r e g a t e s o f E R t a k e on a m o r e c o n v o l u t e d a p p e a r a n c e . T h e cisternae r e m a i n i r r e g u l a r in w i d t h a l t h o u g h the
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K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem
Fig. 4. Higher magnification view of the large ER aggregate shown in Figure 1. The cisternae and intercisternal spaces show a repeating pattern within the aggregate x 29,500 Fig. 5. ER aggregate showing transition from flat to convoluted forms. The cisternae are continuous from the convoluted to the stacked region of the aggregate, x 51,000
densely staining intercisternal spaces remain remarkably u n i f o r m with a width o f a b o u t 25 n m (Fig. 3). Several aggregates of convoluted E R m a y f o r m in a single sieve element (Fig. 1). In some aggregates the cisternae and intercisternal spaces b e c o m e regularly folded in a repeating pattern (Fig. 4). Eventually all the convoluted E R aggregates are f o u n d only in the parietal layer of the sieve element. Flat stacks of ER, which consist of cisternae lying closely appressed and parallel to one another, also occur in i m m a t u r e sieve elements, a l t h o u g h less fre-
quently than the convoluted forms. However, stacked E R predominates in the parietal layer of those sieve elements which are mature e n o u g h to lack tonoplasts (Fig. 9). Our observations o f the developing protop h l o e m sieve elements suggest that some of the convoluted E R o f the i m m a t u r e sieve element gives rise to the stacked forms of E R f o u n d in the mature sieve element. Some of the sieve elements a p p r o a c h i n g maturity a m o n g s t others in a file can be seen to contain E R aggregates which appear intermediate between flat and convoluted forms. A n aggregate of intermedi-
K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem
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Fig. 6. Two crystalline fibrils in transverse section (darts). The upper crystalline fibril is between two ER cisternae, x 107,000 Fig. 7. Longitudinal section through the parietal layer of a mature sieve element, showing crystalline fibril in longitudinal section (dart). x i 11,000 Fig. 8. Crystalline fibril in Iongiludinal sectioninanimrnaturesieveelement~ • 60,500 Fig. 9. Parietal layer of mature sieve element showing stacked ER. • 66,000. F P-protein filament
ate f o r m is shown in F i g u r e 5. A c o n t i n u i t y exists between the cisternae o f the c o n v o l u t e d region a n d the cisternae o f the s t a c k e d region. As c o n v o l u t e d E R c o n v e r t s to s t a c k e d E R the width o f the cisternae b e c o m e s m u c h r e d u c e d a n d they b e c o m e as n a r r o w , or n a r r o w e r , t h a n the intercisternal spaces. The w i d t h o f the i n t e r c i s t e r n a l spaces, however, r e m a i n s as before. S t a c k e d aggregates b e c o m e m o s t c o n s p i c u o u s following the d i s a p p e a r a n c e o f the sieve e l e m e n t nucleus.
Crystalline fibrils were f o u n d in c o n t a c t with the d e v e l o p i n g E R aggregates in the sieve tubes o f the p r o t o p h l o e m . T h e y first a p p e a r e d in the c y t o p l a s m after the E R cisternae h a d lost their r i b o s o m e s a n d h a d aggregated. Crystalline fibrils were n o t f o u n d in y o u n g cells in which the EP. was present as i n d i v i d u a l r o u g h profiles. In transverse section the cystalline fibrils a p p e a r e d as h e x a g o n a l lattices with a r e p e a t p e r i o d o f a b o u t 10 nm. F i g u r e 6 shows two crystalline fibrils in t r a n s v e r s e section. The u p p e r o f the two
26
K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem
fibrils is in the intercisternal space between two dilated E R profiles. In longitudinal section each crystalline fibril appears as a series of electron-dense parallel lines, about 10 nm apart, separated by clear regions. Usually only short lengths of crystalline fibrils appeared in the thickness of a section but longer lengths of fibril were encountered occasionally (Fig. 8). In older sieve elements the crystalline fibrils were frequently found in contact with the stacked ER in the parietal layer of mature sieve elements (Fig. 7). Discussion
The convoluted aggregates of ER found in the petiolar sieve tubes of N. peltata (Johnson, 1969), and shown here in the root, are similar to those reported in the developing protophloem of Phaseolus by Esau and Gill (1971). These authors also found that flat and convoluted forms of ER may occur in the same aggregate and suggested that different arrangements of ER, as well as different orientations of stacked cisternae within the cell, " a r e secondary variations in the basic phenomenon of aggregation of the ER system ". Our observations on the protophloem in N. peltata indicate that convoluted ER is gradually converted to stacked ER during ontogeny. It might be converted when the nucleus breaks down since intermediate aggregates of ER become conspicuous while the nucleus breaks down in the sieve element. A similar change in ER occurs in developing sieve elements of Pisum, where, before and after the nucleus breaks down, dilated aggregates of ER give way to flattened layers of cisternae parallel to the wall (Bouck and Cronshaw, 1965). The ordered convoluted ER aggregates found in the present study resemble those reported by Wooding (1967) to occur in immature sieve elements of Acer. Wooding found mainly stacks of flat cisternae in the mature sieve element and thought that the ordered aggregates degenerated before the cell matures. Perhaps some of the convoluted ER aggregates reported by Wooding were also converted into stacked ER. Behnke (1968) also depicted ordered ER aggregates in sieve elements. In Dioscorea he found that some of the ER membranes in the sieve element became arranged "like sinoidal curves" while the tonoplast degenerated. He was able to construct a 3-dimensional representation of the ordered ER aggregates, The arrangement of cisternae found in the present study also appears to conform to Behnke's model. Interestingly, in mature petiolar sieve elements of Nymphoides a close contact occurs also between ER in the parietal layer and crystalline fibrils (Johnson, 1969). Structures similar to the crystalline fibril are
the hexagonal tubules reported in sieve tubes of apple leaves by Weintraub, Stace-Smith and Schroeder (1975). However, these authors did not report an association between their hexagonal tubules and the ER of the sieve element although they suggested that some of the tubules may have broken away from larger groupings close to the walls. In the present study the crystalline fibrils were always found close to the sieve element ER. Behnke (1968) reported a lattice-like structure in sieve elements of Dioscorea and found the cisternae and intercisternal spaces of the ER to be continuous with the respective electron dense and electron lucent regions of the lattice. In Nymphoides, however, we have been unable to demonstrate continuity between the ER membranes a n d the crystalline fibrils although they occur very close together. It is possible that the crystalline fibrils are independent structures formed as the ER reorganises, either from material produced by the ER or from the actual material of the ER membranes. Parthasarathy (1974a) found tubules varying between 15-20 nm in diameter in contact with the ER in sieve tubes of a number of Palm species and suggested that the tubules might be derived from a conversion of the densely staining material in the intercisternal spaces. Our own micrographs (e.g. Fig. 6) support the idea that this is how the crystalline fibrils originate. Johnson (1969) thought that crystalline fibrils might be precursors of banded P-protein filaments since the two occur close to one another in sieve elements in N. peltata. Thus, in N. peltata, at least, some P-protein filaments might be derived from ER via crystalline fibrils. Conversion of membranes to filaments and vice versa in sieve tubes has been discussed by other authors (Franke, 1971; Melaragno and Walsh, 1976; Parthasarathy, 1974b; Kleinig, D6rr and Kollmann, 1971 ; Weber, Franke and Kartenbeck, 1974; Sauter, 1977). However, the function of the ER retained in the mature sieve element is still obscure. Wooding (1967) suggested that the aggregated ER is in an inactive form. On the other hand Zee (1969b) and Esau and Gill (1972) suggested that it is the site of autolytic enzymes. The possibility that the parietal membranes of sieve elements are able to reorganise into filamentous structures requires further careful scrutiny; the demonstration of such a process might show whether mature sieve elements are able to produce P-protein. References
Behnke, H.-D.: Zum Aufbau gitterartiger Membranstrukturen im Siebelementplasma von Dioscorea. Protoplasma 66, 287-310 (1968) Behnke, H.-D.: Struktur~inderungen des endoplasmatischen Reticulums und Auftreten yon Proteinfilamenten wS_hrend tier
K.J. Oparka and R.P.C. Johnson: ER and Crystalline Fibrils in Protophloem Siebr6hrendifferenzierung bei Smilax excelsa. Protoplasma 77, 279 289 (1973) Bouck, G.B., Cronshaw, J.: The fine structure of differentiating sieve tube elements. J. Cell Biol. 25, 79 96 (1965) Dute, R.R., Evert, R.F. : Sieve-element ontogeny in the root of Equisetum hyemale. Amer. J. Bot. 64, 421-438 (1977) Esau, K. : The Phloem. In: Handbuch der Pflanzenanatomie Histologie, vol. 5, pt. 2. Zimmermann, W., Ozenda, P., Wulff, H.D., eds. Berlin, Stuttgart: Borntraeger 1969 Esau, K., Gill, R.H.: Aggregation of endoplasmic reticulum and its relation to the nucleus in a differentiating sieve element. J. Ultrastruct. Res. 34, 144-158 (1971) Esau, K., Gill, R.H. : Nucleus and endoplasmic reticulum in differentiating root protophloem of Nicotiana tabacum. J. Ultrastruct. Res. 41, 160-175 (1972) Evert, R.F., Deshpande, B.F.: Electron microscope investigation of sieve-element ontogeny and structure in Ulmus americana. Protoplasma 68, 403 432 (1969) Franke, W.W. : Relationship of nuclear membranes with filaments and microtubules. Protoplasma 73,,263-292 (1971) Johnson, R.P.C. : Crystalline fibrils and complexes of membranes in the parietal layer in sieve elements. Planta 84, 68-80 (1969) Kleinig, H., D6rr, I., Kollmann, R. : Vinblastine-induced precipitation of phloem proteins in vitro. Protoplasma 73, 293 302 (1971) Melaragno, J.E., Walsh, M.A. : Ultrastructural features of developing sieve elements in Lemna minor L. the protoplast. Amer. J. Bpt. 63, 1145-1157 (1976) Parthasarathy, M.V. : Ultrastructure of phloem in palms. II Struc-
27
tural changes and fate of the organdies in differentiating sieve elements. Protoplasma 79, 93-125 (1974a) Parthasarathy, M.V.: Ultrastructure of phleom in palms, lII. Mature phloem. Protoplasma 79, 265-315 (1974b) Salema, R., Brandao, I.: The use of PIPES buffer in the fixation of plant cells for electron microscopy. J. Submicr. Cytol. 5, 79-96 (1973) Sauter, J.J.: Electron microscopial localisation of adenosine triphosphatase in sieve cells of Pinus nigra var. austriaca (HOESS) BADOUX. Z. Pflanzenphysiol. 81,438 458 (1977) Srivastava, L.M., O'Brien, T.P. : Secondary phloem ofPinus strobas L. Protoplasma 61, 277 293 (1966) Weber, C., Franke, W.W., Kartenbeck, J. : Structure and biochemistry of phloem-proteins isolated from Cucurbita maxima. Exp. Ceil Res. 87, 79-106 (1974) Weintraub, M., Stace-Smith, R., Schroeder, B. : Hexagonal tubular structures in sieve tubes of apple leaves. Phytopath. 65, 660 663 (I975) Wooding, F.B.P.: Endoplasmic reticulum aggregates of ordered structure Planta 76, 205-208-(1967) Zee, S.-Y.: The developmental fate of endoplasmic reticulum in the sieve element of Pisum. Aust. J. Biol. Sci. 22, 257-259 (1969a) Zee, S.-Y. : The localisation of acid phosphatase in the sieve element of Pisum. Aust. J. Biol. Sci. 22, 1051 1054 (1969b)
Received 28 April; accepted 26 June 1978
