structure and development of sieve cells in the secondary phloem of larix decidua mill. as related...

Trees (1989) 4:192-209 Trees �9 Springer-Verlag 1989

Structure and development of sieve cells in the secondary phloem of Larix decidua Mill. as related to function

Klaus S c h m i t z ~ and Andrea Schne ider 2

Botanisches Institut der Universit~it zu K61n, Gyrhofstrasse 15, D-5000 K61n 41, Federal Republic of Germany 2 Fraunhofer-Institut fiir Atmosph/irische Umweltforschung, Kreuzeckbahnstrasse 19, D-8000 Garmisch-Partenkirchen, Federal Republic of Germany

S u m m a r y . Only one or two layers of sieve cells of the previous year's phloem in lateral branches of Larix decidua persist as fully mature cells. Imma- ture sieve cells or cambial derivatives that have not completed differentiation may also overwinter. Periclinal cell divisions of the vascular cambium were first observed by mid-April. Dur- ing the short period of greatest cambium activity (mid-April to mid-May), the early phloem is laid down. Late phloem is formed over a much longer period, from mid-May to late September. Micro- autoradiography revealed that only mature sieve cells of the early phloem are involved in translocation of 14C assimilates in June. The fine structure of actively translocating sieve cells is described. The impact of structure on long-distance transport of assimilates is discussed.

Key words: A u t o r a d i o g r a p h y - Fine structure - Larch - Larix decidua - Sieve cells

Introduct ion

The increasing body of transport physiological data and the knowledge of sieve element structure indicate that in all probability a pressure-flow mechanism is applicable to sieve tube transport in angiosperms. From sieve-cell fine structure it is questionable, however, whether the same mechanism applies to transport in sieve cells of conifers. Studies on Metasequoia glyptostroboides by Koll- mann (1965, 1967), Willenbrink and Kollmann (1966) and on Juniperus communis by Kollmann

Offprint requests to: K. Schmitz

and D6rr (1966) have shown that young, immature sieve cells in an intermediary state of differentiation are able to translocate. This has been shown by histoautoradiography and by the dem- onstration that aphids feed on immature sieve cells, next to the cambium. The fine structure of actively translocating sieve cells can be deduced from the careful studies of Kollmann and Schu- macher (1961, 1962, 1964) on Metasequoia. Ac- cording to general opinion, sieve elements lack a nucleus at maturity, but in a light microscopic study, Evert et al. (1970) observed that all mature functional sieve cells of the secondary phloem of Metasequoia glyptostroboides, Sequoia sempervirens and Taxodium distichum contain a nucleus of "normal appearance"; yet the function of these cells was not proven.

Indirect localization of translocating sieve cells in the secondary phloem of Larix was provided by Sauter and Braun (1968, 1972), who demonstrated increased enzyme activities in Strasburger cells, associated with fully differentiated sieve cells. No such activity could be detected in those Strasburger cells connected to immature sieve cells (Sauter and Braun 1972).

The structure of sieve cells in the phloem of Larix is poorly known. The anatomy and seasonal development of secondary phloem of Larix was briefly described by Huber (1939) and Evert and Alfieri (1965). Electron micrographs of secondary phloem of Larix were first published by Huber and Liese (1963), but do not provide much infor- mation, due to inadequate fixation at that time. We attempted to correlate structure and function of sieve cells in the secondary phloem of Larix decidua and report here on the autoradiographic localization of translocating sieve cells, their fine

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structure and aspects of their ontogenetic development.

Materials and methods

Plant material of Larix decidua Mill. and the 14C-labelling procedure by pulse feeding were the same as described by Schneider and Schmitz (1989).

Microscopy. Samples for light- and electron-microscopic ex- amination were obtained from 2- or 3-year-old lateral branches which were fixed at different months of the year. The branches were submerged in aldehyde fixative and cut into 2-cm pieces, which were then split in half. After a prefixation of 45 min, small pieces were cut out and fixed for 5 h at room temperature under slight vacuum. Aldehyde fixative contained 1.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M so- dium cacodylate buffer, pH 6.8. Samples were further treated with 1% buffered osmium tetroxide for 12 h at 4 ~ C, followed by a 1-h treatment with 2% aqueous uranyl acetate. A c e t o n e dehydrated samples were embedded in Spurr's resin (Spurr 1969). Semithin sections (0.5-1.5 p.m) were stained with tolu- idine blue O - pyronine G (Ito and Winchester 1963) for light microscopy. Thin sections were post-stained with uranyl acetate and lead citrate (Reynolds 1963) and examined in a trans- mission electron microscope (Type 101, Siemens, Berlin, FRG).

Histoautoradiography. Following a pulse-chase experiment (20 min Jac pulse with 3.7 MBq of NaHI4CO3, followed by a 4-h chase period in 12CO2), the basal part of a lateral branch (transport zone) was rapidly dissected and alternate samples for autoradiography and ~4C counting were taken. ~4C was ex- tracted and analysed as described by Schneider and Schmitz (1989). Samples for autoradiography were freeze-substituted accord- ing to Steinbil~ (1978) and embedded in resin. Semithin sections (1-2 p.m), dried on to glass slides, were coated with a photoemulsion (K5 Ilford, Sussex, UK) by dipping. Prepara- tion of the film emulsion, the dipping technique and the devel- oping procedure were described by Rogers (1979).

Results

Annual development of secondary phloem as revealed by light microscopy

It is assumed that sieve cells of the deciduous tree L. decidua are short lived and function for only one growing season. Larix apparently stops translocating when leaf abscission occurs. All mature sieve cells of the annual growth increment, except one or two next to the cambium zone, accumulate callose and collapse in autumn.

The sieve-cell development is shown in Fig. 1. During winter, only one or two none collapsed persisting sieve cells o f the previous year's growth increment, are discernible. Their transport activity was not determined, but it seems reasonable to assume that those cells are the first to become active in translocation in spring. The dormant cambium zone consists of 3 - 4 layers of undifferentiated cells. Initial cambium activity, i.e. pericli-

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Fig. 1. Scheme of seasonal development of secondary phloem in the stem of Larix decidua. Cell division in cambium cells (C) producing cambial derivatives towards the phloem (CS) and towards the xylem (CX) are indicated by dashed lines. The last late-phloem sieve cell (S) is marked by a solid black cell wall. The early phloem (ePh) is composed of sieve cells (S). Tangential bands of phloem parenchyma (P) or crystal cells (CC) are late-phloem elements (1Ph) and delimit early phloem. Callose is indicated by stippled cell wall areas. For further details see text

nal cell divisions, was recognized by mid-April. It can be seen from Fig. 1 that most phloem elements are produced in a rather short period between mid-April and the end of May. The first cambium derivatives differentiate into thin- walled, rather voluminous sieve cells, which can easily be recognized in cross-sections throughout the growing season. They provide a marker to rec- ognize and identify the annual growth increments of secondary phloem. Those thin-walled sieve elements are followed by a series of 5 - 6 sieve cells orderly arranged in radial seriation from the cambium. Their cell walls become progressively thicker from the first to the last-formed sieve cell of this early phloem. The formation of early phloem is completed by a band of parenchyma cells which delimits the early phloem from the late phloem. Late phloem is composed of two more or less interrupted tangential bands of crystal cells or parenchyma cells, separated by one or two sieve cells and followed by not more than three sieve cells, of which at least one can persist throughout the winter in a mature state. Formation of late phloem starts by the end of May. At that time, the previous year's sieve cells have collapsed and have stopped translocation. By mid-June, first- formed sieve cells of the current growing season reveal callose deposits at their sieve areas in the

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lateral walls. Formation and differentiation of new xylem was not recognized until early phloem was completed. By mid-June, frequently observed periclinal cell divisions in the cambium zone and differentiating cambial derivatives indicate an extremely active formation of new xylem elements.

Differentiation of new sieve cells and xylem elements may continue until late September. Most sieve cells of the early phloem reveal massive callose deposits at that time and the first-formed sieve cells have already collapsed by then.

Autoradiography

Previous studies of long-distance transport of 14C photoassimilate in Larix (Schneider and Schmitz 1989) have shown that the highest rate of assimilate export from the leaves of lateral branches occurs in summer (June), when early phloem is fully developed.

Histoautoradiographs of the lowermost part of a lateral branch in cross-section show that in June radioactive label is confined to the recent growth increment of secondary phloem (Fig. 2). There is no radioactivity in the secondary phloem of previous years, nor in the xylem.

A higher magnification (Fig. 3) clearly demonstrates that in June only the sieve cells of early phloem are involved in assimilate transport. Slight radioactivity is indicated by silver grains confined to the cambial zone and ray parenchyma cells, especially those that are in contact with actively translocating sieve cells (Figs. 3, 4). The quality of cell preservation by the freeze substitution method employed may be seen from Figs. 3 and 4. Only well-preserved tissue samples resulted in trustworthy and reliable autoradiographs. Autoradiographs of transport regions from experiments with extended chase periods (up to 12 h) show lateral transport of t4C-labelled compounds via ray parenchyma cells into parenchyma cells of the bark and the wood, as well as into the cambial zone (results not presented).

Radial longitudinal sections confirm the re- suits derived from cross-sections. Figures 5 and 6 show very clearly that radioactivity is mainly pre-

sent in the wide sieve cells of the early secondary phloem of the pertinent growth increment. A very low level of radioactivity is indicated by silver grains slightly above background in the cambium zone, the ray parenchyma and the previous year's phloem. Autoradiographs of sections cut through the plain of radial longitudinal sieve cell walls show clusters of silver grains associated with lateral sieve fields (Fig. 5).

Ultrastructure and development o f sieve cells Winter material. The cambial zone and the adjacent secondary xylem and phloem of a Larix stem fixed in December is presented in Figs. 7 and 8. Usually, there are 6 -7 cells intercalated between late xylem elements and a tangential band of crystal cells of the previous year's late phloem. For the sake of description, these cells are numbered consecutively. Remnants of the protoplasts are still retained in late-formed tracheids (Fig. 7). Cells 1 and 2 can be considered fusiform xylary derivatives of the cambium. This becomes obvious from longitudinal sections rather than the cross-sections, because these cells are characterized by electron-dense bodies in the vacuoles (Fig. 8). It is speculated that these osmiophilic bodies are tan- nins. Additional characteristic structural features are the very long-stretched plastids (Fig. 9) and the abundance of small vesicles or inflated tubular endoplasmic reticulum (ER, Fig. 10).

Cells 3 and 4 (Figs. 7, 8)are fusiform cambium cells. One of them is the cambium initial, which cannot be recognized here by the fine structure alone. Undifferentiated cambium cells can easily be distinguished from partly differentiated young sieve cells (cell 5 in Fig. 8) by distinct changes in the fine structure of plastids and thickening of the cell wall. The first visible indication for the differentiation of a fusiform cambium derivative into a sieve cell is the appearance of filamentous protein in the plastids at a time when cell wall thickening was not yet apparent. Such plastids have only a few thylakoids and a reticulate tubular membrane system, as shown in Fig. 11. The number of protein filaments apparently increases very rapidly but the thylakoids do not persist. The filaments later show a ring-like arrangement, en-

Figs. 2, 3. Microautoradiographs of the transport zone at the base of a 3-year-old lateral branch in June. The 14C feeding zone was 10 cm away. Fig. 2. Cross-section depicting the anatomy and the distribution of radiocarbon in the secondary phloem. ~4C is confined to the early phloem of the pertinent growth increment. Scale 50 p,m. Fig. 3. Detail of a cross-section demonstrating the localization of ~4C-labelled compounds, indicated by developed silver grains, and the cell preservation by the freeze substitution technique, labelled early-phloem sieve cells (asterisk) are delimited by late-phloem crystal cells (CC) or vascular parenchyma cells (P) and the previous year's late-phloem sieve cells (LS). Note the well-preserved cytoplasm and the nuclei (N) in ray parenchyma (RP) and cambium cells (C). Scale 20 Ixm

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For legend please see p. 200

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circling small starch grains (Fig. 12). The plastid envelope was often ruptured in mature sieve cells and the protein filaments were more or less dispersed in the sieve-cell lumen (Figs. 19, 21). The fine structure of mitochondria, components of the endomembrane system and the nucleus of young sieve elements appeared not to be different from fusiform cambium cells.

Cambium cells and young sieve cells of winter material have a dense population of cortical microtubules in a more or less parallel arrangement, oriented circumferentially (Fig. 13). Bund- les of protein filaments running parallel to the long axis of the cells, resembling microfilament bundles (Figs. 13, 14), were also detected. Both protein structures were present in young, immature sieve cells at all times of the year. Microtu- bules were also infrequently detected in sieve cells that appeared to be in a mature state.

Cells 6 and 7 (Figs. 7, 8) are sieve cells of the previous year which have retained a cell membrane and a variable amount of cytoplasm. A comparison between Figs. 7 and 8 demonstrates that sieve cells can overwinter in different states of development. While the sieve cell lumina in the cross - section (Fig. 7, cells 6 and 7) are filled with vesicles and protoplasmic structures, indicating an immature state, sieve cells 6 and 7 in the longitudinal section (Fig. 8) have only retained a cell membrane, some scattered remnants of cytoplasm

and a degenerated osmiophilic nucleus. The latter cells are fully mature sieve cells. We often observed in the lumen of mature sieve cells of winter material tubular structures with an outer diameter of 40-50 nm. These tubular elements appeared to be rather rigid, sometimes branched, either in parallel arrangement or irregularly dispersed (Fig. 15).

Summer material. Stems of Larix, fixed in June at a time of active carbon translocation as shown by autoradiography, had completed differentiation of early phloem. Early phloem was delimited from late phloem elements by a tangential band of phloem parenchyma cells. There was intensive formation of xylem elements at that time (compare Figs. 1-4) and the very hydrated cells of the cambial zones were extremely difficult to fix.

It seems that the ontogenetic development of individual sieve cells occurs in a very short period because thick-walled sieve cells were commonly observed next to undifferentiated cambium cells. Occasionally, sieve cells in an intermediary state of development were also found in summer material. In grazing sections through sieve cells that had not yet completed cell wall thickening, dense parallel groupings of cortical microtubules and microfilament bundles, as demonstrated in Figs. 13 and 14, were observed. In addition, nu- merous dictyosomes, coated viscles, polyribo-

4 Figs. 4-6 . Microautoradiographs of the 14C translocation pathway in the stem of Larix in June. Fig. 4. Cross-section of a different part at the base of a labelled lateral branch than in Figs. 2 and 3. The photograph shows the restriction of radioactive labelling to the early-phloem sieve cells. Sieve cells of the previous year and cells of the cambial zone are essentially free of radioactivity. The last-formed sieve cells of the previous year are marked by arrows. Scale 20 p.m. Figs. 5, 6. Autoradiographs of serial radial longitudinal sections of Larix cut through the cambial zone (C) and the early phloem of a 3-year-old lateral branch. Xylem elements (X) and ray parenchyma cells (RP) as well as the cambial elements and late-phloem elements of the previous year (asterisk) are nearly free of radioactivity, if the background is subtracted. Intensive radiation is confined to lateral sieve areas (arrows) in the radial longitudinal walls of early-phloem sieve cells (S) of pertinent growth. Crystal cells (CC); late-phloem vascular parenchyma cell (P). Scales 25 Ixm

Fig. 7. Cross-section of the cambial zone and part of late-phloem elements of a Larix stem fixed in December. Protoplasts of all cells in the cambial zone and adjacent sieve cells (S) are characterized by abundant small vacuoles. Sieve cells are recognized by conspicuously thickened cell walls and P-type plastids (arrows). CC Crystal cells; CS phloem derivative of cambium; CX xylem derivative of cambium; X, xylem element. Scale 5 I.tml

Fig. 8. Radial longitudinal section through the cambial zone of a Larix stem fixed in December. Note the dark stained vacuoles in the xylary derivatives (CX) of the cambium (C). A phloem derivative (CP) is characterized by modified plastids (P), containing densely packed protein filaments. Two fully mature late-phloem sieve cells of the previous year contain intact plasmalemma, highly condensed nuclei (N) and parietal membrane structures (unlabelled arrows). Scale 5 ktm

Figs. 9, 10. Fusiform xylary derivatives of cambium fixed in December. Fig. 9. Cells are characterized by abundant dense granular vacuole content and long-streched plastids (P) with poorly developed thylakoids. Scale 400 nm. Fig. 10. Tangential sections show a membrane system that might be a tubular or vesicular ER. Lipid vesicles L. Scale 1.5 Ixm

Figs. 11, 12. P-type plastids of sieve cells. Fig. l l . Plastid of a young immature sieve cell that had not yet developed a thickened cell wall. Note the poorly developed thylakoids (arrows), the reticulate tubular membrane system and the densely packed protein filaments. M Mitochondrion. Scale 500 nm. Fig. 12. Advanced P-type plastid, surrounded by the plastid envelope, containing ring-like arranged protein filaments which enclose small starch grains (S). Scale 500 nm

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Figs. 13, 14. Grazing longitudinal section through young sieve cells shows cortical parallel-arranged microtubules (Mt), running essentially across to the long axis of the cell, and bundles of filaments (Mj), running parallel to the cell axis. Coated vesicles (arrows) and polyribosomes (arrowhead) were frequently observed. Scale 500 nm. Fig. 14. A higher magnification of filament bundles. The fine structure suggests that they are bundles of microfilaments (M/). Scale 200 nm

Fig. 15. Tubular structures frequently observed in mature sieve cells in December. Scale 300 nm

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somes as well as vesicles and vacuoles of different size were present (Fig. 16). The same cell revealed a non-condensed nucleus and mitochondria of normal structure but clearly modified plastids, with abundant protein filaments.

Sieve cells of early phloem, involved in long- distance transport, as shown by autoradiography (Figs. 2-4), appeared all to be in a mature state. Such sieve cells, usually 6 -8 in number and radially diposed, are all characterized by conspicuously thickened walls, lined by a cell membrane and a parietal layer of cytoplasma components such as vesicles, ER aggregates and mitochondria. In addition, mature sieve cells contain modified plastids, loaded with protein filaments, and a pycnotic degenerated nucleus. The predom- inant impression, however, is that mature sieve cells are thick-walled, plasmalemma-lined tubes, with empty, structureless lumina. This is illus- trated in Fig. 18. A higher magnification of the ER membrane system (Fig. 17) demonstrates that the cytoplasmic phase is restricted to the very narrow space between the adjoined membranes, recognized by trapped ribosomes which may persist for a rather long time. The extraplasmatic phase is electron translucent. The sieve cell lumen may be occupied by protein filaments which have been relased from plastids (Figs. 19, 21). Plastid en- velopes may either be decomposed as a normal

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event during the development of sieve cells or may have been ruptured by inadequate fixation. Figure 20 presents the parietal layers of protoplasmic structures in mature sieve cells. One or more layers of membrane structures, tubular and vesicular in appearance, probably inflated ER, and mitochondria are associated with the intact plasmalemma on both sides of a longitudinal wall. Dictyosomes and larger vacuoles are lacking; P-protein was never observed.

Most vesicles and ER occur at the sieve areas of mature sieve cells. This can be shown in cross- sections (Fig. 21) and in radial longitudinal sections cut through lateral sieve areas (Fig. 22). Sieve cells in Fig. 21 are consecutively numbered, starting with the latest-formed sieve cell of early phloem, next to the band of phloem parenchyma that delimits the early phloem from the late phloem. Sieve cells are connected by sieve areas in the radial longitudinal walls. Sieve pores of adjacent cells merge in median cavities. Compound median cavities seem to contain the same convo- luted membrane systems as seen in the sieve tubes, associated mainly with the lateral sieve areas (Fig. 22). Electron micrographs always showed very densely stained sieve pores but did not provide a clear view of the membranes inside the pores. Sieve pores of all mature early sieve cells were lined with callose when fixed in June

Fig. 16. Longitudinal section of an immature sieve cell during the phase of cell wall thickening in June, showing abundant dictyosomes (D), microfilament bundles (MJ), microtubules, coated vesicles (arrow) and polyribosomes. Scale 500 nm

Fig. 17. ER membrane system in a nearly mature sieve cell in June. The protoplasmic phase, recognized by ribosomes, is restricted to the narrow space between the membranes. P Plastid with protein filaments but without a recognizable envelope. Scale 500 nm

Figs. 18, 19. Mature sieve cells cut longitudinally. Conspicuously thickened cell walls are lined by intact plasmalemma. M Mito- chondr ion; P protein filaments released from a plastid; N condensed nucleus; ER structures (arrows). Fig. 18. Scale 1 txm, Fig. 19. Scale 3 ~tm

Fig. 20. Mature early-phloem sieve cells in longitudinal section. The thickened cell walls (IV) are lined by intact plasmalemma parietal layes of ER membrane structures and associated mitochondria (M). Scale 1 ~tm

Fig. 21. Transection of mature sieve cells 5 - 8 of early phloem in June. Sieve cells were counted from the last-formed sieve cell at the cambium. Sieve areas with compound median cavities (arrows) and associated vesicles and ER are shown. P Protein filaments released from plastids; C callose. Scale 2 Ixm

Fig. 22. Tangential section of a lateral sieve area. Sieve pores are surrounded by callose cylinders; ER is seen in the median cavity and in the sieve cell, associated with the sieve area. P Protein filaments. Scale 1.5 I-tm

Fig. 23. Transection of cell connection between sieve cell and Strasburger cell (SC) showing a sieve pore, lined by callose (C), on the sieve cell side and plasmodesmata, merging into a median cavity, on the Strasburger cell side. Scale 500 nm

Fig. 24. Transection showing plasmodesmata between a Strasburger cell (SC) and a ray parenchyma cell (RP). Branched plasmodesmata merge in median cavities (arrows). M Mitochondrion; D dictyosome. Scale 500 nm

Fig. 25. Cross-section of Strasburger cells. Pp las t id ; M mitochondrion; L lipid vacuole; Nnucleus. Scale 1.5 Ixm

Fig. 26. Very irregularly shaped mitochondria, frequently observed in Strasburger cells. Scale 1 l-tm

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pound median cavity (Fig. 23). While the sieve pore contents are constricted by callose and cannot be identified, no callose was associated with plasmodesmata. Strasburger cells were connected to each other as well as to other parenchyma cells by plasmodesmata, united in primary pit fields. Here again, large compound median cavities were present (Fig. 24).

Although Strasburger cells contain all the protoplasmic components, typical of parenchyma cells in general, those associated with translocating sieve cells are characterized by a rather dense protoplast with abundant small vacuoles, numer- ous mitochondria and a dense population of ribosomes (Fig. 25). It is also noteworthy that such cells do not contain starch. While some of the vacuoles appear empty, others seem to be lipid vesicles, as judged from their staining behaviour. Mitochondria are often lobed or branched and form irregularly shaped complex structures (Fig. 26). The rather large nucleus is often lobed and contains fairly condensed heterochromatin. Plastids revealed poorly developed thylakoids. At the periphery, however, a tubular reticulate membrane system, comparable to plastids in immature sieve cells, was apparent. Occasionally, bundles of protein filaments, also comparable to sieve cell plastids, were observed (Fig. 27; compare Fig. 11).

Fig. 27. Portion of Strasburger cell protoplast presenting details of plastids (P) with reticulate membrane system and protein filaments (arrows). M Mitochondrion; V vacuoles. Scale 400 nm

(Figs. 21, 22). The sieve area in cell 8 in, Fig. 21 is heavily covered with callose. Callose deposits at lateral sieve areas of early formed sieve cells of the pertinent growth increment, were also observed by light microscopy and are indicated in Fig. 1. Callose deposits increase in late summer and are most conspicuous in all early sieve cells in autumn.

Strasburger cells occur at the margins of phloem rays as well as in the axial phloem parenchyma of Larix stems. We have not studied the development of Strasburger cells and comment only on the fine structure of those cells, which are closely associated with actively translocating sieve cells. Strasburger cells and adjacent sieve cells are connected by plasmodesmata and sieve pores re- spectively, both of which merge into a large com-

Discussion

The work of Kollmann (1965, 1967), Kollmann and D6rr (1966) and Willenbrink and Kollmann (1966) has provided strong evidence that young immature sieve cells of Metasequoia and Juni- perus may be actively translocating phloem elements. In spring and autumn, when these experiments were performed, the only functioning sieve cells are found in a very narrow band close to the cambium, while all older sieve cells were probably obliterated (Kollmann and Schumacher 1964; Alfieri and Kemp 1983). These inner layers of sieve cells persist and stay functional throughout the winter in a number of conifer trees (Alfieri and Evert 1968, 1973).

Studies of seasonal development of secondary phloem in Larix have shown that only one or two layers of late-phloem sieve cells overwinter (Huber 1939; Alfieri and Evert 1973). We have demonstrated here that sieve cells can persist in a state of structural immaturity or as fully mature cells or both, but their function was not proven. These observations are at variance with the clear statement of Alfieri and Evert (1973) that "mature

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late-phloem sieve cells overwinter outside the cambium and constitute the first functional sieve cells in spring; and no sieve cells overwinter in a partially differentiated condition" in Larix la- ricina as well as in species of Abies, Picea and Pinus.

Conducting sieve cells of secondary phloem can be recognized by associated active Strasbur- ger cells (Sauter and Braun 1968). It was demonstrated that such Strasburger cells are confined to a very narrow band of sieve cells in Larix in April and November, but Strasburger cells, which were associated with differentiating immature sieve cells, did not reveal much increased enzyme activity, indicating that young sieve cells in an early state of development may not be involved in long- distance transport (Sauter and Braun 1972). Highly increased enzyme activity was only detected in those Strasburger cells which were associated and in symplastic connection with fully differentiated sieve cells; it is assumed that only these sieve elements are actively translocating elements (Sauter and Braun 1968, 1972).

Functional sieve cells occupy a wide band of secondary phloem of Larix during summer. We have confirmed here that these sieve cells com- prise the early-phloem elements and we have shown by microautoradiography that only the mature early-phloem elements translocate 14C assimilate in June. This is in accordance with the observations of Sauter (1976) and Sauter and Braun (1968, 1972) on L. decidua. It also agrees with microautoradiographic studies of Langen- feld-Heyser (1987) on Picea abies. In contrast to these studies, the aim of the present investigation was to detect and localize the export of recently fixed 14C in lateral branches; the experiment time had therefore to be kept as short as possible, to avoid lateral or radial transport of labeled water soluble compounds. Hence, 14C sucrose, the main translocate (Schneider and Schmitz 1989), was confined to the mature sieve cells of early phloem.

Considering the fine structure of conducting mature sieve cells, .we recognized great confor- mity with sieve tubes, rather than unique conifer- specific structural features. Mature plasmalemma- lined sieve cells of Larix contain a degenerate nucleus, intact mitochondria, membrane systems of smooth ER, modified plastids and sparse vesicles, all structures arranged in a thin parietal layer, leaving an open unimpeded cell lumen for translocation. A comparable fine structure was re- ported from mature sieve cells of several different conifer tree species (Murmanis and Evert 1966, Pinus strobus; Srivastava and O'Brien 1966,

P. strobus; Wooding 1966, P. pinea; Neuberger and Evert 1974, P. resinosa); there are, however, also conflicting reports in the literature.

The presence of nuclei of "normal appearance" in translocating sieve cells and the statement that nuclear disintegration does not occur until the sieve cells of Sequoia sempervirens, Taxo- dium distichum and Metasequoia glyptostroboides begin to degenerate or cease functioning was claimed by Evert et al. (1970). This observation is certainly not applicable to the early phloem of Larix in June because all mature sieve cells contained degenerate condensed nuclei which gener- ally were necrotic in appearance (see also Evert and Alfieri 1965; Alfieri and Evert 1973). Huber and Liese (1963) as well as Sauter and Braun (1968, 1972) even claimed that mature sieve cells of Larix lack a nucleus or may contain only remnants of a very degenerate nucleus. Remnants of pycnotic disorganized nuclei seem to persist in mature sieve cells of Larix until the ceils oblite- rate, but a physiological function is not known.

The lumen of mature sieve cells of Larix may in part be occluded by protein filaments and con- voluted ER structures, but we cannot confirm previous observations that the lumen is filled with a "ground substance", composed of finely fi- brillar material or a meshwork of vesiculated ER, as decribed by Kollmann and Schumacher (1964), Srivastava and O'Brien (1966), Parameswaran (1971) and Murmanis (1974).

Sieve cells of L. decidua have P-type plastids with ring-like arranged protein filaments. In contrast to Behnke's (1974) observations, we always found protein filaments in abundance but rarely plastid starch. We cannot decide whether the breakdown of the P-type plastid envelope and the release of protein filaments is a normal process during sieve cell development as proposed by Par- ameswaran (1971) and Murmanis (1974) or whether it is a fixation artefact (Behnke 1974; Neuberger and Evert 1974; see discussion in Sauter 1977). It is noteworthy, however, that free protein filaments are more frequently observed in ontogenetically older than in the young mature sieve cells. No defined orientation of free protein filaments in cell lumina, like P-protein arrangement in sieve tubes, was observed. Protein filaments like those in sieve cell plastids were also detected in Strasburger cells of Larix. These protein structures are clearly different from tubular structures observed by Sauter et al. (1976) in Strasbur- ger cells of Pinus nigra. The latter are more comparable to protein tubules, detected here in mature sieve cells during winter, although their

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diameter is different and the possibility cannot be excluded that these structures are artefacts.

The arrangement of the complex ER-membrane system in mature sieve cells of Larix is comparable to other conifer tree species (Wooding 1966; Srivastava and O'Brien 1966; Neuberger and Evert 1974); beside a parietal layer of ER tubules, most of the ER membrane system was always associated with the sieve areas. It was observed by Sauter (1976) and Sauter and Braun (1968, 1972) that activities of acid phosphatases and enzymatic splitting of glycerophosphate, ATP and UTP were enriched and localized at sieve areas and were probably associated with the abundant ER, as demonstrated by electron microscopic localization (Sauter 1977). It can only be speculated that the ER system in mature sieve cells may somehow be involved in translocation; the accumulation of laC at the lateral sieve areas, as revealed by microautoradiography, supports this view. Intact mitochondria were always present and enmeshed between ER membranes. Their respiratory activity could not be detected in mature sieve cells with cytochemical methods (Sauter and Braun 1972) and their function re- mains to be determined.

Based on critical evaluation of the literature and their own observations on P. resinosa, Neuberger and Evert (1975) concluded that sieve areas in mature sieve cells of gymnosperms are re- markably similar in structure. Sieve areas of Larix are certainly no exception because pores and median cavities are lined by plasmalemma and seem to be traversed by ER membranes. Although the membrane structures within the pores were not clearly resolved here and appeared to be constricted by surrounding callose, a distinct membrane system in the median cavity, probably smooth ER, was observed. Callose was present at the sieve areas of all mature active translocating early-phloem sieve cells. No experiments were performed to find out whether the presence of callose is artificial and induced by the fixation procedure or whether sieve areas lack callose in vivo. Trying to correlate the presented structural findings and our transport physiological data (Schneider and Schmitz 1989) raises the question of what the mechanism of long-distance transport in Larix might be. There must be a conductivity along the sieve cells that allows long-distance transport with a velocity of 1 0 - 2 0 c m - h -1 (Schneider and Schmitz 1989) which cannot be explained by diffusion. The much higher transport velocity of up to 60 cm- h- 1 as determined in Metasequoia by Willenbrink and Kollmann

(1966) and the results of translocation studies us- ing aphids (Ziegler and Mittler 1959; Kollmann and D6rr 1966) indicate that a volume flow may be possible along mature sieve cells of gymnos- perm trees. The observed fine structure of sieve areas with associated ER membranes apparently contradicts a pressure-flow-type mechanism of translocation in sieve cells.

Acknowledgements. This research was supported by the Deut- sche Forschungsgemeinschaft. We gratefully acknowledge the excellent technical assistance of Ms Britta Mfiller.

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Received April 24, 1989/June 25, 1989

structure and development of sieve cells in the secondary phloem of larix decidua mill. as related...

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