THE FINE STRUCTURE OF DIFFERENTIATING SIEVE TUBE ELEMENTS

G. BENJAMIN BOUCK, Ph.D., and JAMES CRONSHAW, Ph.D. From the Department of , Yale University, New Haven, Connecticut

ABSTRACT The developmental sequences leading to the formation of mature sieve tube elements were studied in pea by electron . From this study it has been found that the peripheral layer of cytoplasm in the mature element is composed of flattened cisternae which are apparently derived from a tubular form of (ER) and possibly the nuclear envelope. These flattened cisternae, designated in this report as sieve tube reticula, are attached perpendicularly to the wall surface and are oriented in a pre- dominantly longitudinal direction. Cisternae of the sieve tube reticulum are frequently associated with the slime in mature elements, and tubular ER may be associated with slime- like material in the developing . During differentiation mitochondria become reduced in size and either fail to develop stroma and grana lamellae or lose them early in development. In agreement with other workers it is found that the sieve plate pores appear to be plugged with a finely fibrous material, presumably "slime." Nacreous wall formation is well established before reorganization of cytoplasmic compo- nents. Microtubules are prevalent during these early stages, but are lost as the element ma- tures.

The sequence of cytoplasmic events culminating possibly endoplasmic reticulum (ER) may persist in the formation of mature sieve tubes and sieve in these elements, and transport itself is plates is only partially understood. In general, it is very likely metabolically controlled (cf. Kendall, both a degenerative process involving at least the 1955; Esau, Currier, and Cheadle, 1957). In order breakdown of the nucleus, and a synthetic process to fully evaluate the "live" quality and ultimately resulting in increased wall deposition and often the function of these cytoplasmic components, it is the formation of callose. Both light and electron of prime importance to understand the ontogenetic microscope studies have demonstrated in the steps leading to the formation of the mature ele- mature sieve tube element a peripheral layer of ments. Such a developmental study should thus cytoplasm. This peripheral layer is semipermeable provide a firm structural basis for final interpreta- (Rouschal, 1941; Currier, Esau, and Cheadle, tion of sieve tube operation. 1955) and consists structurally of membranes and MATERIALS AND METHODS vesicles (Duloy, Mercer, and Rathgeber, 1961). As is well known, the sieve tube cytoplasm appears Pisum sativum Alaska were germinated and grown in many ways unique (Esau and Cheadle, 1961). in vermiculite under continuous illumination. After 14 days portions of Some criteria of living protoplasm such as vital the whole were fixed di- rectly, or 5-mm segments were cut from the first staining, streaming, and capacity for division internode and placed in Petri dishes containing 1 per are not met in mature sieve tube elements. On the cent sucrose and 0.025 Mphosphate adjusted to a pH other hand, intact mitochondria, proplastids, and of 6.1. The Petri dishes with the internode segments

79 were then placed on a shaking table and given con- area of the wall adjacent to the plasma membrane tinuous illumination. After 17 hours whole segments which is frequently more electron-opaque than were transferred to either 6.5 per cent glutaraldehyde the walls of adjacent cells (Figs. and 2). This area (Sabatini, Bensch, and Barrnett, 1963) or 10 per cent surrounding the differentiating sieve tube element aldehyde) buffered with potassium acrolein (acrylic probably corresponds to the "nacr6" wall (cf. phosphate (Millonig, 1961 b) at a pH of 6.8. The Esau and Cheadle, 1958). The plasma membrane segments were evacuated several times during this differentiating sieve tube cell retains its preliminary fixation. Glutaraldehyde fixation for 4 of the hours or acrolein fixation for 45 minutes was followed usual triple-layered structure (i.e., two electron- by rinsing in three changes of buffer over a 3 hour opaque lines separated by a less opaque region), period. During the buffer rinse the segments were but differs from the plasma membrane of adjacent cut into smaller pieces. Postfixation was accomplished cells in that the two electron opaque lines appear with 2 per cent phosphate-buffered osmium tetroxide thicker and stain more heavily than their counter- and was followed by a rapid rinsing in distilled water, parts in adjacent cells (Fig. 2). Closely associated dehydration in a graded acetone series, propylene with the plasma membrane on its cytoplasmic side oxide (3 changes), and embedding in an Epon mix- are numerous microtubules (Ledbetter and Por- ture containing 50 ml Epon, 44 ml Methyl Nadic ter, 1963). These microtubules appear especially Anhydride and 1.6 ml DMP-30 (Luft, 1961). Sections in the early stages of sieve tube differ- cut with a diamond knife and stained in Millonig's prevalent (1961 a) lead stain (preceded on occasion by 1 hour entiation, but in later stages can be found only with immersion in an uranyl acetate solution) were ex- difficulty. Acrolein fixation as used here fails to amined and photographed in a JEM 6C electron demonstrate them in any cells. microscope. Segments and whole stems gave es- Mitochondria characteristically undergo a re- sentially similar images, but fixation was uniformly duction in size early in the differentiation se- more satisfactory with segments. quence, but otherwise appear structurally similar to mitochondria elsewhere in the stem (Fig. 2). RESULTS Chloroplasts either fail to develop stroma and Mature and differentiating sieve elements and grana lamellae or lose these structures during the phloem cells of pea stem were ex- initial stages of differentiation. Structurally the amined in both longitudinal and transverse section. plastids are similar to the proplastids found in cells of the pea (Sitte, 1958; Bouck, 1963). Differentiation of Sieve Tube Cells Internally, these proplastid-like bodies (Fig. 3) The early stages in sieve tube differentiation retain the tubules known to be extensions of the are characterized by the formation of a thicker inner of the two double membranes of the en-

Key to Labeling

C, callose NP, nuclear pore ER, endoplasmic reticulum P, plastid IB, irregular body PM, plasma membrane L, lysosome-like body S, sieve plate LP, lateral sieve area SI, slime M, STR, sieve tube reticulum Mi, microtubule T, tube N, nucleus V, Na, nacre wall W, wall NE, nuclear envelope

F1GURE 1 Transverse section through a differentiating sieve tube element. The nucleus with its chromatin and nucleolus is still intact. The ER is predominantly of a tubular anastomosing form, while in adjacent cells it is predominantly of a flattened cisternal variety. Note electron opaque region of the wall surrounding the differentiating element (arrow). X 14,500.

80 THE JOURNAL OF CELL BIOLOGY VOLUME 25, 1965 G. BENJAMIN BOUCK AND JAMES CRONSHAW Differentiating Sieve Tube Elements 81 velope, and they also retain starch, although this differ in that there is no crystalline core in the starch may be chemically and physically different bodies of the pea stem cells. from starch found in other types of cells (Esau, Expanded elements of ER containing fibrous 1950; Currier, Esau, and Cheadle, 1955; Mc- material in several regular orientations (imparting Givern, 1957). a characteristic "herringbone" appearance) may Irregular bodies with angular outlines composed be found in several kinds of pea stem cells (Fig. 7) of electron-opaque material are a frequent com- including the developing sieve tube elements. ponent of differentiating and fully differentiated Hepler and Newcomb (1964) believe that por- phloem elements (Fig. 3). Internally they may tions of ER containing similar fibrous material have fracture lines or fine channels, but otherwise are contributing to wall formation. However, appear homogeneous. These irregular bodies are this fibrous material within the ER has thus far found in phloem parenchyma and companion been found only in cultured stem segments in cells as well (eg., Fig. 2), but their larger size in the present study and apparently only in excised sieve elements seems to be unmatched in com- material in Hepler and Newcomb's study. Further- panion and parenchyma cells. Initially these more, where found in pea stem cells, these struc- structures are seen in the ground cytoplasm of the tures show no consistent orientation with respect maturing sieve tube elements, but after the cell to the . becomes generally reorganized they are found in The later stages in sieve tube development in- the lumen of the sieve tube element. volve the reorientation of some of the membranous Large amounts of anastomosing tubular struc- components of the cytoplasm to form marginal tures, probably a form of endoplasmic reticulum, regions of paired membranes or flattened cisternae. appear in the cytoplasm apparently soon after During this process the contents of the nucleus initiation of sieve tube differentiation (Figs. 2 and disintegrate and ultimately the chromatin and 3). In these developing sieve tube cells this ER is nucleolus can no longer be recognized within the often greatly dilated, suggesting increased ac- nucleus. However, the nucleus is still bounded by tivity or increased susceptibility to distortion its usual envelope (Fig. 5). Soon after or concur- during fixation. Kollmann and Schumacher rently with the dissolution of the nuclear contents, (1962) have also reported large amounts of dilated vesicles or cisternae appear along the inner margin ER in reactivated phloem of Metasequoia. of the nuclear membrane (Fig. 4), and this is ap- Other cytoplasmic elements, i.e. nuclei and parently followed shortly by breakdown of the dictyosomes (Golgi apparatus), appear struc- envelope itself. Prior to and after the breakdown turally similar, in the early stages of differentia- of the intact nucleus the ER aggregates in loose tion, to their counterparts in other cells. However, patches along the surface of the wall (Fig. 3). structures bounded by a single membrane (mor- Eventually these patches may be compressed to phologically similar to animal lysosomes) and form flattened layers of cisternae parallel to the with granular contents (Fig. 7) are frequently wall (Fig. 5). These cisternae are separated by a seen in most cells of the pea stem, but appear space and this space initially seems to be occupied absent from differentiating sieve tube elements. in part by a layer of fine tubules or granules. Thus, These bodies resemble structures found in cells of the layer of marginal cytoplasm of the sieve tube oat coleoptiles (Thornton and Thimann, 1964; element appears to be composed of membranes Cronshaw, 1964: Cronshaw and Bouck, 1965) but derived from the endoplasmic reticulum and

FIGURE Transverse section through a portion of a differentiating sieve tube element (left) and a probable companion cell (right). Various profiles of a tubular ER may be seen in the sieve tube element, and associated with this ER are patches of granular slime (Sl). Irregular bodies (IB) may be seen in both cells. Note the difference in the plasma mem- branes of the companion cell and the sieve tube element (double-headed arrow). The more electron opaque region of the wall (Na) is presumably the nacreous wall of the dif- ferentiating sieve tube element. P, plastid, N, nucleus, M, mitochondrion. X 27,000.

82 THE JOURNAL OF CELL BIOLOGY · VOLUME 5, 1965 G. BENJAMIN Boucx AND JAMES CRONSHAW Differentiating Sieve Tube Elements 83 from vesiculation and breakdown of the nuclear Tube formation and then callose deposition prob- envelope. It is proposed that this system of mem- ably occur sequentially since surface sections of branes be designated as "sieve tube reticulum" developing sieve plates never show callose ex- to distinguish it from the usual forms of ER found tending beyond the point of the penetrating tube. in other plant cells. The validity of an association of portions of ER Dictyosomes may retain their identity until with the callose plate (Esau, Cheadle, and Risley, well after the marginal layer of ER is formed and 1962) cannot be clarified in these pea stems. Such the nuclear envelope is broken down. The subse- associations do occasionally exist, but seem to be quent fate of these is unknown. no more frequent than ER associations with other Plastids similar to proplastids (see above) may regions of the cell wall. persist until well after the formation of the sieve The callose plates are paired, one on each side plate. of the developing sieve plate (see also Esau, may be membrane-bounded up to Cheadle, and Risley, 1962), and from these the time of sieve plate formation (Fig. 6) after callose plates additional callose deposition occurs, which the tonoplast is lost and presumably dis- apparently pushing aside or mixing with cellulose integrates (cf. Currier, Esau, and Cheadle, 1955). microfibrils (Frey-Wyssling and Muiller 1957). Lipid droplets seem absent in all later stages of The two separate callose plates ultimately form a development. continuum, joining at the region of the middle lamella, and the tubes now show continuity from Differentiation of Sieve Plates and Lateral one sieve tube cell to the adjacent sieve tube cell. Sieve Connections Ultimately the cells lose their smaller tubules and a perforation is established (Esau and Cheadle, A. SIEVE PLATES 1961), but as the pore increases in size it becomes During the period of ER reorganization areas of filled with fine fibrillar material (Figs. 11 and 12). electron-transparent material characteristic of B. LATERAL SIEVE AREAS callose (Esau, Cheadle, and Risley, 1962) form local isolated patches (Fig. 10). In surface view Lateral sieve areas may be found between sieve each of these patches is transversed by one tube tubes and adjacent cells. As with the sieve plates, bounded by a fine electron opaque line, and con- they are formed usually during the differentiation tains one or several tubular structures similar in of the sieve tube cell and seem to produce a local size to the "microtubules" (Ledbetter and Porter, swelling of the wall which is subsequently oc- 1963) found in the peripheral region (Fig. 9). It cupied largely by callose (Fig. 5). Callose deposi- would appear that the larger tubes of the sieve tion may proceed simultaneously from both sides plate are often formed during the later stages of of the wall or more commonly it may be laid down sieve tube differentiation, and that these tubes first on the sieve tube side of the wall, and then commonly branch or anastomose near the middle progress through the wall. The area of callose is lamella (see also Esau, Cheadle, and Risley, 1962). occupied by one or more tubes containing smaller

FIGURE 3 Transverse section through a portion of a differentiating sieve tube element at a stage later than Fig. 2. Vesicular ER is now collecting adjacent to the wall (W), and irregular bodies (IB) are greatly expanding. Plastids (P) lack grana and stroma lamellae. X 20,000.

FIGUREs 4 and 5 Transverse sections through sieve tube elements at a more advanced stage in differentiation than Fig. 3. In Fig. 4 vesiculation of the inner portion of the nu- clear envelope is apparent (arrow). In Fig. 5 the lack of chromatin and a nucleolus within the nucleus is evident. Note, however, that the nuclear membrane (NE) is still intact. Also seen in Fig. 5 are marginal layers of ER which are believed to have been similar to the ER of Fig. 3 but at this later stage appear as uniformly flattened cisternae. From this stage to the mature sieve tube element the ER is designated as sieve tube reticulum (STR). NP, nuclear pore. LP, lateral sieve area. Figs. 4 and 5, X 27,000.

84 THE JOURNAL OF CELL BIOLOGY - VOLUME 25, 1965 G. BENJAMIN BOUCK AND JAMES CRONSHAW Differentiating Sieve Tube Elements 85 tubules similar in construction to those of the Whorls or stacks of cisternae are seen in many terminal sieve plates. Although large deposits of sieve tube elements, and often are localized in the callose may occur in the lateral sieve areas, dis- region of the sieve plates. are not asso- solution of the central tubes does not follow. Con- ciated with the surfaces of the cisternae, but an tact between a sieve tube cell and an adjacent electron-opaque fibrous substance may collect cell is established through the wall by means of between adjacent cisternae (Fig. 12). the tubes and smaller tubules, but obviously DISCUSSION passage is considerably more restricted in the region of lateral sieve areas than through the end Although the structure of sieve tube elements has sieve plates. been extensively examined by numerous workers, several fundamental and basic problems have re- The Structure of the Mature Sieve Tube mained unsettled. The origin and function of ER- Element like cisternae in the mature element, the origin In the fully differentiated sieve tube, the periph- and nature of slime, the manner of construction eral layer of cytoplasm consists of at least one of the sieve plate, and the method of formation of layer of cisternae (originally derived from the ER the nacre wall are among these problems. Studies and nuclear membrane). In general, these cis- utilizing electron microscopy have been under- ternae appear to be oriented in a longitudinal taken primarily to clarify the construction of the direction and their lateral margins attached per- mature sieve tube element. The surprising plugged pendicularly to the cell surface. This attachment is nature of the mature sieve plate, in particular, has particularly noticeable in transverse sections. The now been reported for a number of higher plants. plasma membrane is difficult to preserve by the However, the peculiar structure and unique func- techniques of preservation utilized in this investi- tion of sieve tubes precludes a direct analysis of gation, and only occasionally does acrolein or the components of the mature element without glutaraldehyde fixation demonstrate a convincing some understanding of events during maturation. triple-layered plasma membrane of the usual A step in this direction has ,been taken by Esau, structure (Fig. 13). With these fixatives such a Cheadle, and Risley (1962) in a fine structural membrane can be more routinely identified in study of sieve plate ontogeny in Curcubita. adjacent cells. The vacuolar membrane has ap- The present investigation has been undertaken parently been lost. Small mitochondria are fre- to further clarify the ontogeny of the sieve plate quently seen associated with the marginal cis- and to extend these observations to other sieve ternae. Except for a reduction in size the tube components. From this study the following mitochondria appear to retain their original facts now seem clear. (a) The ER-like cisternae form throughout sieve tube differentiation found in the mature element are derived from the (Duloy, Mercer, and Rathgeber, 1961). "normal" form of ER and probably also from The sieve plate in the mature sieve tube shows the nuclear membrane (a modification of ER; perforations which are laterally bordered by a cf. Watson, 1959; Porter and Machado, 1960). thin membrane. These perforations are filled with Thus, they are indeed 'cytoplasmic'. () Slime is a finely fibrous material possibly derived from closely associated with ER cisternae both in the adjacent ER cisternae (Fig. 12). Lateral sieve initial stages of differentiation and in the mature areas lack perforations and appear in an arrested element. (c) The sieve plate is derived from a state of perforation. Callose is usually found normal cross-wall which develops pores, ultimately surrounding the tubes of the lateral sieve areas as surrounded by callose, which are rapidly filled well as around the perforation of the sieve plate. with a plugging material. (d) Nacr6 wall formation

FIGURE 6 Transverse section through an advanced stage in sieve tube differentiation. The sieve tube reticulum (arrows) is scattered in patches of varied thickness. These cis- ternae are usually attached by their lateral margins perpendicularly to the surface of the wall. Mitochondria (M) are often associated with the cisternal elements. Vacuoles (V) may still be seen at this stage. X 14,000.

86 THE JOURNAL OF CELL BIOLOGY VOLUME 25, 1965 G. BENJAMIN BOUCK AND JAMES CRONSHAW Differentiating Sieve Tube Elements 87 is completed early in sieve tube differentiation, plasma membrane was to be expected. However, and is followed by the disappearance of micro- the role of the sieve tube reticulum just within tubules from the cytoplasm. Microtubules can be the plasma membrane is less clear. This complex routinely identified in adjacent parenchyma cells of flattened cisternae is oriented primarily in a (Fig. 8) even when they are absent in sieve tube longitudinal direction with its lateral margins elements. usually attached perpendicularly to the long It now seems well established in a variety of axis of the cell. If this attachment accurately plants that the pores of the sieve plate are occupied reflects the living condition, then the free ends with a finely fibrous electron opaque material of the sieve tube reticulum may well extend into (e.g. Buvat, 1960; Kollmann, 1960; Ziegler, 1960; the sieve tube lumen and give rise to the reported Hepton and Preston, 1960; cf. however, Eschrich, transcellular strands (Thaine, 1962). The con- 1963). In those cases in which open pores have struction and location of the sieve tube reticulum been demonstrated (e.g. Esau, Cheadle, and Ris- seems ideally suited for surface transport. While ley, 1962) usually potassium permanganate has phloem transport is generally believed to be more been used as the fixative (cf. Esau and Cheadle, rapid than can be accounted for by the usually 1961). Thus it would seem that while mass flow observed streaming rates, the mature sieve tube (Miinch, 1930) or one of its many variations may conceivably have a modified streaming pat- may best explain the results obtained from trans- tern, or transport may be accelerated through the port studies, the plugged nature of the sieve region of the sieve plate (see above). Streaming, plate appears a severe obstacle to bulk flow of per se, in sieve tubes is still somewhat a matter of phloem materials. Hence, most of the cytological controversy (cf. Thaine, 1962; Esau, Engleman, data available is at variance with the most popular and Bisalputra, 1963) but streaming along mem- current theory for phloem transport. The fre- brane surfaces is not unknown elsewhere in plant quently cited observation of Esau and Cheadle cells (cf. Kamiya, 1959). Energy requirements of (1955) that some secondary phloem sieve elements such a process in sieve tubes could presumably be may form additional sieve plates during matura- supplied by adjacent companion and parenchyma tion has suggested that the sieve plate is endowed cells or from mitochondria persisting in the sieve with a special metabolic function (cf. Zimmer- tube element. mann, 1960; Kursanov, 1963). There is no doubt The origin and function of slime within the that the sieve plate is a carefully constructed sieve tube and within the surrounding parenchyma region of the cell, and that all wall material is and companion cells remains obscure. It is of some removed from the region of the pore and probably interest, however, that slime is usually associated replaced with a fibrous plug. It seems highly likely, with ER cisternae in the developing sieve tube therefore, that this plugged region is the site of element and is often found between adjacent rapid transport through some active process. Then sieve tube cisternae in the mature element. The from present data a reasonable explanation for slime of mature elements appears fibrous while phloem transport might propose a two-phase that of differentiating sieve elements and other system-that is, an active pumping through the phloem cells seems coarsely granular. Since slime sieve plate and perhaps a cytoplasm-mediated and ER are both present in mature and immature transport through the sieve cell (Kursanov, 1963). sieve tube cells, and since there seems to be a The plasmolysis studies of Rouschal (1941) topographical association between slime and ER, and Currier, Esau, and Cheadle, (1955) have it appears probable that ER is actually synthesiz- demonstrated a permeability barrier at the mar- ing slime. There is no evidence in the material gin of the sieve cell, and thus the presence of a examined here to support the interesting ob-

FIGcRE 7 Transverse section of a portion of a phloem parenchyma cell illustrating a lysosome-like body (L) and a portion of expanded ER containing fibrous material in a characteristic "herringbone" pattern. X 57,000.

FIGURE 8 Glancing section through 4 phloem parenchyma cells showing distribution of microtubules (Mi). X 28,000.

88 THE JOURNAL OF CELL BIOLOGY VOLUME 25, 1965 G. BENJAMIN BOUCK AND JAMES CRONSHAW Differentiating Sieve Tube Elements 89 servation of Falk (1964) that slime may be de- with wall deposition and/or cellulose microfibril rived from degenerating plastids. orientation. Their prevalence during the early The cellulose nacreous wall (Schneider, 1945; stages of nacreous wall formation in pea stem Esau, 1950) seems to be well formed before the phloem cells would seem to support this con- general reorganization of cell contents. Micro- tention. tubules (Ledbetter and Porter, 1963) are often associated with the wall at this time but are lost The authors wish to express their appreciation to as the sieve cell differentiates. From present in- Professor A. W. Galston for supplying the pea seg- formation it is uncertain whether this thinning ments and whole stems used in this investigation. out of tubules in later stages is the consequence of a The work was supported from funds supplied by decrease in their function as a result of slowing of grant GB-279 from the National Science Foundation, wall deposition, or results from the general reor- and grants GM11100 and GM11215-01 from the ganization of cell components during sieve tube National Institutes of Health, United States Public differentiation. Ledbetter and Porter (1963) Health Service. suggest that the microtubules may be associated Receivedfor publication, April 3, 1964.

BIBLIOGRAPHY

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FIGURE 9 Oblique longitudinal section through differentiating sieve tube element (lower cell) and an adjacent cell. The intervening wall presumably will become the sieve plate of the mature element. The tube (T) probably represents the first indication of the sieve plate pore. Note at this stage the tube contains finer tubules and is surrounded by a fine electron-opaque line. Little callose seem to be associated with the tube. Microtubules (arrows) may be seen in the corners of both cells. X 34,000.

FIGURE 10 Oblique longitudinal section of a developing sieve plate at a later stage than Fig. 9. Note accumulation of callose (C) around tubes. X 32,000.

90 THE JOURNAL OF CELL BIOLOGY VOLUME 25, 1965 G. BENJAMIN BOUCK AND JAMES CRONSHAW Differentiating Sieve Tube Elements 91 tubules and Fibrils in the Cytoplasm of Coleus cells MILLONIG, G., 1961 b, Advantages of a phosphate undergoing secondary wall deposition, J. Cell Biol., buffer for OsO04 solutions in fixation, J. Appl. 20, 529. Physics., 32, 1637. HEPTON, C. E. L., and PRESTON, R. D., 1960, E/M MiNCH, E., 1930, Die Stoffbewegungen in der Observations of the structure of sieve connexions Pflanze, Jena, Germany, Gustav Fischer. in the phloem of angiosperms and , PORTER, K. R., and MACHADO, R. D., 1960, Studies J. Exp. Bot., 11, 381. on the endoplasmic reticulum. IV. Its form and KAMIYA, N., 1959, Protoplasmic streaming, in Proto- distribution during mitosis in cells of onion root plasmatologia-Handbuch der Protoplasmafor- tip, J. Biophysic. and Biochem. Cytol., 7, 167. schung, (L. V. Heilbrunn and F. Weber, editors), ROUSCHAL, E., 1941, Untersuchungen fiber die Berlin, Springer, 8, 3a. Protoplasmatik und Funktion der Siebrohren. KENDALL, W. A., 1955, Effect of certain metabolic Flora 35, 135.

inhibitors on translocation of P32 in bean plants, SABATINI, D. D., BENSCH, K., and BARRNETT, R. J., Plant Physiol., 30, 347. 1963, Cytochemistry and electron microscopy. KOLLMANN, R., 1960, Untersuchungen fiber das The preservation of cellular ultrastructure and protoplasma der siebrohren von Passiflora coerulea. enzymatic activity by aldehyde fixation, J. Cell. II. Elektronenoptische Untersuchungen, Planta, Biol., 17, 19. 55, 67. SCHNEIDER, H., 1945, The anatomy of peach and KOLLMANN, R., and SCHUMACHER, W., 1962, Uber cherry phloem, Bull. Torrey Bot. Club, 72, 137. die Feistrnuktur des Phloems von Metasequoia SITTE, P., 1958, Die Ultrastruktur von Wurzel- glyptostroboides und seine Jahreszeitlichen Verin- meristemzellen der Erbse (Pisum sativum), Proto- derungen. III. Mitteilung. Die Reaktiuierung der plasma, 49, 447. Phloemzellen im Frihjahr, Planta, 59, 195. THAINE, R., 1962, A translocation hypothesis based KURSANOV, A. L., 1963, Metabolism and the transport on the structure of plant cytoplasm, J. Exp. Bot., of organic substances in the phloem, Adv. Bot. 13, 152. Research, 1, 209. THORNTON, R. M., and THIMANN, K. V., 1964, On LEDBETTER, M. C., and PORTER, K. R., 1963, A a crystal-containing body in cells of the oat coleop- "microtubule" in plant cell fine structure, J. Cell tile, J. Cell Biol., 20, 345. Biol., 19, 239. WATSON, M. L., 1959, Further observations on the LUFT, J. H., 1961, Improvements in epoxy resin embedding methods, J. Biophysic. and Biochem. nuclear envelope of the animal cell, J. Biophysic. Cytol., 9, 409. and Biochem. Cytol., 6, 147. McGrvERN, M. J., 1957, Mitochondria and plastids ZIEGLER, H., 1960, Untersuchungen fiber die Fein- in sieve tube cells, Am. J. Bot., 44, 37. struktur des Phloems I. Die S:ebplatten bei Hera- MILLONIG, G., 1961 a, A modified procedure for cleum mantegazzianum, Plan!a, 55, 1. lead staining of thin sections, J. Biophysic. and ZIMMERMANN, M. H., 1960, Transport in the phloem, Biochem. Cytol., 11, 736. Ann. Rev. Plant. Physiol., 11, 167.

FIGURE 11 Longitudinal section through 2 sieve tubes showing mature sieve plates (S) Plastids (P) contain granules of storage . Mitochondria also persist, and slime (S1) may be seen in large accumulations near the sieve plates. X 1,100.

92 THE JOURNAL OF CELL BIOLOGY VOLUME 25, 1965 G. BENJAMIN BOUCK AND JAMES CRONSIAW Differentiating Sieve Tube Elements 93 FIGURE 19 Longitudinal section through a mature sieve plate. Pores of the sieve plate are filled with a finely fibrous material (arrows). Note accumulation of cisternae near the sieve plate and the similarity of these cisternae to the sieve tube reticulum (STR) along the margin of the sieve tube element. The sieve tube reticulum is reduced to a single flat- tened layer in this section. The fibrous material in the sieve pore appears to be associated with the free cisternae. C, callose. X 53,000.

FIGURE 13 Portion of a transverse section through a mature phloem element showing presence of plasma membrane (PM) and sieve tube reticulum (STR). X 58,000

94 THE JOURNAL OF CELL BIOLOGY VOLUME 5, 1965 G. BENJAMIN BOUCK AND JAMES CRONSHAW Differentiating Sieve Tube Elements 95