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The Differentiation of Contact Cells and Isolation Cells in the Xylem Ray Parenchyma of Populus maximowiczii
Annals of Botany 84 : 429–435, 1999 Article No. anbo.1999.0931, available online at http:\\www.idealibrary.com on
The Differentiation of Contact Cells and Isolation Cells in the Xylem Ray Parenchyma of Populus maximowiczii Y. M U R A K A M I, R. F U N A DA*, Y. S A N O and J. O H T A N I Department of Forest Science, Faculty of Agriculture, Hokkaido Uniersity, Kita 9, Nishi 9, Kita-ku, Sapporo 060-8589, Japan Received : 24 March 1999
Returned for revision : 27 April 1999
Accepted : 11 June 1999
A histochemical analysis was made of the differentiation of contact cells and isolation cells in the xylem ray parenchyma of Populus maximowiczii. The contact cells formed secondary walls at approximately the same time as adjoining vessel elements. The lignification of the cell walls of contact cells and vessel elements began earlier than that of wood fibres and isolation cells. Thus, the formation of the secondary wall, including lignification, of the contact cells might occur at the same time as that of the vessel elements to which they are directly connected. By contrast, the isolation cells began to form secondary walls later than the vessel elements and wood fibres in the vicinity of the isolation cells. After the deposition of the secondary wall, a protective layer was formed in contact cells but no isotropic layer was observed in isolation cells. The results suggest the importance of vessel elements in the determination of the differentiation of adjoining ray parenchyma cells. # 1999 Annals of Botany Company Key words : Contact cell, isolation cell, vessel element, xylem differentiation, Populus maximowiczii Henry.
INTRODUCTION Ray parenchyma cells play an important role in the storage, conductive and secretory systems of woody plants. Mostly, they maintain radial continuity between the phloem and xylem, thereby facilitating the radial and lateral transport of materials (Larson, 1994). The ray parenchyma cells of xylem are of two types, namely contact cells and isolation cells, and they differ with respect to their contacts, through pits, with vessel elements. Contact cells are ray cells that make direct connections with adjacent vessel elements through half-bordered pit pairs, and the isolation cells are ray cells that make no pitmediated direct connections with adjacent vessel elements (Braun, 1967). Sauter and Kloth (1986) proposed that isolation cells might be more specialized for radial translocation than contact cells. By contrast, tyloses develop from contact cells and not from isolation cells. Therefore, contact cells might function to protect living ray parenchyma cells from hydrolases released by dead vessel elements (Schmid, 1965 ; O’Brien, 1970). In contrast, Chafe (1974) proposed that contact cells might function as ‘ transfer cells ’ because both types of cell have a similar structure. It appears that contact cells and isolation cells differ in terms of both form and function, even though both types of cell are derived from the same cambial ray cells (Braun, 1970). Thus, it seems possible that these two types of cell might be formed via different processes of differentiation from cambial ray cells.
* For correspondence. Fax j81 117361791, e-mail funada!for. agr.hokudai.ac.jp
0305-7364\99\100429j07 $30.00\0
Ray parenchyma cells generally form an additional layer on the inner side of the secondary wall (Schmid, 1965). This layer contains high levels of polysaccharides (Czaninski, 1973). Chafe (1974) reported that, in Populus tremuloides, such an additional layer in contact cells was unlignified during the first growing season (this layer was referred to as the protective layer), while that of isolation cells was lignified within one growing season (this layer was referred to as the isotropic layer). The protective layer was finally lignified when the formation of heartwood began. Using histochemical techniques, Fujii, Harada and Saiki (1981) found that there was little difference between the protective layer and the isotropic layer in terms of their ultrastructure and chemical compositions prior to lignification. Thus, they concluded that these two layers might have the same origin and that lignification might begin at different times in the two layers. These observations indicate that the formation of the secondary wall, including lignification, differs between the two types of ray parenchyma cell. Such differences might be dependent on the disposition of ray parenchyma cells with respect to the vessel elements. The purpose of the present study was to monitor the differentiation of contact cells and isolation cells in Populus maximowiczii by histochemical staining. The rays of Populus have a simple composition, being homogeneous and uniseriate (Panshin and de Zeeuw, 1980). Therefore, it is easy to determine, by observation, whether or not ray parenchyma cells make direct contact with adjacent vessel elements. We postulated that the deposition and lignification of cell walls of the two types of ray parenchyma cell would provide a suitable model for clarification of the mechanism of differentiation of secondary xylem cells derived from cambial cells. # 1999 Annals of Botany Company
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Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus
F. 1. A differentiating vessel element (V) and other elements around it, at a distance of approximately 500 µm from the cambium, in radial section. Arrows indicate transverse walls of contact cells with birefringence. Arrowheads indicate ray-vessel pits of contact cells. No birefringence was visible in cell walls of isolation cells (asterisks). The left side of each micrograph corresponds to the outer side of the tree. Light micrograph (A) and polarizing light micrograph (B). Bar l 50 µm.
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F. 2. Electron micrographs showing tangential sections of differentiating ray parenchyma cells at a similar stage to that shown in Fig. 1. Contact cell with a secondary wall (A) and an isolation cell without a secondary wall (B). V, Vessel element ; F, wood fibre ; CC, contact cell ; IC, isolation cell. Bars l 2 µm.
MATERIALS AND METHODS Samples from a straight, healthy tree (approx. 30 years old) Populus maximowiczii Henry (height, 18 m ; diameter at breast height, 30 cm) growing in the Tomakomai Experimental Forest of Hokkaido University, were used. Small blocks containing phloem, cambial zone and differentiating xylem were taken from the stem at breast height in July 1995, when cambial activity was very high. At that time, the successive stages of differentiation of ray parenchyma cells could be observed within a single radial section. All sample blocks were immediately placed in a 4 % solution of glutaraldehyde in 0n1 phosphate buffer (pH 7n2) and fixed overnight at room temperature. After washing with the same buffer, some blocks were postfixed in 1 % osmium tetroxide in the same buffer for 2 h at room temperature. Other sample blocks for ultraviolet microscopy were not postfixed in 1 % osmium tetroxide. Samples were then washed with buffer and dehydrated through a graded ethanol series. They were then embedded in epoxy resin.
For standard light microscopy and polarizing light microscopy, radial and tangential sections (1 µm thick) were cut with a glass knife on an ultramicrotome (Ultracut J ; Reichert, Austria). Sections were stained with a 1 % aqueous solution of safranin or a 1 % aqueous solution of gentian violet. They were observed with a light microscope (BHS-2 ; Olympus, Japan) and a polarizing microscope (BHS-751P ; Olympus). For determination of the extent of lignification, radial and tangential sections (1 µm thick) were cut with a glass knife, placed on quartz slides and mounted in glycerine with quartz coverslips. Photographs were taken under an ultraviolet (UV) microscope (MPM-800 ; Carl Zeiss, Germany) at a wavelength of 280 nm with a bandwidth of 5 nm (Fukazawa, 1992 ; Sano and Nakada, 1998). For transmission electron microscopy, ultra-thin tangential sections (70 nm thick) were cut with a diamond knife and stained with uranyl acetate and lead citrate. The ultrathin sections were then observed with a transmission electron microscope (JEM-100C ; JEOL, Japan) operated at an accelerating voltage of 100 kV.
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Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus RESULTS
Figure 1 shows a differentiating vessel element, which was located approx. 500 µm from the cambium, and the other elements around it. In this region, the birefringence of the cell walls of the axial xylem cells was detectable with a polarizing light microscope (Fig. 1 B). This birefringence indicated that the deposition of secondary walls had started in these cells (Abe et al., 1997). Ray parenchyma cells, which were oriented transversely, were also observed. The parenchyma cells with ray-vessel pits (arrowheads in Fig. 1 A and B) were considered to be contact cells. The birefringence of transverse walls of contact cells was clearly visible (arrows in Fig. 1 B). Several thin-walled ray parenchyma cells (asterisks in Fig. 1 A and B) were observed between contact cells. These cells were considered to be isolation cells because they had no ray-vessel pits. Serial, radial, semithin sections revealed that isolation cells were longer than contact cells and that there were many intercellular spaces between isolation cells (data not shown). The walls of isolation cells were not birefringent under the polarizing light microscope, and indication that the deposition of secondary walls had not yet started in these cells. Figure 2 shows the ultrastructure of cell walls in differentiating ray parenchyma cells in tangential section of a sample at a similar stage to that shown in Fig. 1. The contact cells formed ray-vessel pits (Fig. 2 A). The thick secondary walls of contact cells and vessel elements had already formed. The contact cells were present at the upper and lower margins of the ray. By contrast, the isolation cells had thin cell walls (Fig. 2 B), and the thickness was similar to that of the cell walls of cambial cells. The UV absorption of cell walls at 280 nm was apparent on radial sections of a region similar to that shown in Fig. 1.
F. 4. UV photomicrograph showing a tangential section of a differentiating vessel element and other elements around it at a similar stage to that shown in Fig. 3. Arrows indicate cell walls of contact cells and vessel elements that absorbed UV light. Arrowheads indicate cell walls of isolation cells (asterisks) that did not absorb UV light. V, Vessel element ; F, wood fibre. Bar l 10 µm.
F. 3. UV photomicrograph showing a radial section of a differentiating vessel element (V) and other elements around it at a similar stage to that shown in Fig. 1. UV absorption of cell walls is visible in vessel elements (large arrows) and contact cells (small arrows). The cell walls of wood fibres absorbed UV light very weakly (arrowheads). The left side of this micrograph corresponds to the outer side of the tree. Bar l 50 µm.
The UV absorption of cell walls was visible first in vessel elements (large arrows in Fig. 3). This absorption indicated that the lignification of these cell walls had begun. UV absorption by the secondary walls of contact cells was as strong as that by cell walls of vessel elements (small arrows in Fig. 3). By contrast, the cell walls of wood fibres absorbed UV light only very weakly (arrowheads in Fig. 3). Figure 4 shows differentiating axial elements and ray parenchyma cells at a similar stage to that shown in Fig. 3 on a tangential section. The UV absorption of cell walls of vessel elements and contact cells was stronger than that of wood fibres in the vicinity of vessel elements (arrows in Fig. 4). Strong UV absorption was observed at the middle lamella in contact cells, but no UV absorption was detected
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F. 5. Electron micrographs showing tangential sections of differentiating ray parenchyma cells at distances from the cambium that ranged from approx. 1500–2000 µm. Contact cell with a protective layer (arrows) (A) and an isolation cell without an isotropic layer (B). V, Vessel element ; CC, contact cell ; IC, isolation cell. Bars l 2 µm.
at pit membranes of ray-vessel pits. Moreover, the cell walls (arrowheads in Fig. 4) of isolation cells (asterisks in Fig. 4) did not absorb UV light. Various terms have been used for the additional layers of parenchyma cells. Schmid (1965) names these layers the ‘ protective layer ’. Later Chafe (1974) and Chafe and Chauret (1974) used the term ‘ protective layer ’ in the case of vessel-associated ray parenchyma cells and the term ‘ isotropic layer ’ in the case of axial parenchyma cells and non-vessel-associated ray parenchyma cells. By contrast, Fujii, Harada and Saiki (1980, 1981) proposed both layers be called the ‘ amorphous layer ’ because there appeared to be little difference between them in their original chemical composition and fine structure. In this report, we use separate designations for the protective layer and the isotropic layer because these layers appear to have different
functions. Figure 5 shows ray parenchyma cells at distances from the cambium that range from approx. 1500–2000 µm. The protective layer was formed (arrows in Fig. 5 A) in the contact cells after the deposition of the secondary wall. In agreement with the observations reported by Chafe (1974), this layer was not limited only to the side where vessel elements were located, but was also present on the opposite side. However, the protective layer on the opposite side was much thinner than that on the side on which vessel elements were located. Czaninski (1973) and Chafe (1974) reported that the deposition of the protective layer began at the site of secondary wall thickening. Figure 6 shows ray parenchyma cells at a similar stage to that shown in Fig. 5 in tangential section. The protective layer of contact cells (arrows in Fig. 6) and pit membranes of ray-vessel pits (arrowheads in Fig. 6) did not absorb UV light. The
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Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus
F. 6. UV photomicrograph showing a tangential section of differentiating ray parenchyma cells at a similar stage to that shown in Fig. 5. No UV absorption by the protective layer (arrows) and pit membranes of ray-vessel pits (arrowheads) is evident in the contact cells. UV absorption of cell walls is evident in the isolation cells (asterisks). V, Vessel element ; F, wood fibre. Bar l 10 µm.
secondary walls of isolation cells absorbed UV light (asterisks in Fig. 6), but no isotropic layer was apparent in isolation cells (Fig. 5 B). DISCUSSION The cell walls of isolation cells in the regions shown in Figs 1 and 2 were primary walls because they were thin and exhibited no birefringence under polarized light. By contrast, contact cells formed secondary walls at approximately the same time as vessel elements. Chafe (1974) observed that the initiation of the deposition of the secondary wall in vessel elements occurred slightly earlier than in contact cells. At this stage, in our samples, UV absorption of cell walls of vessel elements and contact cells appeared to be stronger
than that of the wood fibres in the vicinity of vessel elements (Figs 3 and 4). By contrast, no UV absorption of cell walls of isolation cells was detected (arrowheads in Fig. 4). These results indicated that the lignification of the cell walls of contact cells and vessel elements began earlier than that of wood fibres and isolation cells. Thus, the timing of the formation of the secondary walls, including lignification, of the contact cells was similar to that of the vessel elements with which they were directly connected. The similarity in timing of differentiation between contact cells and vessel elements suggests that differentiation of contact cells might be influenced by vessel elements through ray-vessel pits. By contrast, the differentiation of isolation cells, which make no direct pit-mediated connections with adjacent vessel elements, might not be directly affected by the differentiation of vessel elements. Our results indicate that contact cells and vessel elements began to form lignified secondary walls earlier than the wood fibres in the vicinity of the vessel elements. By contrast, the pit membranes of ray-vessel pits and the protective layers in contact cells were not lignified soon after deposition of secondary walls (Fig. 6). Barnett, Cooper and Bonner (1993) suggested that maintenance of an apoplastic pathway around a lignified cell with living protoplast was the primary function of the protective layer. The contact cells might specialize in the translocation of water and materials between ray and vessel. In contrast, the differentiating isolation cells with unlignified tangential walls might become pathways for temporary radial continuity between the phloem and xylem. In this way, easy radial translocation is maintained through the rows of isolation cells. We observed no isotropic layer in isolation cells after deposition of secondary walls (Fig. 5 B). Chafe (1974) reported that isolation cells of Populus tremuloides formed secondary walls and isotropic layers, all of which became lignified, within a single growth season. By contrast, Fujii, Harada and Saiki (1979) reported that the cell wall in differentiated isolation cells of Populus maximowiczii consisted of only a cellulosic secondary wall. The reason for the discrepancy among species that belong to the same genus is that the isotropic layer might be only partially deposited in the isolation cells and, thus, the occurrence of an isotropic layer in ultra-thin sections would depend on the place in which the isolation cells were sectioned. In addition, there is the possibility that isolation cells had not yet begun to form the isotropic layer in the present study because our specimens were collected in July. Chafe (1974) reported that deposition of the protective layer began after completion of the deposition of the cellulosic secondary wall. However, the isotropic layer might not be fully developed soon after completion of the deposition of the cellulosic secondary wall in isolation cells. Our results indicate that the structure of the ray parenchyma cells of the secondary xylem differs considerably between contact cells and isolation cells, which are derived from the same cambial ray cells. Therefore, these ray parenchyma cells are formed by different processes of differentiation, depending on whether or not they make direct connections, through pits, with adjoining vessel elements. Our observations indicate the importance of
Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus neighbouring cells in controlling xylem differentiation. Expanding vessels might send some signals to adjoining cells through the plasma membrane and primary wall. These signals determine the direction of differentiation of ray parenchyma cells. Although the nature of these putative signals remains to be characterized, some specific proteins or polysaccharides of the cell wall, which trigger cell differentiation, might be secreted and reach future contact cells from differentiating vessel elements. In contrast, future isolation cells might not receive such signals. Therefore, future contact cells and isolation cells might differ originally in both structure and the nature of the primary wall and plasma membrane. They might have different numbers and distributions of plasmodesmata with different structures since it has been proposed that plasmodesmata play an important role in the fate of xylem cells (see Gunning, 1978 ; Barnett, 1981, 1982). More detailed information about plasmodesmata in cambial ray cells is needed to clarify the mechanisms of differentiation of xylem ray cells. Cambial cells and their derivatives have been proposed to serve as a model system for studies of cytodifferentiation in secondary tissues in situ because it is possible to follow the process of differentiation in a radial direction (Abe et al., 1995 ; Chaffey, Barlow and Barnett, 1997 ; Chaffey, Barnett and Barlow, 1997 ; Funada et al., 1997). The successive differentiation of contact cells and isolation cells, both of which are derived from cambial ray cells, is also a useful model system for studies of the mechanism of control of the formation and lignification of secondary walls in secondary xylem. A C K N O W L E D G E M E N TS This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (Nos 08760157 and 09760152) and from the Japan Society for the Promotion of Science (no. JSPSRFTF 96L00605). LITERATURE CITED Abe H, Funada R, Imaizumi H, Ohtani J, Fukazawa K. 1995. Dynamic changes in the arrangement of cortical microtubules in conifer tracheids during differentiation. Planta 197 : 418–421. Abe H, Funada R, Ohtani J, Fukazawa K. 1997. Changes in the arrangement of cellulose microfibrils associated with the cessation of cell expansion in tracheids. Trees 11 : 328–332. Barnett JR. 1981. Secondary xylem cell development. In : Barnett JR, ed. Xylem cell deelopment. Tunbridge Wells, Kent : Castle House Publications, 47–95. Barnett JR. 1982. Plasmodesmata and pit development in secondary xylem elements. Planta 155 : 251–260.
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Barnett JR, Cooper P, Bonner LJ. 1993. The protective layer as an extension of the apoplast. IAWA Journal 14 : 163–171. Braun HJ. 1967. Development and structure of wood rays in view of contact-isolation-differentiation to hydrosystem. Holzforschung 21 : 33–37. Braun HJ. 1970. Functionelle histologie der sekunda$ ren sproßachse. I. Das Holz. In : Zimmermann W, Ozenda P, Wulff HD, eds, Handbuch der Pflanzenanatomie, ol. IX, pt. 1. Berlin : Gebru$ der Borntra$ ger, 1–190. Chafe SC. 1974. Cell wall formation and ‘ protective layer ’ development in the xylem parenchyma of trembling aspen. Protoplasma 80 : 335–354. Chafe SC, Chauret G. 1974. Cell wall structure in the xylem parenchyma of trembling aspen. Protoplasma 80 : 129–147. Chaffey NJ, Barlow PW, Barnett JR. 1997. Cortical microtubules rearrange during differentiation of vascular cambial derivatives, microfilaments do not. Trees 11 : 333–341. Chaffey NJ, Barnett JR, Barlow PW. 1997. Cortical microtubule involvement in bordered pit formation in secondary xylem vessel elements of Aesculus hippocastanum L. (Hippocastanaceae) : a correlative study using electron microscopy and indirect immunofluorescence microscopy. Protoplasma 197 : 64–75. Czaninski Y. 1973. Observations sur une nouvelle couche parie! tale dans les cellules associe! es aux vaisseaux du Robinier et du Sycomore. Protoplasma 77 : 211–219. Fujii T, Harada H, Saiki H. 1979. The layered structure of ray parenchyma secondary wall in the wood of 49 Japanese angiosperm species. Mokuzai Gakkaishi 25 : 251–257. Fujii T, Harada H, Saiki H. 1980. The layered structure of secondary walls in axial parenchyma of the wood of 51 Japanese angiosperm species. Mokuzai Gakkaishi 26 : 373–380. Fujii T, Harada H, Saiki H. 1981. Ultrastructure of ‘ amorphous layer ’ in xylem parenchyma cell wall of angiosperm species. Mokuzai Gakkaishi 27 : 149–156. Fukazawa K. 1992. Ultraviolet microscopy. In : Lin SY, Dence CW, eds. Methods in lignin chemistry. Berlin : Springer-Verlag, 110–121. Funada R, Abe H, Furusawa O, Imaizumi H, Fukazawa K, Ohtani J. 1997. The orientation and localization of cortical microtubules in differentiating conifer tracheids during cell expansion. Plant and Cell Physiology 38 : 210–212. Gunning BES. 1978. Age-related and origin-related control of the number of plasmodesmata in cell walls of developing Azolla roots. Planta 143 : 181–190. Larson PR. 1994. Rays. In : Larson PR, ed. The ascular cambium : deelopment and structure. Berlin : Springer-Verlag, 367–391. O’Brien TP. 1970. Further observations on hydrolysis of the cell wall in the xylem. Protoplasma 69 : 1–14. Panshin AJ, de Zeeuw C. 1980. The minute structure of hardwoods (porous woods). In : Panshin AJ, de Zeeuw C, eds. Textbook of wood technology. 4th edn. New York : McGraw-Hill. Sano Y, Nakada R. 1998. Time course of the secondary deposition of incrusting materials on bordered pit membranes in Cryptomeria japonica. IAWA Journal 19 : 285–299. Sauter JJ, Kloth S. 1986. Plasmodesmatal frequency and radial translocation rates in ray cells of poplar (Populusicanadensis Moench ‘ robusta ’). Planta 168 : 377–380. Schmid R. 1965. The fine structure of pits in hardwoods. In : Co# te! WA Jr, ed. Cellular ultrastructure of woody plants. New York : Syracuse University Press, 291–304.
The Differentiation of Contact Cells and Isolation Cells in the Xylem Ray Parenchyma of Populus maximowiczii Y. M U R A K A M I, R. F U N A DA*, Y. S A N O and J. O H T A N I Department of Forest Science, Faculty of Agriculture, Hokkaido Uniersity, Kita 9, Nishi 9, Kita-ku, Sapporo 060-8589, Japan Received : 24 March 1999
Returned for revision : 27 April 1999
Accepted : 11 June 1999
A histochemical analysis was made of the differentiation of contact cells and isolation cells in the xylem ray parenchyma of Populus maximowiczii. The contact cells formed secondary walls at approximately the same time as adjoining vessel elements. The lignification of the cell walls of contact cells and vessel elements began earlier than that of wood fibres and isolation cells. Thus, the formation of the secondary wall, including lignification, of the contact cells might occur at the same time as that of the vessel elements to which they are directly connected. By contrast, the isolation cells began to form secondary walls later than the vessel elements and wood fibres in the vicinity of the isolation cells. After the deposition of the secondary wall, a protective layer was formed in contact cells but no isotropic layer was observed in isolation cells. The results suggest the importance of vessel elements in the determination of the differentiation of adjoining ray parenchyma cells. # 1999 Annals of Botany Company Key words : Contact cell, isolation cell, vessel element, xylem differentiation, Populus maximowiczii Henry.
INTRODUCTION Ray parenchyma cells play an important role in the storage, conductive and secretory systems of woody plants. Mostly, they maintain radial continuity between the phloem and xylem, thereby facilitating the radial and lateral transport of materials (Larson, 1994). The ray parenchyma cells of xylem are of two types, namely contact cells and isolation cells, and they differ with respect to their contacts, through pits, with vessel elements. Contact cells are ray cells that make direct connections with adjacent vessel elements through half-bordered pit pairs, and the isolation cells are ray cells that make no pitmediated direct connections with adjacent vessel elements (Braun, 1967). Sauter and Kloth (1986) proposed that isolation cells might be more specialized for radial translocation than contact cells. By contrast, tyloses develop from contact cells and not from isolation cells. Therefore, contact cells might function to protect living ray parenchyma cells from hydrolases released by dead vessel elements (Schmid, 1965 ; O’Brien, 1970). In contrast, Chafe (1974) proposed that contact cells might function as ‘ transfer cells ’ because both types of cell have a similar structure. It appears that contact cells and isolation cells differ in terms of both form and function, even though both types of cell are derived from the same cambial ray cells (Braun, 1970). Thus, it seems possible that these two types of cell might be formed via different processes of differentiation from cambial ray cells.
* For correspondence. Fax j81 117361791, e-mail funada!for. agr.hokudai.ac.jp
0305-7364\99\100429j07 $30.00\0
Ray parenchyma cells generally form an additional layer on the inner side of the secondary wall (Schmid, 1965). This layer contains high levels of polysaccharides (Czaninski, 1973). Chafe (1974) reported that, in Populus tremuloides, such an additional layer in contact cells was unlignified during the first growing season (this layer was referred to as the protective layer), while that of isolation cells was lignified within one growing season (this layer was referred to as the isotropic layer). The protective layer was finally lignified when the formation of heartwood began. Using histochemical techniques, Fujii, Harada and Saiki (1981) found that there was little difference between the protective layer and the isotropic layer in terms of their ultrastructure and chemical compositions prior to lignification. Thus, they concluded that these two layers might have the same origin and that lignification might begin at different times in the two layers. These observations indicate that the formation of the secondary wall, including lignification, differs between the two types of ray parenchyma cell. Such differences might be dependent on the disposition of ray parenchyma cells with respect to the vessel elements. The purpose of the present study was to monitor the differentiation of contact cells and isolation cells in Populus maximowiczii by histochemical staining. The rays of Populus have a simple composition, being homogeneous and uniseriate (Panshin and de Zeeuw, 1980). Therefore, it is easy to determine, by observation, whether or not ray parenchyma cells make direct contact with adjacent vessel elements. We postulated that the deposition and lignification of cell walls of the two types of ray parenchyma cell would provide a suitable model for clarification of the mechanism of differentiation of secondary xylem cells derived from cambial cells. # 1999 Annals of Botany Company
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Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus
F. 1. A differentiating vessel element (V) and other elements around it, at a distance of approximately 500 µm from the cambium, in radial section. Arrows indicate transverse walls of contact cells with birefringence. Arrowheads indicate ray-vessel pits of contact cells. No birefringence was visible in cell walls of isolation cells (asterisks). The left side of each micrograph corresponds to the outer side of the tree. Light micrograph (A) and polarizing light micrograph (B). Bar l 50 µm.
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F. 2. Electron micrographs showing tangential sections of differentiating ray parenchyma cells at a similar stage to that shown in Fig. 1. Contact cell with a secondary wall (A) and an isolation cell without a secondary wall (B). V, Vessel element ; F, wood fibre ; CC, contact cell ; IC, isolation cell. Bars l 2 µm.
MATERIALS AND METHODS Samples from a straight, healthy tree (approx. 30 years old) Populus maximowiczii Henry (height, 18 m ; diameter at breast height, 30 cm) growing in the Tomakomai Experimental Forest of Hokkaido University, were used. Small blocks containing phloem, cambial zone and differentiating xylem were taken from the stem at breast height in July 1995, when cambial activity was very high. At that time, the successive stages of differentiation of ray parenchyma cells could be observed within a single radial section. All sample blocks were immediately placed in a 4 % solution of glutaraldehyde in 0n1 phosphate buffer (pH 7n2) and fixed overnight at room temperature. After washing with the same buffer, some blocks were postfixed in 1 % osmium tetroxide in the same buffer for 2 h at room temperature. Other sample blocks for ultraviolet microscopy were not postfixed in 1 % osmium tetroxide. Samples were then washed with buffer and dehydrated through a graded ethanol series. They were then embedded in epoxy resin.
For standard light microscopy and polarizing light microscopy, radial and tangential sections (1 µm thick) were cut with a glass knife on an ultramicrotome (Ultracut J ; Reichert, Austria). Sections were stained with a 1 % aqueous solution of safranin or a 1 % aqueous solution of gentian violet. They were observed with a light microscope (BHS-2 ; Olympus, Japan) and a polarizing microscope (BHS-751P ; Olympus). For determination of the extent of lignification, radial and tangential sections (1 µm thick) were cut with a glass knife, placed on quartz slides and mounted in glycerine with quartz coverslips. Photographs were taken under an ultraviolet (UV) microscope (MPM-800 ; Carl Zeiss, Germany) at a wavelength of 280 nm with a bandwidth of 5 nm (Fukazawa, 1992 ; Sano and Nakada, 1998). For transmission electron microscopy, ultra-thin tangential sections (70 nm thick) were cut with a diamond knife and stained with uranyl acetate and lead citrate. The ultrathin sections were then observed with a transmission electron microscope (JEM-100C ; JEOL, Japan) operated at an accelerating voltage of 100 kV.
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Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus RESULTS
Figure 1 shows a differentiating vessel element, which was located approx. 500 µm from the cambium, and the other elements around it. In this region, the birefringence of the cell walls of the axial xylem cells was detectable with a polarizing light microscope (Fig. 1 B). This birefringence indicated that the deposition of secondary walls had started in these cells (Abe et al., 1997). Ray parenchyma cells, which were oriented transversely, were also observed. The parenchyma cells with ray-vessel pits (arrowheads in Fig. 1 A and B) were considered to be contact cells. The birefringence of transverse walls of contact cells was clearly visible (arrows in Fig. 1 B). Several thin-walled ray parenchyma cells (asterisks in Fig. 1 A and B) were observed between contact cells. These cells were considered to be isolation cells because they had no ray-vessel pits. Serial, radial, semithin sections revealed that isolation cells were longer than contact cells and that there were many intercellular spaces between isolation cells (data not shown). The walls of isolation cells were not birefringent under the polarizing light microscope, and indication that the deposition of secondary walls had not yet started in these cells. Figure 2 shows the ultrastructure of cell walls in differentiating ray parenchyma cells in tangential section of a sample at a similar stage to that shown in Fig. 1. The contact cells formed ray-vessel pits (Fig. 2 A). The thick secondary walls of contact cells and vessel elements had already formed. The contact cells were present at the upper and lower margins of the ray. By contrast, the isolation cells had thin cell walls (Fig. 2 B), and the thickness was similar to that of the cell walls of cambial cells. The UV absorption of cell walls at 280 nm was apparent on radial sections of a region similar to that shown in Fig. 1.
F. 4. UV photomicrograph showing a tangential section of a differentiating vessel element and other elements around it at a similar stage to that shown in Fig. 3. Arrows indicate cell walls of contact cells and vessel elements that absorbed UV light. Arrowheads indicate cell walls of isolation cells (asterisks) that did not absorb UV light. V, Vessel element ; F, wood fibre. Bar l 10 µm.
F. 3. UV photomicrograph showing a radial section of a differentiating vessel element (V) and other elements around it at a similar stage to that shown in Fig. 1. UV absorption of cell walls is visible in vessel elements (large arrows) and contact cells (small arrows). The cell walls of wood fibres absorbed UV light very weakly (arrowheads). The left side of this micrograph corresponds to the outer side of the tree. Bar l 50 µm.
The UV absorption of cell walls was visible first in vessel elements (large arrows in Fig. 3). This absorption indicated that the lignification of these cell walls had begun. UV absorption by the secondary walls of contact cells was as strong as that by cell walls of vessel elements (small arrows in Fig. 3). By contrast, the cell walls of wood fibres absorbed UV light only very weakly (arrowheads in Fig. 3). Figure 4 shows differentiating axial elements and ray parenchyma cells at a similar stage to that shown in Fig. 3 on a tangential section. The UV absorption of cell walls of vessel elements and contact cells was stronger than that of wood fibres in the vicinity of vessel elements (arrows in Fig. 4). Strong UV absorption was observed at the middle lamella in contact cells, but no UV absorption was detected
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F. 5. Electron micrographs showing tangential sections of differentiating ray parenchyma cells at distances from the cambium that ranged from approx. 1500–2000 µm. Contact cell with a protective layer (arrows) (A) and an isolation cell without an isotropic layer (B). V, Vessel element ; CC, contact cell ; IC, isolation cell. Bars l 2 µm.
at pit membranes of ray-vessel pits. Moreover, the cell walls (arrowheads in Fig. 4) of isolation cells (asterisks in Fig. 4) did not absorb UV light. Various terms have been used for the additional layers of parenchyma cells. Schmid (1965) names these layers the ‘ protective layer ’. Later Chafe (1974) and Chafe and Chauret (1974) used the term ‘ protective layer ’ in the case of vessel-associated ray parenchyma cells and the term ‘ isotropic layer ’ in the case of axial parenchyma cells and non-vessel-associated ray parenchyma cells. By contrast, Fujii, Harada and Saiki (1980, 1981) proposed both layers be called the ‘ amorphous layer ’ because there appeared to be little difference between them in their original chemical composition and fine structure. In this report, we use separate designations for the protective layer and the isotropic layer because these layers appear to have different
functions. Figure 5 shows ray parenchyma cells at distances from the cambium that range from approx. 1500–2000 µm. The protective layer was formed (arrows in Fig. 5 A) in the contact cells after the deposition of the secondary wall. In agreement with the observations reported by Chafe (1974), this layer was not limited only to the side where vessel elements were located, but was also present on the opposite side. However, the protective layer on the opposite side was much thinner than that on the side on which vessel elements were located. Czaninski (1973) and Chafe (1974) reported that the deposition of the protective layer began at the site of secondary wall thickening. Figure 6 shows ray parenchyma cells at a similar stage to that shown in Fig. 5 in tangential section. The protective layer of contact cells (arrows in Fig. 6) and pit membranes of ray-vessel pits (arrowheads in Fig. 6) did not absorb UV light. The
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F. 6. UV photomicrograph showing a tangential section of differentiating ray parenchyma cells at a similar stage to that shown in Fig. 5. No UV absorption by the protective layer (arrows) and pit membranes of ray-vessel pits (arrowheads) is evident in the contact cells. UV absorption of cell walls is evident in the isolation cells (asterisks). V, Vessel element ; F, wood fibre. Bar l 10 µm.
secondary walls of isolation cells absorbed UV light (asterisks in Fig. 6), but no isotropic layer was apparent in isolation cells (Fig. 5 B). DISCUSSION The cell walls of isolation cells in the regions shown in Figs 1 and 2 were primary walls because they were thin and exhibited no birefringence under polarized light. By contrast, contact cells formed secondary walls at approximately the same time as vessel elements. Chafe (1974) observed that the initiation of the deposition of the secondary wall in vessel elements occurred slightly earlier than in contact cells. At this stage, in our samples, UV absorption of cell walls of vessel elements and contact cells appeared to be stronger
than that of the wood fibres in the vicinity of vessel elements (Figs 3 and 4). By contrast, no UV absorption of cell walls of isolation cells was detected (arrowheads in Fig. 4). These results indicated that the lignification of the cell walls of contact cells and vessel elements began earlier than that of wood fibres and isolation cells. Thus, the timing of the formation of the secondary walls, including lignification, of the contact cells was similar to that of the vessel elements with which they were directly connected. The similarity in timing of differentiation between contact cells and vessel elements suggests that differentiation of contact cells might be influenced by vessel elements through ray-vessel pits. By contrast, the differentiation of isolation cells, which make no direct pit-mediated connections with adjacent vessel elements, might not be directly affected by the differentiation of vessel elements. Our results indicate that contact cells and vessel elements began to form lignified secondary walls earlier than the wood fibres in the vicinity of the vessel elements. By contrast, the pit membranes of ray-vessel pits and the protective layers in contact cells were not lignified soon after deposition of secondary walls (Fig. 6). Barnett, Cooper and Bonner (1993) suggested that maintenance of an apoplastic pathway around a lignified cell with living protoplast was the primary function of the protective layer. The contact cells might specialize in the translocation of water and materials between ray and vessel. In contrast, the differentiating isolation cells with unlignified tangential walls might become pathways for temporary radial continuity between the phloem and xylem. In this way, easy radial translocation is maintained through the rows of isolation cells. We observed no isotropic layer in isolation cells after deposition of secondary walls (Fig. 5 B). Chafe (1974) reported that isolation cells of Populus tremuloides formed secondary walls and isotropic layers, all of which became lignified, within a single growth season. By contrast, Fujii, Harada and Saiki (1979) reported that the cell wall in differentiated isolation cells of Populus maximowiczii consisted of only a cellulosic secondary wall. The reason for the discrepancy among species that belong to the same genus is that the isotropic layer might be only partially deposited in the isolation cells and, thus, the occurrence of an isotropic layer in ultra-thin sections would depend on the place in which the isolation cells were sectioned. In addition, there is the possibility that isolation cells had not yet begun to form the isotropic layer in the present study because our specimens were collected in July. Chafe (1974) reported that deposition of the protective layer began after completion of the deposition of the cellulosic secondary wall. However, the isotropic layer might not be fully developed soon after completion of the deposition of the cellulosic secondary wall in isolation cells. Our results indicate that the structure of the ray parenchyma cells of the secondary xylem differs considerably between contact cells and isolation cells, which are derived from the same cambial ray cells. Therefore, these ray parenchyma cells are formed by different processes of differentiation, depending on whether or not they make direct connections, through pits, with adjoining vessel elements. Our observations indicate the importance of
Murakami et al.—Differentiation of Ray Parenchyma Cells of Populus neighbouring cells in controlling xylem differentiation. Expanding vessels might send some signals to adjoining cells through the plasma membrane and primary wall. These signals determine the direction of differentiation of ray parenchyma cells. Although the nature of these putative signals remains to be characterized, some specific proteins or polysaccharides of the cell wall, which trigger cell differentiation, might be secreted and reach future contact cells from differentiating vessel elements. In contrast, future isolation cells might not receive such signals. Therefore, future contact cells and isolation cells might differ originally in both structure and the nature of the primary wall and plasma membrane. They might have different numbers and distributions of plasmodesmata with different structures since it has been proposed that plasmodesmata play an important role in the fate of xylem cells (see Gunning, 1978 ; Barnett, 1981, 1982). More detailed information about plasmodesmata in cambial ray cells is needed to clarify the mechanisms of differentiation of xylem ray cells. Cambial cells and their derivatives have been proposed to serve as a model system for studies of cytodifferentiation in secondary tissues in situ because it is possible to follow the process of differentiation in a radial direction (Abe et al., 1995 ; Chaffey, Barlow and Barnett, 1997 ; Chaffey, Barnett and Barlow, 1997 ; Funada et al., 1997). The successive differentiation of contact cells and isolation cells, both of which are derived from cambial ray cells, is also a useful model system for studies of the mechanism of control of the formation and lignification of secondary walls in secondary xylem. A C K N O W L E D G E M E N TS This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (Nos 08760157 and 09760152) and from the Japan Society for the Promotion of Science (no. JSPSRFTF 96L00605). LITERATURE CITED Abe H, Funada R, Imaizumi H, Ohtani J, Fukazawa K. 1995. Dynamic changes in the arrangement of cortical microtubules in conifer tracheids during differentiation. Planta 197 : 418–421. Abe H, Funada R, Ohtani J, Fukazawa K. 1997. Changes in the arrangement of cellulose microfibrils associated with the cessation of cell expansion in tracheids. Trees 11 : 328–332. Barnett JR. 1981. Secondary xylem cell development. In : Barnett JR, ed. Xylem cell deelopment. Tunbridge Wells, Kent : Castle House Publications, 47–95. Barnett JR. 1982. Plasmodesmata and pit development in secondary xylem elements. Planta 155 : 251–260.
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