ANALYSIS of CAMBIUM and DIFFERENTIATING VESSEL ELEMENTS in KALOPANAX PICTUS USING RESIN CAST REPLICAS By

IAWA Journal, Vol. 22 (1), 2001: 15–28

ANALYSIS OF CAMBIUM AND DIFFERENTIATING VESSEL ELEMENTS IN KALOPANAX PICTUS USING RESIN CAST REPLICAS by

Peter Kitin1, 2, Yuzou Sano1 & Ryo Funada1*

SUMMARY A resin-casting method with subsequent scanning electron microscopy (SEM) was used to examine the three-dimensional (3-D) shapes of cells and the cell walls of cambium and differentiating xylem. Glutaralde- hyde-fixed and dehydrated specimens were embedded in polystyrene and then organic material was removed by digestion with acidic solutions or enzymes. The acidic solutions used for treatment were sulphuric acid and a mixture of acetic acid and hydrogen peroxide and the enzymes used for treatment were pectinase and cellulase, with a final treatment with sodium hypochlorite. Both methods could be used for studies of the differentiation of cambial cells; however, digestion with enzymes allowed better preservation of the 3-D organisation of the tissue. Negative replicas of inner surfaces of cell walls of differentiating vessel elements revealed the sequential stages of the development of bordered pits and perforation plates. Future bordered pits at the early stages of the differentiation of cell walls were demarcated by the accumulation of organic material between adjacent pit membranes. Subse- quent deposition of cell wall material resulted in formation of pit cavities and the rims of perforation plates. Key words: Cambium, differentiating vessel elements, Kalopanax pictus, pit formation, resin casting.

INTRODUCTION

Differentiating vessel elements are turgid cells with soft walls and large vacuoles. If a specimen is not embedded in resin, the cells often break or shrink during sectioning. In addition, it is difficult to observe cambial cells and differentiating xylem elements from one end to the other in a single histological section by conventional light microscopy because both ends might not appear on the same section or in the same focal plane (Kitin et al. 1999).

1) Department of Forest Science, Faculty of Agriculture, Hokkaido University, Sapporo 060- 8589, Japan. 2) Department of Dendrology, University of Forestry, Kliment Ohridski str. 10, Sofia 1756, Bulgaria. *) Corresponding author. E-mail: [email protected]

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Data on the three-dimensional (3-D) shapes of cambial and differentiating xylem cells in dicotyledonous species have been obtained from analyses of serial thin sections of resin-embedded specimens (Fujita et al. 1984; Fujita 1993). Preparation of such sections is, however, time-consuming and impractical for studies of the dimensions and shapes of large numbers of cells. In addition, due to the uneven thickness of sections and to errors during the manual ordering and stacking of serial images, mis- takes in the reconstruction of the 3-D images are easily made. The 3-D shapes and the lengths of fusiform cambial cells in Kalopanax pictus have been studied by confocal laser scanning microscopy (CLSM) with reconstruction from serial images of optical sections (Kitin et al. 1999, 2000). However, the resolution of CLSM is limited and is far below the level achieved by electron microscopy. Therefore, it is not possible to examine the details of micromorphological changes in differentiating cells, such as the changes in the minute structures associated with sculpturing of the cell wall. The resin-casting method allows the 3-D shapes of matured xylem cells and their spatial arrangement to be clearly reproduced as negative replicas (Fujii 1993; Mauseth & Fujii 1994; André 1995, 1998; Fujii & Hatano 1996, 2000; André et al. 1999; Kitin et al. 1999). Low-viscosity resins can penetrate the minute apoplastic spaces in plant tissues. Casts have high fidelity and, thus, negative replicas of the cell wall structures, such as pits, perforation plates and warts, can be studied in detail with the scanning electron microscope (Stieber 1981; Fujii 1993; Mauseth & Fujii 1994; André et al. 1999; Xu & Liese 1999). The potential of this method for plant anatomical studies seems to be considerable: it has been reported that not only xylem elements but all cell types can be infiltrated with styrene (Mauseth & Fujii 1994). However, preparation of resin cast replicas of differentiating tissues, which might provide important information for studies of developmental anatomy, has not yet been reported, to our knowledge. Kalopanax pictus (Araliaceae) is a ring-porous hardwood that grows in the tem- perate forests of East Asia. In this species, uniseriate, large-diameter earlywood vessels contrast strongly in terms of size with the small-diameter vessels and vascular tracheids of the latewood (Ohtani 2000). The process of differentiation of the secondary xylem in Kalopanax pictus has been studied by light microscopy (Imagawa & Ishida 1972; Kitin et al. 1999). We examined the cambial and differentiating xylem cells using a resin-casting method with scanning electron microscope in an attempt to analyse their shapes, as well as cell wall structures, such as pits and perforation partitions, during sequential stages of differentiation.

MATERIAL AND METHODS Plant material A single specimen of Kalopanax pictus (diameter at breast height, 76 cm), grow- ing on the campus of Hokkaido University, was used for all experiments. Samples, including cambium and adjacent phloem and xylem, were taken by chisel from the stem at breast height during the periods of cambial reactivation (April 3) and cambial activity (June 5). Radial slivers (of approximately 20 mm, 10 mm and 3 mm in the axial, radial and tangential directions, respectively) were cut from the sample blocks

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and immediately fixed in a 4% solution of glutaraldehyde. The solution was degassed briefly under a vacuum and then samples were left overnight in the fixative.

Embedding in resin Glutaraldehyde-fixed slivers were trimmed into small blocks (of approximately 5 mm, 5 mm and 3 mm in the axial, radial and tangential directions, respectively) and dehydrated through a graded ethanol series. The resin was prepared by mixing styrene with benzoyl peroxide (99 : 1, v/v). Then the tissue blocks were infiltrated by immer- sion in increasing concentrations of the resin in ethanol (25%, 50%, 75%) for one hour per solution. Finally, they were incubated in 100% resin three times for one hour each. The specimens were then inserted into gelatine capsules filled with resin and baked in an oven at 60°C for three days for polymerisation of the resin.

Removal of cell walls from embedded tissue Embedded tissues were exposed on the surface of individual resin blocks either by polishing or by splitting the block. Then the organic material was digested by alter- nate treatments with 95% sulphuric acid and a mixture of equal volumes of hydrogen peroxide and glacial acetic acid, as described by Fujii (1993) and Mauseth and Fujii (1994). Alternatively, digestion was performed with pectinase (pectinase from mould, 0.01 U mg -1; Fluka, Buchs, Switzerland), and then with cellulase (ʻOnouzukaʼ RS; Yakult Co. Ltd., Tokyo, Japan) and sodium hypochlorite. Samples were immersed in 2% pectinase in phosphate buffer (0.1 M, pH 6.5), then in 2% cellulase in phosphate buffer (0.1 M, pH 5.8) and, finally, in sodium hypochlorite solution (available chlo- rine, min. 5%). Treatment was continued for 72 h at 35 °C in each reagent. Solutions were renewed at 24 h-intervals. The surfaces of specimens were cleaned after digestion either by agitation of specimens in water or with sticky tape.

Preparation of samples and observations The casts were rinsed in distilled water. Then they were dehydrated by passage through a graded ethanol series and drying in an oven at 35 °C for 1–2 h. The blocks were mounted on specimen stubs and coated with carbon and gold by vacuum evapo- ration. They were observed with a field-emission scanning electron microscope (JSM- 6301F; Jeol Co. Ltd., Tokyo, Japan) at an accelerating voltage of 2 to 3 kV (Sano et al. 1999).

RESULTS AND DISCUSSION Infiltration of tissues with styrene The extent of infiltration and the stability of the resin are both factors of crucial importance for successful resin casting. We selected polystyrene for our experiments because Taneda et al. (1979) and Fujii (1993) showed that polystyrene casts were stable against the chemical reagents used for digestion of organic materials. Styrene was able to penetrate pit membranes of cells and was successfully used to produce polystyrene casts of various types of xylem and cortical cells by Mauseth and Fujii (1994).

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Fig. 1 — For legends, see page 20

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Fig. 2 — For legends, see page 20

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Our observations showed that styrene had fully infiltrated the lumens of cambial cells and differentiating cambial derivatives (Fig. 1A & 1B). By contrast, styrene did not infiltrate the cell walls. Thus, after digestion of cell walls, there were empty spaces between the cell casts. As observed in other studies (Mauseth & Fujii 1994; André et al. 1999; Kitin et al. 1999), styrene fully infiltrated the lumens of differentiated xylem elements (Fig. 1C). Minute cavities in cell walls, such as pits, were filled with resin and the casts replicated cell wall structures with high fidelity. No styrene infiltrated the crystals (Fig. 1A, arrow) or starch grains (data not shown).

Exposure of surfaces of specimen The method used to expose embedded tissues, namely, polishing or splitting, influ- enced the quality of casts. Polished samples often had rough surfaces that were due to eroded casts (Fig. 2 & 3A). The eroded casts on the polished surfaces of the samples were partially removed with sticky tape or by agitation of samples in water. These procedures revealed small areas with preserved casts, usually located beneath the polished plane, which were appropriate for observation (Fig. 3). Exposure of larger areas for observation by agitation in water or removal with tape of eroded casts from the surfaces resulted in deformation of samples. Mauseth and Fujii (1994) showed that individual casts of cells that were not con- nected via perforations were normally separated from casts of adjacent cells after complete digestion of cell walls. Thus, the 3-D organisation of the tissue was easily destroyed. In our samples, when cell casts had been entirely exposed on the surface of a specimen, they were easily displaced during washing and it was difficult to obtain radial files of differentiating cells for analysis. For example, as seen in Figure 4A, the

←← Fig. 1. Polystyrene casts showing cambium and different stages of differentiation of cambial derivatives. – A: Radially split specimen obtained on April 3 at the time of cambial reactivation. Enzymatic digestion of organic material. Cambium (ca) and the adjacent phloem (ph) and xylem (xy) are seen with cells in their original arrangement. In some phloem cells, cavities, which are traces of crystals (arrow), can be seen. Casts of intercellular spaces between the ray phloem cells are also visible. Some of the phloem ray cells have been obliquely split. There are wide spaces at sites of digested thick cell walls between the casts of the xylem cells. Replicas of pit cavities of the xylem cell walls are visible. – B: Radial surface of a specimen of cambium obtained on June 5 at a time when the cambium was active. Part of the cambium (on the left of the micrograph) and differentiating vessel elements arranged in a radial file can be seen. The sequential numbering of casts of vessel elements (1, 2 and 3) indicates the progression of cell differentiation. – C: Casts of differentiated xylem elements from the previous annual ring. The tail of an earlywood vessel element and sculpturing of cell walls that indicates bordered pits are visible. Spaces between casts of cells indicate thick secondary walls. — Scale bars = 50 μm. ← Fig. 2. Radial face, exposed by polishing, of a specimen obtained on June 5. Digestion with sulphuric acid and a mixture of glacial acetic acid and hydrogen peroxide. Polished casts of cambium (in the centre) and adjacent differentiating tissues are visible. The left side of the micrograph corresponds to the phloem side. — Scale bar = 50 μm.

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Fig. 3. Radial face, exposed by polishing, of a specimen obtained on June 5. – A: General view of the sample, showing the cambium. Differentiating phloem is visible on the left of the micrograph. The surface of the sample was not clean because many of the casts had been eroded during polishing. Arrows show areas with undamaged casts, exposed after cleaning the surface of the specimen, that allow examination of the shapes and spatial arrangements of cells. The exposed face of the specimen is slightly oblique. Thus, only one of the two tips of the long fusiform cambial cells is visible. Scale bar = 50 μm. – B: Detail of A showing casts of differentiating cells on the xylem side. The upper ends of the axially oriented cells are clearly visible. Scale bar = 25 μm. – C: Another detail of A. Fusiform and ray cambial cells. The tips of two fusiform cells can be seen and, next to them, the narrow ray cambial cells that had recently divided. The smooth surface of the casts indicates the absence of sculpturing of cell walls. The narrow spaces between the casts indicate thin cell walls. Casts of intercellular spaces between ray cambial cells are visible. Scale bar = 25 μm.

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Fig. 4. Radial view of differentiating latewood, exposed by polishing of a polystyrene-embedded specimen. The sample was obtained on June 5. Cambium is on the left side of each micrograph. – A: A latewood vessel element at an early stage of differentiation is visible on the left. The upper tip of the cell cast has been preserved but the lower tip was broken. The position of a future perforation plate (asterisk) is indicated. Beneath this cell and on the right of the micrograph there are vessel ele-ments at later stages of differentiation. – B: Casts of differentiating vessel elements adjacent to the cambium. The tip of the cell cast on the left is visible but the tip of the cell cast on the right is not. The position and shape of a future perforation plate (asterisk) are visible. Sequential stages of the differentiation of cell walls are indicated by the increasing depths of bordered pit cavities. – C: Casts of differentiating latewood vessel elements that are arranged in radial files. Perforations had not yet developed. So, the casts of the cells are separated from one another. — Scale bars = 50 μm.

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cast of a single differentiating vessel element remained on the surface of the specimen but the casts of adjacent cells were missing from the surface. The polished faces of samples were not precisely oriented along cell axes but were slightly oblique to these axes and, thus, only one tip of each cell cast was visible at the surface (Fig. 3A). A disadvantage of such obliquely exposed faces was that the cells could not be observed in their entirety from one tip to the other. However, such samples were stable to the chemical treatment and the cell casts retained their original arrangement in radial files (Fig. 3, 4B & C).

Methods for digestion of organic materials The protocol for digestion of organic materials is an important factor in the suc- cess of the final observations. Treatment with strong chemical reagents is necessary for the rapid and complete extraction of organic materials. By contrast, for better preservation of the fine structures of casts and their spatial arrangement, mild conditions for the extraction of organic materials seem more reasonable. The method for digestion with sulphuric acid and then a mixture of hydrogen peroxide and glacial acetic acid was relatively rapid. However, it was not easy to control the conditions for appropriate treatment with these reagents. The extent of extraction of the cell wall and the preservation of casts depended not only on the duration of treatment but also on other factors, such as the size of the exposed area of the sample and its orientation along the cell axes. When the area of exposed tissue was small, samples were better preserved after the digestion of organic materials. The major problem associated with the treatment with sulphuric acid was deformation by swelling of the embedded tissue. This problem was reported by Fujii (1993) in samples with ʻlow packing densityʼ, in which swelling of cell walls during chemical treatment appeared to be responsible for the deformation. Swelling of cell walls that pushed neighbouring casts apart was also reported by Mauseth and Fujii (1994). They suggested that, if precise cell-to-cell orientation is required, sulphuric acid should be replaced by another chemical reagent that would cause less extensive swelling of cell walls and less of an exothermic reaction during treatment. In our experiments, treatment with sulphuric acid caused the unequal swelling of the cells in the cambium, phloem and xylem, with subsequent shrinking during drying, which caused cracks in the resin and deformation of the casts. By contrast, no swelling of samples was apparent after digestion with enzymes. The samples were less deformed than those prepared with strong acids (Fig. 1A). The casts obtained allowed the visualisation and study of larger areas of cambium and the original arrangements of cells were preserved (Fig. 1A). Enzymatic digestion required more time than digestion with acid solutions. However, this drawback might be outweighed by the better preservation of cell casts.

Morphology and arrangement of cells in resin casts Figure 1A shows a cast of radially split tissue at and around the cambium. The border between cambium and xylem was clearly evident from the abrupt changes in the shapes of cell casts and in the widths of spaces between adjacent cell casts.

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The microcasts revealed the tips and 3-D shapes of both cambial and differentiating cells, which are difficult to observe on single histological sections under the light microscope (Fig. 3B, C, 4 & 5A). Although entire cells can be observed in macerated tissues (Kitin et al. 1999), cambial and differentiating xylem cells in such tissues are frequently broken or reduced in volume during maceration because of their fragile walls. Infiltration of resin into lumens can protect soft cells from shrinkage (Fig. 3B, C & 4A). Sequential stages of differentiation of cell walls were easily visualised with gradual enlargement of the spaces between adjacent cell casts and gradual increases in the depths of pit cavities. The progression of the differentiation of cells from the cambium towards the xylem was evident in casts of cells arranged in radial files (Fig. 1B & 4). The cast designated no. 1 in Figure 1B, which was close to the cambium, was the cast of a vessel element at an early stage of differentiation that had a predominantly smooth wall. In the cast of the next cell, designated no. 2 in the same Figure, it was clear that the positions of pits had been already determined and demarcation of the pit membranes was apparent (a cell at a similar stage of differentiation is shown in Fig. 5A). The cast designated no. 3 replicated the adjacent vessel element in this radial file, in which the formation of pit cavities had progressed still further. Casts of cells with pits at consecutive stages of formation are also seen in Figures 4B & C and 5. A cast of a latewood vessel element at an early stage of differentiation is shown in Figure 4A (cast with asterisk). The cast had a wavy rather than a rounded side wall. Studies by transmission electron microscopy (TEM) and by light microscopy have suggested that the formation of bordered pits begins early in the ontogenesis of xylem cells, namely, during formation of the primary wall (Frey-Wyssling et al. 1956; Bauch et al. 1968; Fengel 1972; Barnett 1981; Uehara & Hogetsu 1993; Abe et al. 1995; Fu- nada et al. 1997; Chaffey et al. 1997, 1999). No clear evidence of pit formation was visible in the vessel element shown in Figure 4A (cast with asterisk). Therefore, this vessel element might possibly have been in the process of expanding. Figures 4B and 4C show differentiating vessel elements at sequential stages of development of the cell wall. By contrast to the vessel element in Figure 4A, the side walls of these cells were more or less rounded and replicas of pit cavities at different stages of differentiation could be seen. Regions at which pits are forming in the primary walls of developing xylem elements can be recognised from the accumulation of cell wall materials at the periphery of the pit membranes (Imamura & Harada 1973; Barnett & Harris 1975; Barnett 1981; Hirakawa & Ishida 1981; Chaffey et al. 1997). Negative replicas of accumulated or-

Fig. 5. Details of casts of vessel elements. – A: The tip of a differentiating vessel element and sculpturing of the cell wall (enlarged portion of Fig. 4B). The bordered pits are at the initial stages of formation. Arrows indicate traces of organic material that had accumulated between adjacent pit membranes of future pits. – B: Pit cavities that had differentiated further than those in Figure 3A. – C: Details of casts of differentiated xylem elements from the previous annual ring. Spaces between adjacent casts indicate thick secondary walls. Replicas of differentiated bordered pits are visible. The faces of the casts of pits replicate the pit membranes. Long pit canals are evident in casts visualised from the side. — Scale bars = 10 μm.

Downloaded from Brill.com10/05/2021 07:44:30AM via free access 26 IAWA Journal, Vol. 22 (1), 2001 Kitin, Sano & Funada — Resin casting of differentiating vessel elements 27 ganic material, which delineated the sites of pits, could also be detected in our resin casts (Fig. 4B & 5A), confirming the results of the studies mentioned above. In addition, it was evident that, initially, the accumulation of cell wall material did not occur uniformly around the periphery of pit membranes. Such material was deposited predominantly in the narrow spaces between adjacent pit membranes (Fig. 5A, arrows). As the differentiation of vessel elements proceeded, the casts of pits became more distinct with deeper pit cavities and well-defined borders. The cast shown in Figure 5B revealed a vessel element with a still thin cell wall, as suggested by the narrow spaces between adjacent casts. However, pit differentiation had clearly progressed since the pit cavities were slightly deeper and the pit membranes were better delineated than those of the cell shown in Figure 5A. In differentiated bordered pits, the cell walls were thick and the pit borders and pit canals were fully formed (Fig. 5C). Progression of the differentiation of perforation plates was also clearly revealed. The perforation partition in the cell cast in Figure 4A (asterisk) was less differentiated than that in the cell cast in Figure 4B (asterisk). Deposition of cell wall material led to the formation of a rim at the periphery of the perforation partition (Fig. 4B). When membranes at the perforation partitions of cells were present, the casts of individual vessel elements were separated from one another (Fig. 4). Then the tips of cells could be seen and it was apparent that tips of several adjacent vessel elements in a radial file were positioned at a similar horizontal level. It was also evident that the end walls of differentiating latewood vessel elements were oblique (Fig. 4). By contrast, in mature vessel elements, which were perforated, the resin formed continuous casts and it was difficult to distinguish the end walls of cells (Fig. 1C). Our results show that polystyrene casting has high potential utility for the visualisation and study of apoplastic spaces in cambium and differentiating vascular tissues. The shapes of cells were relatively well preserved but the possibility of some deformation during the fixation of tissue and embedding in resin cannot be excluded. The fine structure of the casts could be examined by SEM. Thus, such resin-casting might be particularly useful for studies of the minute details of the structures inside cell walls, such as the structure of pit cavities and the development of perforation plates during cell differentiation. The ability to visualise cell tips and the microstructures of cell walls in three dimensions provides much additional information that supplements the results obtained by light microscopy and TEM, which have previously been used in studies of the developmental anatomy of woody plants.

ACKNOWLEDGEMENTS

The authors thank Dr. J. Ohtani, Dr. T. Fujii and Dr. Y. Uraki for their valuable comments. This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (no. JSPS–RFTF 96L00605).

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