Electron Microscope Observations on the Root Hair Cell of Azolla imbricata Nakai

Transcription

1 12 Cytologia 28 Electron Microscope Observations on the Root Hair Cell of Azolla imbricata Nakai Sigenobu Kawamatu Biological Institute, Aichi Gakugei University, Okazaki, Japan Received May 7, 1962 Recently electron microscope studies on plant cells have been carried out as extensively as on animal cells. But as far as the present author knows, electron microscope studies on root hair cells have not been published. The author has found that there are granules in the root hair cell of Azolla imbricata Nakai (an aquatic fern) that look like mitochondria, but it could not be decided definitely by various techniques using optical microscope whether they are mitochondria or not (Kawamatu 1960). By electron micro scope observations, however, the granules in question could be obviously discriminated from the true mitochondria. In the present paper, the structure of these bodies and other cytoplasmic components observed with electron microscope will be described. Materials and methods Root hairs of Azolla imbricata Nakai were used in this study. Young roots with root-cap were collected, bundled by about tens with fine wool threads, blotted with pieces of filter paper, and fixed in 1% solution of osmium tetroxide for 1-2 hours at about 2 Ž, the ph of which was adjusted to 7.4 with veronal-acetate buffer. Some materials were fixed in 2% potassium permanganate for 2-3 minutes at room temperature (15-25 Ž). The fixed materials were then washed in water, dehydrated through a graded series of ethanol, suspended in equal mixture solution of ethanol and monomer (a mixture of N-butyl methacrylate 8: methyl methacrylate 2, containing 2% benzoyl peroxide as catalyst), and then washed three times with small quanti ties of the monomer. The suspension was transferred into gelatin capsules and the plastic was polymerized at 45 Ž for 8-10 hours or above a fluore, scent lamp of 6 W for hours. Sections were made with glass knives on an ultrathin microtome of JUM-5 type. Observations were made without removal of the embedding plastic, using JEM-5G or JEM-T6 type electron microscope. The electron micrographs were taken at original magnifications of 2000 to 7000 times and photographically enlarged.,

2 1963 Electron Microscope Observations on the Root Hair Cell 13 Observations When the root hairs are observed with optical microscope, the cytoplasm is rich in the apical region where the nucleus, the yellowish green plastids, and numerous granules of various sizes are embedded (Fig. 1). The mito chondria also seem to be comprised in these granules, but identification of them is nearly impossible by optical means. In the young root hairs large vacuoles are seen, in which there exist granules stainable with basic dyes Figs , fresh material of root hairs observed with optical microscope. The boundary between vacuole (v) and cytoplasm is clearly discriminated, the nucleus (n) being embedded in the latter. ~650. 2, electron-dense granules (gl) in vacuole (v). Fixed with 1% OsO4. ~ such as Janus green B as well as the granules in the cytoplasm. These granules react positively to various routine staining reagents applied to identify the mitochondria. In a thin layer of the cytoplasm between the vacuole and the cell wall, the cytoplasmic streaming can be conspicuously observed, and plastids are rarely found in this part. Small granules are moving, conveyed by the cytoplasmic streaming. Occasionally low refractive strand-shaped components like so-called mitochondria are also seen moving more slowly than the above-mentioned granules and changing gradually their shapes. In electron micrographs, the cytoplasm is obviously distinguished from the vacuole. In the cytoplasm generally mitochondria, plastids, endoplasmic

3 14 S. Kawamatu Cytologia 28 reticulum, and Golgi-like structures are found, and in the vacuoles many electron-dense bodies. 1. Vacuoles: In the apical portion of the young root hairs, the to noplast membrane seems to be present, because a discernible surface boundary is obviously seen between the cytoplasm and the vacuole under the optical microscope (Fig. 1). In the electron micrographs of the sections of the cell portion where a large vacuole occupies nearly all of the cell diameter, there are found some fragments of cytoplasm in which mitochondria, plastids and other cytoplasmic components are included. On the other hand, in the sections of the cell portion densely filled with the cytoplasm, there are cases in which vacuoles are seen distributed here and there in the cytoplasm. In the vacuoles, especially in those of the apical portion, heavily electron-dense bodies are found scattered (Fig. 3). They are somewhat rodshaped and appear to be composed of closely packed small granules of ca. 30mƒÊ in diameter (Fig. 2). 2. Mitochondria: The mitochondria can be distinctly identified on electron micrographs and are distributed here and there in the cytoplasm. They show the features of the typical structure of mitochondria in plant cells. They consist of the outer and the inner membrane; the latter have internal foldings, i.e., cristae mitochondriales, of various appearances in section (Figs. 5 and 8). 3. Plastids: In optical microscope observations, the plastids show slightly yellowish green color and some of them include starch grains which are stained with I2-KI reaction or with Lillie's polysaccharide-staining method. In electron micrographs, some of the plastids include large elliptic electron transparent bodies which are considered to be starch grains. In Figs. 3 and 6, the plastids show several lamellar structures, each set of which appears to be composed of two lamellae and a grana-like structure. Among the lamellae, a few small electron-dense spherical bodies are observed. In permanganate-fixed cells (Figs. 4 and 9), the plastids show no typical lamellar structure, but contain piles of several short ones, i.e., grana-like structures. The plastids are mostly encircled with endoplasmic reticulum. 4. Endoplasmic reticulum: In the cytoplasm, there is a system of endoplasmic reticulum which is irregularly winding in a shape of discrete layers (Figs. 4, 5, 6 and 10) or is in stacks of lamellar layers (Fig. 7). Each set of the layers is composed of a pair of membranes. In osmium-fixed cells, Palade granule-like bodies appear to be attached on the outer surface of the double layer (Figs. 5, 6 and 7), but in permanganate-fixed cells, the endo plasmic reticulum is more plentifully seen and Palade granule-like bodies are absent on the surface of the layer (Figs. 4 and 10). The endoplasmic reticulum has a tendency to develop relatively well in the vicinity of the tonoplast membrane. Around the edges of some layers, small vesicles are seen. 5. Golgi-like structures: In Fig. 6, a disc-shaped body is found in the

4 1963 Electron Microscope Observations on the Root Hair Cell 15 Figs Two examples of cross-sections of the root hair cell near the apical region. 3, nucleus (n), plastids (p) in the cytoplasm and electron-dense granules (gl) in the vacuoles (v). Some of the plastids contain starch grains (s). Fixed with 1% OsO4. ~6000 4, mito chondria (m), plastids (p), endoplasmic reticulum (er) and Golgi-like structures (g) in the cytoplasm. Fixed with 2% KMnO4. ~10000

5 16 S. Kawamatu Cytologia 28 Figs , large vacuole (v), mitochondria (m), plastid (p) and endoplasmic reticulum (er) in which Palade granule-like bodies are seen. cw, cell wall: pm; protoplasmic mem brane. Fixed with 1% OsO4. ~ a part of the cytoplasm among the vacuoles. A plastid (p1) contains a few small electron-dense spherical bodies, and another one (p2) a large starch grain (s). Golgi-like structure (g) has four cisternae. Electron-dense granules (g)1 are eeen in the vacuole. er, endoplasmic reticulum. Fixed with 1% OsO4. ~30000

6 1963 Electron Microscope Observations on the Root Hair Cell 17 cytoplasm. The components of this system can be termed "cisternae", and often there are small, discrete vesicles at the edges of the cisternae. The number of the cisternae is commonly four, but counted about eight in those of large sizes (Fig. 8). On a survey of low magnification (Fig. 4), Golgi-like structures are found numerously here and there in the cytoplasm. They show a tendency to be abundant in the periphery of the cell and various shapes in section. Discussion In the vacuole of the root hair cell of Azolla, no other substance except heavily electron-dense bodies can be found in the electron micrographs, so that it is probable that these bodies are the same as the granules observed with optical microscope in the vacuole. They show heavy electron-density after fixation with osmium tetroxide, and are supposed to contain lipids. As to the low refractive strand-shaped cytoplasmic components like mitochondria, the author has observed similar ones in the epidermal cells of Allium cepa under optical microscope. Sorokin (1938) has already identified them in Allium cepa as mitochondria with Janus green B staining. However, when the author investigated the granules in the epidermis of Allium and also in the root hair of Azolla according to Sorokin's methods, they showed no staining with Janus green B. In electron micrographs, however, mito chondria are found numerously in the cytoplasm of the root hair of Azolla. They show the feature of the typical structure of mitochondria in plant cells. The plastids in the root hair cell as well as in the cell of root itself show distinctly a lamellar and grana-like structure (Figs. 3, 5, 6 and 7), and some of them contain starch grains. Accordingly, they have the appearance of both chloroplasts and amyloplasts, but tentatively they may be called "chloroplasts" similarly to those in the cells of root proper (Kawamatu 1961). Because the fern used in this study always grows on the water surface, their roots are exposed to day light, hence they seem very likely to perform pho tosynthetic activity. Besides, in Fig. 9 (fixed with potassium permanganate), the plastids show several short grana-like structures, but no so-called lamellar structure. This is likely to imply the description by Muhlethaler et al. (1959), "... zuerst entwickeln sich die Granen, die geldrollenartig ubereinander liegen und erst wenn diese nahezu fertig ausgebildet sind, wachsen dazwischen die Stromaschichten.". Therefore, it is also possible to assume that the differentiation of grana in the root hair cells of Azolla may take place much earlier than that of stroma in lamellar form, so far as discussed from the results of observations on the cells fixed with permanganate. Moreover, in these cells, the majority of the plastids are encircled by endoplasmic reticulum, but no mitochondria encircled. Accordingly, the formation and/or the charac teristic function (e.g., photosynthesis) of the plastids may have some relation ships with the endoplasmic reticulum surrounding them. Cytologia 28,

7 18 S. Kawamatu Cytologia 28 Figs , endoplasmic reticulum (er) consisting of stacks of many lamellar layers -dense spherical body, and a plastid (p) contai a s. Fixed with 1% OsO4. x , mitochondria (m) showing the outher and the inner membranes, the latter h aving internal foldings. A large Golgi-like structure (g) appears to have about eight cisternae. Fixed with 2% KMn04 x , plastids (p) showing short grana-like scruc. tures. s, starch grain; m, mitochondrion. Fixed with 2% KMn04 x , endo plasmic reticulum (er) winding in a shape of many lamellar layers. Small vesicles (sv) _??_ seen about the edges of some double layers. Fixed with 2% KMnO4 x40000

8 1963 Electron Microscope Observations on the Root Hair Cell 19 Many investigators (Luft 1956, Whaley et al. 1959, Mollenhauer 1959 and Whaley et al. 1960, and others) have found that Palade granule-like bodies observed in osmium-fixed cells are not demonstrated in permanganate-fixed cells, and that the cisternae of the Golgi-like structures and other membrane bound cytoplasmic inclusions appear more clearly in the cells fixed with the latter fixative. Similar facts were found also to the root hair cells of Azolla. Whaley et al. (1960) has postulated of the possibility that the clusters of large Golgi-vesicles represent a stage in secretion of substances from the epidermal cells. It is assumed also in the plant studied here that Golgi-like structures may have a relationship to absorbtion and secretion of substances in the root hair cell, as the Golgi-like structures are found more numerously in the periphery of the cell. Summary 1. In the electron micrographs of root hair cells of Azolla imbricata, the following intracellular organellae were observed. 2. In the vacuoles, a number of heavily electron-dense bodies are seen. They appear to be aggregates of particles of ca. 30mƒÊ in diameter and probably of lipid nature. 3. The mitochondria show features of the typical structure of those described in plant cells. 4. The plastids in cells treated with osmium fixatives appear to be composed of lamellae and grana-like structures. The plastids in cells fixed with permanganate show the piles of several short lamellar structures, i.e., grana-like structures, but show no so-called lamellar structures. 5. The endoplasmic reticulum is seen irregularly winding in a shape of discrete layers or in stacks of lamellar layers. Palade granule-like bodies are seen in osmium-fixed cells, but not demonstrated in permanganate-fixed cells. 6. The Golgi-like structures are composed of cisternae, and small discrete vesicles are often found at the edges of the cisternae, which appear more clearly in permanganate-fixed cells than in osmium-fixed ones. Acknowledgment The author wishes to thank Dr. C. Matsui of Faculty of Agriculture, Nagoya University, for the excellent guidance in this study, and Dr. K. Ito, Dr. M. Oikawa, Dr. A. Tanaka and Mr. M. Inoue of Japan Electron Optics Laboratory Co. for the facilities to prepare the electron micrographs. Literature cited Kawamatu, S On the granules in the root hair of Azolla imbricata Nakai. (in Japa nese). Bull. Aichi Gakugei Univ. 9: Electron micrographs on the plastids in the root of Azolla imbricata. Experientia 2*

9 20 S. Kawamatu Cytologia 28 17: Luft, J. H Permanganate-a new fixation for electron microscopy. J. Biophs. and Biochem. Cytol. 2: Mollenhauer, H. H Permanganate fixation of plant cells. J. Biophs. and Biochem. Cytol. 6: Muhlethaler, K. and Frey-Wyssling, A Entwicklung und Struktur der Proplastiden. J. Biophs. and Biochem. Cytol. 6: Sorokin, H Mitochondria and plastids in living cells of Allium cepa. Amer. Jour. Bot. 25: Whaley, W. G., Mollenhauer, H. H., and Kephart, J. E The endoplasmic reticulum and the Golgi structures in maize root cells. J. Biophs. and Biochem. Cytol. 5: , Kephart, J. E. and Mollenhauer, H. H Developmental changes in the Golgi apparatus of maize root cells. Amer. Jour. Bot. 46: , Mollenhauer, H. H. and Leech, J. H The ultra-structure of the meristematic cell. Amer. Jour. Bot. 47: