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ABSTRACT. The fine structure of young root hairs of radish was studied, with special attention to cyto- plasm-wall relationships. Hairs up to 130/~ in length were ...
CYTOPLASMIC

MICROTUBULE

WALL MICROFIBRIL IN ROOT HAIRS

AND

ORIENTATION

OF RADISH

E L D O N H. N E W C O M B

and H O W A R D T. B O N N E T T , J R.

From the Department of Botany, University of Wisconsin, Madison, Wisconsin. Dr. Bonnett's present address is the Department of Biology, University of Oregon, Eugene, Oregon

ABSTRACT The fine structure of young root hairs of radish was studied, with special attention to cytoplasm-wall relationships. Hairs up to 130/~ in length were examined after fixation of root tips in glutaraldehyde followed by osmium tetroxide. Microtubules occur axially aligned in the cytoplasm just beneath the plasmalemma, and extend from the base of the hair to within 2 to 3 ~ of the tip. Poststaining with uranyl acetate and lead citrate clearly reveals in thin sections the presence of the two layers of cellulose microfibrils known from studies on shadowed wall preparations: an outer layer of randomly arranged microfibrils arising at the tip, and a layer of axially oriented microfibrils deposited on the inside of this layer along the sides. The youngest microfibrils of the inner, oriented layer first appear at a distance of about 25 /z from the tip. Although the microfibrils of the inner layer and the adjacent microtubules are similarly oriented, the oriented microtubules also extend through the 20- to 25-/z zone near the tip where the wall structure consists of random microfibrils. This suggests that the role of microtubules in wall deposition or orientation may be indirect. INTRODUCTION In problems of cell wall structure and growth currently receiving considerable attention, questions concerning the origin and deposition of the cellulose microfibrils, which form the framework of the wall, occupy a prominent position (14, 15, 23, 24). The changes in pattern which microfibrils undergo in the walls of cells elongating by generalized surface growth are described in the theory of multinet growth proposed by Roelofsen and Houwink (17). In this common type of growth, microfibrils are deposited on the inner surface of the expanding wall in a direction approximately transverse to that of cell elongation, but, as cell elongation and the deposition of new microfibrils proceeds, are gradually reoriented to an oblique and eventually an axial arrangement while occupying first an intermediate and later an outside position in the wall.

Although considerable evidence has accumulated favoring multinet growth as an acceptable description of microfibrillar reorientation in the wall, little progress has yet been made in our' understanding of a number of other basic aspects of microfibril participation in wall growth, including the site of microfibril synthesis and the mechanism of control over the oriented deposition of microfibrils outside the cytoplasm. Several cell types are known to possess distinctive patterns of wall growth and microfibril deposition which differ from those in the above mentioned generalized type of wall growth (16). Among these are cells characterized by extreme elongation, including certain hairs, germinating pollen grains, and root hairs. In pollen robes and root hairs, growth in surface area is confined strictly to the tip; in the cotton hair, on the other

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h a n d , extension growth occurs along the sides as well as a t the tip. A study of these cell types should provide valuable c o m p a r a t i v e information. R o o t hairs provide attractive material for a n investigation of cytoplasm-cell wall relationships with the electron microscope. T h e y are localized p r o t u b e r a n c e s of single cells, their extension growth is restricted to the apex, a n d their rate of elongation is quite high, being in the vicinity of 100 # per hour. W i t h few exceptions (Sievers, 19-21), investigation of their fine structure has been performed principally o n shadowed wall preparations. R e c e n t technical advances, including the introduction of glutaraldehyde as a fixative a n d h e a v y metal salts as electron stains, have m a d e the investigation of cytoplasmic structures in relation to microfibril deposition considerably more promising. O f greatest encouragem e n t to this a p p r o a c h has b e e n the discovery of microtubules in higher p l a n t cells (8, 1 l) a n d the d e m o n s t r a t i o n t h a t they are aligned in the cytoplasm n e a r growing walls with a n orientation m a t c h e d by t h a t of the microfibrits of the i n n e r wall surface (4, 8, 11, 25). T h e present study explores the distribution of microtubules a n d their a l i g n m e n t with respect to t h a t of the wall microfibrils in radish root hairs. MATERIALS

AND METHODS

Seeds of radish, Raphanus sativus L., variety "White Icicle," were surface sterilized in a l0 per cent solution of Clorox and gcrminated at 22-23°C in Petri plates containing moist filter paper. Forty-four hours after placing the seeds in the Petri plates, the seedling radiclcs were fixed, at which time a large quantity of root hairs had developed, representing a range of lengths from small protuberances to hairs 1 or 2 m m

in length. Fixation, rinsing, and postfixation in osmium tetroxide were carried out at room temperature in 0.025 • phosphate buffer at pH 6.8. Intact seedling radicles were immersed in 3 per cent glutaraldehyde (18), segmented into 2-mm portions, and fixed for 1.5 hours. Following washing, segments were treated with 2 per cent osmium tetroxide for 2 hours, dehydrated in an acetone series, and ei-nbedded in Araldite-Epon (13). Silver sections were cut on a Servall MT-1 Ultramicrotome, stained with uranyl acetate and lead citrate, and viewed in a Hitachi HU-I 1A microscope at 50 or 75 kv. RESULTS T h e root hairs used in the present study varied in length from slight bulges of the epidermal cell to protuberances as long as 130 #. Since radish root hairs c o m m o n l y reach several millimeters in length when fully grown, the observations reported here concern only quite young stages. O l d e r hairs h a v e also been examined, a l t h o u g h they are difficult to fix b y the procedures satisfactory for younger hairs. T h e nucleus of the epidermal cell bearing a young root hair of the length studied here is generally located at the base of the hair; later, it migrates o u t into the hair. A large central vacuole does not develop in the hair until it is s o m e w h a t older a n d longer t h a n those used in this study. T h e cytoplasm of the young hair contains a n a b u n d a n c e of r o u g h endoplasmic reticulum. This c o m p o n e n t is frequently sectioned normally, revealing the profiles of the m e m b r a n e s b e a r i n g ribosomes o n their outer surfaces (Figs. 1 a n d 2). I n tangential sections of the reticulum in which the m e m b r a n e s are seen in face view, the ribosomes are c o m m o n l y observed disposed over the

Abbreviations for Figures The vertical arrow in the upper right hand corners of Figs. 6 to 9 runs in the direction of the root hair axis and points toward the apex. The position and orientation of the sections represented ia Figs. 4 to 9 are shown in Fig. 10.

RMf, randomly arranged microfibrils AMf, axially oriented mlcrofibrils CV, coated vesicle CW, cell wall

]fit, microtubule P1, plasmalemma Pr, polyribosome TI, tubular incursion of the wall by the cytoplasm

FIGURES 1 and ~ Transections of two different root hairs showing microtubules in transection beneath the plasmalemma. The microfibrillar structure of the wall is evident. Several coated vesicles can be seen in Fig. ~. X 67,000.

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E. H. NEWCOMB AND H. T. BOI'~NETT, J'R.

Cytoplasmic Microtubule

577

surface in the form of coils or spirals (Fig. 3). These aggregates, or polyribosomes, commonly include 15 to 25 ribosomes (3). Unattached ribosomes are also abundant (Figs. 1, 7, and 8). A m o n g the prominent components of the root hair cytoplasm are dictyosomes and associated vesicles (Fig. 1). " C o a t e d " vesicles about 85 m # in diameter, including coat, are encountered throughout the cytoplasm and are particularly evident near the wall, both at the tip of the hair and along its length. They are present in most of the figures, but are best seen in Figs. 2, 3, and 5. The properties and contribution of dictyosomes and coated vesicles to the growth of the hair will be considered in detail in a later paper. ORIENTATION OF CYTOPLASMIC MICROTUBULES

Microtubules similar to those previously described for higher plant cells (4, 8, l l, 25) are found in the peripheral cytoplasm near the plasmalemma of the root hair. They are markedly restricted to this position and are only infrequently observed elsewhere in the cytoplasm. The microtubules lie parallel to the cell surface and are axially aligned. This is evident in transections of the hairs, in which the microtubules are also observed in transection (Figs. 1 and 2). W h e n hairs are sectioned longitudinally near the surface, the unevenness of the cytoplasmic surface results in differences in the amounts of the plasmalemma and cytoplasm included in a section, and in the extent to which individual microtubules can he traced. Fig. 3 shows a longisection i0 # in length; the inner part of the tangentially sectioned wall is visible in some areas, the plasmalemma and immediately adjacent cytoplasm in others, and the cytoplasm slightly deeper in the hair elsewhere. The thickness of the section is sufficient, however, to permit a number of microtubules to be followed for distances of a few microns despite the variations in the cytoplasmic contour. The microtubules are not perfectly straight, but exhibit slight curvatures or small angular changes

in direction as they course through the cytoplasm (Fig. 3). In median longitudinal sections, in which the plasmalemma is sectioned normally, it is apparent that at least some of the angular changes in microtubule direction reflect irregularities in the outline of the plasmalemma. The distribution of microtubules along the root hair was determined in several steps which included, first, facing the block with the knife and measuring the length of an entire hair lying near the surface with an ocular micrometer in a light microscope at a magnification of 300. Then, as an oblique section of the hair was cut and micrographed, its location was determined with the electron microscope from the number of plate lengths or widths at a defined magnification spanning the distance either to the tip of the hair or to the epidermal wall at t h e base of the hair fi'om a preselected region within the graze. The position and orientation of the sections represented in Figs. 4 to 9 are schematically shown in Fig. 10. Inspection of transverse and oblique sections through the cytoplasm within 1 to 2 Iz of the extreme tip of the hair fails to reveal microtubules. However, microtubules can be observed in sections within the apical dome only 3 to 4 ~ from the tip. The transverse section shown in Fig. 5 reveals the cytoplasmic structure at a distance from the tip estimated from serial sections to be only 1 to 2 /~. No microtubules can be detected. While these might be difficult to distinguish in transverse section, it is at least evident that microtubules do not traverse the apex from one side of the hair to the other, since in this event they would have been sectioned longitudinally and readily seen. Fig. 6 is an oblique section through the side of the dome only a few microns from the tip of a hair (see diagram, Fig. 10). Several microtubules are observed in longitudinal section, a fact taken to indicate that they extend into the base of the dome and curve in conformity to it. In median longitudinal sections, microtubules can

FIGURE 3 Longitudinal graze, 10 # in length, located approximately 100 lZ from the apex of a root hair lS0/~ long. The randomly arranged microfibrils in the outer part, and the axia]ly oriented microfibrils in the inner part of the tangentially sectioned wall are clearly evident. When the plane of section passes through the wall into the surface layers of the cytoplasm, numerous axially aligned microtubules lying just beneath the wa]l can be traced for distances of several microns. X 88,000.

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THE JOURNAL OF CELL BIOLOGY • VOLUME 27, 1965

be detected within several microns of the apex, although it is difficult to determine their presence in the dome itself because of the greater density of the background cytoplasm in this region. The extent of individual microtubules has not been determined; possibly they run the length of the hair and are extended at their apical end as the apex of the root hair itself pushes forward. Although microtubules have also been observed in the main body of the epidermal cell from which the root hair arises, what their orientation may be at the base of the hair and whether they are continuous with the microtubules in the hair are aspects which have not been determined. ORIENTATION OF WALL MICROFIBRILS

It is well established that thc root hair clongates by tip growth only (6, 16), and that the wall of the hair contains two distinct laycrs of cellulose microfibrils (2, 5, 9, 22). The outer layer, which arises at the tip and completcly covers the hair, contains a random network of micro fibrils. The microfibrils of the inner layer do not arise at the tip, but are deposited on the inside of thc wall along the sidcs of the hair with a strictly axial oricntation. The poststaining procedure used in this investigation clearly reveals prominent microfibrils in the root hair wall. These exhibit the microfibrillar organization expected in the wall as described above, and are believed to constitute its cellulosic framework. This organization and the position and orientation of the sections represcntcd in most of the figures are diagrammed in Fig. 10. Figs. 4 and 5 represent closely succcssive but not consccutive transverse sections through the same hair; Fig. 6, an oblique section through a second hair; Fig. 7, a scction through a third hair; and Figs. 8 and 9, oblique sections through a fourth hair at distances from the tip of approximately 26 and 36 #, respectively. It should be noted that the orientation of the oblique sections with respect to the root hair apex is also shown in the diagram. This orientation, indicating whether cytoplasm or wall is closer to the root hair apex, is also given in each figure by means of an arrow running in the direction of the hair axis and pointing toward the apex. The oblique sections (Figs. 6 to 9) have been cut at a low angle with respect to the hair axis, and consequently are particularly useful in corn-

paring the orientation of the wall microfibrils with that of the microtubules. In these sections, the structures in question are advantageously displayed as the plane of section passes gradually from the peripheral cytoplasm through the inner and finally the outer structure of the wall. In Fig. 4, which shows a transverse section of the wall beyond the cytoplasm at the tip of a hair, the arrangement of the microfibrils is clearly random. This random structure is also evident in Figs. 5 and 6, which are of transverse and oblique sections, respectively, through the cytoplasm in the apical domes of two different hairs. An oblique section estimated to be 15 /z from the apex of a hair is shown in Fig. 7. The random arrangement seen here is present throughout the wall up to a distance of approximately 25 # from the apex. Beginning at about 25 # from the tip of a hair, axially oriented microfibrils become detectable on the inside of the wall, while the random network of microfibrils discussed above becomes the outer layer. Fig. 8 is an electron micrograph of a section 26 /z from the hair tip, in which axially oriented microfibrils are unmistakably present on the inner wall surface, though they are not yet prominent. The microfibrils of this inner layer become somewhat coarser and more numerous as the distance from the apex increases. At a distance of 36 /~ (Fig. 9), the randomly arranged microfibrils in the outer and central regions of the wall and the prominent, axially oriented microfibrils in the inner part next to the cytoplasm are readily apparent. The difference in these two layers is also clearly seen in Fig. 3, which shows the microfibrillar organization of the wall in that portion of the hair about 100/z from the apex. From the above results, it is evident that throughout most of the length of the root hair both the microfibrils of the inner wall surface and the microtubules in the nearby cytoplasm are axially, and thtls similarly, aligned. There is, however, a zone extending back from the tip of the root hair for about 25/z in which there is a different set of relationships. Although axial microtubules extend through this region into the dome, no axial microfibrils are deposited, and there is only a random microfibrillar structure to the wall. As a consequence, there is a lack of coincidence in orientation between the microtubules and the microfibrils in this zone of the hair. These relationships are illustrated diagrammatically in Fig. 10.

E. H. NEWCOMBAND H. T. BONNETT, JR.

CytoplaxmivMicrotubule

579

FIGURE 4 Transection of the wall at the tip of a root hair, showing the random arrangement of the microfibrils. Several tubular incursions of the wall by the cytoplasm are present. X 71,000.

FIGURE 5 Transection of a root hair estimated to be only 1 to ~//. from the tip. The section is closely successive, but not consecutive, to that of Fig. 4. The cytoplasm contains numerous vesicles. The large, membrane-limited, vesiculate bodies are inpocketings of the plasmalemma and include the inner region of the wall, which is nearly devoid of stained contents. )< 45,000.

E. H. NEWCOMB AND H. T. BONNETT, JR.

Cytoplasmic Microtubule

581

FIGURE 6 Oblique section through the side of the dome of a root hair a few microns from the tip. Five nearly parallel microtubu]es are seen in longisection, demonstrating that aligned microtubules extend into the tip region. X 56,000.

]?IGURE 7 Oblique section through a root hair approximately 15/z from the tip. Numerous axially aligned microtubules are present in the cytoplasm just beneath the wall, but the microfibrillar pattern in the wall is random. X 55,000. FIGURE 8 Oblique section through a root hair approximately 26 # from the tip. Axially oriented microfibrils are now present in the inner region of the wail next to the cytoplasm but are not yet numerous.

X 66,000.

FXCURE 9 Oblique section approximately 36 # from the tip of the same root hair as t h a t shown in Fig. 8. Axially oriented microfibrils are prominent on the inner wall surface while a r a n d o m pattern of microfibrils can be observed in the outer and central regions of the wall. Numerous tubular incursions of the wall by the cytoplasm are seen in oblique and longitudinal section. X 55,000.

MICRONS FROM TIP

--O

FiG4 FIG. 5 ~

--

-5 MICROTU BULE

-10 FIG. 7 \

\

\ \

\ FIG. 6

--15

\

-20 COATED VESICLE

\

-25

\ \

FIG. 8

AXIALLY ORIEN1

7

MICROFIBRILS

t

--30

\ RANDOMLY

.

ARRANGED

--35

MICROFIBRILS

I \ I~IG. 9

~

--40

FIGURE 10 Diagrammatic representation of a median longitudinal section through the apical 40 # of a young root hair showing the position and orientation of the sections represented in Figs. 4 to 9. TI~E CYTOPLASMIC SURFACE

extend into the inner region of the wall. These

The cytoplasmic surface of the young root hair is greatly increased by irregularities, including numerous tubular or finger-like processes which

incursions, which are found throughout the hair, are present in all of the figures, including that of the microfibrils at the tip (Fig. 4), in which a few

E. H. NEWCOMS AND H. T. BONNETT, JR.

Cytoplasmic Microtubule

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processes can be distinguished in transection. Figs. 7 and 8 show m a n y of these tubules sectioned obliquely, while in Fig. 9 several are observed more or less in longisection. They are delimited by a unit membrane which is frequently identifiable as an extension of the plasmalemma. The tubes twist and turn in a random manner, so that a single structure is sectioned both longitudinally and transversely. Although tube diameter is variable, it is commonly between 30 and i00 m#. The contents are of a rather uniform, moderately electron-opaque nature within which very little structure can be distinguished. Intensely electron-opaque spots sometimes present along the cytoplasmic margin or within the wall (see especially Figs. 6 and 7) apparently represent sections of these structures which have stained excessively. The region of the wall into which the tubules extend is electron transparent and contains a delicate network of fine fibrils which frequently appear to converge on or arise from the plasmalemma surrounding the tubules. In many cases, these fine fibrils can be traced to an apparent merger with the coarser wall microfibrils (Fig. 9). DISCUSSION The present investigation clearly discloses that a random network of microfibrils covers the dome of the radish root hair and extends back along the sides on the outer surface of the wall. There is, in addition, a layer of axially oriented microfibrils covering the inner wall surface to within about 25 # of the tip. Thus, these results demonstrate in thin-sectioned material a microfibrillar pattern in the root hair wall similar to that already established with shadowed wall material. Several lines of evidence indicate that this pattern represents the cellulosic framework of the wall. It is comprised of outer and inner layers with characteristically different microfibrillar arrangements, in agreement with the arrangement of the cellulose microfibrils reported by several groups of investigators using shadowed preparations (see below). Moreover, cellulose is the only component of the root hair wall known to have a microfibrillar texture. The other components are present as amorphous substances. These include mucilaginous material and cutin on the outer surface (5), and the pectic substances and non-cellulosic polysaccharides which form a matrix in which the microfibrils are embedded (2). The possibility

536

that the observed microfibrillar structure is due to a coating of amorphous material on the cellulose microfibrils seems unlikely since this material is not confined to the microfibrils, and should stain in the interstices as well. Furthermore, the observed microfibrils are approximately 35 A in diameter, whereas a considerably greater diameter and the presence of a lighter core would be expected if the observed structure were due to staining of a coat of non-cellulosic material. Finally, as shown by Belford and Preston (2), the amorphous incrusting substances are located predominantly in the outer layer of microfibrils in the root hair wall, and are largely absent from the highly oriented and more compact inner layer. In view of this, the prominent and sharply delineated structure of the inner layer observed in the present study seems clearly to represent stained cellulose microfibrils. Visualization of the microfibrillar wall structure is due to poststaining achieved with the heavy metal salts. Sections fixed in the usual way with glutaraldehyde followed by osmium tetroxide, but not poststained with heavy metal salts, show virtually no wall structure, although, by overexposure during the printing process, a very faint pattern of microfibrils can be detected in some places. The microfibrils are stained by uranyl acetate alone, but not so intensely as when lead citrate is also used. T h a t lead acetate acts on cellulose fibers as an electron stain was shown some years ago by Kinsinger and Hock (10). Belford et al. (1), using a range of dilute aqueous salt solutions, have demonstrated the adsorption of a variety of metal ions onto cellulose microfibrils obtained from higher and lower plants. Such an effect of the heavy metals might be enhanced by the postfixation in osmium tetroxide, creating additional reactive groups through oxidation and allowing greater penetration of the stains by loosening the microfibril structure. Although there have been a number of previous studies of the growth of the root hair wall, the extent to which the sides of the apex are involved in extension growth and the site at or below the tip at which the axially oriented microfibrils first appear are not yet well defined. It was established by earlier investigators (cf. 6) that growth of root hairs is confined to the tip, but the techniques used, employing particles as markers on the surface of the root hair, do not allow certainty as to whether this growth is con-

TttE JOURNAL OF CELL BIOLOGY" VOLUME27, 1965 •

fined strictly to the apical 3 to 5 # or whether it tapers off more gradually along the sides below the dome over a distance of 5 to 10 # or more. More recently, both electron microscopy and autoradiography have been used in the study of the structure and growth of the root hair wall. The random orientation of the microfibrils at the apex and in the outer wall was first observed in maize by Frey-Wyssling and Mfihlethaler (7). The layer of axially oriented microfibrils on the inner wall surface was discovered by Houwink and Roelofsen (9), who studied it in root hairs of maize with the polarizing microscope. They concluded that this inner layer was deposited "from just below the tip downwards," and that its presence as well as the absence of transversely oriented microfibrils was consistent with the fact that growth of the root hair is limited to the tip and does not involve the tubular portion. Wardrop (22), in an autoradiographic study of the growth of root hairs of oat seedlings, obtained results indicating that the deposition of the oriented microfibrils on the inner wall surface probably goes on simultaneously throughout the growing hair, except for the tip region. Even when hairs were kept for only 3.5 hours in C14-glucose, the radioactivity in the wall was not localized at the tip, but appeared to be uniformly distributed along the hairs irrespective of their length. Since other labeling results were consistent with the occurrence of growth only at the tip, the fact that labeling became pronounced in the wall at a considerable distance from the tip pointed to the continued synthesis and deposition of axially oriented microfibrils on the inner wall surface. Using shadowed preparations, Dawes and Bowler (5) observed that walls of young radish hairs about 50 # in length showed only a random microfibrillar pattern, while walls from hairs 100 /z or more in length were thickened by the presence of longitudinally arranged microfibrils on the inner surface. Although it was not determined how far toward the tip these axial microfibrils extended, they were apparently not observed in preparations from the young hairs about 50 # in length. In a study employing white mustard (Sinapis alba), Belford and Preston (2) also concluded that root hair walls consist of two distinct layers, the outer composed of randomly arranged microfibrils with which amorphous wall constituents are associated, and the inner, of axially oriented mi-

crofibrils with little or no amorphous substances. The outer layer extends continuously over the root hair, while the inner extends along the length of the hair to just below the tip. In recent fine structure investigations reporting on the presence of cytoplasmic microtubules in plants, the possibility that the microtubules may play a role in cellulose microfibril deposition has been raised, since the microtubular elements occur in the cytoplasm adjacent to regions of actively growing wall, and their orientation coincides with that of the newly deposited microfibrils. In their study of growing walls of meristematic root tip cells of Phleum and Juniperus, Ledbetter and Porter (11) found that the microtubules run transversely next to the lateral walls like the microfibrils in these walls, while next to the end walls they are randomly arranged, as are the end wall microfibrils. In parenchymal cells of Coleus undergoing differentiation into tracheary elements, Hepler and Newcomb (8) found that the microtubules are confined to the regions where the bands of secondary wall are forming, and are aligned parallel to the band axes and at right angles to the long axis of the cell as are the microfibrils in the bands. A similar correspondence of alignment between microtubules and the microfibrils of the developing secondary wall has subsequently been reported for differentiating xylem of sycamore by Wooding and Northcote (25) and the oat coleoptile by Cronshaw and Bouck (4). Finally, in the young radish root hairs investigated here, both the microtubules in the peripheral cytoplasm and the microfibrils which lie on the inner wall surface adjacent to these microtubules are similarly aligned--in this case axially. This coincidence of alignment again raises the question whether the microtubules may not be directly involved in microfibril deposition or orientation. In the radish root hairs, a lack of coincidence in the alignment between microtubules and microfibrils is also apparent. Since the microtubules extend into the tip of the hair and the axially oriented microfibrils are not found closer to the tip than about 25 # (Fig. 10), the microtubules run for some 20 to 25 # in oriented array near a region of the wall in which the microfibrils are randomly arranged. T o our knowledge, this is the first report of the occurrence of numerous microtubules uniformly oriented near a cell wall in which the microfibrils of the inner wall surface are not correspondingly aligned.

E. H. NEWCOMBAND H. T. BONNETT,JR.

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It must be recognized, however, t h a t the rand o m wall structure present t h r o u g h o u t most of this region is p r o b a b l y the result of microfibril deposition at the tip, a n d t h a t these microfibrils have p r o b a b l y been left along the sides as further tip growth occurred, so t h a t their r a n d o m arrangem e n t represents not the influence of the a d j a c e n t cytoplasm, b u t merely the persistence of the irregularity with which they were deposited. If this interpretation is correct, a n interesting situation exists in which n u m e r o u s microtubules are aligned a d j a c e n t to a quiescent region of the wall in w h i c h neither microfibril deposition nor surface growth is occurring. T h e possibility t h a t i m p o r t a n t additions to, or changes in, the a m o r p h o u s substances of the wall are taking place in this region is not excluded, however. These observations raise the possibility t h a t the microtubules m a y not be directly engaged in controlling microfibril orientation, b u t m a y influence it t h r o u g h their role in a more general process, such as cytoplasmic streaming, as suggested by L e d b e t t e r a n d Porter (11, 12). A m o n g alternative explanations is the possibility t h a t the microtubules directly control microfibril orientation, a n d t h a t due to the rapidity of root hair elongation, they c a n be observed in process of formation in

the region immediately b e h i n d the tip, b u t only become functional in microfibril orientation some 12 to 15 minutes later, by w h i c h time they are situated 20 to 25 ~ farther back. I n view of this a n d other alternative possibilities, the observations on lack of coincidence of a l i g n m e n t reported herein obviously can only be considered suggestive. I n n u m e r o u s micrographs o b t a i n e d in the present study, a network of fine fibrils can be detected on the i n n e r wall surface. M a n y of these a p p e a r to join or arise from the p l a s m a l e m m a , a n d c a n be traced deeper into the wall, where they a p p e a r to merge with the coarser wall microfibrils. A l t h o u g h their r a n d o m n e s s a p p e a r s as a n inconsistency in view of the oriented n a t u r e of b o t h the microtubules a n d the inner wall microfibrils, the possibility is being investigated t h a t these fine fibrils are early stages in the aggregation of cellulose microfibrils, or proteinaceous threads concerned in microfibril synthesis. This work was supported in part by grant RG-628 from the National Science Foundation to E. H. Newcomb, and grant GM-10493 from the National Institutes of Health, United States Public Health Service, to H. T. Bonnett, Jr. Received for publication, July 30, 1965.

REFERENCES I. BELFORD, D. S., MYERS, A., and PRESTON, R. D., A study of the ordered adsorption of metal ions on the surface of cellulose microfibrils, Biochim. et Biophysica Acta, 1959, 34, 47. 2. BELFORD, D. S., and PRESTON, R. D., The structure and growth of root hairs, J. Exp. Bot., 1961, 12, 157. 3. BONNETT, H. T., JR., and NEWCOMB, E. H., Polyribosomes and cisternal accumulations in root hairs of radish, J. Cell Biol., 1965, 27, 423. 4. CRONS~AW, J., and BoucK, G. B., The fine structure of differentiating xylem elements, J. Cell Biol., 1965, 24, 415. 5. DAWES, C. J., and BOWLER, E., Light and electron microscope studies of the cell wall structure of the root hairs of Raphanus sativus, Am. J. Bot., 1959, 46, 561. 6. EKI)AnL, I., Studies on the growth and the osmotic condition of root hairs, Symb. Bot. Upsalienses, 1953, 11, 5. 7. FREY-WYSSLINO, A., and M/SHLET~ALER, K., t ) b e r den Feinbau der Zellwand Yon Wurzelhaaren, Mikroskopie, 1949, 4, 257. 8. HEPLER, P. K., AnD NEWCOMB, E. H., Microtubules and fibrils in the cytoplasm of Coleus

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