Microtubules and Fibrils in the Cytoplasm of Coleus Cells Undergoing Secondary Wall Deposition

Much attention is currently being devoted to the nature of the mechanisms in the cytoplasm which control and orient the deposition of polysaccharide microfibrils on the inner surface of the plant cell wall (1-4). We have approached the question of cytoplasmic participation in wall deposition by studying the fine structure of cells undergoing rapid secondary wall formation. Our study has demonstrated the presence of two different cytoplasmic components which appear to be associated with the rapidly developing secondary wall: (1) microtubular structures aligned in the cytoplasm adjacent to the secondary wall; and (2) clusters of fibrillar elements within cisternae of the endoplasmic reticulum. We believe these two components are different from one another, and, in order to distinguish clearly between them, will hereafter refer to them as "tubules" and "fibrils," respectively. The plant material used in the investigation consisted of stem segments of Coleus blumei Benth. which had been excised, wounded, cultured on agar, and supplied with indoleacetic acid at the apical end (5, 6). Parenchyma cells interior to a vascular bundle severed by the wounding were induced by this treatment to redifl~rentiate into tracheary elements, and, in the process, to lay down numerous bands or reticulations of secondary wall. These redifferentiating cells were used in the investigation of fine structure. They were excised after 4 days of culture, fixed in glutaraldehyde followed by osmium tetroxide (7), stained in a solution of uranyl acetate (8), and embedded in an Epon-Araldite resin mixture. Sections were cut with a diamond knife and mounted on bare 400-mesh grids. In some cases sections were poststained with lead hydroxide (9). Secondary wall deposition in the redifferentiat ing cells is preceded by the appearance of thick bands of cytoplasm in the thin peripheral cytoplasmic layer of the large, vacuolate cells (10). These cytoplasmic bands are rich in elements of the endoplasmic reticulum, Golgi bodies, vesicles, and mitochondria (6). Deposition of secondary wall material results within 4 to 5 days in the appearance of scalariform, anastomosing bands consisting of massive depositions of cellulose, lignin, and other wall components. The tubules were localized beneath the plasmalemma in the cytoplasm next to the developing bands of secondary wall (Fig. I). In sections normal to the wall bands, the tubules were observed in cross-section arranged around the bands (Fig. 1, inset). Similar structures were not positively identified adjacent to the primary wall, an observation which may be related to the fact that the primary wall was no longer growing. Tubules associated with a particular wall band ran parallel to its surface and were usually aligned roughly parallel to one another in the direction taken by the band. They were sectioned longitudinally and could be traced for some distance through the cytoplasm when the region of transition between wall and cytoplasm lay within the plane of section (Figs. 1 and 5). The tubules were generally straight, although some exhibited a slight curvature or small angular changes in direction. They measured from 220 to 280 A in diameter, and consisted of a dense wall 55 to 70 A thick and a light interior 110 to 140 A in diameter. Their lengths were indeterminate, although some of the tubules were traced through the cytoplasm for at least 2 g. They were not observed in material fixed in permanganate. The nature and function of the tubules are unknown. Microtubular structures have been widely reported in animals and among the protista (reviewed by Slautterback, 11), and have been demonstrated by Manton in the spermatozoids of a moss (12) and of a fern (13). Quite recently, cytoplasmic tubules resembling those reported here have been found in higher plants by Ledbetter and Porter (14) in close association with the growing primary walls of root tips. An association of similar structures with the secondary wall has not been previously reported, so far as we are aware. We conjecture that the tubules may be concerned in some way with the development of the wall, e.g., by determining the place of deposition and the orientation of the