Observations on the Fine Structure of Plant Cell Walls III. The Sieve Tube Wall in Cucurbita

The sieve tube wall in Cucurbita was examined in ultra-thinsections of petioles treated in different ways for the removalof non-cellulosic wall components. The sections were stainedwith permanganate. The microfibrillar components of the wallare arranged in concentric lamellae. The earliest (outermost)part of the wall is similar to that of ordinary parenchyma inhaving its lamellae composed of thinly-distributed microfibrilsreadily separated from one another by certain treatments suchas pectinase extraction. In the characteristically-thickenedinner (nacreous) layer the microfibrils are very densely packedand the lamellae do not separate readily. The microfibrils inthis layer of the wall are very close to transverse and the‘crossed fibrillar’ orientation is not easily discernible.     

Observations on the Fine Structure of Plant Cell Walls II. The Microfibrillar Framework of the Parenchymatous Cell Wall in Cucurbita

The microfibrillar framework of parenchymatous walls in Cucurbitawas observed in petioles treated so as to remove various non-cellulosiccell wall components. Such extraction typically results in separationof the microfibrillar components into concentric lamellae. Thenumber and thickness of these lamellae vary according to theage and type of cell wall. The microfibrils appear to be orientatedwithin the plane of their lamellae but the orientation may varyin successive lamellae, and in many walls the crossed polylamellatecondition was detected. The collenchyma—and the outerepidermal cell walls show an alternation of lamellae with almostvertical microfibrils with those with a practically transverseorientation. In ordinary parenchymatous walls the alternationis not so extreme and is revealed only by the occasional presenceof the ‘herring bone pattern’ in non-radial sections.As a rule the lamellae are continuous around the circumferenceof a cell though individual lamellae may vary in thickness andsometimes appear to ‘fade out’. The present observationssuggest that growth of the primary wall occurs by depositionof microfibrils in successive lamellae thus confirming the basicpremise of the multinet theory of growth.     

Electron Microscopic Observations on the Fine Structure of Cell Walls of Chlamydia psittaci

L-cell cultures were infected with elementary bodies (EB) of meningopneumonitis organisms. Cell walls were prepared from reticulate bodies (RB), which are the intracellular developmental forms into which EB are converted, and from EB at appropriate times after infection. When fragmented EB cell walls were shadowcast with platinum palladium alloy, about one-half of the fragments were seen to be composed of hexagonally arrayed structures on the inner side of the cell wall. When EB cell walls were negatively stained with phosphotungstic acid, they all showed this fine structural array. These macromolecular units were estimated to be about 18 nm in diameter. RB cell walls, harvested at various times after infection, were similarly stained; about 20% of RB walls at 15 hr after infection showed traces of these regular structures, but only 2% of them had the structures at 24 hr. When RB cell walls prepared from penicillin-containing culture were examined, they were observed to be similar to RB without penicillin. When EB cell walls were treated with formamide at 160 C, and then centrifuged in a 10 to 40% potassium tartrate density gradient, hexagonal particles about 20 nm in diameter were obtained as a middle band in the gradient column. These particles were not obtained from RB cell walls harvested from cultures with or without penicillin. It is concluded that the particles are macromolecular subunits located on the inner side of the EB cell walls, that the subunits probably provide the structural rigidity found in the EB, and that their synthesis is inhibited by penicillin.     

Differential Staining for Cellulosic and Modified Plant Cell Walls

A simple method to enhance the staining of cell wall components for fluorescence microscopy is described. In stems of Nicotiana tabacum and needles of Pinus eldarica lignin, the cuticle and unsaturated lipids are indicated by a purple-red fluorescence while pectocellulosic components fluorescc pale blue.     

The Structure of Plant Cell Walls: VI. A Survey of the Walls of Suspension-cultured Monocots

The primary cell walls of six suspension-cultured monocots and of a single suspension-cultured gymnosperm have been investigated with the following results: (a) the compositions of all six monocot cell walls are remarkably similar, despite the fact that the cell cultures were derived from diverse tissues; (b) the cell walls of suspension-cultured monocots differ substantially from those of suspension-cultured dicots and from the suspension-cultured gymnosperm; (c) an arabinoxylan is a major component (40% or more by weight) of monocot primary cell walls; (d) mixed β-1,3; β-1,4-glucans were found only in the cell wall preparations of rye grass endosperm cells, and not in the cell walls of any of the other five monocot cell cultures nor in the walls of suspension-cultured Douglas fir cells; (e) the monocot primary cell walls studied contain from 9 to 14% cellulose, 7 to 18% uronic acids, and 7 to 17% protein; (f) hydroxyproline accounts for less than 0.2% of the cell walls of monocots. Similar data on the soluble extracellular polysaccharides secreted by these cells are included.     

STUDIES ON THE OXIDATIVE METABOLISM OF SACCHAROMYCES CEREVISIAE : I. Observations on the Fine Structure of the Yeast Cell

Vegetative cells of Saccharomyces cerevisiae were fixed with potassium permanganate followed by uranyl nitrate, embedded in methacrylate, and studied in electron micrographs of thin sections. Details of the structure of the cell wall, cytoplasmic membrane, nucleus, vacuole, and mitochondria are described. Cell membranes, about 70 to 80 A thick, have been resolved into two dense layers, 20 to 25 A thick, separated by a light layer of the same dimensions, which correspond in thickness and appearance to the components of the "unit membrane" as described by Robertson (15). The cell wall is made up of zones of different electron opacity. Underlying the cell wall is the cytoplasmic membrane, a sinuous structure with numerous invaginations. The nucleoplasm, often of uneven electron opacity, is enclosed in a pair of unit membranes in which nuclear pores are apparent. The vacuole, limited by a single unit membrane, is usually irregular in outline and contains some dense material. Rod-shaped mitochondria, 0.4 to 0.6 µ in length and 0.2 to 0.3 µ in diameter, are smaller in size, but similar in structure to some of those described in plant and animal cells. Attempts to use osmium tetroxide as fixative were unsuccessful, a result similar to that obtained by other workers. It is suggested that yeast cells are impermeable to osmium tetroxide, except when grown under specific conditions.     

Structure of Plant Cell Walls: VIII. A New Pectic Polysaccharide

This paper describes the isolation and characterization of rhamnogalacturonan II, a hitherto unobserved component of the primary cell walls of dicotyledonous plants. Rhamnogalacturonan II constitutes 3 to 4% of the primary cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells. Rhamnogalacturonan II is a very complex polysaccharide yielding, upon hydrolysis, 10 different monosaccharides including the rarely observed sugars apiose, 2-O-methylxylose, and 2-O-methylfucose. In addition, rhamnogalacturonan II is characterized by the rarely observed glycosyl interconnections of 2-linked glucuronosyl, 3,4-linked fucosyl, and 3-linked rhamnosyl residues. These glycosyl linkages have never previously been detected in primary sycamore cell walls. Evidence is presented which suggests that polysaccharides similar to rhamnogalacturonan II are present in the primary cell walls of the three other dicotyledonous plants examined.     

The Structure of Plant Cell Walls: VII. Barley Aleurone Cells

The walls of barley (Hordeum vulgare var. Himalaya) aleurone cells are composed of two major polysaccharides, arabinoxylan (85%) and cellulose (8%). The cell wall preparations contain 6% protein, but this protein does not contain detectable amounts of hydroxyproline. The arabinoxylan has a linear 1,4-xylan backbone; 33% of the xylosyl residues are substituted at the 2 and/or 3 position with single arabinofuranosyl residues. The results of in vitro cellulose binding experiments support the hypothesis that noncovalent bonds between the arabinoxylan chains and cellulose fibers play a part in maintaining wall structure. It is suggested that bonding between the arabinoxylan chains themselves is also utilized in forming the walls.     

The Structure of Plant Cell Walls: II. The Hemicellulose of the Walls of Suspension-cultured Sycamore Cells

The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed “amyloid” xyloglucans.     

The Structure of Plant Cell Walls: I. The Macromolecular Components of the Walls of Suspension-cultured Sycamore Cells with a Detailed Analysis of the Pectic Polysaccharides

This is the first in a series of papers dealing with the structure of cell walls isolated from suspension-cultured sycamore cells (Acer pseudoplatanus). These studies have been made possible by the availability of purified hydrolytic enzymes and by recent improvements in the techniques of methylation analysis. These techniques have permitted us to identify and quantitate the macromolecular components of sycamore cell walls. These walls are composed of 10% arabinan, 2% 3,6-linked arabinogalactan, 23% cellulose, 9% oligo-arabinosides (attached to hydroxyproline), 8% 4-linked galactan, 10% hydroxyproline-rich protein, 16% rhamnogalacturonan, and 21% xyloglucan.     

Observations on the Fine Structure of the Gastric Parietal Cell of the Rat

The structure of the rat parietal cell was examined by electron microscopy. The intercellular and intracellular canalculi are lined by microvilli which are more numerous and larger than those of other gastric cells. The numerous mitochondria have closely packed cristae and a dense matrix containing opaque particles. The cytoplasmic vacuoles typical of parietal cells are part of a network of smooth surfaced tubules and vacuoles (the endoplasmic reticulum) which is intimately associated with the mitochondria and probably connected with the lumen of the canaliculi. Only a few dense particles are found attached to the surface of these tubules. The structure of the parietal cell is compared with that of other cells whose function also is transport of inorganic ions and water. Evidence is presented supporting the hypothesis that parietal cells differentiate from a less structurally specialized cell in the neck region of the gastric gland.     

Observations on the Structure of the Cell Walls of Valonia ventricosa and of Dictyosphaeria favulosa

The dissection of partially macerated piem of the cell wallof Valonia ventricosa is described. Laminae comprising a veryfew, or only one, of the component lamellae of the wall havebeen readily obtained. The occurrence of three directions offibrillar orientation is confirmed. In Dictyosphaeria favulosa the cell walls show three equallywell developed directions of fibrillar orientation, one forminga set of great circle meridians about the cell, so definingtwo diametrically opposite ‘poles’, and the otherstwo series of spirals, right- and left-handed, lying obliquelyto the meridians at approximately equal angles in the regionof 70°–80°. Single lamellae of Valonia walls show only one direction offibril orientation, which is parallel to the major extinctiondirection when viewed between crossed Nicol prisms. When stretchedtransversely to the fibrils these isolated lamellae open outinto networks in a characteristic fashion, indicating the presenceof some system of lateral linkages between the strands. The strands of such networks are shown by electron microscopyto be composed of aggregates of microfibrils of indefinite lengthand of diameter apparently about 100 Å. This is much lessthan the diameter (300 Å.) of similar fundamental microfibrilspreviously reported; the difference may arise from the differentmethode of preparation of the material. Electron-micrographsdo not reveal any structural basis for the lateral linkagesbetween fibrils. The mechanics of the growth of these multi-lamellate walls isbriefly considered.     

Structure of Plant Cell Walls : XXVI. The Walls of Suspension-Cultured Sycamore Cells Contain a Family of Rhamnogalacturonan-I-Like Pectic Polysaccharides

Considerable information has been obtained about the primary structures of suspension-cultured sycamore (Acer pseudoplatanus) cell-wall pectic polysaccharides, i.e. rhamnogalacturonan I, rhamnogalacturonan II, and homogalacturonan. However, these polysaccharides, which are solubilized from the walls by endo-α-1,4-polygalacturonase, account for only about half of the pectic polysaccharides known to be present in sycamore cell walls. We now report that, after exhaustive treatment with endo-α-1,4-polygalacturonase, additional pectic polysaccharides were extracted from sycamore cell walls by treatment with Na₂CO₃ at 1 and 22°C. These previously uncharacterized polysaccharides accounted for ~4% of the cell wall. Based on the glycosyl and glycosyl-linkage compositions and the nature of the products obtained by treating the quantitatively predominant NaCO₃-extracted polysaccharides with lithium metal dissolved in ethylenediamine, the polysaccharides were found to strongly resemble rhamnogalacturonan I. However, unlike rhamnogalacturonan I that characteristically had equal amounts of 2- and 2,4-linked rhamnosyl residues in its backbone, the polysaccharides extracted in Na₂CO₃ at 1°C had markedly disparate ratios of 2- to 2,4-linked rhamnosyl residues. We concluded that polysaccharides similar to rhamnogalacturonan I but with different degrees of branching are present in the walls of suspension-cultured sycamore cells.     

The Structure of Plant Cell Walls: III. A Model of the Walls of Suspension-cultured Sycamore Cells Based on the Interconnections of the Macromolecular Components

Degradative enzymes have been used to obtain defined fragments of the isolated cell walls of suspension-cultured sycamore cells. These fragments have been purified and structurally characterized. Fragments released from endopolygalacturonase-pretreated cell walls by a purified endoglucanase and the fragments extracted from these walls by urea and alkali provide evidence for a covalent connection between the xyloglucan and pectic polysaccharides. Fragments released by a protease from endopolygalacturonase-endoglucanase-pretreated cell walls provide evidence for a covalent connection between the pectic polysaccharides and the structural protein of the cell wall. Based on these interconnections and the strong binding which occurs between the xyloglucan and cellulose, a tentative structure of the cell wall is proposed.     

Structure of Plant Cell Walls : XVIII. An Analysis of the Extracellular Polysaccharides of Suspension-Cultured Sycamore Cells

The water-soluble polysaccharides (SEPS) secreted into the medium by suspension-cultured sycamore cells were examined to determine whether the polysaccharides were the same as those present in the walls of sycamore cells. The SEPS were made more amenable to fractionation by treatment with a highly purified α-1,4-endopolygalacturonase (EPG). The EPG-treated SEPS were fractionated by anion-exchange and gelpermeation chromatography. The following polysaccharides were found: xyloglucan, arabinoxylan, at least two arabinogalactans, a rhamnogalacturonan-II-like polysaccharide, and a polygalacturonic acid-rich polysaccharide. The oligogalacturonide fragments expected from EPG-digested homogalacturonan were also identified. Evidence was obtained for the presence of a rhamnogalacturonan-I-like polysaccharide. All of the above polysaccharides have been isolated from or are believed to be present in sycamore cell walls. Furthermore, all of the noncellulosic polysaccharides known to be present in sycamore cell-walls appear to be present in the SEPS.