Tuatara: Volume 19, Issue 1, November 1971
Plant Cell Wall Structure and Cell Wall Growth
Plant Cell Wall Structure and Cell Wall Growth
The plant cell wall is a product of protoplasmic activity and in the higher plants its development begins with the formation of the cell plate, immediately after nuclear division. This thin cell plate quickly acquires the form of a primary cell wall, which is defined by Wardrop (1962) ‘as the structure which encloses the protoplasts during the period of cell enlargement’. Once the period of cell enlargement is over the cell wall becomes thickened to become the secondary wall. The secondary wall is regarded as the structural component of the plant (that is the plant skeleton). Such walls are also the major components of the conducting vessels.
The interrelationships of the protoplast and the cell wall, and the processes whereby the former maintains and extends the structure of the latter, thus present fundamental botanical problems which have been of interest for more than a hundred years.
Cell Wall Constituents
The principal constituents of the cell wall are cellulose, hemi-cellulose, pectic substances, lignin and proteins. Waxes, cutin, suberin and sporopollenin are also found.
(a) Structural Components
Cellulose: This is the most abundant substance in the plant kingdom, and is a polymer of -b-glucose residues joined in long chains by 1-4 links. Over certain parts of their length these chains lie parallel to each other and are very regularly spaced, to form long crystalline microfibrils. These microfibrils may be arranged randomly or in a regular fashion (Probine, 1963). Meyer and Misch (1937) proposed the ‘unit cell’ of cellulose. They considered that the cellulose chains lie antiparallel in such a way that alternate chains point in opposite directions. Within the microfibrils themselves are smaller units, the micelles, which are small aggregations of cellulose molecules that lie parallel to one another and thus confer a crystalline structure upon the microfibrils. More recently it has been claimed that the ultimate structural units of the cell wall are elementary fibrils about 35 A in diameter which are not aggregated into larger strands (Muhlethaler, 1967). The microfibrils are necessary to bear the stress in the wall due to turgor pressure.page 44
(b) Amorphous Components
The Pectic Substances: These consist of polymers of d-galacturonic acid, l-arabinose, d-galactose and l-rhamnose. These substances are found mainly in the middle lamella of primary walls. (Stace, 1963.)
Northcote (1969) has studied the metabolic changes which occur in the pectic substances deposited in the wall. He showed that the strongly acidic polygalacturonic acid was formed first. He found that pectin in the cell walls of a mature tissue such as in apple fruit was composed mainly of neutral and weakly acidic material and only traces of the strongly acidic polymer could be detected.
Hemi Celluloses: These are amorphous and consist of linear or branched polymers of d-xyloses, d-galactose, d-mannose, l-arabinose, and l-rhamnose. In contrast to cellulose the hemicelluloses are not crystalline in their natural condition although they have been found in a crystalline state after extraction (Roelofsen, 1959).
The distinction between the hemicelluloses and the pectins is not absolutely one of chemical constituents, but rather of physical properties, mainly solubility, which depends on their degree of methylation, cross-linkage and the extent to which different sugars are joined in the same molecule.
Lignin: This is an amorphous substance which occurs as an incrustation between cellulose microfibrils. The concentration is highest in the middle lamella and falls off towards the lumen. It is an important structural material and it is this substance that gives strength to wood. page 45 Because of this deposition of lignin between the existing cellulose framework there is always a swelling of the cell wall during lignification.
Protein: Recent work has demonstrated the occurrence of a group of proteins containing hydroxyproline in the primary walls of various tissues. The amount present increases during growth and it is thought that the protein may serve enzymatic as well as structural functions.
The primary and secondary walls differ in their chemical composition and in fine structure. In most cases the secondary walls have a higher percentage of cellulose and lignin while the pectic substances are present only in trace quantities as compared to the primary walls.
Thus the general cell wall consists of the structural components of the wall, the cellulose microfibrils, orientated in the amorphous component comprising the pectic substances, hemicelluloses, lignin and the protein substances.
Biosynthesis of Cell Wall Material
The endoplasmic reticulum, Golgi bodies and microtubules have all been implicated in the synthesis and organised deposition of the material of the plant cell wall on the basis of their association in electron micrographs with developing cell walls (Abersheim, 1965). Direct chemical evidence of a role for these organelles in the synthesis of wall materials is almost entirely lacking except for a demonstration by Northcote and Picket-Heaps (1966) that pectic substances are formed in the Golgi apparatus and are transported to the cell wall in the Golgi vesicles.
The possible role that the endoplasmic reticulum plays in the mechanism for laying down cell walls has been investigated but as yet ribosomes have not been identified in electron micrographs of the cell walls. However, Kivilaon et al. (1959) have detected trace amounts of ribosomal RNA (rRNA) in a preparation of cell walls of Zea mays coleoptiles. Phetheon et al. (1968) considered that the rRNA found in Zea mays was bacterial contamination, but they have isolated highly purified preparations of cell walls from a number of plants and have found that RNA was invariably present.
The possible role of rRNA in the cell wall may be concerned with the synthesis of protein in situ (Albersheim, 1965). However, Phetheon et al. (1968) consider that protein synthesis requires such an elaborate system of enzymes and nucleic acids that it is unlikely that such a synthesis would occur in the wall. The process of biosynthesis and orientated deposition of cellulose fibrils is considered by Ben-Hayyim and Ohad (1965) to consist of four steps: (a) polymerisation (of the activated monomeric precursor) to form a cellulose molecule of high molecular weight; (b) transport of the molecule from the site of synthesis to that of crystallisation; (c) crystallisation or fibril formation; (d) orientation of fibrils during deposition.page 46
These steps are not necessarily isolated from one another. Frei and Preston (1961) believe the polymerisation, fibril formation and orientation occur simultaneously in plants. They propose that a catalytically active protein moves around the cell membrane and deposits the formed cellulose fibrils in pre-determined directions, the whole process being under genetic control.
Preston and Goodman (1968) considered the hypothesis of end synthesis of microfibrils through ordered enzyme-complex granules as a satisfactory explanation for the organisation and biosynthesis of the microfibrils. They suggested that the cytoplasmic surface is covered by layers of granules in cubic packing each containing cellulose synthesising enzymes. When these granules come into contact with a microfibril end they are stimulated and start synthesising the microfibril. The microfibrils are maintained straight both by intra-chain hydrogen bonding and mutual orientation of the granules. The packing of the granules are such that they would restrict the direction of growth of the microfibrils in such a way that only two directions at right angles to another and an occasional departure along the diagonals to these directions would be possible. It is considered that this hypothesis may explain the observations of Setterfield and Bailey (1958) that cellulose synthesis can occur in outer wall layers away from the cytoplasm, as the granules which come to be buried in the wall could continue to synthesise cellulose. Where or how the microfibril ends are produced is not known; however it is proposed that they could be produced by enzyme degradation of the cellulose or by stresses in the wall during cell elongation.
Frey-Wyssling (1962) has suggested that extrusions of the plasmolemma inside the cell wall are responsible for the synthesis and orientation of cellulose fibrils in plants.
Opposed to this is evidence that the synthesis, transport and crystallisation may be separate processes in bacterial systems (Ohad et al., 1962) and probably in some plants (Setterfield and Bayley, 1968). Many workers still regard the process of cellulose fibril orientation in the secondary wall of plant cells as a passive one, rearrangement being the result of mechanical strain or stress (Roelofsen and Houwinh, 1953). Northcoate (1963) noted that the synthesis of material and the extent of enlargement of the cell wall during development can be influenced by nutrition of the growing cell. Factors affecting growth are thus important in affecting the structure and growth of the cell wall.
The microfibrils are orientated in various ways in the cell wall, usually more regularly in the secondary wall. In the primary wall the microfibrils are often orientated in a direction transverse to the page 47 long axis. They become arranged more longitudinally during growth of the cell as the subsequent wall layers are formed. This transition is gradual and the change in direction of the microfibrils in successive wall layers may be about 120° (Cutler, 1969). For example in cotton root hairs there is a gradual transition from an approximately axial orientation of microfibrils on the outer surface through a central region of crossed microfibrils to the transverse orientation of the inner layer (Roelofsen, 1959). Also in the cells of different regions of a developing organ the wall may show differential orientations of the microfibrils. In the root of Allium cepa the cell walls of the apical initials show a loosely woven mesh of microfibrils; in slightly older cells the microfibrils are mainly aligned horizontally, and this is the case during active elongation.
Jensen (1961) revealed that in the onion root these changes in cell structure can apparently be correlated with changes in the selective amount of cell wall components both in cells of different stages of development, i.e. at different distances from the root tip and in cells of different tissues at any one level. In particular the transitional region between radial enlargement and rapid elongation of the root is characterised by changing relationships between the wall components.
Ben Hayyim and Ohad (1965) have suggested a possible model for the orientation of microfibrils in the cell wall. They introduced a synthetic cellulose called Ma-carboxymethyl cellulose (CMC) into the microfibrils. This, they considered, played a similar role to that of charged polysaccharides. The parallel orientation of fibrils containing CMC could be explained partially in terms of charged interactions and mechanical stress. Non-charged cellulose is considered to form a random mesh, the rigidity of which would depend on the number of interfibriller cross links, i.e. H bonds. Electrostatic repulsion between fibrils containing CMC can weaken the interfibriller links occurring in the vicinity of adjacent carboxyls. Stress or strain applied to this kind of mesh could result in an easier deformation of the matrix in the direction of the applied force. The most stable state would be achieved when the fibrils are parallel and the overall numbers of links formed between fibrils exceed the number of weak bonds due to electrostatic repulsion. From a similar consideration it is conceivable that a second layer of fibrils formed above the first would reach a more stable position when oriented at an angle with the first, when the repulsion between the layers would be minimal.
Growth of Cell Wall
Formerly two theories were held regarding the method of cell wall growth in thickness: that of growth by intussusception, where new microfibrils were held to be laid down between existing microfibrils and that of growth by apposition where new microfibrils were laid down on top of the existing ones, forming a new layer.page 48
It is now considered that the formation of both primary and secondary cell walls occurs principally by the mechanism of apposition (Muhlethaler, 1961). It is probable, however, that some growth by intussusception does occur. Deposition of cellulose uniformly over the whole surface of the cell has been demonstrated by the use of the radio-active isotope C14. This was incorporated into the whole length of the primary cell wall (Roelofsen, 1959).
With respect to longitudinal growth the theory now most widely held is the multi-net theory of cell growth (first proposed by Roelofsen and Houwinh, 1953). This accounts also for the observed orientation of the microfibrils in successive layers of the wall. In this theory microfibrils are first deposited transversely to the long axis of the cell, and this layer is later pushed outwards as a result of the formation by apposition of a layer internal to it. During cell elongation the first formed layers of microfibrils are stretched and thus become orientated in a progressively more longitudinal plane.page 49
Recent studies of cell wall formation in fibres and tracheids using the electron microscope and also the technique of autoradiography whereby the path of a radio-active isotope is followed are consistent with the multi-net theory of cell wall growth (Wardrop et al., 1965).
As in early work with the primary cell walls, labelled carbon was found to be deposited more or less uniformly over the secondary wall fibres and tracheids. In fibres, however, the formation of the secondary wall may begin near the centre of the cell and progress towards the tips. The wall is thus thicker near the centre (Cutter, 1969). In some cells, e.g. root hairs, pollen tubes, tracheids and fibres, growth occurs only at the tip (Roelofsen, 1965). This tip growth is regarded as a localised type of multi-net growth. The role of determining whether the wall of the whole cell will grow or only a localised part of it, as for example in root hairs or stellate parenchyma cells, is attributed to the cytoplasm (Roelofsen, 1965).
These are formed after cell expansion has stopped. The secondary walls, particularly of fibres and tracheids, show microscopic layering. The microscopic layers are commonly known as the S1 (outer), S2 (middle) and S3 (inner). The S3 layer is usually thinner than either the S1 or S2 and may be absent altogether. The S1 layer normally consists of four submicroscopic lamellae, alternate ones having microfibrils in opposed helices. The S2 layer, the middle of the secondary wall, consists of numerous lamellae in which the orientation of the microfibrils is at only a small angle to the long axis of the cell. There seems to be a tendency for the microfibrils of this very thick and conspicuous wall layer to be aggregated into macrofibrils. The S3 layer is always poorly developed in contrast to the S2 layers and there is evidence too that the S3 layer may differ chemically in some way from S1 and S2 (Clowes and Juniper, 1968).
The hypothesis that the Golgi apparatus is involved in the bio-synthesis of the amorphous components of the cell wall and the ordered granules in the biosynthesis and orientation of the cellulose microfibrils is now supported by a great deal of evidence and accords well with the known geometrical requirements of the cell wall.
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