G., 1970: Ultrastructural aspects of cell wall synthesis inSpirogyra.
G.: Ultrastructural aspects of cell wall synthesis in Spirogyra.
Ultrastructural aspects of cell wall synthesis in Spirogyra.
To determine the approximate, if not the precise, conditions under which cellulose synthesis takes place in the cell it is important to demonstrate cellulose synthesis in vitro using purified or partially purified cellular components. This is not unlike any other cellular reaction being mimicked in vitro; however, demonstration of in vitro synthesis of cellulose using extracts from plants has been notoriously difficult. Numerous reasons can be cited for the difficulties in determining cellulose synthase activities from plant extracts, not the least of which is the large amount of callose (β-1,3-glucan) being produced under most reaction conditions (). Other reasons for failure in determining synthesis of cellulose in vitro from plant extracts could just be that the proper reaction conditions have not yet been determined and the difficulty in characterizing the in vitro cellulose product. At this point it has to be mentioned that in vitro cellulose synthesis has been routinely achieved using membrane preparations and detergent-solubilized proteins (including partially pure preparations) from the bacterium A. xylinum (). Whereas the cellulose produced in vitro generally is obtained as cellulose II, cellulose I microfibrils have also been observed under specific conditions. Although in vitro cellulose products using cell-free preparations of A. xylinum were described in 1958 (), conditions for obtaining high rates of cellulose synthesis in vitro were not defined until a much later date (). As we now know, these conditions allowed the formation of the activator c-di-GMP (). By manipulating the use of detergents and reaction conditions, in vitro cellulose synthesis was demonstrated using extracts from cotton fibres (; ; ), mung bean (), blackberry () and cell suspension cultures of hybrid aspen (). Although callose still makes a large part of the in vitro product, were able to separate the callose synthase activity from cellulose synthase activity by native gel electrophoresis. However, no conclusive evidence regarding the similarity or differences between callose synthase activity and cellulose synthase activity could be obtained by analyzing the polypeptide composition in these two fractions. In certain cases, the cellulose I microfibrils obtained in vitro were shown to have dimensions similar to microfibrils obtained from primary cell walls (), suggesting that synthesis of native cellulose microfibrils can be mimicked in vitro. Interestingly, the same cellulose product (cellulose I) is obtained when cellobiosyl fluoride and cellulase are used in an in vitro reaction (). As with A. xylinum, no unique effector has so far been identified for modulating in vitro cellulose synthase activity in vascular plants (). Hopefully, identification of this missing link would allow determination of the optimal conditions under which cellulose synthesis occurs not only in vitro but also in plant cells.
Filaments ofSpirogyra were fixed in 2% osmium tetroxide dehydrated in alcohol and embedded in Araldite. The fine structure of cells with regard to wall synthesis was studied. The cell wall was shown to have four layers. The inner one contains microfibrils and is considered to be the cell wall proper. The outer three layers are components of the slime layer. The innermost of these, the second layer of the wall, was shown to be between 1μm to 3μm and the third 0.3μm to 1μm. The fourth layer appears as no more than a dark black line measuring 10 nm across. In the cytoplasm two types of vesicles were seen. The largest of these has contents similar in appearance to the slime layer of the wall. This same material was also seen in the large vesicles attached to the Golgi bodies. It is suggested that the smaller vesicles are derived from the larger vesicles and later fuse with the cell membrane. The Golgi bodies were found to be fairly large measuring up to 5μm across. Small electron opaque blobs and flecks on the outside of the plasmalemma and in between the microfibrils of the cell wall proper are considered to be mucilage droplets travelling to the slime layer. It cannot be excluded that some of the material of the large vesicles is released directly into the cytoplasm and is transferred without vesicles through the plasma membrane. The negative contrast appearance of the microfibrils seen in the cell wall is thought to be due to the spaces between them being filled with this electron opaque mucilage.
Ultrastructural aspects of cell wall ..
One of the most important and intriguing aspects of plant cell wall ultrastructure is the orientation of the cellulose microfibrils within the wall (Albersheim, 1965). Cellulose microfibrils are deposited (or possibly synthesized) at the inner surface of the wall, adjacent to the plasmalemma. They have a particular orientation when deposited. On the other side of the plasmalemma there is a thin layer of microtubules. The orientation of the microtubules in this layer exactly parallels the orientation of the cellulose microfibrils being deposited on the opposite side of the plasmalemma (Newcomb, 1969). Disruption of the microtubules (e.g. by colchicine) affects the orientation of the microfibrils.
A number of arabidopsis mutants altered in their growth and development have been characterized, and changes in some of them are related to the decreased amount of cellulose produced in these mutants (see ). In these strains, mutations are observed in genes predicted to have a role in cellulose biosynthesis, including those that encode cellulose synthase. Gene expression of CesA genes in different tissues, developmental stages and under different environmental conditions has been analysed in a number of plants including arabidopsis (), maize () and hybrid aspen (). In most cases, no significant differences have been observed in the expression of the different CesA genes in different tissues. However, different groups of genes are co-expressed in cells that synthesize cellulose in the primary cell wall versus those that are active in the synthesis of cellulose in the secondary cell wall. A relationship between these genes has also been obtained from mutant analysis as well as phylogenetic analysis. In arabidopsis, AtCesA1, AtCesA3 and AtCesA6 are proposed to be required for primary cell wall cellulose synthesis (; ; ) and AtCesA4, AtCesA7 and AtCesA8 are proposed to be required for secondary cell wall cellulose synthesis (). Similar sets of genes have also been identified in other plants (). These observations have led to the suggestion that three different CesA gene products may be required for the formation of a functional rosette TC in plants (). Although the three different CesA gene products encode cellulose synthase, they are non-redundant. A mutation in any one results in the loss of cellulose microfibril formation. Hypothetical models showing the arrangement of the different CesA sub-units have been proposed, but as yet there is no experimental evidence as to how the different CesA sub-units are arranged in the rosette TC (). Rosettes associated with cellulose microfibrils have a six-fold symmetry and each particle in the rosette is believed to contain six CesA sub-units allowing for an assembly of 36 CesA sub-units in a rosette. The number of CesA sub-units in a rosette is predicted from the number of glucan chains present in a cellulose microfibril. Interaction between the three cellulose synthases (AtCesA4, AtCesA7 and AtCesA8) that are required for cellulose synthesis in the secondary cell wall has been demonstrated (). Moreover, the interaction between the different cellulose synthase sub-units to give rise to a multimeric rosette structure has been suggested to take place via intermolecular disulfide bridges formed in the N-terminal zinc finger regions of cellulose synthases (). At this point it is important to consider that only a part of the rosette structure is exposed to the extracellular side of the plasma membrane with a significantly larger proportion of this complex being present in the cytoplasm ().
CONJUGATION IN SPIROGYRA, Journal of Phycology | …
Knowledge concerning microtubules has been derived mainly from studies involving animal tissues or cells of lower plants. Elucidation of the roles of microtubules in higher plant cells has not proceeded so rapidly. Ultrastructural investigations have confirmed the ubiquity of the organelle and have shown its oriented presence to be correlated with a variety of processes. While it is clear that microtubules are involved in various stages of cell division, their precise role in the earlier events which predict the plane of cleavage is unclear. Similarly, their functioning in other aspects of plant development, including cell wall growth and differentiation, remains conjectural. There are, of course, practical difficulties in studying microtubules in the cells of higher plants. Such problems vary from the general, i.e., lack of tissue specialization and presence of a vacuole and cell wall, to the more specific paucity of microtubules in the meagre protoplasm of the differentiated higher plant cell. In addition, there would seem to be a problem in the resistance of certain plant cytoplasmic microtubules to depolymerizing agents. Since this includes, in some cases, stability to such physical agents as temperature and pressure, it would seem that it is a real property of the microtubule rather than an indirect effect of, say, cellular impermeability towards a drug. This property may be related to the stability of flagellar micro-
A further important aspect of hormonal effects on plant cell wall biosynthesis and differentiation has been revealed by studies on the changes in microtubule orientation caused by exogenously supplied hormones (Shibaoka, 1974). In bean epicotyl segments, kinetin inhibits elongation and promotes a thickening or lateral expansion of the cells. Gibberellins, on the other hand, promote elongation and inhibit lateral expansion. The microtubules adjacent to the plasmalemma in cells of epicotyl sections supplied with kinetin and auxin are found to be oriented parallel to the cell axis. However, in sections supplied with gibberellin and auxin the microtubules are oriented transverse to the cell axis. The microtubules are randomly oriented in sections supplied with auxin alone. Thus, kinetin and gibberellins (but not auxins) appear to be able to control the direction of cell growth by somehow determining the orientation of the microtubules. The orientation of the microtubules, in turn, determines the orientation of the cellulose microfibrils being deposited in the cell wall—which determines the direction in which the walls can most easily expand. (See Chapter 13 for further discussion of hormone action.)
Ultrastructural changes in Spirogyra submargaritata growing ..
An ultrastructural study of Spirogyra
The cell ..
Interactions among Cytoplasm, Endomembranes, and ..
contraction of the male gamete from its cell wall and may be responsible ..
Ultrastructural observations of cell wall ..
wall thickening in differentiating cambial derivatives ..
Cellulose is often referred to as the most abundant macromolecule on earth () and most of the cellulose is produced by vascular plants. Apart from these plants, cellulose synthesis also occurs in most groups of algae, the slime mold Dictyostelium, a number of bacterial species (including the cyanobacteria), and tunicates in the animal kingdom. Cellulose is an extracellular polysaccharide and, with the exception of bacteria and the tunicates, it is part of the cell wall in plants, algae and Dictyostelium. The function of cellulose in these different groups of organisms reflects the diverse roles associated with this simple structural polysaccharide. Whereas it is possible for some of these organisms, specifically bacteria, to survive in the absence of cellulose synthesis, it may not be true for most vascular plant cells to survive in the absence of cellulose synthesis. As such, the importance of cellulose in the life of a plant cannot be overemphasized since it not only provides the necessary strength to resist the turgor pressure in plant cells but also has a distinct role in maintaining the size, shape and division/differentiation potential of most plant cells and ultimately the direction of plant growth (). In the authors' view, deposition of cellulose microfibrils in a specific orientation for determining the direction of plant cell elongation, in a sense, is a stage of commitment akin to the S phase and M phase in eukaryotic cell cycle. Once the cellulose microfibrils are ordered in a specific orientation, the direction of cell elongation is essentially fixed. There are a very large number of questions related to cellulose biosynthesis that need to be addressed; however, at this point it is important to recognize that after a long hiatus there is an exponential increase in the number of research articles that discuss the molecular aspects of cellulose biosynthesis, and many of these advances have been made with the identification of genes, specifically for cellulose synthases, and cellulose-deficient mutants in plants. A comprehensive view of cellulose synthesis and the plant cell wall is provided in reviews by , , and . Excellent articles on individual topics related to cellulose biosynthesis are provided in a special recent issue of the journal Cellulose (Vol. 11, no. 3/4, September/December 2004). In this review, selective topics in cellulose biosynthesis will be discussed with the goal of providing a timely and unique view of this rather exciting field of study from the authors' perspective.
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