Brigitte Vian

The Wall of the Growing Plant Cell: Its Three-Dimensional Organization

Publisher Summary This chapter summarizes successive steps involved in the formation of a growing cell wall: (1) polymerization and release of wall subunits, (2) three-dimensional assembly of the subunits in the periplasm; positioning, and cross-linking of subunits probably involving self-assembly or transmembrane control, and (3) anisotropic surface expansion of the wall implying selective loosening and sliding of the subunits when the wall is under stress. The two cellular pathways necessary for growth are (1) the exocytic route, which contributes to plasmalemma enlargement and produces the subunits of the wall and (2) the endocytic route, which contributes to tonoplast enlargement and turgor pressure regulation. In this dichotomic flow, the endoplasmic reticulum and the Golgi body play a key role. Thus, the main elements of growth regulation and wall morphogenesis, though acting at different levels in the cell, should result in a homogeneous sequential process. In the future, closer cooperation among cytologists, biochemists, and physiologists seems highly desirable for a better understanding of the characteristic modalities of plant growth.

Further observations on cell wall morphogenesis and polysaccharide arrangement during plant growth

SummaryThe three-dimensional arrangement of the polysaccharide chains in cell walls was investigated, using ultracryotomy and cytochemistry, in order to test the validity of the previously postulated “ordered fibril hypothesis” and to analyze the characteristics of the primary wall morphogenesis.Both in mung bean hypocotyl (Phaseolus aureus) and pea root (Pisum sativum) cultured in defined conditions, cell to cell endogenous specificity is marked by differences in the numbers of layers, thickness, rhythm and direction of deposition. The occurrence of bow-shaped arrangements and of strata of orientation intermediate between the main crisscrossed multifibrillar layers suggests that the sequential changes of the morphogenetic activity of the cells is progressive. The twisted polysaccharide disposition evokes certain mesomorphic states; a part of the mechanism responsible for the wall arrangement may result from a self-assembly process as in the orientation of the molecules in a liquid cristal. This possibility finds experimental support in the fact that a three-dimensional association of the hemicellulose chains spontaneously appears when precipitated in acellular conditions.Polysaccharide removal associated with shadowing indicates that the ordered disposition within the wall is extensively altered by even a slight extraction. These data may invalidate diverse results which are generally brought forward to explain the wall organization during growth.

Cellulose-glucuronoxylans and plant cell wallstructure

Abstract The plant cell wall is a composite made of cellulose microfibrils associated with matrix components, among which the xylanfamily is the most represented. The review focuses on two types of biological models that are cellulose/glucuronoxylan (GX) composites. They present both a basic helicoidal organization but their behaviours are opposite: hard model and consolidation, soft model and dispersion. Hard models correspond to stony tissues and related structures. They are permanent constructions and ensure a role of support and protection. Stone cells and endocarps are characterized by thick and ordered secondary walls showing a cholesteric-like pattern (helicoidal structure). Typical defects emerging in the stacks of microfibrils (disclinations, distortions, saddle-like figures) are a diagnostic of an actual liquid crystal behaviour under mechanical constraints. Lignification follows the helicoidal orders and provokes its hardening. Among the hard models, hardwood provides an example in which the helicoidal motion of microfibrils is intermittent and can be correlated with a preferential localization of GX. Soft models correspond to cellulosic mucilages. They are never consolidated and their behaviour is a complete dispersion. Quince mucilage presents a typical helicoidal organization which is relevant to a true cholesteric liquid crystal state. Cytochemical labelling reveals the close association between cellulose and GX, the latter constituting a strong acidic environment around the microfibrils. The importance of GX in helicoidal ordering of cellulose microfibrils is demonstrated by means of in vitro experiments. Spontaneous self-assembly of isolated mucilage components occurs in acellular conditions only if GX is present. In the last section of the review, a simple hypothesis is proposed which emphasizes that GX could play a possible key roles as well in the composite cholesteric assembly (twisting agent) as in the hardening by lignification process (host structure).

The helicoidal plant cell wall as a performing cellulose-based composite☆

The helicoidal plant cell wall can be considered as a composite in which cellulose is the constant reinforcing fiber. In order to strengthen the analogy with cholesteric liquid crystals, and taking into account a range of data, we describe a progressive series showing that cellulosic helicoidal systems are versatile and multifunctional. The following examples were considered: a) the cellulose microfibrils, with their rigid backbone possibly coated with a plastifying matrix; b) actual cholesteric cellulosic derivatives, such as in vitro liquid crystals and in vitro cellulosic mucilages; c) viscoplastic. growing cell walls; d) consolidated “stony” cell walls with their adaptation to intercellular communications. The series shows a dramatic progression from a liquid construction to what is the hardest in the plant cells, i.e. the sclerified walls.

Affinodetection of the sites of formation and of the further distribution of polygalacturonans and native cellulose in growing plant cells

Summary— The object of the present paper is to complement the cytochemical detection of the polysacharides of the plant cell wall and of its precursors, taking benefit of two kinds of affinity methods: the enzyme‐gold technique and immunocytochemistry. Cellobiohydrolase (CBH 1, EC 3.2.1.91) was used to target native crystalline cellulose and two monoclonal antibodies, JIM 5 and JIM 7, were used to target homogalacturonan sequences with various degrees of esterification. Observations were performed at the light microscope level (UV epifluorescence, enzyme‐gold silver staining) and at the electron microscope level. Two types of biological specimens, both in steady state of growth, were chosen: in vitro cultures of melon cells (thin, unidirectional primary walls, loosely associated cells), and elongating zone of mung bean hypocotyl (thick walled and tightly associated cells). The following points were examined successively: the labelling at the histological level, the detection of cellulose and of polygalacturonan components in muro, the visualization of the emerging sites of the polymers along the endomembrane flow and their post‐synthetic modifications (crystallization and methylation respectively). JIM antibodies showed the early labelling of homogalacturonans on the bulging margins of the dictyosomes. The labelled vesicles appeared as sites of polymerization, cytoplasmic transport and beginning of molecular maturation with likely an early action of methyl transferases. The first labelling of cellulose occurred only on the outer face of the plasma‐membrane. Later on, CBH 1‐gold complexes remained distributed throughout the width of the growing wall, despite the surface expansion and the dispersion of the ordered framework. No significant change of the cellulose crystallinity was noticed. A co‐localization of polygalacturonan and cellulose markers was seen from the assembly to the deassembly of the cell wall. In complement, subtractive cytochemistry was performed using PATAg in association with an endopolygalacuronase to split the pectic chains or chelators (EDTA, EGTA, oxalate) to solubilize the calcium‐connected polyuronic acid chains. All the attacks exposed the individual microfibrils of the cellulose framework revealing uniformly the helicoidal organization and confirming that cellulose and polygalacturonans remain closely associated spatially during growth.

Réactivité du plasmalemme végétal Etude cytochimique

SummarySeveral cytochemical methods converge to indicate a reactivity of the plasmalemma, which makes it different from others cytomembranes. 1. Positive and constant results are obtained with two kinds of methods. Some are performed on specimen blocks, using the binding of colloidal metals with electronegative groups; some are performed on ultrathin sections: procedure derivating from PAS reaction, and method using phosphotungstic acid at a low pH in different embedding resins (epon, araldite, methacrylate, glycol-methacrylate). 2. Rather sporadic results are obtained with other procedures, specially those which demonstrate inorganic ions.These cytochemical data tend to indicate the existence, at the level of the plasmalemma, of a kind of glycocalyx similar to that described in animal cells. It is however different in some characteristics (neuraminidase has no effect on its reactivity).After a comparison of the results with those obtained from enzymatic tests or autoradiographic studies, the idea of a special activity of the plasmalemma is enhanced.These results give some informations on the ways the differenciation of cytomembranes occurs, and on their relations during exchanges between the cell and the exterior (pinocytosis and emission of secretory vesicles).RésuméPlusieurs méthodes cytochimiques utilisées convergent pour indiquer lexistence dune réactivité du plasmalemme, qui le distingue des autres cytomembranes. 1. Des résultats positifs constants ont été obtenus avec deux sortes de méthodes. Certaines sont effectuǵes sur pièces, utilisant la capture de métaux colloïdaux par les groupements électronégatifs. Dautres sont réalisées sur coupes ultrafines: technique dérivant de lAPS et méthode utilisant lacide phosphotungstique à bas pH, dans différents milieux dinclusion. 2. Dautres méthodes fournissent des résultats plus sporadiques, en particulier les techniques de détection dions inorganiques.Ces données cytochimiques tendent à indiquer lexistence dune sorte de glycocalyx au niveau du plasmalemme végétal, évoquant celui des cellules animales. Il sen distingue toutefois par certains caractères (absence daction de la neuraminidase). La confrontation de ces résultats avec ceux obtenus après les tests dactivité enzymatique ou après des études histoautoradiographiques, confirme lidée dune organisation du plasmalemme, liée à son activité.Ces résultats fournissent des précisions sur les modalités de différenciation des membranes et sur leurs relations dans les phénomènes déchange entre la cellule et le milieu extérieur (pinocytose, émission de vésicules sécrétrices).

Organized Microfibril Assembly in Higher Plant Cells

The cell wall represents one of the most characteristic features of plant cells. Its occurrence around the protoplasts accounts for a great difference between plant cells and animal cells, the latter being considered as “naked cells” limited only by the plasmalemma and its sugar-rich coat, the glycocalyx. In plants, the thick wall enclosing the cells can be seen in two different ways that, at first sight, may appear antinomic. On the one hand, the wall is an essentially rigid formation, a skeleton responsible for the shape of the cell and for the strength of the plant. On the other hand, the wall must be plastic and extensible so that a surface increase during extension may occur.

Co‐localization of the cellulose framework and cell‐wall matrix in helicoidal constructions

Affinity methods (enzymes, lectins and antibodies used as specific probes) were applied in order to target cellulose and the major matrix components in cell walls of dicotyledons built up as helicoids. The probes were the enzyme cellobiohydrolase, CBH1, for cellulose, polyclonal antibodies and the lectin RCA (Ricinus communis agglutinin) for xyloglucans, and monoclonal antibodies JIM5 or JIM7 for homogalac‐turonan sequences (according to their degree of esterification). Observations were performed: (i) in muro and/or on heteromolecular fractions following controlled cell‐wall dissociation experiments; and (ii) at the light microscope level and/or at the electron miscroscope level by means of various visualization markers. Affinity labelling was complemented by subtractive cytochemistry and by the labelling of available anionic groups by means of cationic gold particles. Data confirm the importance of using a variety of probes, the combination of which allows the acquisition of convergent and complementary results. Concerning the particular case of helicoidal walls of elongating cells, it was shown that cellulose was always co‐localized with xyloglucans and homogalacturonan polymers in zones where the cholesteric order was well defined. Cellulose was always associated with compatible hemicellulose polymers capable of binding tightly. Moreover, residual charges were always present along the microfibrils, forming an anionic coat able to repel the adjacent cellulose microfibrils. A possible role of the heteromolecular association of xyloglucan and pectate as a surfactant allowing the cholesteric assembly is hypothesized.

La technique des coupes au froid appliquée au matériel végétal

SummaryCompared to conventional methods of ultrastructural microscopy, ultracryotomy has developed for two main intentions: to avoid the effects of all chemical treatments necessary for plastic embedding and to minimize or even omit the aldehyde fixation which inactivates most of the biological structures. Moreover, owing to the absence of plastic, all ultrastructures become directly accessible to markers or exogenous agents.Various procedures have been proposed to obtain ultrathin frozen sections mainly for animal tissues. Concerning plant cells, the difficulty due to the high water content of vacuoles is actually balanced by the existence of stiff walls which constitute a natural encapsulation of cells and give a good plasticity to tissues. A method is proposed for plant tissues and the problems which arise along the different steps are analyzed.The paper reviews the fields of application of ultracryotomy. They are discussed or illustrated from various types of tissues (algae, yeasts, higher plant cells...