Danièle Reis

Liquid crystal-type assembly of native cellulose-glucuronoxylans extracted from plant cell wall.

Summry— In numerous plant cell walls, the cellulose microfibrils are arranged in a helicoidal pattern which has been considered as an analog to a cholesteric order. Here, we report on the spontaneous helicoidal organization which occurs in acellular conditions from aqueous suspensions of cellulose. The cellulosic mucilage of mature seeds of quince (Cydonia oblonga L) was studied both in situ (pre‐release mucilage) and after water extraction and in in vitro re‐assembly (prolonged high speed ultracentrifugation, further progressive dehydration and embedding in LR White methacrylate or hydrosoluble melamine resin). The cellulosic component was characterized by the use of cellobiohydrolase (CBH1) bound to colloidal gold, and the glucuronic acid residues of the xylan matrix were characterized by the use of cationised gold. Inside the seeds, the pre‐release mucilage is mostly helicoidal, with the occurrence of more or less ordered domains, which indicate a fluid organization relevant to an actual liquid crystal state. Cytochemical tests revealed the tight association between cellulose and glucuronoxylans, the latter constituting a charged coat around each microfibril. Following the hydration of the seed, a cellulosic suspension was extracted in which microfibrils were totally dispersed. The progressive dehydration of the suspension gave rise to concentrated viscous drops. Ultrastructural observations revealed the occurrence of multidomain organization, from non‐ordered to cholesteric‐like regions, revealing that the mucilage is at the same time crystalline and liquid. This constitutes the first demonstration that liquid crystal type assemblies can arise from crystalline and biological cellulose in aqueous suspension. It strengthens the hypothesis that a transient liquid crystal state must occur during the cellulose ordering. The possible morphogenetic role of the glucuronoxylans in the cholesteric organization of the cellulose is discussed.

Probes and Microscopical Methods for the Localization of Pectins in Plant Cells

It is well accepted that the cell wall is not only a complex network of polysaccharides and other macromolecules but also an exciting dynamic structure that plays key roles in plant development, cell differentiation, and the perception of the environment (Roberts 1990, 1994; Carpita and Gibeaut 1993; Albersheim et al. 1994). As underlined by Knox (1992), the precise deposition of cell wall polymers dictates the anatomy and the form of all plants. Thus, of central importance is to elucidate the molecular arrangement of the cell wall components and to understand their roles in the wall structure and other physiological functions, describing in detail the subtle changes that occur during cell differentiation or in response to environmental stresses. This review deals with available methods to localize pectins within plant cells. We have focused our attention on pectic polymers because of their crucial importance in cell wall ontogeny and the considerable progress made in visualizing them. Our contribution does not intend to compile all the techniques for visualization of pectins. Rather, we have focused on microscopical methods that are sensitive enough to generate data at the level of a single cell and to investigate subtle changes within the pectin network.

Primary cell wall texture and its relation to surface expansion

Recent data have revealed the frequent occurrence of helicoidal and cholesteric-like structures in various primary and secondary walls. The present article deals with the texture specificity and the texture changes in the primary wall during the rapidly growing phase. First we define the characteristics of the cholesteric pattern and the mesophases and discuss two possible sources of misinterpretations: one technical, the fragility of constructions, and one biological, the gradients, differentiation, and short-lived organization. Both features explain why the liquid crystal concept has emerged recently for the growing wall. Two examples of cells showing highly oriented surface growth are considered and compared. (a) Mung bean seedlings are often used for the study of expansion. In this model the external and growth-limiting tissues have primary walls with early and transient planar twisted assemblies. The latter are progressively sheared and dispersed, and growth stops when the helicoidal pattern is completely randomized. (b) In the differentiated cell wall of collenchyma the bundles of supporting cells assemble walls in which similar planar twists are found. The helicoids are likewise randomized during the surface growth. The difference is that they are vigorously renewed and the last arced layers are no longer involved in the elongation process and persist without apparent modification of their cholesteric arrangement. Finally, we discuss the question of the intermolecular interrelations and degree of freedom of the wall components through the helicoids.

XYLANASE-MEDIATED HYDROLYSIS OF WHEAT BRAN: EVIDENCE FOR SUBCELLULAR HETEROGENEITY OF CELL WALLS

Previous work has shown that (1→4)‐β‐D‐endoxylanase‐mediated hydrolysis of wheat bran leads to solubilization of 50% of arabinoxylans. However, xylanase efficiency on the individual bran tissues is unequal because of histological and chemical heterogeneity. We describe here the results of an immunocytochemical study that is aimed at an understanding of in situ enzyme action on bran xylans at different hydrolysis kinetic stages. Two polyclonal antibodies were used, one against xylanase and the other against (1→4)‐β‐D‐unsubstituted xylopyranosyl residues, to target poorly substituted arabinoxylans. These antibodies were used on optical microscopy or transmission electron microscopy sections of xylanase‐treated and water‐treated wheat bran. After 30 min of xylanase treatment, xylanase distribution was found to be confined to the aleurone cell walls close to the endosperm, and arabinoxylan labeling had been lost. After 75 min, xylanase had progressed throughout the aleurone and had begun to attack the nucellar layer. After 24 h, the aleurone was completely lost, while some remnants of the nucellar layer were still observable. In contrast, the morphology of the pericarp and the testa was unaltered, and no xylanase labeling in these tissues was detected. Xylanase localization was correlated to the level of arabinoxylan substitution. That way, we showed that xylanase is preferentially localized and degrades poorly substituted arabinoxylans, as shown by visible subcellular heterogeneity of aleurone and nucellus walls.

Differential cell wall degradation byErwinia chrysanthemi in petiole ofSaintpaulia ionantha

SummaryErwinia chrysanthemi is a soft-rot pathogenic enterobacterium that provokes maceration of host plant tissues by producing extracellular cell-wall-degrading enzymes, among which are pectate lyases, pectin methyl esterases, and cellulases. Cell wall degradation in leaves and petiole tissue of infectedSaintpaulia ionantha plants has been investigated in order to define the structural and temporal framework of wall deconstruction. The degradation of major cell wall components, pectins and cellulose, was studied by both classical histochemical techniques (Calcofluor and periodic acid-thiocarbohydrazide-silver proteinate staining) and immunocytochemistry (tissue printing for detection of pectate lyases; monoclonal antibodies JIM5 and JIM7 for detection of pectic substrates). The results show that the mode of progression of the bacteria within the host plant is via the intercellular spaces of the parenchyma leaf and the petiole cortex. Maceration symptoms and secretion of pectate lyases PelA, -D, and -E can be directly correlated to the spread of the bacteria. Wall degradation is very heterogeneous. Loss of reactivity with JIM5 and JIM7 was progressive and/or clearcut. The primary and middle lamella appear to be the most susceptible regions of the wall. The innermost layer of the cell wall frequently resists complete deconstruction. At the wall intersects and around intercellular spaces resistant domains and highly degraded domains occurred simultaneously. All results lead to the hypothesis that both spatial organisation of the wall and accessibility to enzymes are very highly variable according to regions. The use of mutants lacking pectate lyases PelA, -D, -E or -B, -C confirm the important role that PelA, PelD, and PelE play in the rapid degradation of pectins from the host cell walls. In contrast, PelB and PelC seem not essential for degradation of the wall, though they can be detected in leaves infected with wild-type bacteria. With Calcofluor staining, regularly localised cellulose-rich and cellulose-poor domains were observed in pectic-deprived walls.

Expression of the Ferrioxamine Receptor Gene of Erwinia amylovora CFBP 1430 During Pathogenesis

Mutants of Erwinia amylovora CFBP 1430 lacking a functional high-affinity iron transport system mediated by desferrioxamine are impaired in their ability to initiate fire blight symptoms (A. Dellagi, M.-N. Brisset, J.-P. Paulin, and D. Expert. Mol. Plant-Microbe Interact. 11:734-742, 1998). In this study, a chromosomal transcriptional lacZ fusion was used to analyze the expression in planta of the E. amylovora ferrioxamine receptor gene foxR. LacZ activity produced by the strain harboring the fusion was highly induced in iron-restricted conditions and in inoculated apple leaf tissues. Microscopic observation revealed differential expression of this gene in relation to the localization and density of bacterial cells within the diseased tissue. Thus, the ability of bacterial cells to express their iron transport system in accordance with environmental conditions is likely important for disease evolution.

Distribution and Possible Morphogenetic Role of the Xylans Within the Secondary Vessel Wall of Linden Wood

The topochemistry of xylans was studied at the ultrastructural level in vessels of the wood of linden (Tilia plaryphyllos Scop.), by using an affinodetection method (xylan asegold labelling) and the cationic gold labelling for acidic charges.

IMMUNOLOCALIZATION OF A PURIFIED XYLANASE DURING HYDROLYSIS OF WHEAT STRAW STEMS

Hydrolysis of wheat stems and delignified wheat stems with a purified 20.7 kDa xylanase (EC 3.2.1.8) isolated from a Bacillus sp. yielded 18% and 70% of xylans, respectively. In order to evaluate the accessibility of the xylans and the extent of diffusion of the xylanase in wheat stems, antixylan and antixylanase polyclonal antibodies in conjunction with immunogold labeling and microscopic studies were used on stem fragments degraded by the xylanase. After 30 min of hydrolysis, the labeling of the xylanase was abundant for both lignified and delignified stems and occurred within all cell walls, demonstrating a fast penetration of xylanase in stem fragments. However, the distribution of immunolabel differed between the two types of straw. Xylanase was unevenly distributed in nondelignified straw, especially around the lumen side of cell walls and often grouped in clusters but homogeneous within delignified straw. Localization of xylans in both stem fragments showed that xylans were distributed homogeneously within the entire cell walls. These results indicated that xylans were accessible to the xylanase. The presence of lignins did not prevent, but modulated, the diffusion of the xylanase into stem fragments.