B. Vian

Cellulose and pectin localization in roots of mycorrhizalAllium porrum: labelling continuity between host cell wall and interfacial material

Two different types of contacts (or interfaces) exist between the plant host and the fungus during the vesicular-arbuscular mycorrhizal symbiosis, depending on whether the fungus is intercellular or intracellular. In the first case, the walls of the partners are in contact, while in the second case the fungal wall is separated from the host cytoplasm by the invaginated host plasmamembrane and by an interfacial material. In order to verify the origin of the interfacial material, affinity techniques which allow identification in situ of cell-wall components, were used. Cellobiohydrolase (CBH I) that binds to cellulose and a monoclonal antibody (JIM 5) that reacts with pectic components were tested on roots ofAllium porrum L. (leek) colonized byGlomus versiforme (Karst.) Berch. Both probes gave a labelling specific for the host cell wall, but each probe labelled over specific and distinct areas. The CBH I-colloidal gold complex heavily labelled the thick epidermal cell walls, whereas JIM 5 only labelled this area weakly. Labelling of the hypodermis was mostly on intercellular material after treatment with JIM 5 and only on the wall when CBH I was used. Suberin bands found on the radial walls were never labelled. Cortical cells were mostly labelled on the middle lamella with JIM 5 and on the wall with CBH I. Gold granules from the two probes were found in interfacial material both near the point where the fungus enters the cell and around the thin hyphae penetrating deep into the cell. The ultrastructural observations demonstrate that cellulose and pectic components have different but complementary distributions in the walls of root cells involved in the mycorrhizal symbiosis. These components show a similar distribution in the interfacial material laid down around the vesicular-arbuscular mycorrhizal fungus indicating that the interfacial material is of host origin.

The glucuronoxylans and the helicoidal shift in cellulose microfibrils in linden wood: Cytochemistryin muro and on isolated molecules

SummaryIn fibres of wood, the classical S1 and S2 layers are connectedvia a transition zone where a helicoidal texture occurs. In order to understand the actual mechanism of cellulose microfibril rotation in this zone, the study of relationship between cellulose and matrix was undertaken cytochemically at the ultrastructural level.Glucuronoxylans,i.e., the main hemicellulose component of hardwood, were studied in cell walls of linden tree. Xylanase-gold complexes were used as a new cytochemical tool to directly and specifically label glucuronoxylans within the wall of fibres. Subtractive localization (KOH or DMSO extraction and PATAg test or shadowing) associated with chemical analysis was carried out as control. The study of isolated glucuronoxylan molecules was undertaken in parallel.Both from direct (xylanase-gold labeling) and indirect techniques (extractions), glucuronoxylans appear preferentially concentrated in the transition zone which overlaps the layers S1 and S2. A comparison between KOH and DMSO extraction indicates a difference in accessibility of glucuronoxylans distributed across the whole wall and those located in the transition zone. Isolated molecules have a rodlike aspect and show a tendancy to spatially organize in parallel alignment. Cytochemical labeling of the isolated molecules concerns covalent linkages, vic-glycol groups and acid side groups along the main chain.The preferential localization indicates that in the helicoidal zone glucuronoxylans constitute a thick matrix embedding the cellulose microfibrils in the course of rotation. This data leads to a discussion of how these localized matrix molecules could intervene in the assembly and the twisted morphogenesis of the fibre cell wall.

Wall texture in the spore of a vesicular-arbuscular mycorrhizal fungus

SummaryInGlomus epigaeum Daniels and Trappe, a vesicular-arbuscular mycorrhizal fungus, the mature spore has a complex multi-layered wall containing a regular pattern of wall subunits.The outer wall (2–4 μm thick) consists of a simple layer of parallel microfibrils. The inner wall (5–6 μm thick) is built from two layers possessing different organization. The innermost layer, near the plasmalemma has a texture of apparently dispersed fibrils, whereas the second layer is regularly organized with an arced texture. Ten to twelve bundles of fibrils connected by apparently bow-shaped fibrils are consistently observed. The appearance of this arced organization depends on the section plane and on the angle of observation in the electron microscope as confirmed by tilting experiments. Wall subunits are evident as straight electron transparent fibrils; particularly well-defined in negatively stained frozen sections: their diameter is about 3.5nm.The regular pattern of wall subunits in this fungal cell wall is compared with the textures shown by cellulose fibrils in algae or higher plants and by chitin fibrils in arthropod cuticle.

Immunocytochemical location of hydroxyproline rich glycoproteins at the interface between a mycorrhizal fungus and its host plants

SummaryWhen vesicular-arbuscular mycorrhizal (VAM) fungi colonize the cortical cells of their host plant roots, the hyphae are separated from the host cytoplasm by the invaginated host plasmalemma and interfacial material. The presence of hydroxyproline rich glycoproteins (HRGPs) at the interface was investigated with a polyclonal antibody obtained against melon callus HRGP2b. By using a combination of cytochemical methods, antigens were detected in pea, in both the presence and absence ofGlomus versiforme, a mycorrhizal fungus. For comparison, observations were performed in parallel with leek as a monocot host. Antigens were localized over the pea cell wall in root tissues. At the ultrastructural level, gold granules were mostly present in the periplasmic space. In mycorrhizal plants, the most substantial deposition occurred at the interface between the fungal wall and the host membrane. Dot blot experiments revealed HRGP2b antigens in soluble root fractions from both uninfected and mycorrhizal samples.The results demonstrate that HRGP2b antigens can be localized over the cell wall of both dicot and monocot hosts, although they mostly occur in the contact zone in infected samples. Their presence-in the company of localized glucans and pectins-means that the contact zone can be regarded as an apoplastic space presenting a structural response to the symbiotic mycorrhizal status.

Sequential patterns of intramural digestion of galactoxyloglucan in tamarind seedlings.

The structure and breakdown of galactoxyloglucan (GXG)-rich cells was studied from cotyledons of Tamarindus indicus L. The depletion of GXG was followed at different levels: quantitative, histochemical and cytochemical. At the ultrastructural level two probes were used: one general for polysaccharides (periodic acid — thiocarbohydrazide — silver proteinate test), the other specific for the terminal galactosyl residues of GXG (β-galactosidase-gold complex). They were complemented by water-extraction of the GXG and analysis of the constituting monosaccharides by gas chromatography. Despite their collenchymateous aspect and the chemical similarity of the reserve GXG with the structural xyloglucan of growing walls, the thickened storage walls are not interpretable as being an hypertrophied primary wall. The tamarind cells produce an original type of wall construction in which GXGs are sequestered in a sort of homomolecular bulk. There is no evidence for intussusception of the molecules within a network of cellulose. The bulk of GXG is sandwiched between two thin layers: the outer is comparable to a regular primary wall, the inner behaves like a barrier during GXG withdrawal. Temporal and spatial patterns of GXG-mobilisation lead to the definition of a sequence of stages of cell activities (premobilising, mobilising, postmobilising). They are synchronized with the growth of the seedling axis, the duration and characteristics of the stages being subordinated to the location of the cells within the organ. Cell lysis is initiated in close relationship with intramural cavities. The development of digestion pockets results in a highly digested wall. The barrier prevents any engulfing of the cytoplasm in the wall clefts and creates an increasing free space. The attack front of digestion is always sharp. During all steps, the monosaccharide composition remains stable. At the end of GXG depletion, the storage wall is withdrawn and cells are rendered in a parenchyma-like state. The breakdown is not a complete wall collapse but an original controlled and limited wall-thinning. The data lead to the speculation that the hydrolytic activities result from a complementation between precursors relinquished by the cytoplasm and factors already present in the storage wall.

The sustained and warped helicoidal pattern of a xylan-cellulose composite: the stony endocarp model

SummaryThe study was devoted to the microstructure of the thick walled cells of the endocarp of prune (Prunus domestica L.), cherry (Prunus cerasus L.), walnut (Juglons regia L.). The tissue is formed of closely associated cells showing a homogeneous development characterized by an intense constructive activity of ordered walls with a typically twisted pattern (cholesteric-like). The arced layers are produced in tens, each corresponding to a 180° full rotation of the molecules (axis of rotation oriented radially) and their succession gives rise to a basic regular and monotonous periodicity. On the other hand, observation of the tissue revealed the large capacity of the helicoidal morphogenesis to adjust itself under the influence of two topological contingent constraints: (1) the spherical shape (and derivated shapes) of the cell and (2) the numerous pit canals which maintain the symplastic transport and produce a recess during the construction of the wall. Spherical shape (closed surfaces) and recess both introduce additional internal strains which are relieved by deviations of the molecular array in the basic pattern (moiré and knotty aspects). Special attention was given to the defects integrated in the spherical twist. The defects emerging in the angled stacks of microfibrils (disclinations, distortions) were a diagnostic feature of an actual liquid crystal behaviour under mechanical constraints. The abundance of such defects, of cusps and spiral motions strengthened the hypothesis that a transient fluid phase, later on consolidated and stiffened, operates during the cellulose ordering. The saddle-like figures developed in the complex polylobed situation of walnut were particularly demonstrative. The fractionation of the secondary wall yielded the glucidic matrix in the same ratio as cellulose. The bulk of this embedding matrix was composed of acidic xylans more or less tightly bound to the microfibrils. The coat of negatively charged polysaccharides visualized by the binding of cationic gold to wall strips might be expected to act as a surfactant generating an electrostatic repulsion between microfibrils. This could be a cooperative mechanism for the self-positioning (aligment in sheets and progressive rotation) of the composite.

Morphogenesis of twisted cell wall: chronology following an osmotic shock

SummaryThe effect of an osmotic shock on the subsequent growth and cell wall texture was studied at 0, 1/2, 1, 2, 4 and 24 hours. Cells were taken at the beginning of their exponential growth from mung bean hypocotyl.The shock reveals the instability and the fragility of the assembly mechanisms. It induces a rupture in the texture (formation of a loose layer) or, occasionally, the apparition of a swirling pattern. After the shock, the twisting positioning can be restored. The “post-shock” deposit appears similar to the “pre-shock” deposit. The loose layer provides a visible guide-mark (time marker) within the wall. It allows one to evaluate the oscillatory period (i.e., the duration necessary for a 180° rotation of the microfibrils). This period was found to be ca. 3 hours following a lag period of ca. 1 hour. It confirms the endogenous ultradian character of the rhythm of the assembly.

Transmural exocytosis in maize root cap : visualization by simultaneous use of a cellulose-probe and a fucose-probe

SummaryThe maize root cap is a tissue known for its high production of a fucose-rich slime. At the cellular periphery, two kinds of components exist which are indistinguishable: the cell wall barrier and the slime which passes through. Two complementary probes were used, both at the light and the electron microscope level, in order to distinguish the different components. The lectinUlex europaeus agglutinin I was used as a probe targetting the slime and the enzyme cellulose 1,4-β-D-glucan cellobiohydrolase I was used to probe the cellulose framework. Both probes were used either alone or sequentially for double labeling. The cytochemical PATAg test was optionally used with the enzyme-gold complex labeling. After several technical improvements (multistep method, increase in accessibility), UeA I was used to follow the exocytic pathway of the slime from the Golgi apparatus to the exterior of the cell. The results indicate the occurrence of at least two populations of Golgi apparatus vesicles, and one is directly engaged in the transport of the fucoserich slime. The slime accumulated in pockets between the plasmamembrane and the outer tangential cell wall. The CBH I-gold complex showed the existence and the maintenance of a thin but continuous cellulosic layer, even when the cells slough. The double labeling showed the fucose-rich compounds within the cell wall. Data emphasize the role of the cell wall as a filtering barrier and a mechanical protection in the course of differentiation.

Cholesteric-like crystal analogs in glucuronoxylan-rich cell wall composites: Experimental approach of acellular re-assembly from native cellulosic suspension

SummaryMany plant cell walls are constructed according to a helicoidal pattern that is analog to a cholesteric liquid crystal order. This raises the question whether the wall assembly passes through a true but temporary liquid crystal state. The paper focuses on experiments performed from aqueous suspensions of extracted quince slime, i.e., a cellulose/glucuronoxylan wall composite that presents a helicoidal order when observed in situ, within the enlarged periplasm of the seed epidermal cells. Experiments carried out in acellular conditions showed that a spontaneous reassociation into a helicoidal order can be obtained from totally dispersed suspensions. The ultrastructural aspect of the reassembled mucilage suspension was different according to the resin used (LR White or nanoplast, a water-soluble melamin resin). It was always typically polydomain, and when an order was visible it was cholesteric-like and similar to the in situ native organization. Transition states with many imperfections expressed the difficulty of the system to reassemble in the absence of constraining surfaces. The possible intervention of glucuronoxylan (GX) in the ordered assembly of the microfibrils was checked by: (1) progressive extraction of GX by trifluoroacetic acid (TFA). The extraction was associated to a control of the fraction by analysis of uronic acid contents and observation at the electron microscope level. Extraction of GX provoked the formation of a flocculent mass, the flocculation being more intense when the TFA was more concentrated; (2) progressive change of pH in order to analyze the influence of pH on flocculation. Low pH (ca. pH 3) led also to a flocculation of the suspension, but the floc was reversibly lost after dialysis against distilled water. The results indicate the antifloc role of the GX due to the anionic charges carried by the side-chains. However, the function of GX as helper twisting agent in the cholesteric-like reassembly must not be ruled out.

On the interpretation of twisted patterns in elongating plant cell wall: Informations obtained with ultracryotomy

SummaryUltracryotomy, which gives anin situ visualization of wall subunits, was used to study the texture of elongating wall of mung bean hypocotyl (Phaseolus aureus). The criss-crossed texture of the wall was confirmed. The controversed disposition of the subunits in twisted patterns was analyzed on the basis of observations of oblique sections on which a mild extraction may be performed. Despite the twisted appearance within one sectioning plane, no curved subunits were seen running between the criss-crossed layers. The results confirmed that the appearance of arcs is an illusion due to thin sectioning of successive strata in which the orientation of subunits rotates.