Alain Jauneau

A plasma membrane-bound putative endo-1,4-β-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis

Endo‐1,4‐β‐D‐glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non‐crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane‐anchored member of the EGase family, which is highly conserved between mono‐ and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane–cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark‐grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR‐mRNA levels. However, reduced KOR‐mRNA levels were observed in det2, a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose–hemicellulose network in the expanding cell wall.

Altered pectin composition in primary cell walls of korrigan, a dwarf mutant of Arabidopsis deficient in a membrane-bound endo-1,4-β-glucanase

Abstract.Korrigan (kor) is a dwarf mutant of Arabidopsis thaliana (L.) Heynh. that is deficient in a membrane-bound endo-1,4-β-glucanase. The effect of the mutation on the pectin network has been studied in kor by microscopical techniques associated with various probes specific for different classes of pectic polysaccharides. The localisation of native crystalline cellulose was also examined using the cellobiohydrolase I-gold probe. The investigations were focused on the external cell walls of the epidermis, a cell layer that, in a number of plant species, has been shown to be growth limiting. Anionic sites associated with pectic polymers were quantified using the cationic gold probe. Homogalacturonans were quantified using polyclonal anti-polygalacturonic acid/rhamnogalacturonan I antibodies recognising polygalacturonic acid, and monoclonal JIM7 and JIM5 antibodies recognising homogalacturonans with a high or low degree of methyl-esterification, respectively. Rhamnogalacturonans were quantified with two monoclonal antibodies, LM5, recognising β-1,4 galactan side chains of rhamnogalacturonan I, and CCRCM2. Our results show a marked increase in homogalacturonan epitopes and a decrease in rhamnogalacturonan epitopes in kor compared to the wild type. A substantial decrease in cellobiohydrolase I-gold labelling was also observed in the mutant cell walls. These findings demonstrate that a deficiency in an endo-1,4-β-glucanase, which is in principle not directly implicated in pectin metabolism, can induce important changes in pectin composition in the primary cell wall. The changes indicate the existence of feedback mechanisms controlling the synthesis and/or deposition of pectic polysaccharides in primary cell walls.

Methyl-esterification, de-esterification and gelation of pectins in the primary cell wall

Abstract This paper deals with the enzymes controlling the extent and pattern of methylesterification in pectins within the primary walls of plant cells. It also reviews the consequences of methyl-esterification for gel formation within the cell wall and for the resistance of the wall to mechanical stress. Methyl ester groups are added to pectic galacturonans by pectin methyltransferase (PMT) enzymes during pectin synthesis. Later, within the cell wall, some methyl esters may be removed by pectin methylesterase (PME) enzymes. These enzymes activities were examined in three systems, in each of which growing or dividing cells were compared with inactive cells: suspension-cultured cells of flax, mung bean hypocotyls and poplar cambium. In each system the pectins in the walls of actively growing or dividing cells were more highly methyl-esterified than those of inactive cells. Microsomal PMT activity was characterized throughout the growth cycle of suspension-cultured flax cells. The total PMT activity was maximal during the phase of rapid growth and declined when growth ceased. It was stimulated by exogenous pectins, the optimum type depending on pH. Several basic, neutral and acidic isoforms were solubilised. A number of PME isoforms were present in all three plant systems. In munge bean hypocotyls and poplar stems, neutral isoforms predominated in active cells and basic isoforms in non-growing cells. Three mung bean PMEs were characterised and the most basic one sequenced. It is suggested that as the cells pass beyond the stage of active growth, the pectins are less methyl-esterified at the point when they are exported into the wall and causing stronger bounding of the basic PME isoforms which predominate at that growth stage. The interactions of non-esterified pectic carboxyls with cations control the gelation of pectins and the mechanical properties of the gels. A new ‘cable’ model is presented for the structure of calcium pectate gels at the high concentrations typical of cell walls. The cable model is based on conformational analysis of galacturonans by solid-state NMR, and incorporates not only the accepted 21 helical ‘egg-box’ structures but also 31 helical and intermediate regions. Similar NMR experiments on cell walls revealed still more complex gels with methyl-esterified chain regions participating in both junction zones and inter-junction segments. Because of this structural complexity the stability of cation binding covers a wide range within and between cell walls. The chelating agents most often used to extract pectins have a high enough affinity for calcium ions to remove them completely from cell walls, although imidazole is weaker than the rest. SIMS microscopy of flax hypocotyls showed that calcium ions, bound to low-ester galacturonan segments, were concentrated in the epidermal cell walls particularly at the tricellular junctions. Since the tricellular junctions are stressed by turgor pressure and contain only these pectins, they are a good example of in muro load-bearing by mechanically strong pectate gels. However low-ester pectins with a high affinity for calcium ions also appear to stiffen the Vigna epidermal cell wall longitudinally and may contribute to the cessation of growth as the hypocotyl matures.

Immunolocalization of β-(1→4) and β-(1→6)-D-galactan epitopes in the cell wall and Golgi stacks of developing flax root tissues

SummaryMost cell wall components are carbohydrate including the major matrix polysaccharides, pectins and hemicelluloses, and the arabinogalactan-protein proteoglycans. Both types of molecules are assembled in the Golgi apparatus and transported in secretory vesicles to the cell surface. We have employed antibodies specific to β-(1→6) and β-(1→4)-D-galactans, present in plant cell wall polysaccharides, in conjunction with immunofluorescence and electron microscopy to determine the location of the galactan-containing components in the cell wall and Golgi stacks of flax root tip tissues. Immunofluorescence data show that β-(1→4)-D-galactan epitopes are restricted to peripheral cells of the root cap. These epitopes are not expressed in meristematic and columella cells. In contrast, β-(1→6)-D-galactan epitopes are found in all cell types of flax roots. Immunogold labeling experiments show that both epitopes are specifically located within the wall immediately adjacent to the plasma membrane. They are also detected in Golgi cisternae and secretory vesicles, which indicates the involvement of the Golgi apparatus in their synthesis and transport. These findings demonstrate that the synthesis and localization of β-(1→4)-D-galactan epitopes are highly regulated in developing flax roots and that different β-linked D-galactans associated with cell wall polysaccharides are expressed in a cell type-specific manner.

Galactans and cellulose in flax fibres: putative contributions to the tensile strength

The proton spin-spin relaxation time, T2, measured from solid-state NMR, indicates a greater rigidity for cellulose than for the adhesive matrix between the microfibrils of flax ultimate fibres. Cytochemical and biochemical analyses allow the identification of: (1) EDTA-soluble RG I-polymers in the primary walls and cell junctions of fibres; (2) long 1 --> 4-beta-D-galactan chains between primary and secondary wall layers; and (3) arabinogalactan-proteins throughout the secondary walls. These polymers in the adhesive matrix between microfibrils and/or cellulose layers ensure that cracks propagate along the matrix rather than across the fibres and play an important role in allowing flax fibres to approach the tensile strength of advanced synthetic fibres like carbon and Kevlar.

Micro-heterogeneity of pectins and calcium distribution in the epidermal and cortical parenchyma cell walls of flax hypocotyl

SummaryPectic polysaccharides are major components of the plant cell wall matrix and are known to perform many important functions for the plant. In the course of our studies on the putative role of pectic polysaccharides in the control of cell elongation, we have examined the distribution of polygalacturonans in the epidermal and cortical parenchyma cell walls of flax seedling hypocotyls. Pectic components have been detected with (1) the nickel (Ni2+) staining method to visualize polygalacturonates, (2) monoclonal antibodies specific to low (JIM5) and highly methylesterified (JIM7) pectins and (3) a combination of subtractive treatment and PATAg (periodic acid-thiocarbohydrazide-silver proteinate) staining. In parallel, calcium (Ca2+) distribution has been imaged using SIMS microscopy (secondary ion mass spectrometry) on cryo-prepared samples and TEM (transmission electron microscopy) after precipitation of calcium with potassium pyroantimonate. Our results show that, at the tissular level, polygalacturonans are mainly located in the epidermal cell walls, as revealed by the Ni2+ staining and immunofluorescence microscopy with JIM5 and JIM7 antibodies. In parallel, Ca2+ distribution points to a higher content of this cation in the epidermal walls compared to cortical parenchyma walls. At the ultrastructural level, immunogold labeling with JIM5 and JIM7 antibodies shows a differential distribution of pectic polysaccharides within cell walls of both tissues. The acidic polygalacturonans (recognized by JIM5) held through calcium bridges are mainly found in the outer part of the external wall of epidermal cells. In contrast, the labeling of methylesterified pectins with JIM7 is slightly higher in the inner part than in the outer part of the wall. In the cortical parenchyma cells, acidic pectins are restricted to the cell junctions and the wall areas in contact with the air-spaces, whereas methylesterified pectins are evenly distributed all over the wall. In addition, the pyroantimonate precipitation method reveals a clear difference in the Ca2+ distribution in the epidermal wall, suggesting that this cation is more tightly bound to acidic pectins in the outer part than in the inner part of that wall. Our findings show that the distribution of pectic polysaccharides and the nature of their linkages differ not only between tissues, but also within a single wall of a given cell in flax hypocotyls. The differential distribution of pectins and Ca2+ in the external epidermal wall suggests a specific control of the demethylation of pectins and a central role for Ca2+ in this regulation.

Immunogold localization of pectin methylesterases in the cortical tissues of flax hypocotyl

SummaryPectin methylesterases (PMEs, EC 3.1.1.11) catalyse the deesterification of pectins. Up to now, most information concerning their location was obtained from biochemical analyses. Taking advantage of specific anti-PME antibodies, we report the precise localization of PMEs at the electron microscopy level within the different cortical tissues of flax hypocotyl. Quantitative data on the densities of immunolabelling have been collected, using anti-PME antibodies as well as JIM5 and JIM7 monoclonal antibodies. Our findings show a co-localization of PMEs and acidic pectins (as revealed by JIM5 antibodies) within specific cell wall microdomains. Moreover, PME epitopes are associated with the cellular membranes, particularly with the plasmalemma.

Endopolygalacturonase‐gold complex: Parameters influencing the labelling of pectic acid in the cell walls of flax hypocotyl

An enzyme/colloidal‐gold complex was prepared using a purified endopolygalacturonase (endoPGH, E.C. 3.2.1.15). Gold particles with a radius of 4·8 nm were prepared. To stabilize the gold particles, it was necessary to add a minimal amount of enzyme (approximately 0·5 μg) at a pH higher than its isoelectric pH. Under these conditions, the number of protein molecules adsorbed per gold particle was approximately one.

Pectate lyase production by Bacillus subtilis in a membrane bioreactor

SummaryAn environmental strain ofBacillus subtilis was cultivated in a membrane bioreactor. Microbial cells were trapped by an upright membrane module fitted in the vessel reactor. Cell biomass and pectate lyase activity were increased about 5–6 times in comparison to a batch process. Clogging of the membrane module appears to be a major problem.