Immunolocalisation of arabinogalactan proteins and pectins in Actinidia deliciosa pollen

Summary. The cell wall composition of germinating pollen grains of Actinidia deliciosa was studied by immunolocalization with monoclonal antibodies against arabinogalactan proteins (AGPs) and pectins. In ungerminated pollen, the JIM8 epitope (against a subset of AGPs) was located in the intine and in the cytoplasm, while the MAC207 epitope (against AGPs) was only located in the exine. After germination, the JIM8 and MAC 207 epitopes were located in the cytoplasm and in the pollen tube wall. The Yariv reagent that binds to AGPs was added to the germination medium inducing a reduction or inhibition in pollen germination. This indicates that AGPs are present in the growing pollen tube and play an important role in pollen germination. To identify the nature of the pectins found in pollen grains and tubes, four monoclonal antibodies were used. The JIM5 epitope (against unesterified pectins) was located in the intine, more intensely in the pore region, and along the pollen tube wall, and the JIM7 epitope (against methyl-esterified pectins) was also observed in the cytoplasm. After germination, the JIM5 epitope was located in the pollen tube wall; although, the tube tip was not labelled. The JIM7 epitope was located in the entire pollen tube wall. LM5 (against galactans) showed a labelling pattern similar to that of JIM5 and the pattern of LM6 (against arabinans) was similar to that of JIM7. Pectins show different distribution patterns when the degree of esterification is considered. Pollen tube wall pectins are less esterified than those of the pollen tube tip. The association of AGPs with pectins in the cell wall of the pollen grain and the pollen tube may play an important role in the maintenance of cell shape during pollen growth and development.Correspondence and reprints: Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.     

An extensin-rich matrix lines the carinal canals in Equisetum ramosissimum, which may function as water-conducting channels

Background and Aims

The anatomy of Equisetum stems is characterized by the occurrence of vallecular and carinal canals. Previous studies on the carinal canals in several Equisetum species suggest that they convey water from one node to another.

Methods

Cell wall composition and ultrastructure have been studied using immunocytochemistry and electron microscopy, respectively. Serial sectioning and X-ray computed tomography were employed to examine the internode–node–internode transition of Equisetum ramosissimum.

Key Results

The distribution of the LM1 and JIM20 extensin epitopes is restricted to the lining of carinal canals. The monoclonal antibodies JIM5 and LM19 directed against homogalacturonan with a low degree of methyl esterification and the CBM3a probe recognizing crystalline cellulose also bound to this lining. The xyloglucan epitopes recognized by LM15 and CCRC-M1 were only detected in this lining after pectate lyase treatment. The carinal canals, connecting consecutive rings of nodal xylem, are formed by the disruption and dissolution of protoxylem elements during elongation of the internodes. Their inner surface appears smooth compared with that of vallecular canals.

Conclusions

The carinal canals in E. ramosissimum have a distinctive lining containing pectic homogalacturonan, cellulose, xyloglucan and extensin. These canals might function as water-conducting channels which would be especially important during the elongation of the internodes when protoxylem is disrupted and the metaxylem is not yet differentiated. How the molecularly distinct lining relates to the proposed water-conducting function of the carinal canals requires further study. Efforts to elucidate the spatial and temporal distribution of cell wall polymers in a taxonomically broad range of plants will probably provide more insight into the structural–functional relationships of individual cell wall components or of specific configurations of cell wall polymers.     

Immunolocalization of the cell wall components inPinus densiflora pollen

Summary In order to compare cell wall formation in gymnosperm pollen with that in angiosperm pollen, the distribution of cell wall constituents in the pollen grain and pollen tube ofPinus densiflora was studied immunocytochemically with monoclonal antibodies JIM 5 (against non- or poorly esterified pectin), JIM 7 (against highly esterified pectin), JIM 13 (against arabinogalactan proteins, AGPs), and LM 2 (against AGPs containing glucuronic acid). In the pollen grain wall, only the outer layer of the intine was labeled with JIM 5 and weakly with JIM 7. The tube wall was scarcely labeled with JIM 5 and very weakly labeled with JIM 7. In contrast, the whole of both the intine and the tube wall was strongly labeled with JIM 13 and LM 2, and the generative-cell wall was also labeled only with LM 2. The hemicellulose B fraction, which is the main polysaccharide fraction from the pollen tube wall, reacted strongly with JIM 13 and especially LM 2, but not with antipectin antibodies. These results demonstrate that the wall constituents and their localization inP. densiflora pollen are considerably different from those reported in angiosperm pollen and suggest that the main components of the cell wall ofP. densiflora pollen are arabinogalactan and AGPs containing glucuronic acid.Abbreviations AGPs arabinogalactan proteins - ELISA enzymelinked immunosorbent assay - MAbs monoclonal antibodies     

Immunogold localization of pectins and glycoproteins in tissues of peach with reference to deep supercooling

Living xylem tissues and floral buds of several species of woody plants survive exposure to freezing temperatures by deep supercooling. A barrier to water loss and the growth of ice crystals into cells is considered necessary for deep supercooling to occur. Pectins, as a constituent of the cell wall, have been implicated in the formation of this barrier. The present study examined the distribution of pectin in xylem and floral bud tissues of peach (Prunus persica). Two monoclonal antibodies (JIM5 and JIM7) that recognize homogalacturonic sequences with varying degrees of esterification were utilized in conjunction with immunogold electron microscopy. Results indicate that highly esterified epitopes of pectin, recognized by JIM7, were the predominant types of pectin in peach and were uniformly distributed throughout the pit membrane and primary cell walls of xylem and floral bud tissues. In contrast, un-esterified epitopes of pectin, recognized by JIM5, were confined to the outer surface of the pit membrane in xylem tissues. In floral buds, these epitopes were localized in middle lamellae, along the outer margin of the cell wall lining empty intercellular spaces, and within filled intercellular spaces. JIM5 labeling was more pronounced in December samples than in July/August samples. Additionally, epitopes of an arabinogalactan protein, recognized by JIM14, were confined to the amorphous layer of the pit membrane. The role of pectins in freezing response is discussed in the context of present theory and it is suggested that pectins may influence both water movement and intrusive growth of ice crystals at freezing temperatures.     

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

Summary Pectic 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.Abbreviations Cdta diamino-1,2-cyclohexane tetra-acetic acid - PATAg periodic acid-thiocarbohydrazide-silver proteinate - PGA polygalacturonic acid - PME pectin methylesterase - RG I rhamnogalacturonan I - SIMS secondary ion mass spectrometry - TEM transmission electron microscopy     

Local differentiation of cell wall matrix polysaccharides in sinuous pavement cells: its possible involvement in the flexibility of cell shape

• The distribution of homogalacturonans (HGAs) displaying different degrees of esterification as well as of callose was examined in cell walls of mature pavement cells in two angiosperm and two fern species. We investigated whether local cell wall matrix differentiation may enable pavement cells to respond to mechanical tension forces by transiently altering their shape.
• HGA epitopes, identified with 2F4, JIM5 and JIM7 antibodies, and callose were immunolocalised in hand‐made or semithin leaf sections. Callose was also stained with aniline blue. The structure of pavement cells was studied with light and transmission electron microscopy (TEM).
• In all species examined, pavement cells displayed wavy anticlinal cell walls, but the waviness pattern differed between angiosperms and ferns. The angiosperm pavement cells were tightly interconnected throughout their whole depth, while in ferns they were interconnected only close to the external periclinal cell wall and intercellular spaces were developed between them close to the mesophyll. Although the HGA epitopes examined were located along the whole cell wall surface, the 2F4‐ and JIM5‐ epitopes were especially localised at cell lobe tips. In fern pavement cells, the contact sites were impregnated with callose and JIM5‐HGA epitopes. When tension forces were applied on leaf regions, the pavement cells elongated along the stretching axis, due to a decrease in waviness of anticlinal cell walls. After removal of tension forces, the original cell shape was resumed.
• The presented data support that HGA epitopes make the anticlinal pavement cell walls flexible, in order to reversibly alter their shape. Furthermore, callose seems to offer stability to cell contacts between pavement cells, as already suggested in photosynthetic mesophyll cells.

     

In situ, chemical and macromolecular study of the composition of Arabidopsis thaliana seed coat mucilage

A comprehensive analysis was carried out of the composition of seed coat mucilage from Arabidopsis thaliana using the Columbia-0 accession. Pectinaceous mucilage is released from myxospermous seeds upon imbibition, and in Arabidopsis consists of a water-soluble, outer layer and an adherent, inner layer. Analysis of monosaccharide composition in conjunction with digestion with pectolytic enzymes conclusively demonstrated that the principal pectic domain of both layers was rhamnogalacturonan I, and that in the outer layer this was unbranched. The macromolecular characteristics of the water-soluble mucilage indicated that the rhamnogalacturonan molecules in the outer layer were in a slightly expanded random-coil conformation. The inner, adherent layer remained attached to the seed, even after extraction with acid and alkali, suggesting that its integrity was maintained by covalent bonds. Confocal microscopy and monosaccharide composition analyses showed that the inner layer can be separated into two domains. The internal domain contained cellulose microfibrils, which could form a matrix with RGI and bind it to the seed. In effect, in the mum5-1 mutant where most of the inner and outer mucilage layers were water soluble, cellulose remained attached to the seed coat. Immunolabeling with anti-pectin antibodies indicated the presence of galactan and arabinan in the inner layer, with the latter only present in the non-cellulose-containing external domain. In addition, JIM5 and JIM7 antibodies labeled different domains of the inner layer, suggesting the presence of stretches of homogalacturonan with different levels of methyl esterification.     

An extended set of monoclonal antibodies to pectic homogalacturonan

Three novel rat monoclonal antibodies, designated LM18, LM19 and LM20, were isolated from screens for binding to Arabidopsis thaliana seed coat mucilage. The binding of these antibodies to mucilage subject to enzyme and high pH pre-treatments and to a series of model homogalacturonan-rich pectins with defined levels of methyl-esterification indicated their recognition of pectic homogalacturonan epitopes. The binding capacities of these monoclonal antibodies to cell walls in sections of tobacco stem pith parenchyma were also differentially sensitive to equivalent treatments with high pH buffers and pectate lyase. The epitopes bound by these antibodies display some similarities and some differences to the epitopes recognized by the previously isolated and established pectic homogalacturonan probes JIM5 and JIM7.     

A cytochemical and immunocytochemical analysis of the wall labyrinth apparatus in leaf transfer cells in Elodea canadensis

Background and Aims

Transfer cells are plant cells specialized in apoplast/symplast transport and characterized by a distinctive wall labyrinth apparatus. The molecular architecture and biochemistry of the labyrinth apparatus are poorly known. The leaf lamina in the aquatic angiosperm Elodea canadensis consists of only two cell layers, with the abaxial cells developing as transfer cells. The present study investigated biochemical properties of wall ingrowths and associated plasmalemma in these cells.

Methods

Leaves of Elodea were examined by light and electron microscopy and ATPase activity was localized cytochemically. Immunogold electron microscopy was employed to localize carbohydrate epitopes associated with major cell wall polysaccharides and glycoproteins.

Key Results

The plasmalemma associated with the wall labyrinth is strongly enriched in light-dependent ATPase activity. The wall ingrowths and an underlying wall layer share an LM11 epitope probably associated with glucuronoarabinoxylan and a CCRC-M7 epitope typically associated with rhamnogalacturonan I. No labelling was observed with LM10, an antibody that recognizes low-substituted and unsubstituted xylan, a polysaccharide consistently associated with secondary cell walls. The JIM5 and JIM7 epitopes, associated with homogalacturonan with different degrees of methylation, appear to be absent in the wall labyrinth but present in the rest of cell walls.

Conclusions

The wall labyrinth apparatus of leaf transfer cells in Elodea is a specialized structure with distinctive biochemical properties. The high level of light-dependent ATPase activity in the plasmalemma lining the wall labyrinth is consistent with a formerly suggested role of leaf transfer cells in enhancing inorganic carbon inflow. The wall labyrinth is a part of the primary cell wall. The discovery that the wall ingrowths in Elodea have an antibody-binding pattern divergent, in part, from that of the rest of cell wall suggests that their carbohydrate composition is modulated in relation to transfer cell functioning.     

Distribution of pectins in cell walls of maize coleoptiles and evidence against their involvement in auxin-induced extension growth

Summary Aiming to elucidate the possible involvement of pectins in auxin-mediated elongation growth the distribution of pectins in cell walls of maize coleoptiles was investigated. Antibodies against defined epitopes of pectin were used: JIM 5 recognizing pectin with a low degree of esterification, JIM 7 recognizing highly esterified pectin and 2F4 recognizing a pectin epitope induced by Ca²⁺. JIM 5 weakly labeled the outer third of the outer epidermal wall and the center of filled cell corners in the parenchyma. A similar labeling pattern was obtained with 2F4. In contrast, JIM 7 densely labeled the whole outer epidermal wall except the innermost layer, the middle lamellae, and the inner edges of open cell corners in the parenchyma. Enzymatic de-esterification with pectin methylesterase increased the labeling by JIM 5 and 2F4 substantially. A further increase of the labeling density by JIM 5 and 2F4 and an extension of the labeling over the whole outer epidermal wall could be observed after chemical de-esterification with alkali. This indicates that both methyl- and other esters exist in maize outer epidermal walls. Thus, in the growth-controlling outer epidermal wall a clear zonation of pectin fractions was observed: the outermost layer (about one third to one half of wall thickness) contains unesterified pectin epitopes, presumably cross-linked by Ca²⁺ extract. Tracer experiments with³H-myo-inositol showed rapid accumulation of tracer in all extractable pectin fractions and in a fraction tightly bound to the cell wall. A stimulatory effect of IAA on tracer incorporation could not be detected in any fraction. Summarizing the data a model of the pectin distribution in the cell walls of maize coleoptiles was developed and its implications for the mechanism of auxin-induced wall loosening are discussed.Abbreviations CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acid - CWP cell-wall pellet - IAA indole-3-acetic acid - LSE low-salt extract - TCA trichloroacetic acid; Tris tris-(hydroxy-methyl)aminoethane     

Integument cell gelatinisation—the fate of the integumentary cells in <Emphasis Type="Italic">Hieracium</Emphasis> and <Emphasis Type="Italic">Pilosella</Emphasis> (Asteraceae)

Members of the genera Hieracium and Pilosella are model plants that are used to study the mechanisms of apomixis. In order to have a proper understanding of apomixis, knowledge about the relationship between the maternal tissue and the gametophyte is needed. In the genus Pilosella, previous authors have described the specific process of the “liquefaction” of the integument cells that surround the embryo sac. However, these observations were based on data only at the light microscopy level. The main aim of our paper was to investigate the changes in the integument cells at the ultrastructural level in Pilosella officinarum and Hieracium alpinum. We found that the integument peri-endothelial zone in both species consisted of mucilage cells. The mucilage was deposited as a thick layer between the plasma membrane and the cell wall. The mucilage pushed the protoplast to the centre of the cell, and cytoplasmic bridges connected the protoplast to the plasmodesmata through the mucilage layers. Moreover, an elongation of the plasmodesmata was observed in the mucilage cells. The protoplasts had an irregular shape and were finally degenerated. After the cell wall breakdown of the mucilage cells, lysigenous cavities that were filled with mucilage were formed.     

Early local differentiation of the cell wall matrix defines the contact sites in lobed mesophyll cells of Zea mays

Background and Aims

The morphogenesis of lobed mesophyll cells (MCs) is highly controlled and coupled with intercellular space formation. Cortical microtubule rings define the number and the position of MC isthmi. This work investigated early events of MC morphogenesis, especially the mechanism defining the position of contacts between MCs. The distributions of plasmodesmata, the hemicelluloses callose and (1 → 3,1 → 4)-β-d-glucans (MLGs) and the pectin epitopes recognized by the 2F4, JIM5, JIM7 and LM6 antibodies were studied in the cell walls of Zea mays MCs.

Methods

Matrix cell wall polysaccharides were immunolocalized in hand-made sections and in sections of material embedded in LR White resin. Callose was also localized using aniline blue in hand-made sections. Plasmodesmata distribution was examined by transmission electron microscopy.

Results

Before reorganization of the dispersed cortical microtubules into microtubule rings, particular bands of the longitudinal MC walls, where the MC contacts will form, locally differentiate by selective (1) deposition of callose and the pectin epitopes recognized by the 2F4, LM6, JIM5 and JIM7 antibodies, (2) degradation of MLGs and (3) formation of secondary plasmodesmata clusterings. This cell wall matrix differentiation persists in cell contacts of mature MCs. Simultaneously, the wall bands between those of future cell contacts differentiate with (1) deposition of local cell wall thickenings including cellulose microfibrils, (2) preferential presence of MLGs, (3) absence of callose and (4) transient presence of the pectins identified by the JIM5 and JIM7 antibodies. The wall areas between cell contacts expand determinately to form the cell isthmi and the cell lobes.

Conclusions

The morphogenesis of lobed MCs is characterized by the early patterned differentiation of two distinct cell wall subdomains, defining the sites of the future MC contacts and of the future MC isthmi respectively. This patterned cell wall differentiation precedes cortical microtubule reorganization and may define microtubule ring disposition.     

Isolation and Characterization of Mutants Defective in Seed Coat Mucilage Secretory Cell Development in Arabidopsis

In Arabidopsis, fertilization induces the epidermal cells of the outer ovule integument to differentiate into a specialized seed coat cell type producing extracellular pectinaceous mucilage and a volcano-shaped secondary cell wall. Differentiation involves a regulated series of cytological events including growth, cytoplasmic rearrangement, mucilage synthesis, and secondary cell wall production. We have tested the potential of Arabidopsis seed coat epidermal cells as a model system for the genetic analysis of these processes. A screen for mutants defective in seed mucilage identified five novel genes (MUCILAGE-MODIFIED [MUM]1–5). The seed coat development of these mutants, and that of three previously identified ones (TRANSPARENT TESTA GLABRA1, GLABRA2, and APETALA2) were characterized. Our results show that the genes identified define several events in seed coat differentiation. Although APETALA2 is needed for differentiation of both outer layers of the seed coat, TRANSPARENT TESTA GLABRA1, GLABRA2, and MUM4 are required for complete mucilage synthesis and cytoplasmic rearrangement. MUM3 and MUM5 may be involved in the regulation of mucilage composition, whereas MUM1 and MUM2 appear to play novel roles in post-synthesis cell wall modifications necessary for mucilage extrusion.     

The structure and biochemistry of charophycean cell walls: I. Pectins of Penium margaritaceum

Summary. Plant cell walls are essential for proper growth, development, and interaction with the environment. It is generally accepted that land plants arose from aquatic ancestors which are sister groups to the charophycean algae (i.e., Streptophyta), and study of wall evolution during this transition promises insight into structure–function relationships of wall components. In this paper, we explore wall evolutionary history by studying the incorporation of pectin polymers into cell walls of the model organism Penium margaritaceum, a simple single-cell desmid. This organism produces only a primary wall consisting of three fibrillar or fibrous layers, with the outermost stratum terminating in distinct, calcified projections. Extraction of isolated cell walls with trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid yielded a homogalacturonan (HGA) that was partially methyl esterified and equivalent to that found in land plants. Other pectins common to land plants were not detected, although selected components of some of these polymers were present. Labeling with specific monoclonal antibodies raised against higher-plant HGA epitopes (e.g., JIM5, JIM7, LM7, 2F4, and PAM1) demonstrated that the wall complex and outer layer projections were composed of the HGA which was significantly calcium complexed. JIM5 and JIM7 labeling suggested that highly methyl esterified HGA was secreted into the isthmus zone of dividing cells, the site of active wall secretion. As the HGA was displaced to more polar regions, de-esterification in a non-blockwise fashion occurred. This, in turn, allowed for calcium binding and the formation of the rigid outer wall layer. The patterning of HGA deposition provides interesting insights into the complex process of pectin involvement in the development of the plant cell wall. Correspondence and reprints: Department of Biology, Skidmore College, 815 North Broadway, Saratoga Springs, NY 12866, U.S.A.     

Characterization of Laurencia arbuscula spore mucilage and cell walls with stains and FITC-labelled lectins

In most red algae, spores are liberated without a cell wall, within a sheath of mucilage that is responsible for its primary attachment. Utilizing fluorescent-labelled lectins, we identified carbohydrate residues and their location in the mucilage and cell walls of spores of Laurencia arbuscula. Cell wall formation and mucilage composition were studied with Calcofluor, Toluidine Blue (AT-O), Alcian Blue (AB) and periodic acid-Schiff (PAS). In the mucilage, we identified α-d-mannose, α-d-glucose, N-acetyl-glucosamine, N-acetyl-galactosamine and β-d-galactose. All sugar residues were found in the cell wall, in the spore body rather than in the rhizoid, which suggests that the residues may be related to initial substrate adhesion. A cell wall is produced soon after the spore's attachment, beginning with a deposition of cellulose around the spore, as indicated by Calcofluor. A polarization of the cell wall triggers the process of germination. The cell-wall matrix was positive to AB and metachromatic to AT-O, indicating acidic polysaccharides, while neutral polysaccharides were positive to PAS.     

Mucilage on the Root Surface and Root Hairs of Sorghum: Heterogeneity in Structure, Manner of Production and Site of Accumulation

Small drops of a mucilaginous character near the tip of roothairs were seen by light microscopy in several species of Sorghumand the Sorghum hybrid Vidan. Electron microscopy revealed thatthe drops are formed from at least two distinct substances,both apparently secreted from the endoplasmic reticulum. In addition, a patchy, fibrillar mucilaginous layer, also withat least two components, was found on the cell wall of the roothairs and on the outer wall of ordinary root epidermal cells.Golgi bodies as well as mitochondria take part in its production.As a rule, the mucilaginous patches are colonized by bacteria. Sorghum, root hairs, mucilage     

Electrophoretic profiling and immunocytochemical detection of pectins and arabinogalactan proteins in olive pollen during germination and pollen tube growth

Background and Aims

Cell wall pectins and arabinogalactan proteins (AGPs) are important for pollen tube growth. The aim of this work was to study the temporal and spatial dynamics of these compounds in olive pollen during germination.

Methods

Immunoblot profiling analyses combined with confocal and transmission electron microscopy immunocytochemical detection techniques were carried out using four anti-pectin (JIM7, JIM5, LM5 and LM6) and two anti-AGP (JIM13 and JIM14) monoclonal antibodies.

Key Results

Pectin and AGP levels increased during olive pollen in vitro germination. (1 → 4)-β-d-Galactans localized in the cytoplasm of the vegetative cell, the pollen wall and the apertural intine. After the pollen tube emerged, galactans localized in the pollen tube wall, particularly at the tip, and formed a collar-like structure around the germinative aperture. (1 → 5)-α-l-Arabinans were mainly present in the pollen tube cell wall, forming characteristic ring-shaped deposits at regular intervals in the sub-apical zone. As expected, the pollen tube wall was rich in highly esterified pectic compounds at the apex, while the cell wall mainly contained de-esterified pectins in the shank. The wall of the generative cell was specifically labelled with arabinans, highly methyl-esterified homogalacturonans and JIM13 epitopes. In addition, the extracellular material that coated the outer exine layer was rich in arabinans, de-esterified pectins and JIM13 epitopes.

Conclusions

Pectins and AGPs are newly synthesized in the pollen tube during pollen germination. The synthesis and secretion of these compounds are temporally and spatially regulated. Galactans might provide mechanical stability to the pollen tube, reinforcing those regions that are particularly sensitive to tension stress (the pollen tube–pollen grain joint site) and mechanical damage (the tip). Arabinans and AGPs might be important in recognition and adhesion phenomena of the pollen tube and the stylar transmitting cells, as well as the egg and sperm cells.     

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

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.     

Isolation and characterisation of the homogalacturonan from type II cell walls of the commelinoid monocot wheat using HF-solvolysis

In contrast to the typical type I cell wall of the dicot plants, the type II cell wall of the commelinoid monocot plants is known to be relatively poor in pectins. Assuming a critical role for the remaining pectins in terms of cell wall architecture and/or as a reservoir of signalling molecules, we have compared different protocols for the isolation of the main pectin polymer, homogalacturonan, from wheat leaf cell walls. Pectin was detected in these cell walls immunochemically using the monoclonal antibodies JIM5 and JIM7, and biochemically by monosaccharide analysis. The Ca(++)-chelators CDTA and imidazole extracted a pectin rich fraction from isolated cell walls which was however contaminated with significant amounts of hemicelluloses. Pretreatment of the cell walls with anhydrous hydrogen fluoride at controlled low temperatures followed by HF/ether- and water-extraction prior to imidazole-extraction of pectins yielded a purer homogalacturonan fraction. The near absence of rhamnosyl residues proved that the isolated homogalacturonan fraction was free of rhamnogalacturonans. If HF-solvolysis was performed at -23 degrees C, the resulting homogalacturonan had a degree of methyl esterification identical to that of the pectins in the initial wheat cell wall. The antibodies JIM5 and JIM7 as well as PAM1 and LM5 proved that the isolated homogalacturonan had a low methyl ester content, was polymeric and free of galactan side chains. We can thus isolate native homogalacturonan from the type II wheat cell walls with the original in muro pattern of methyl esterification still intact, to further investigate e.g., its degradability by plant or microbial pectic enzymes.