Fucosylated arabinogalactan-proteins are required for full root cell elongation in arabidopsis

The Arabidopsis thaliana mutant mur1 is affected in the biosynthesis of l-fucose and has less than 2% of the normal amounts of this sugar in the cell walls of its aerial parts. Although in roots the reduction of l-fucose is only 40%, this causes a decrease of about 50% in root cell elongation. Since arabinogalactan-proteins (AGPs) are known to play a role in plant cell expansion we studied the composition of mur1 root AGPs. Arabidopsis root AGPs were shown to contain l-fucose, which was reduced in level in mur1 AGPs. In wild-type plants, an l-fucose containing epitope is present in AGPs in the cell wall of differentiating root cells. Addition of eel lectin, which specifically recognizes this epitope, and not fucose in other wall polymers, can phenocopy mur1 roots. Several lines of evidence are presented to support the contention that l-fucose containing root AGPs are required for the full elongation of root cells.     

Xyloglucan endo-transglycosylase-mediated xyloglucan rearrangements in developing wood of hybrid aspen

Xyloglucan endo-transglycosylases (XETs) encoded by xyloglucan endo-transglycosylases/hydrolase (XTH) genes modify the xyloglucan-cellulose framework of plant cell walls, thereby regulating their expansion and strength. To evaluate the importance of XET in wood development, we studied xyloglucan dynamics and XTH gene expression in developing wood and modified XET activity in hybrid aspen (Populus tremula × tremuloides) by overexpressing PtxtXET16-34. We show that developmental modifications during xylem differentiation include changes from loosely to tightly bound forms of xyloglucan and increases in the abundance of fucosylated xyloglucan epitope recognized by the CCRC-M1 antibody. We found that at least 16 Populus XTH genes, all likely encoding XETs, are expressed in developing wood. Five genes were highly and ubiquitously expressed, whereas PtxtXET16-34 was expressed more weakly but specifically in developing wood. Transgenic up-regulation of XET activity induced changes in cell wall xyloglucan, but its effects were dependent on developmental stage. For instance, XET overexpression increased abundance of the CCRC-M1 epitope in cambial cells and xylem cells in early stages of differentiation but not in mature xylem. Correspondingly, an increase in tightly bound xyloglucan content was observed in primary-walled xylem but a decrease was seen in secondary-walled xylem. Thus, in young xylem cells, XET activity limits xyloglucan incorporation into the tightly bound wall network but removes it from cell walls in older cells. XET overexpression promoted vessel element growth but not fiber expansion. We suggest that the amount of nascent xyloglucan relative to XET is an important determinant of whether XET strengthens or loosens the cell wall.     

Differential Localization of Carbohydrate Epitopes in Plant Cell Walls in the Presence and Absence of Arbuscular Mycorrhizal Fungi

Two monoclonal antibodies (McAbs) generated against rhamnogalacturonan I and characterized as specific for a terminal [alpha]-(1->2)-linked fucosyl-containing epitope (CCRC-M1) and for an arabinosylated [beta]-(1,6)-galactan epitope (CCRC-M7) were used in immunogold experiments to determine the distribution of the epitopes in four plants. Allium porrum, Zea mays, Trifolium repens, and Nicotiana tabacum plants were chosen as representatives of monocots and dicots with different wall structures. Analyses were performed on root tissues in the presence and absence of arbuscular mycorrhizal fungi. A differential localization of the two cell wall epitopes was found between tissues and between species: for example, in leek, CCRC-M1 labeled epidermal and hypodermal cells, whereas CCRC-M7 labeled cortical cells only. Clover walls were labeled by both McAbs, whereas maize and tobacco were only labeled by CCRC-M7. In the presence of the arbuscular mycorrhizal fungi, labeling was additionally found in an apoplastic compartment typical of the symbiosis (the interface) occurring around the intracellular hyphae. Epitopes binding both McAbs were found in the interfacial material, and their distribution mirrored the pattern found in the host cell wall. These findings demonstrate that the composition of the interface zone in a fungus-plant symbiosis reflects the composition of the wall of the host cell.     

The reb1-1 mutation of Arabidopsis. Effect on the structure and localization of galactose-containing cell wall polysaccharides

The Arabidopsis (Arabidopsis thaliana) root epidermal bulger1-1 (reb1-1) mutant (allelic to root hair defective1 [rhd1]) is characterized by a reduced root elongation rate and by bulging of trichoblast cells. The REB1/RHD1 gene belongs to a family of UDP-D-Glucose 4-epimerases involved in the synthesis of D-Galactose (Gal). Our previous study showed that certain arabinogalactan protein epitopes were not expressed in bulging trichoblasts of the mutant. In this study, using a combination of microscopical and biochemical methods, we have investigated the occurrence and the structure of three major Gal-containing polysaccharides, namely, xyloglucan (XyG), rhamnogalacturonan (RG)-I, and RG-II in the mutant root cell walls. Our immunocytochemical data show that swollen trichoblasts were not stained with the monoclonal antibody CCRC-M1 specific for alpha-L-Fucp-(1-->2)-beta-D-Galp side chains of XyG, whereas they were stained with anti-XyG antibodies specific for XyG backbone. In addition, analysis of a hemicellulosic fraction from roots demonstrates the presence of two structurally different XyGs in reb1-1. One is structurally similar to wild-type XyG and the other is devoid of fuco-galactosylated side chains and has the characteristic of being insoluble. Similar to anti-XyG antibodies, anti-bupleuran 2IIC, a polyclonal antibody specific for galactosyl epitopes associated with pectins, stained all root epidermal cells of both wild type and reb1-1. Similarly, anti-RG-II antibodies also stained swollen trichoblasts in the mutant. In addition, structural analysis of pectic polymers revealed no change in the galactosylation of RG-I and RG-II isolated from reb1-1 root cells. These findings demonstrate that the reb1-1 mutation affects XyG structure, but not that of pectic polysaccharides, thus lending support to the hypothesis that biosynthesis of Gal as well as galactosylation of complex polysaccharides is regulated at the polymer level.     

Effects of the mur1 mutation on xyloglucans produced by suspension-cultured Arabidopsis thaliana cells

Mutation of the Arabidopsis thaliana (L.) Heynh. gene MUR1, which encodes an isoform of GDP-D-mannose-4,6-dehydratase, affects the biosynthetic conversion of GDP-mannose to GDP-fucose. Cell walls in the aerial tissues of mur1 plants are almost devoid of alpha-L-fucosyl residues, which are partially replaced by closely related alpha-L-galactosyl residues. A line of suspension-cultured A. thaliana cells was generated from leaves of mur1 plants and the structure of the xyloglucan in the walls of these cells was structurally characterized. Xyloglucan fractions were prepared from the walls of both wild-type (WT) and mur1 cells by sequential extraction with a xyloglucan-specific endoglucanase (XEG) and aqueous KOH. Structural analysis of these fractions revealed that xyloglucan produced by cultured mur1 cells is similar, but not identical to that isolated from leaves of mur1 plants. As previously reported for mur1 leaves, the xyloglucan from cultured mur1 cells contains less than 5% of the fucose present in the xyloglucan from WT cells. Fucosylation of the xyloglucan is substantially restored when mur1 cells are grown in medium supplemented with L-fucose. Xyloglucan isolated from leaves contains more oligosaccharide subunits in which the central sidechain is terminated with a beta-D-galactosyl residue than does xyloglucan prepared from cultured cells. This was observed for both mur1 and WT plants, indicating that this correlation is independent of the mur1 mutation and that it is possible to distinguish changes due to genetic mutation from those due to the physiological state of the cells in culture. Suspension-cultured cells thus provide a convenient source of genetically altered cell wall material, facilitating the biochemical characterization of mutations that affect cell wall structure.     

Tensile properties of Arabidopsis cell walls depend on both a xyloglucan cross-linked microfibrillar network and rhamnogalacturonan II-borate complexes

The mechanical properties of plant organs depend upon anatomical structure, cell-cell adhesion, cell turgidity, and the mechanical properties of their cell walls. By testing the mechanical responses of Arabidopsis mutants, it is possible to deduce the contribution that polymers of the cell wall make to organ strength. We developed a method to measure the tensile parameters of the expanded regions of turgid or plasmolyzed dark-grown Arabidopsis hypocotyls and applied it to the fucose biosynthesis mutant mur1, the xyloglucan glycosyltransferase mutants mur2 and mur3, and the katanin mutant bot1. Hypocotyls from plants grown in the presence of increasing concentrations of dichlorobenzonitrile, an inhibitor of cellulose synthesis, were considerably weakened, indicating the validity of our approach. In order of decreasing strength, the hypocotyls of mur2 > bot1 and mur1 > mur3 were each found to have reduced strength and a proportionate reduction in modulus compared with wild type. The tensile properties of the hypocotyls and of the inflorescence stems of mur1 were rescued by growth in the presence of high concentrations of borate, which is known to cross-link the pectic component rhamnogalacturonan II. From comparison of the mechanical responses of mur2 and mur3, we deduce that galactose-containing side chains of xyloglucan make a major contribution to overall wall strength, whereas xyloglucan fucosylation plays a comparatively minor role. We conclude that borate-complexed rhamnogalacturonan II and galactosylated xyloglucan contribute to the tensile strength of cell walls.     

Developmental and Tissue-Specific Structural Alterations of the Cell-Wall Polysaccharides of Arabidopsis thaliana Roots

The plant cell wall is a dynamic structure that plays important roles in growth and development and in the interactions of plants with their environment and other organisms. We have used monoclonal antibodies that recognize different carbohydrate epitopes present in plant cell-wall polysaccharides to locate these epitopes in roots of developing Arabidopsis thaliana seedlings. An epitope in the pectic polysaccharide rhamnogalacturonan I is observed in the walls of epidermal and cortical cells in mature parts of the root. This epitope is inserted into the walls in a developmentally regulated manner. Initially, the epitope is observed in atrichoblasts and later appears in trichoblasts and simultaneously in cortical cells. A terminal [alpha]-fucosyl-containing epitope is present in almost all of the cell walls in the root. An arabinosylated (1->6)-[beta]-galactan epitope is also found in all of the cell walls of the root with the exception of lateral root-cap cell walls. It is striking that these three polysaccharide epitopes are not uniformly distributed (or accessible) within the walls of a given cell, nor are these epitopes distributed equally across the two walls laid down by adjacent cells. Our results further suggest that the biosynthesis and differentiation of primary cell walls in plants are precisely regulated in a temporal, spatial, and developmental manner.     

Inducible expression of <Emphasis Type="Italic">Pisum sativum</Emphasis> xyloglucan fucosyltransferase in the pea root cap meristem,and effects of antisense mRNA expression on root cap cell wall structural integrity

Mitosis and cell wall synthesis in the legume root cap meristem can be induced and synchronized by the nondestructive removal of border cells from the cap periphery. Newly synthesized cells can be examined microscopically as they differentiate progressively during cap development, and ultimately detach as a new population of border cells. This system was used to demonstrate that Pisum sativum L. fucosyl transferase (PsFut1) mRNA expression is strongly expressed in root meristematic tissues, and is induced >2-fold during a 5-h period when mitosis in the root cap meristem is increased. Expression of PsFut1 antisense mRNA in pea hairy roots under the control of the CaMV35S promoter, which exhibits meristem localized expression in pea root caps, resulted in a 50-60% reduction in meristem localized endogenous PsFut1 mRNA expression measured using whole mount in situ hybridization. Changes in gross levels of cell wall fucosylated xyloglucan were not detected, but altered surface localization patterns were detected using whole mount immunolocalization with CCRC-M1, an antibody that recognizes fucosylated xyloglucan. Emerging hairy roots expressing antisense PsFut1 mRNA appeared normal macroscopically but scanning electron microscopy of tissues with altered CCRC-M1 localization patterns revealed wrinkled, collapsed cell surfaces. As individual border cells separated from the cap periphery, cell death occurred in correlation with extrusion of cellular contents through breaks in the wall.     

Polysaccharide and glycoprotein distribution in the epidermis of cotton ovules during early fiber initiation and growth

The cotton fiber is a model system to study cell wall biosynthesis because the fiber cell elongates (∼3 cm in ∼20 days) without mitosis. In this study, developing cotton ovules, examined from 1 day before anthesis (DBA) to 2 days post-anthesis (DPA), that would be difficult to investigate via classical carbohydrate biochemistry were probed using a battery of antibodies that recognize a large number of different wall components. In addition, ovules from these same stages were investigated in three fiberless lines. Most antibodies reacted with at least some component of the ovule, and several of the antibodies reacted specifically with the epidermal layer of cells that may give clues as to the nature of the development of the fibers and the neighboring, nonfiber atrichoblasts. Arabinogalactan proteins (AGPs) labeled the epidermal layers more strongly than other ovular tissue, even at 1 DBA. One of the AGP antibodies, CCRC-M7, which recognizes a 1➔6 galactan epitope of AGPs, is lost from the fiber cells by 2 DPA, although labeling in the atrichoblasts remained strong. In contrast, LM5 that recognizes a 1➔4 galactan RGI side chain is unreactive with sections until the fibers are produced and only the fibers are reactive. Dramatic changes also occur in the homogalacturonans (HGs). JIM5, which recognizes highly de-esterified HGs, only weakly labels epidermal cells of 1 DBA and 0 DPA ovules, but labeling increases in fibers cells, where a pectinaceous sheath is produced around the fiber cell and stronger reaction in the internal and external walls of the atrichoblast. In contrast, JIM7-reactive, highly esterifed HGs are present at high levels in the epidermal cells throughout development. Fiberless lines displayed similar patterns of labeling to the fibered lines, except that all of the cells had the labeling pattern of atrichoblasts. That is, CCRC-M7 labeled all cells of the fiberless lines, and LM5 labeled no cells at 2 DPA. These data indicate that a number of polysaccharides are unique in quantity or presence in the epidermal cell layers, and some of these might be critical participants in the early stages of initiation and elongation of cotton fibers.     

Dynamic changes in cell surface molecules are very early events in the differentiation of mesophyll cells from Zinnia elegans into tracheary elements

The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to transdifferentiate semi-synchronously into tracheary elements (TEs). This system has been used to establish the precise time point at which the TE cell fate becomes determined, and then changes have been looked for in cell-wall composition and architecture that are associated with the establishment of competence, determination, and differentiation with the transition from primary to secondary cell wall formation. At very early stages in this time course, changes in the repertoire of proteins and polysaccharides both in the cell wall and secreted into the culture medium were found. Changes in the secretion of pectic polysaccharides, xyloglucans and arabinogalactan proteins (AGPs) have been detected using the monoclonal antibodies JIM 7, CCRC-M1 and JIM 13, that recognize these three classes of cell-wall molecule, respectively. Twenty-four hours before secondary thickenings are visible, an AGP is present in the primary walls of a subpopulation of cells, and is secreted into the culture medium. This molecule is present in the secondary thickenings of mature TEs but not in their surrounding primary walls. Methyl-esterified pectic polysaccharides are present in all cell walls and are secreted into the culture medium throughout the time course of differentiation, though at an increased rate in inductive medium. However, sugar and linkage analysis of culture media shows that a relatively unbranched rhamnogalacturonan is enriched in inductive medium around the time of determination and increases rapidly in concentration. The amount of fucosylated xyloglucan in cell walls increases during the time course, but appears in inductive medium 24 h earlier than in control medium and may have a subtly different structure. The fucose-containing epitope on the xyloglucan disappears abruptly and entirely from inductive medium 6 h before any secondary thickenings are visible in the cells. The disappearance of the epitope is correlated with secretion of several hydrolytic enzyme activities. In Zinnia leaves, the mesophyll cell walls contain neither the fucosylated xyloglucan nor the AGP, although methylesterified pectin is present. All three epitopes are expressed in the vascular bundles, and the AGP is specifically localized in the xylem cells. Fucosylated xyloglucan is also present in the epidermal tissue, and the AGP is present in guard cells. The dynamic behaviour of these specific cell-wall molecules is tightly correlated with differentiation events in vitro, and can be clearly distinguished from the production of new wall material found in expanding and elongating cells. The precise timing of the appearance and disappearance of these proteins and polysaccharides compared with the point of cell-fate determination provides us with a series of cell-surface markers for cell states at very early times in the transdifferentiation pathway.     

Localization of cell wall polysaccharides in nonarticulated laticifers ofAsclepias speciosa Torr.

Summary Asclepias speciosa Torr, has latex-containing cells known as nonarticulated laticifers. In stem sections of this species, we have analyzed the cell walls of nonarticulated laticifers and surrounding cells with various stains, lectins, and monoclonal antibodies. These analyses revealed that laticifer walls are rich in (1→4) β-D-glucans and pectin polymers. Immunolocalization of pectic epitopes with the antihomogalacturonan antibodies JIM5 and JIM7 produced distinct labeling patterns. JIM7 labeled all cells including laticifers, while JIM5 only labeled mature epidermal cells and xylem elements. Two antibodies, LM5 and LM6, which recognize rhamnogalacturonan I epitopes distinctly labeled laticifer walls. LM6, which binds to a (l→5) α-arabinan epitope, labeled laticifer walls more intensely than walls of other cells. LM5, which recognizes a (1→4) β-D-galac-tan epitope, did not label laticifer segments at the shoot apex but labeled more mature portions of laticifers. Also the LM5 antibody did not label cells at the shoot apical meristem, but as cells grew and matured the LM5 epitope was expressed in all cells. LM2, a monoclonal antibody that binds to β-D-glucuronic acid residues in arabinogalactan proteins, did not label laticifers but specifically labeled sieve tubes. Sieve tubes were also specifically labeled byRicinus communis agglutinin, a lectin that binds to terminal β-D-galactosyl residues. Taken together, the analyses conducted showed that laticifer walls have distinctive cytochemical properties and that these properties change along the length of laticifers. In addition, this study revealed differences in the expression of pectin and arabinogalactan protein epitopes during shoot development or among different cell types.     

The GMD1 and GMD2 genes of Arabidopsis encode isoforms of GDP-D-mannose 4,6-dehydratase with cell type-specific expression patterns

l-Fucose (l-Fuc) is a monosaccharide constituent of plant cell wall polysaccharides and glycoproteins. The committing step in the de novo synthesis of l-Fuc is catalyzed by GDP-d-mannose 4,6-dehydratase, which, in Arabidopsis, is encoded by the GMD1 and GMD2 (MUR1) genes. To determine the functional significance of this genetic redundancy, the expression patterns of both genes were investigated via promoter-beta-glucuronidase fusions and immunolocalization of a Fuc-containing epitope. GMD2 is expressed in most cell types of the root, with the notable exception of the root tip where strong expression of GMD1 is observed. Within shoot organs, GMD1::GUS expression is confined to stipules and pollen grains leading to fucosylation of the walls of these cell types in the mur1 mutant. These results suggest that GMD2 represents the major housekeeping gene for the de novo synthesis of GDP-l-Fuc, whereas GMD1 expression is limited to a number of specialized cell types. We conclude that the synthesis of GDP-l-Fuc is controlled in a cell-autonomous manner by differential expression of two isoforms of the same enzyme.     

Generation of monoclonal antibodies against plant cell-wall polysaccharides. I. Characterization of a monoclonal antibody to a terminal alpha-(1-->2)-linked fucosyl-containing epitope.

Monoclonal antibodies (McAbs) generated against rhamnogalacturonan I (RG-I) purified from suspension-cultured sycamore maple (Acer pseudoplatanus) cells fall into three recognition groups. Four McAbs (group I) recognize an epitope that appears to be immunodominant and is present on RG-I from maize and sycamore maple, pectin and polygalacturonic acid from citrus, gum tragacanth, and membrane glycoproteins from suspension-cultured cells of maize, tobacco, parsley, bean, and sycamore maple. A second set of McAbs (group II) recognizes an epitope present in sycamore maple RG-I but does not bind to any of the other polysaccharides or glycoproteins recognized by group I. Lastly, one McAb, CCRC-M1 (group III), binds to RG-I and more strongly to xyloglucan (XG) from sycamore maple but not to maize RG-I, citrus polygalacturonic acid, or to the plant membrane glycoproteins recognized by group I. The epitope to which CCRC-M1 binds has been examined in detail. Ligand competition assays using a series of oligosaccharides derived from or related to sycamore maple XG demonstrated that a terminal alpha-(1-->2)-linked fucosyl residue constitutes an essential part of the epitope recognized by CCRC-M1. Oligosaccharides containing this structural motif compete with intact sycamore maple XG for binding to the antibody, whereas structurally related oligosaccharides, which do not contain terminal fucosyl residues or in which the terminal fucosyl residue is linked alpha-(1-->3) to the adjacent glycosyl residue, do not compete for the antibody binding site. The ligand binding assays also indicate that CCRC-M1 binds to a conformationally dependent structure of the polysaccharide. Other results of this study establish that some of the carbohydrate epitopes of the plant extracellular matrix are shared among different macromolecules.     

Immunohistochemical localization of apyrase during initial differentiation and germination of pea seeds

Localization of the 49-kDa apyrase (ATP diphosphohydrolase, EC3.6.1.5; DDBJ/EMBL/GenBank BAB40230) was investigated during early stages of germination of pea (Pisum sativum L. var. Alaska) at the organ, tissue, cellular, and sub-cellular level using light-microscopical immunohistochemistry. Whole mount tissues were immuno-reacted with anti-APY1 serum, pre-immune serum or anti-actin antibody for control. Antigen to the anti-APY1 serum was not detected until 16 h after sowing (26 h after start of imbibition), when the antigen was detected throughout the tissue, especially in the epidermis and cortex. At 35 h after sowing, the younger regions including the root tip and the tip of the stele were more strongly stained than the control. Both, epidermal and cortical cells of the epicotyl and root tip were stained. The stain was mainly localized in the cytoplasm and around nuclei in the apical meristem and the root tip, while vacuoles and cell walls were not stained. At 62 h, there was major staining in the plumule, hook, and elongating regions of the epicotyl and in the region between cotyledons and the epicotyl. After 84 h, lateral root primordia were stained. The pre-immune serum showed virtually no staining while the anti-actin antibody reacted solely with the cytoplasm. Since the antigen to the anti-APY1 serum was primarily found in the cytoplasm and around nuclei in elongating and differentiating tissues and labeling declined in mature tissues, it is suggested that apyrases may play a role in growth and development of tissues, for example, lateral roots.     

Cell wall polysaccharide distribution in Sandersonia aurantiaca flowers using immuno-detection

The localization of cell wall polysaccharides of the fused petals of monocotyledonous Sandersonia aurantiaca flowers has been identified using antibodies directed to pectin and xyloglucan epitopes and detection by fluorescence microscopy. Cross sections of the petal tissue were taken from cut flowers in bud and at various stages of maturity and senescence. Patterns of esterification in pectin backbones were identified by JIM5 and 2F4 labelling. Pectic galactan and arabinan side branches were detected by LM5 and LM6, respectively, while fucosylated xyloglucan was identified by CCRC-M1. The labelling patterns highlighted compositional differences between walls of the outer/inner epidermis compared to the spongy parenchyma cells of the interior mesophyll for fucosylated xyloglucan and arabinan. Partially esterified homogalacturonan was present in the junction zones of the outer epidermis and points of contact between cells of the mesophyll, and persisted throughout senescence. Pectic galactans were ubiquitous in the outer and inner epidermal cell walls and walls of the interior mesophyll at flower opening, whereas pectic arabinan was found predominantly in the epidermal cells. Galactan was lost from walls of all cells as flowers began to senesce, while fucosylated xyloglucan appeared to increase over this time. Such differences in the location of polysaccharides and the timing of changes suggest distinct combinations of certain polysaccharides offer mechanical and rheological advantages that may assist with flower opening and senescence.     

UPTAKE BY AND FATE OF LYSOZYME IN ROOTS OF IASIONE MONTANA (CAMPANULACEAE)

A basic protein, lysozyme, labeled with fluorescein isothiocyanate, is readily taken up by roots of lasione montana. Most of the protein taken up is tightly bound to cell walls of the roots. Fluorescent protein is diluted in the growing region of a root as cells elongate and divide. Fluorescence remains in mature nongrowing regions and root cap cells for one to two weeks. Redistribution and translocation of the protein within the root is minimal or nil. A layer of chloroform-soluble material that prevents lysozyme from interacting with stem cell walls exposed to fluorescent lysozyme was found on stems of germinating Iasione montana.     

Wall ingrowths in epidermal transfer cells of Vicia faba cotyledons are modified primary walls marked by localized accumulations of arabinogalactan proteins

Despite the importance of transfer cells in enhancing nutrient transport in plants, little is known about how deposition of the complex morphology of their wall ingrowths is regulated. We probed thin sections of mature cotyledon epidermal transfer cells of Vicia faba with affinity probes and antibodies specific to polysaccharides and glycoproteins, to determine the distribution of these components in their walls. Walls of these transfer cells consist of the pre-existing primary wall, a uniformly deposited wall layer and wall ingrowths which are comprised of two regions; an electron-opaque inner region and an electron-translucent outer region. The primary wall reacted strongly with antibodies against esterified pectin, xyloglucan, the side chains of rhamnogalaturonan-1 and a cellulase-gold affinity probe. The electron-opaque inner region of wall ingrowths displayed a similar labeling pattern to that of the primary wall, showing strong cross-reactivity with all antibodies tested, except those reacting against highly de-esterified pectins. The electron-opaque outer layer of developmentally more mature wall ingrowths reacted strongly with anti-callose monoclonal and polyclonal antibodies, but showed no reaction for pectin or xyloglucan antibodies or the cellulase-gold affinity probe. The plasma membrane-wall interface was labeled strongly with anti-arabinogalactan protein (AGP) antibodies, with some AGP-reactive antibodies also labeling the electron-translucent zone. Nascent wall ingrowths were labeled specifically with AGPs but not anti-callose. A reduction in wall ingrowth density was observed when developing transfer cells were exposed to beta-d-glucosyl Yariv reagent compared with controls. Our results indicate that wall ingrowths of transfer cells are primary wall-like in composition and probably require AGPs for localized deposition.     

Root hair-specific disruption of cellulose and xyloglucan in <Emphasis Type="Italic">AtCSLD3</Emphasis> mutants,and factors affecting the post-rupture resumption of mutant root hair growth

The glycosyl transferase encoded by the cellulose synthase-like gene CSLD3/KJK/RHD7 (At3g03050) is required for cell wall integrity during root hair formation in Arabidopsis thaliana but it remains unclear whether it contributes to the synthesis of cellulose or hemicellulose. We identified two new alleles, root hair-defective (rhd) 7-1 and rhd7-4, which affect the C-terminal end of the encoded protein. Like root hairs in the previously characterized kjk-2 putative null mutant, rhd7-1 and rhd7-4 hairs rupture before tip growth but, depending on the growth medium and temperature, hairs are able to survive rupture and initiate tip growth, indicating that these alleles retain some function. At 21°C, the rhd7 tip-growing root hairs continued to rupture but at 5oC, rupture was inhibited, resulting in long, wild type-like root hairs. At both temperatures, the expression of another root hair-specific CSLD gene, CSLD2, was increased in the rhd7-4 mutant but reduced in the kjk-2 mutant, suggesting that CSLD2 expression is CSLD3-dependent, and that CSLD2 could partially compensate for CSLD3 defects to prevent rupture at 5°C. Using a fluorescent brightener (FB 28) to detect cell wall (1 → 4)-β-glucans (primarily cellulose) and CCRC-M1 antibody to detect fucosylated xyloglucans revealed a patchy distribution of both in the mutant root hair cell walls. Cell wall thickness varied, and immunogold electron microscopy indicated that xyloglucan distribution was altered throughout the root hair cell walls. These cell wall defects indicate that CSLD3 is required for the normal organization of both cellulose and xyloglucan in root hair cell walls.