Domain-specific and cell type-specific localization of two types of cell wall matrix polysaccharides in the clover root tip

Using immunocytochemical techniques and antibodies that specifically recognize xyloglucan (anti-XG), polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), and methylesterified pectins (JIM 7), we have shown that these polysaccharides are differentially synthesized and localized during cell development and differentiation in the clover root tip. In cortical cells XG epitopes are present at a threefold greater density in the newly formed cross walls than in the older longitudinal walls, and PGA/RG-I epitopes are detected solely in the expanded middle lamella of cortical cell corners, even after pretreatment of sections with pectinmethylesterase to uncover masked epitopes. These results suggest that in cortical cells XG and PGA/RG-I are differentially localized not only to particular wall domains, but also to particular cell walls. In contrast to their nonoverlapping distribution in cortical cells, XG epitopes and PGA/RG-I epitopes largely colocalize in the epidermal cell walls. The results also demonstrate that the middle lamella of the longitudinal walls shared by epidermal cells and by epidermal and cortical cells constitutes a barrier to the diffusion of cell wall and mucilage molecules. Synthesis of XG and PGA/RG-I epitope-containing polysaccharides also varies during cellular differentiation in the root cap. The differentiation of gravitropic columella cells into mucilage-secreting peripheral cells is marked by a dramatic increase in the synthesis and secretion of molecules containing XG and PGA/RG-I epitopes. In contrast, JIM 7 epitopes are present at abundant levels in columella cell walls, but are not detectable in peripheral cell walls or in secreted mucilage. There were also changes in the cisternal labeling of the Golgi stacks during cellular differentiation in the root tip. Whereas PGA/RG-I epitopes are detected primarily in cis- and medial Golgi cisternae in cortical cells (Moore, P. J., K. M. M. Swords, M. A. Lynch, and L. A. Staehelin. 1991. J. Cell Biol. 112:589-602), they are localized predominantly in the trans-Golgi cisternae and the trans-Golgi network in epidermal and peripheral root cap cells. These observations suggest that during cellular differentiation the plant Golgi apparatus can be both structurally and functionally reorganized.     

Immunocytochemical localization of cell wall polysaccharides in the root tip ofAvena sativa

Summary Two polyclonal antisera, anti-xyloglucan (anti-XG) and anti-polygalacturonic acid/rhamnogalacturonan I (anti-PGA/RG-I), which recognize, respectively, noncellulosic -(14)-D-glucan containing polysaccharides and the unesterified forms of the acidic pectic polysaccharide polygalacturonic acid/rhamnogalacturonan I, were used to localize epitopes recognized by the two antisera in the root tip of oat (Avena sativa). Immunoblot analysis shows that epitopes recognized by the anti-XG antibodies are present in both the mixed linkage -(13)-(14)-D-glucans (MG) and in xyloglucan (XG). Immunogold electron microscopy shows that the cell walls of meristematic, cortical, epidermal, columella, and peripheral cells contain significant amounts of such epitopes. In contrast, the molecules that carry these MG/XG epitopes appear to be sparse in the expanded middle lamella of meristematic cells, but dense in the expanded middle lamella of peripheral root cap cells. This finding suggests that the porosity of the middle lamella is altered in peripheral root cap cells to facilitate mucilage secretion. In contrast, few PGA/RG-I epitopes were detected in any cell walls of any of the cell types examined. Double immunogold labeling experiments revealed an intriguing localization pattern of MG/XG and of PGA/RG-I epitopes in the peripheral mucilage-secreting cells of the root cap. Whereas MG/XG epitopes were abundant in the cell wall, they were sparse in both the secreted mucilage and in intracellular secretory vesicles. In marked contrast, PGA/RG-I epitopes were detected at high density in intracellular secretory vesicles, but unexpectedly, were quite sparse in both the cell wall and in the mucilage. These immunolabeling patterns are consistent with the hypotheses that the synthesis and secretion of particular -D-glucans is subject to both activation and down-regulation during cell development and differentiation and that post-secretory alterations of pectic polysaccharides, such as enzymatic release of RG-I-type mucilage molecules from PGA/RG-I precursors, may occur in the peripheral cell walls of the oat root cap.Abbreviations MG mixed linkage -(13)-(14)-D-glucan - PGA/RG-I polygalacturonic acid/rhamnogalacturonan I - SEPS sycamore extracellular polysaccharides - TGN trans Golgi network - XG xyloglucan     

Immunogold localization of xyloglucan and rhamnogalacturonan I in the cell walls of suspension-cultured sycamore cells

Plant cell walls serve several functions: they impart rigidity to the plant, provide a physical and chemical barrier between the cell and its environment, and regulate the size and shape of each cell. Chemical studies have provided information on the biochemical composition of the plant cell walls as well as detailed knowledge of individual cell wall molecules. In contrast, very little is known about the distribution of specific cell wall components around individual cells and throughout tissues. To address this problem, we have produced polyclonal antibodies against two cell wall matrix components; rhamnogalacturonan I (RG-I), a pectic polysaccharide, and xyloglucan (XG), a hemicellulose. By using the antibiodies as specific markers we have been able to localize these polymers on thin sections of suspension-cultured sycamore cells (Acer pseudoplatanus). Our results reveal that each molecule has a unique distribution. XG is localized throughout the entire wall and middle lamella. RG-I is restricted to the middle lamella and is especially evident in the junctions between cells. These observations indicate that plant cell walls may have more distinct chemical (and functional?) domains than previously envisaged.     

Spatial organization of the assembly pathways of glycoproteins and complex polysaccharides in the Golgi apparatus of plants

The Golgi apparatus of plant cells is the site of assembly of glycoproteins, proteoglycans, and complex polysaccharides, but little is known about how the different assembly pathways are organized within the Golgi stacks. To study these questions we have employed immunocytochemical techniques and antibodies raised against the hydroxyproline-rich cell wall glycoprotein, extensin, and two types of complex polysaccharides, an acidic pectic polysaccharide known as rhamnogalacturonan I (RG-I), and the neutral hemicellulose, xyloglucan (XG). Our micrographs demonstrate that individual Golgi stacks can process simultaneously glycoproteins and complex polysaccharides. O-linked arabinosylation of the hydroxyproline residues of extensin occurs in cis-cisternae, and glycosylated molecules pass through all cisternae before they are packaged into secretory vesicles in the monensin-sensitive, trans-Golgi network. In contrast, in root tip cortical parenchyma cells, the anti-RG-I and the anti-XG antibodies are shown to bind to complementary subsets of Golgi cisternae, and several lines of indirect evidence suggest that these complex polysaccharides may also exit from different cisternae. Thus, RG-I type polysaccharides appear to be synthesized in cis- and medial cisternae, and have the potential to leave from a monensin-insensitive, medial cisternal compartment. The labeling pattern for XG suggests that it is assembled in trans-Golgi cisternae and departs from the monensin-sensitive trans-Golgi network. This physical separation of the synthesis/secretion pathways of major categories of complex polysaccharides may prevent the synthesis of mixed polysaccharides, and provides a means for producing secretory vesicles that can be targeted to different cell wall domains.     

Characterization of the cell-wall polysaccharides of Arabidopsis thaliana leaves.

The cell-wall polysaccharides of Arabidopsis thaliana leaves have been isolated, purified, and characterized. The primary cell walls of all higher plants that have been studied contain cellulose, the three pectic polysaccharides homogalacturonan, rhamnogalacturonan I and rhamnogalacturonan II, the two hemicelluloses xyloglucan and glucuronoarabinoxylan, and structural glycoproteins. The cell walls of Arabidopsis leaves contain each of these components and no others that we could detect, and these cell walls are remarkable in that they are particularly rich in phosphate buffer-soluble polysaccharides (34% of the wall). The pectic polysaccharides of the purified cell walls consist of rhamnogalacturonan I (11%), rhamnogalacturonon II (8%), and homogalacturonan (23%). Xyloglucan (XG) accounts for 20% of the wall, and the oligosaccharide fragments generated from XG by endoglucanase consist of the typical subunits of other higher plant XGs. Glucuronoarabinoxylan (4%), cellulose (14%) and protein (14%) account for the remainder of the wall. Except for the phosphate buffer-soluble pectic polysaccharides, the polysaccharides of Arabidopsis leaf cell walls occur in proportions similar to those of other plants. The structure of the Arabidopsis cell-wall polysaccharides are typical of those of many other plants.     

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.     

Structural characterization of cell wall polysaccharides from two plant species endemic to central Africa,Fleurya aestuans and Phragmenthera capitata

Fleurya aestuans (Linnaeus) Miquel and Phragmenthera capitata (Spreng) are two plants endemic to central Africa that are used in traditional medicine. However, information on their molecular constituents is lacking. In the present study and as part of our research on the structure/bioactivity relationship of plant cell wall molecules, we investigated the structure of polysaccharides isolated from leaf cell walls of both plant species. To this end, we used sequential extraction of polysaccharides, gas chromatography, matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-TOF MS) and immuno-dot assays. Our data indicate the presence of both pectin and hemicellulosic polysaccharides in the cell walls of both plants. In particular, cell wall of F. aestuans leaves appears to contain much more pectin than those of P. capitata. Structural analysis of hemicellulosic polysaccharides revealed differences in the structure of xyloglucan isolated from both species. While only the XXXG-type was found in P. capitata, both XXXG and XXGG types were detected in F. aestuans. No arabinosylated subunits were found in any of the xyloglucan isolated from both plant species. In addition, xylan structure with non methylated-α-d-glucuronic acid on side chains was only detected in F. aestuans leaf cell walls. Finally, structural analysis of rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) shows that unlike RG-II, RG-I is qualitatively different between F. aestuans and P. capitata leaves.     

Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides

Isolated cell walls of Argania spinosa fruit pulp were fractionated into their polysaccharide constituents and the resulting fractions were analysed for monosaccharide composition and chemical structure. The data reveal the presence of homogalacturonan, rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II) in the pectic fraction. RG-I is abundant and contains high amounts of Ara and Gal, indicative of an important branching in this polysaccharide. RG-II is less abundant than RG-I and exists as a dimer. Structural characterisation of xyloglucan using enzymatic hydrolysis, gas chromatography, MALDI-TOF-MS and methylation analysis shows that XXGG, XXXG, XXLG and XLLG are the major subunit oligosaccharides in the ratio of 0.6:1:1.2:1.6. This finding demonstrates that the major neutral hemicellulosic polysaccharide is a galacto-xyloglucan. In addition, Argania fruit xyloglucan has no XUFG, a novel xyloglucan motif recently discovered in Argania leaf cell walls. Finally, the isolation and analysis of arabinogalactan-proteins showed that Argania fruit pulp is rich in these proteoglycans.     

Antibody localization of extensin in cell walls of carrot storage roots

The accumulation and cross-linking of hydroxyproline-rich glycoproteins (HRGPs) in cell walls of dicotyledonous plants has been correlated with a number of wall-strengthening phenomena. Polyclonal antibodies raised against glycosylated extensin-1, the most abundant HRGP in carrot (Daucus carota L.) cell walls, recognize this antigen on gel and dot blots and on thin sections of epoxy-embedded carrot-root cell walls. Since wall labeling can be largely reduced by preincubating the antibodies with purified extensin-1, most labeling can be attributed to recognition of this antigen. The remaining label may be the result of recognition of extensin-2, a second carrot HRGP, or other wall components (cellulose, hemicellulose and pectin are not recognized). Extensin-1 label was distributed quite uniformly across the cell wall but was absent from the expanded middle lamella at the intersection of three or more cells and was reduced in the narrow middle lamella between two cells. This distribution is essentially the same as that of cellulose. Because of limitations of this labeling technique, it is not possible to construct a complete model of the structure of the cross-linked extensin matrix. Nonetheless, short, linear arrays of gold particles may represent small portions of the extensin matrix or of individual extensin molecules as they are exposed on the surface of sections. These and other results presented here indicate that: a) newly synthesized extensin is added to the wall by intussusception; b) extensin cannot cross the middle lamella separating the walls of adjacent cells; and c) incorporation of extensin is a late event in the development of phloem-parenchyma cell walls in carrot.Abbreviations dE-1 antibodies antibodies raised against deglycosylated extensin 1 - ELISA enzyme-linked immunosorbant assay - gE-1 antibodies antibodies raised against glycosylated extensin 1 - HRGP hydroxyproline-rich glycoprotein - PAGE polyacrylamide gel electrophoresis - RG-1 rhamnogalacturonan I - SDS sodium dodecyl sulfate     

Secretion of cell wall polysaccharides in Vicia root hairs

Root hairs of hairy winter vetch ( Vicia villosa Roth) synthesize and secrete abundant cell wall matrix polysaccharides, making this an excellent system for the study of secretion during tip growth. Roots with newly formed hairs were preserved by cryofixation and freeze substitution. Cryofixed root hairs showed excellent structural and antigenic preservation. Ultrastructural analyses showed numerous vesicles near the tip and a concentration of Golgi bodies in the subapical region of the hair. The distribution of polygalacturonic acid and xyloglucan in the endomembrane system and cell wall were revealed by immunolabeling, using previously characterized monoclonal antibodies. De-esterified polygalacturonic acid was present on the external surface of the cell wall, but was not detected within the cell, although chemical de-esterification revealed abundant antigen in Golgi bodies and secretory vesicles. Methyl-esterified polygalacturonic acid epitopes were detected within the medial and trans cisternae of Golgi bodies, in secretory vesicles, and throughout the wall, indicating that pectin is secreted in a neutral form and may then be de-esterified in muro . Xyloglucan was also detected within the trans cisternae of Golgi bodies, secretory vesicles and throughout the cell wall. Double labeling experiments demonstrated that both polysaccharides occur simultaneously in the same Golgi bodies, and that secretory vesicles containing both polygalacturonic acid and xyloglucan deliver the polysaccharides to the cell wall at the growing tip.     

Biophysical consequences of remodeling the neutral side chains of rhamnogalacturonan I in tubers of transgenic potatoes

Two lines of transgenic potato (Solanum tuberosum L.) plants modified in their cell wall structure were characterized and compared to wild type with regard to biomechanical properties in order to assign functional roles to the particular cell wall polysaccharides that were targeted by the genetic changes. The targeted polymer was rhamnogalacturonan I (RG-I), a complex pectic polysaccharide comprised of mainly neutral oligosaccharide side chains attached to a backbone of alternating rhamnosyl and galacturonosyl units. Tuber rhamnogalacturonan I molecules from the two transformed lines are reduced in linear galactans and branched arabinans, respectively. The transformed tuber tissues were found to be more brittle when subjected to uniaxial compression and the side-chain truncation was found to be correlated with the physical properties of the tissue. Interpretation of the force–deflection curves was aided by a mathematical model that describes the contribution of the cellulose microfibrils, and the results lead to the proposition that the pectic matrix plays a role in transmitting stresses to the load-bearing cellulose microfibrils and that even small changes to the rheological properties of the matrix have consequences for the biophysical properties of the wall.     

Assembly of cell-wall glycoproteins of Chlamydomonas reinhardii: Oligosaccharides are added in medial and trans Golgi compartments

A series of monoclonal antibodies and a polyclonal antiserum have been used to investigate the localisation and pathway of biosynthesis of the cell-wall hydroxyproline-rich glycoprotein 2BII in the alga Chlamydomonas reinhardii. Glyco-protein precursors were detected within the endoplasmic reticulum using a polyclonal antiserum raised to the deglycosylated 2BII. Monoclonal antibodies which are known to recognise different carbohydrate epitopes of 2BII were found to label two distinct regions of the Golgi stack. The immunolabelling results demonstrate that there is compartmentation of protein synthesis and glycosylation steps for these O-glycosidically linked glycoproteins. Newly synthesised glycoproteins are transported from the Golgi apparatus to the cell surface via two distinct routes. They then undergo assembly into a cell wall, the inner wall layer being formed first and probably functionaing as a template within which the outer crystalline wall layers are assembled.Abbreviations DGP deglycosylated glycoprotein - ER endoplasmic reticulum - MAC monoclonal antibody centre - M _r relative molecular mass     

Characterization of cell wall polysaccharides from the medicinal plant Panax notoginseng

Panax notoginseng is a commonly used medicinal plant in south-western China. Recent studies indicate that wall polysaccharides are responsible for some of the immunostimulatory activity. Fractionation of the P. notoginseng root powder alcohol insoluble residue (AIR) and its compositional analysis enabled us to deduce the polysaccharide and protein composition of the root cell walls. P. notoginseng walls are composed primarily of polysaccharide (approximately 97% w/w) and some protein. The polysaccharides include pectic polysaccharides (neutral Type I 4-galactan (21%), arabinan (5%), acidic rhamnogalacturonan I (RG I, 2%) and homogalacturonan (HGA, 24%), non-cellulosic polysaccharides (heteroxylan, 3%), xyloglucan (XG, 3%) and heteromannan (1%)) and cellulose (24%). The root AIR also contains Type II AG/AGPs (5% w/w) typically associated with the plasma membrane and extracellular matrix. Thus, P. notoginseng roots contain polysaccharides typical of Type I primary cell walls but are distinguished by their very high levels of Type I 4-galactans and low levels of XGs. The major amino acids in the AIR were Leu (14 mol%), Asx (16 mol%), Glx (10 mol%), Ala (9 mol%), Thr (9 mol%) and Val (9 mol%).     

A distinct class of vesicles derived from the trans‐Golgi mediates secretion of xylogalacturonan in the root border cell

Root border cells lie on the surface of the root cap and secrete massive amounts of mucilage that contains polysaccharides and proteoglycans. Golgi stacks in the border cells have hypertrophied margins, reflecting elevated biosynthetic activity to produce the polysaccharide components of the mucilage. To investigate the three‐dimensional structures and macromolecular compositions of these Golgi stacks, we examined high‐pressure frozen/freeze‐substituted alfalfa root cap cells with electron microscopy/tomography. Golgi stacks in border cells and peripheral cells, precursor cells of border cells, displayed similar morphological features, such as proliferation of trans cisternae and swelling of the trans cisternae and trans‐Golgi network (TGN) compartments. These swollen margins give rise to two types of vesicles larger than other Golgi‐associated vesicles. Margins of trans‐Golgi cisternae accumulate the LM8 xylogalacturonan (XGA) epitope, and they become darkly stained large vesicles (LVs) after release from the Golgi. Epitopes for xyloglucan (XG), polygalacturonic acid/rhamnogalacturonan‐I (PGA/RG‐I) are detected in the trans‐most cisternae and TGN compartments. LVs produced from TGN compartments (TGN‐LVs) stained lighter than LVs and contained the cell wall polysaccharide epitopes seen in the TGN. LVs carrying the XGA epitope fuse with the plasma membrane only in border cells, whereas TGN‐LVs containing the XG and PGA/RG‐I epitopes fuse with the plasma membrane of both peripheral cells and border cells. Taken together, these results indicate that XGA is secreted by a novel type of secretory vesicles derived from trans‐Golgi cisternae. Furthermore, we simulated the collapse in the central domain of the trans‐cisternae accompanying polysaccharide synthesis with a mathematical model.     

GO-PROMTO illuminates protein membrane topologies of glycan biosynthetic enzymes in the Golgi apparatus of living tissues

The Golgi apparatus is the main site of glycan biosynthesis in eukaryotes. Better understanding of the membrane topology of the proteins and enzymes involved can impart new mechanistic insights into these processes. Publically available bioinformatic tools provide highly variable predictions of membrane topologies for given proteins. Therefore we devised a non-invasive experimental method by which the membrane topologies of Golgi-resident proteins can be determined in the Golgi apparatus in living tissues. A Golgi marker was used to construct a series of reporters based on the principle of bimolecular fluorescence complementation. The reporters and proteins of interest were recombinantly fused to split halves of yellow fluorescent protein (YFP) and transiently co-expressed with the reporters in the Nicotiana benthamiana leaf tissue. Output signals were binary, showing either the presence or absence of fluorescence with signal morphologies characteristic of the Golgi apparatus and endoplasmic reticulum (ER). The method allows prompt and robust determinations of membrane topologies of Golgi-resident proteins and is termed GO-PROMTO (for GOlgi PROtein Membrane TOpology). We applied GO-PROMTO to examine the topologies of proteins involved in the biosynthesis of plant cell wall polysaccharides including xyloglucan and arabinan. The results suggest the existence of novel biosynthetic mechanisms involving transports of intermediates across Golgi membranes.     

Structure and function in the ligule ofSelaginella kraussiana 3. The uptake of tritiated glucose

Summary The ligule ofSelaginella kraussiana shows active incorporation of tritiated glucose in the central region; particularly into the Golgi system, but also into endoplasmic reticulum, mitochondria and cell periphery.Two hours chase in unlabelled glucose reveals a small amount of cell wall formation but most of the label remains in the Golgi bodies. The results suggest that the Golgi system in the mature ligule has the capacity to synthesise some complex carbohydrate, but is relatively inactive in its secretion. This is discussed in relation to the ontogeny and phyllogeny of the ligule.     

Polysaccharides from grape berry cell walls. Part I: tissue distribution and structural characterization of the pectic polysaccharides

Buffer-soluble arabinogalactan-proteins (AGPs) and pectins from grape berry skin and pulp tissues have been isolated and their structure has been partly determined. Pectic polysaccharides from the cell wall material were solubilized by treating pulp and skin cell walls with homogeneous glycosyl hydrolases. Homogalacturonans, rhamnogalacturonans I (RG-I), and rhamnogalacturonan II (RG-II) of each tissue have been fractionated by high resolution size exclusion chromatography and their relative distribution and major structural features have been determined. It has been shown that pulp tissue contains two-fold more buffer-soluble AGPs and pectins than skin tissue and we have determined that 75% of the grape berry walls originates from the skin tissue. There is three-fold more RG-I and RG-II in skin tissue than in pulp tissue and three-fold more RG-I than RG-II in the grape berry cell walls.

The results of this study have shown that the grape polysaccharide content of a wine is related to the type of tissue used for wine making and to the solubility of the grape polysaccharides and their resistance to fragmentation by grape and yeast glycanases.     

O-Acetylation of plant cell wall polysaccharides: identification and partial characterization of a rhamnogalacturonan O-acetyl-transferase from potato suspension-cultured cells

 A microsomal preparation from suspension-cultured potato stem cells (Solanum tuberosum L. cv. AZY) was incubated with [¹⁴C]acetyl-CoA resulting in a precipitable radiolabeled product. Analysis of the product revealed that it consisted mostly of acetylated proteins and cell wall polysaccharides, including xyloglucan, homogalacturonan and rhamnogalacturonan I. Thus, acetyl-CoA is a donor-substrate for the O-acetylation of wall polysaccharides. A rhamnogalacturonan acetylesterase was used to develop an assay to measure and characterize rhamnogalacturonan O-acetyl transferase activity in the microsomal preparation. Using this assay, it was shown that the transferase activity was highest during the linear growth phase of the cells, had a pH-optimum at pH 7.0, a temperature optimum at 30 °C, an apparent K _m of 35 μM and an apparent V _max of 0.9 pkat per mg protein. Further analysis of the radiolabeled acetylated product revealed that it had a molecular mass >500 kDa. Received: 3 July 1999; Accepted: 27 September 1999     

Functional characterisation of Nicotiana tabacum xyloglucan endotransglycosylase (NtXET-1): generation of transgenic tobacco plants and changes in cell wall xyloglucan

To study the function of xyloglucan endotransglycosylase (XET) in vivo we isolated, a tomato (Lycopersicon esculentum Mill.) XET cDNA (GenBank AA824986) from the homologous tobacco (Nicotiana tabacum L.) clone named NtXET-1 (Accession no. D86730). The expression pattern revealed highest levels of NtXET-1 mRNA in organs highly enriched in vascular tissue. The levels of NtXET-1 mRNA decreased in midribs with increasing age of leaves. Increasing leaf age was correlated with an increase in the average molecular weight (MW) of xyloglucan (XG) and a decrease in the relative growth rates of leaves. Transgenic tobacco plants with reduced levels of XET activity were created to further study the biochemical consequences of reduced levels of NtXET-1 expression. In two independent lines, total XET activity could be reduced by 56% and 37%, respectively, in midribs of tobacco plants transformed with an antisense construct. The decreased activity led to an increase in the average MW of XG by at least 20%. These two lines of evidence argue for NtXET-1 being involved in the incorporation of small XG molecules into the cell wall by transglycosylation. Reducing the incorporation of small XG molecules will result in a shift towards a higher average MW. The observed reduction in NtXET-1 expression and increase in the MW of XG in older leaves might be associated with strengthening of cell walls by reduced turnover and hydrolysis of XG. Received: 24 January 2000 / Accepted: 21 July 2000     

Pectin esterification is spatially regulated both within cell walls and between developing tissues of root apices

Monoclonal antibodies recognizing un-esterified (JIM5) and methyl-esterified (JIM7) epitopes of pectin have been used to locate these epitopes by indirect immunofluorescence and immunogold electron microscopy in the root apex of carrot (Daucus carota L.). Both antibodies labelled the walls of cells in all tissues of the developing root apex. Immunogold labelling observed at the level of the electron microscope indicated differential location of the pectin epitopes within the cell walls. The un-esterified epitope was located to the inner surface of the primary cell walls adjacent to the plasma membrane, in the middle lamella and abundantly to the outer surface at intercellular spaces. In contrast, the epitope containing methyl-esterified pectin was located evenly throughout the cell wall. In root apices of certain other species the JIM5 and JIM7 epitopes were found to be restricted to distinct tissues of the developing roots. In the root apex of oat (Avena sativa L.), JIM5 was most abundantly reactive with cell walls at the region of intercellular spaces of the cortical cells. JIM7 was reactive with cells of the cortex and the stele. Neither epitope occurred in walls of the epidermal or root-cap cells. These pattern of expression were observed to derive from the very earliest stages of the development of these tissues in the oat root meristem and were maintained in the mature root. In the coleoptile and leaf tissues of oat seedlings, JIM5 labelled all cells abundantly whereas JIM7 was unreactive. Other members of the Gramineae and also the Chenopodiaceae are shown to express similar restricted spatial patterns of distribution of these pectin epitopes in root apices.Abbreviations CDTA 1,2-diaminocyclohexane tetraacetic acid - RG rhamnogalacturonan J.P.K. was supported by the Agricultural and Food Research Council Cell Signalling and Recognition Programme. We thank J. Cooke and N. Stacey for technical assistance, H.A. Schols, Drs. P. Albersheim and A. Darvill for pectic polysaccharides, and Dr. R.R. Selvendran and M. McCann for useful discussions.