Developmental regulation of pectic epitopes during potato tuberisation.

We show, by immunogold labelling, that potato (Solanum tuberosum L. cv Karnico) pectic epitopes are developmentally regulated within regions of the stolon, in addition to showing tissue-specific differences in abundance and localisation. The (1-->4)-beta-D-galactan and (1-->5)-alpha-arabinan epitopes demarcate two distinct zones within stolons; galactans are enriched in primary walls of elongating cells proximal to the stolon hook, whilst arabinans predominate in younger cells distal to the hook. Low-methoxyl homogalacturonan epitopes are concentrated in the middle lamella and show a proximo-distal gradient in stolons similar to that of galactans, whilst high-methoxyl homogalacturonan is uniformly abundant. Calcium pectate is restricted to the middle lamella at cell corners and pit fields. Calcium-binding sites are uniformly present in stolon cell walls, but their total density is reduced and they become localised to a few cell corners in mature tubers, as determined by image-electron energy loss spectroscopy. During the transition from elongation growth to isodiametric expansion during tuberisation of the stolon hook, there were no detectable changes in pectic epitope abundance or localisation. As tubers matured, all epitopes increased in abundance in parenchymal cell walls, except for calcium pectate. We conclude that potentially significant changes in pectic composition occur as young cells distal to the stolon hook move into the zone of cell elongation proximal to the hook.

Cell wall architecture of the elongating maize coleoptile.

The primary walls of grasses are composed of cellulose microfibrils, glucuronoarabinoxylans (GAXs), and mixed-linkage beta-glucans, together with smaller amounts of xyloglucans, glucomannans, pectins, and a network of polyphenolic substances. Chemical imaging by Fourier transform infrared microspectroscopy revealed large differences in the distributions of many chemical species between different tissues of the maize (Zea mays) coleoptile. This was confirmed by chemical analyses of isolated outer epidermal tissues compared with mesophyll-enriched preparations. Glucomannans and esterified uronic acids were more abundant in the epidermis, whereas beta-glucans were more abundant in the mesophyll cells. The localization of beta-glucan was confirmed by immunocytochemistry in the electron microscope and quantitative biochemical assays. We used field emission scanning electron microscopy, infrared microspectroscopy, and biochemical characterization of sequentially extracted polymers to further characterize the cell wall architecture of the epidermis. Oxidation of the phenolic network followed by dilute NaOH extraction widened the pores of the wall substantially and permitted observation by scanning electron microscopy of up to six distinct microfibrillar lamellae. Sequential chemical extraction of specific polysaccharides together with enzymic digestion of beta-glucans allowed us to distinguish two distinct domains in the grass primary wall. First, a beta-glucan-enriched domain, coextensive with GAXs of low degrees of arabinosyl substitution and glucomannans, is tightly associated around microfibrils. Second, a GAX that is more highly substituted with arabinosyl residues and additional glucomannan provides an interstitial domain that interconnects the beta-glucan-coated microfibrils. Implications for current models that attempt to explain the biochemical and biophysical mechanism of wall loosening during cell growth are discussed.

Differential expression of cell-wall-related genes during the formation of tracheary elements in the Zinnia mesophyll cell system.

Plants, animals and some fungi undergo processes of cell specialization such that specific groups of cells are adapted to carry out particular functions. One of the more remarkable examples of cellular development in higher plants is the formation of water-conducting cells that are capable of supporting a column of water from the roots to tens of metres in the air for some trees. The Zinnia mesophyll cell system is a remarkable tool with which to study this entire developmental pathway in vitro. We have recently applied an RNA fingerprinting technology, to allow the detection of DNA fragments derived from RNA using cDNA synthesis and subsequent PCR-amplified fragment length polymorphisms (cDNA-AFLP), to systematically characterize hundreds of the genes involved in the process of tracheary element formation. Building hoops of secondary wall material is the key structural event in forming functional tracheary elements and we have identified over 50 partial sequences related to cell walls out of 600 differentially expressed cDNA fragments. The Zinnia system is an engine of gene discovery which is allowing us to identify and characterize candidate genes involved in cell wall biosynthesis and assembly.

Approaches to understanding the functional architecture of the plant cell wall.

Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins.

COBRA encodes a putative GPI-anchored protein, which is polarly localized and necessary for oriented cell expansion in Arabidopsis.

To control organ shape, plant cells expand differentially. The organization of the cellulose microfibrils in the cell wall is a key determinant of differential expansion. Mutations in the COBRA (COB) gene of Arabidopsis, known to affect the orientation of cell expansion in the root, are reported here to reduce the amount of crystalline cellulose in cell walls in the root growth zone. The COB gene, identified by map-based cloning, contains a sequence motif found in proteins that are anchored to the extracellular surface of the plasma membrane through a glycosylphosphatidylinositol (GPI) linkage. In animal cells, this lipid linkage is known to confer polar localization to proteins. The COB protein was detected predominately on the longitudinal sides of root cells in the zone of rapid elongation. Moreover, COB RNA levels are dramatically upregulated in cells entering the zone of rapid elongation. Based on these results, models are proposed for the role of COB as a regulator of oriented cell expansion.

Xylogenesis: the birth of a corpse.

Xylogenesis is a complex developmental process culminating in programmed cell death as a truly terminal differentiation event. In Arabidopsis, the availability of vascular-patterning mutants, and the identification of genes and their products that are involved in cell specification, secondary-wall deposition and lignification, are providing clues to the functions of some of the sequences in the large expressed sequence tag databases derived from the xylem-rich tissues of trees. An in vitro system, the Zinnia mesophyll cell system, provides an alternative system for those cell-biological experiments that are difficult to tackle in intact plants. In particular, a combination of molecular-genetic and cell-biological approaches has made possible the elucidation of some of the features of plant programmed cell death.

Pectin engineering: modification of potato pectin by in vivo expression of an endo-1,4-beta-D-galactanase.

Potato tuber pectin is rich in galactan (oligomer of beta-1,4-linked galactosyl residues). We have expressed a fungal endo-galactanase cDNA in potato under control of the granule bound starch synthase promoter to obtain expression of the enzyme in tubers during growth. The transgenic plants displayed no altered phenotype compared with the wild type. Fungal endo-galactanase activity was quantified in the transgenic tubers, and its expression was verified by Western blot analysis. The effect of the endo-galactanase activity on potato tuber pectin was studied by Fourier transform infrared microspectroscopy, immuno-gold labeling, and sugar analysis. All analyses revealed alterations in pectin composition. Monosaccharide composition of total cell walls and isolated rhamnogalacturonan I fragments showed a reduction in galactosyl content to 30% in the transformants compared with the wild type. Increased solubility of pectin from transgenic cell walls by endo-polygalacturonase/pectin methylesterase digestion points to other changes in wall architecture.

An abundant TIP expressed in mature highly vacuolated cells.

Aquaporins are water channel proteins found in vacuolar membranes and plasma membranes, and belong to the major intrinsic protein (MIP) family of proteins. In the present study, we purified a 75 kDa MIP protein from a crude fraction of spinach leaf intracellular membranes. Upon urea/SDS-PAGE, the 75 kDa protein appeared as a 21 kDa polypeptide, and the 75 kDa species therefore probably represents a tetramer. The corresponding cDNA was obtained by PCR cloning and had an open reading frame encoding a 25.1 kDa protein. The protein, So-deltaTIP, was most homologous to the tonoplast intrinsic protein (TIP) subfamily of plant MIPs. Using affinity-purified So-deltaTIP-specific peptide antibodies, we investigated the subcellular and tissue distribution of So-deltaTIP. So-deltaTIP was specifically located in the vacuolar membrane. It was abundant in most vacuolated cells in all vegetative organs, but was excluded from the leaf epidermis as well as from the root phloem parenchyma and meristem. In spite of the high sequence homology between delta-TIPs of spinach, Arabidopsis, sunflower and radish, their expression patterns were totally different. However, a comparison of the expression pattern of So-deltaTIP with that of more distantly related TIPs showed similarities with Arabidopsis gamma-TIP, which is expressed in zones of cell elongation/differentiation but excluded from meristematic tissues. Meristematic cells are characterized by many small vacuoles as opposed to elongating and mature cells, which generally harbour a single, large vacuole. Our results indicate that the expression of So-deltaTIP may be induced when the large vacuole is formed.

A pectate lyase from Zinnia elegans is auxin inducible.

The Zinnia mesophyll cell system consists of isolated leaf mesophyll cells in culture that can be induced, by auxin and cytokinin, to reproducibly trans-differentiate into tracheary elements (TE) after 96 h, while in the presence of auxin alone the cells simply elongate. In a search for genes involved in modifications to cell-wall architecture before any overt signs of cell differentiation, a differential hybridization of a 72-h cDNA library with probes from mRNA at time-points of 24 h and 72 h was done revealing a number of transcripts up-regulated between these times. One of these cDNAs shows homology to pectate lyase, a pectin-degrading enzyme. The complete cDNA sequence (ZePel) corresponds to a translated protein of 44 kDa with an N-terminal signal peptide of about 2 kDa, and one potential N-glycosylation site. Northern analysis confirms that the strong expression of this gene during TE induction occurs at a very early stage of the process and is due solely to the presence of auxin in the induction medium. In situ hybridization studies in young Zinnia stems show that ZePel expression is associated with vascular bundles and shoot primordia. Recombinant protein made in Escherichia coli possesses calcium-dependent pectate lyase activity. Pectate lyase activity is detected in elongating and differentiating in vitro cell populations. The role of this enzyme in remodelling the cell wall during cell elongation and differentiation is discussed.

A rapid method to screen for cell-wall mutants using discriminant analysis of Fourier transform infrared spectra.

We have developed a rapid method to screen large numbers of mutant plants for a broad range of cell wall phenotypes using Fourier transform infrared (FTIR) microspectroscopy of leaves. We established and validated a model that can discriminate between the leaves of wild-type and a previously defined set of cell-wall mutants of Arabidopsis. Exploratory principal component analysis indicated that mutants deficient in different cell-wall sugars can be distinguished from each other. Discrimination of cell-wall mutants from wild-type was independent of variability in starch content or additional unrelated mutations that might be present in a heavily mutagenised population. We then developed an analysis of FTIR spectra of leaves obtained from over 1000 mutagenised flax plants, and selected 59 plants whose spectral variation from wild-type was significantly out of the range of a wild-type population, determined by Mahalanobis distance. Cell wall sugars from the leaves of selected putative mutants were assayed by gas chromatography-mass spectrometry and 42 showed significant differences in neutral sugar composition. The FTIR spectra indicated that six of the remaining 17 plants have altered ester or protein content. We conclude that linear discriminant analysis of FTIR spectra is a robust method to identify a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification.

Pectin Modification in Cell Walls of Ripening Tomatoes Occurs in Distinct Domains.

The class of cell wall polysaccharides that undergoes the most extensive modification during tomato (Lycopersicon esculentum) fruit ripening is pectin. De-esterification of the polygalacturonic acid backbone by pectin methylesterase facilitates the depolymerization of pectins by polygalacturonase II (PGII). To investigate the spatial aspects of the de-esterification of cell wall pectins and the subsequent deposition of PGII, we have used antibodies to relatively methylesterified and nonesterified pectic epitopes and to the PGII protein on thin sections of pericarp tissue at different developmental stages. De-esterification of pectins and deposition of PGII protein occur in block-like domains within the cell wall. The boundaries of these domains are distinct and persistent, implying strict, spatial regulation of enzymic activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins strongly associated with cell walls of pericarp tissue at each stage of fruit development show ripening-related changes in this protein population. Western blots of these gels with anti-PGII antiserum demonstrate that PGII expression is ripening-related. The PGII co-extracts with specific pectic fractions extracted with imidazole or with Na2CO3 at 0[deg]C from the walls of red-ripe pericarp tissue, indicating that the strong association between PGII and the cell wall involves binding to particular pectic polysaccharides.

Fourier-Transform Raman and Fourier-Transform Infrared Spectroscopy (An Investigation of Five Higher Plant Cell Walls and Their Components).

Infrared and Raman spectra of sequentially extracted primary cell walls and their pectic polymers were obtained from five angiosperm plants. Fourier-transform Raman spectrometry was shown to be a powerful tool for the investigation of primary cell-wall architecture at a molecular level, providing complementary information to that obtained by Fourier-transform infrared microspectroscopy. The use of an extraction procedure using imidazole instead of cyclohexane trans-1,2-N,N,N[prime],N[prime]-diaminotetraacetate allows the extension of the infrared spectral window for data interpretation from 1300 to 800 cm-1, to 2000 to 800 cm-1, and allows us to obtain Raman spectra from extracted cell-wall material. Wall constituents such as pectins, proteins, aromatic phenolics, cellulose, and hemicellulose have characteristic spectral features that can be used to identify and/or fingerprint these polymers without, in most cases, the need for any physical separation. The Gramineae (rice [Oryza sativa], polypogon [Polypogon fugax steud], and sweet corn [Zea mays]) are spectroscopically very different from the nongraminaceous monocotyledon (onion [Allium cepa]) and the dicotyledon (carrot [Daucus carota]); this reflects differences in chemical composition and cross-linking of the walls. The possibility of a taxonomic classification of plant cell walls based on infrared and Raman spectroscopies and the use of spectral fingerprinting for authentication and detection of adulteration of products rich in cell-wall materials are discussed.

Orientation of macromolecules in the walls of elongating carrot cells.

When round cells from a carrot cell suspension culture are diluted into fresh medium without auxin, the cells elongate to almost 50 times their original diameter within three days. This process of elongation is accompanied by changes in both the composition and the orientation of cell wall polymers. We have obtained information on the orientation of wall polymers in elongating cells by two complementary techniques, one using microscopy and one spectroscopy. Images obtained by the fast-freeze, deep-etch, rotary-shadowed replica technique show that walls of round carrot cells have no net orientation of cellulose microfibrils, and that many thin fibres can be seen cross-linking microfibrils. Walls of elongated carrot cells, in contrast, show a marked net orientation of microfibrils at right angles to the axis of elongation. Fourier Transform Infrared (FTIR) spectra obtained from defined areas of single cell walls show that walls of round carrot cells contain more protein, esters and phenolics in a given area (10 microns x 10 microns) than walls of elongated carrot cells, that contain proportionally more carbohydrate. The orientation of particular functional groups, with respect to the direction of elongation of the cell, can be determined by inserting a polariser into the path of the infrared beam, before it passes through a cell wall sample mounted on the stage of the microscope accessory. In the walls of elongated cells, ester bands, amide bands characteristic of proteins, and stretching frequencies in the carbohydrate region of the spectrum all show a net orientation transverse to the long axis of the cells. In the walls of round carrot cells, however, there is no such net orientation of polymers. Spectra obtained from 25 microns-thick fresh sections of the etiolated stem of a carrot seedling show that different wall components are polarised in different tissue types. These techniques have therefore enabled us to define differences in both the composition and the architecture of walls of elongating cells at the level of a single cell, and to suggest that polymers not previously thought to be ordered, such as pectin and protein, are strictly oriented in some wall types.

A Novel Hydroxyproline-Deficient Arabinogalactan Protein Secreted by Suspension-Cultured Cells of Daucus carota (Purification and Partial Characterization).

Arabinogalactan proteins (AGPs) are secreted or membrane-associated glycoproteins that have been operationally defined as binding to [beta]-glucosyl Yariv artificial antigen, being rich in arabinose and galactose, and containing high levels of alanine, serine, and hydroxyproline. Using an anti-AGP monoclonal antibody (MAC 207) bound to cyanogen bromide-activated Sepharose 4B, we have purified by immunoaffinity chromatography an extracellular AGP from the culture medium of suspension-cultured cells of carrot (Daucus carota). The apparent molecular mass of this highly glycosylated proteoglycan is 70 to 100 kD as judged by sodium dodecyl sulfate-polyacrylamide gels. Although its sugar analysis, [beta]-glucosyl Yariv binding, and high alanine, serine, and proline content are consistent with it being an AGP, the amino acid composition unexpectedly revealed this molecule to have no detectable hydroxyproline. This suggests that this glycoprotein is not a "classical" AGP, but represents the first example of a new class of hydroxyproline-poor AGPs. Deglycosylation of the AGP with anhydrous hydrogen fluoride revealed that the purified proteoglycan contains probably a single core protein with an apparent molecular mass of 30 kD. Direct visualization of the native AGP in the electron microscope showed ellipsoidal putative AGP monomers, approximately 25 nm by 15 nm, that showed a strong tendency to self assemble into higher-order structures. Upon desiccation, the glycosylated AGP formed paracrystalline arrays visible in the light microscope. Polarized Fourier transform infrared microspectroscopy of these arrays demonstrated a high degree of polarization of the sugar moieties under these conditions. These results put possible constraints on current models of AGP structure; a putative role for these novel AGPs as pectin-binding proteins is discussed.

Fourier transform infrared microspectroscopy is a new way to look at plant cell walls.

Highly reproducible Fourier transform infrared (FTIR) spectra from both single onion (Allium cepa) cell walls and their constituent polymers were obtained under a variety of sampling conditions. The specificity of the chemical extraction sequence used in the preparation of the material was confirmed: pectins only are extracted by cyclohexanediaminetetraacetic acid and sodium carbonate, whereas xyloglucans are extracted by increasing concentrations of potassium hydroxide. There was very little contamination of the first potassium hydroxide extract with residual pectin. The low abundance of both phenolics and protein was also confirmed. The first sodium carbonate extraction almost completely removes esters remaining in the cell wall. We have demonstrated that FTIR spectroscopy can detect large conformational changes in pectic polymers on removal from the cell wall and on drying. FTIR spectroscopy provides a powerful and rapid assay for wall components and putative cross-links by identifying polymers and functional groups nondestructively in muro. The availability of micro-sampling and data acquisition techniques that permit subtraction of the blanket absorption of water make FTIR spectroscopy particularly suitable for studies of cell wall architecture. The use of polarizers with the microscope accessory permits determination of the orientation of particular functional groups with respect to the direction of cell elongation in carrot suspension cells.

Stability of murine monoclonal anti-A, anti-B and anti-A,B ABO grouping reagents and a multi-centre evaluation of their performance in routine use.

We have previously reported the production of 3 murine monoclonal reagents for ABO typing (designated ES-9, ES-4 and ES-15). This study presents results of tests of stability of these 3 reagents, together with a fourth murine monoclonal antibody (LM103/107). In addition, data are also presented from a multi-centre evaluation of the performance of the murine monoclonal reagents in routine ABO typing of both donors and patients using a wide variety of techniques, both manual and automated. The potency and stability of the 4 monoclonal antibody based reagents is compared with a broad selection of monoclonal and polyclonal ABO typing reagents. The reagents used for comparison were produced by European and United States manufacturers in both the public and private sector and are widely used in routine ABO typing. The Scottish monoclonal reagents have been used successfully to ABO type over 500,000 blood samples in 7 centres within the UK, with no discrepant results.

Production and use of human monoclonal anti-D antibodies.

Rhesus haemolytic disease of the newborn is a condition which can result in intrauterine or perinatal death. Although the passive administration of therapeutic anti-D post-partum is a most effective method for the prevention of this condition, there is currently a shortage of immune plasma for the preparation of the therapeutic anti-D immunoglobulin product. In addition the availability of anti-D for use in blood grouping has also been reduced. The advances made in recent years in the techniques for the production of human monoclonal antibodies raise the possibility that human monoclonal anti-D-based products may provide solutions to both of these problems. There are now a number of reports of the production of stable cell lines secreting high titre human anti-D. In this review we consider the various strategies used in the production of human monoclonal anti-D-secreting cell lines, the basic properties of these reagents and their potential usefulness in blood grouping, in therapy and as research tools.

The use of rat mixed-thymocyte culture-conditioned medium for hybridoma production, cloning and revival.

Allogeneic rat mixed-thymocyte 48 h culture-conditioned medium (MTM) was used successfully in place of feeder cells for hybridoma production with the NS-1 and NS-0 plasmacytoma lines. It permitted lower concentrations of fused cells to be seeded, and supported the transition from 96 to 24 well plates. MTM improved the performance of poor sera during cloning. It also assisted the survival of cells that were sensitive to thawing from liquid nitrogen storage, and cells that had inadvertently been allowed to overgrow. Two rats could produce the equivalent of 1500-5000 ml feeder cell suspension according to the dilution used; 150-500 mice would be required to produce such a quantity of cells. Thus use of MTM entailed a considerable saving in mice and provided a secure supply of 'reagent', since a batch could be prepared, checked for sterility, frozen and stored indefinitely.