Control of mannose/galactose ratio during galactomannan formation in developing legume seeds

Galactomannan deposition was investigated in developing endosperms of three leguminous species representative of taxonomic groups which have galactomannans with high, medium and low galactose content. These were fenugreek (Trigonella foenum-graecum L.; mannose/galactose (Man/Gal) = 1.1), guar (Cyamopsis tetragonoloba (L.) Taub.; Man/Gal = 1.6) and Senna occidentalis (L.) Link. (Man/Gal = 3.3), respectively. Endosperms were analysed at different stages of seed development for galactomannan content and the levels, in cell-free extracts, of a mannosyltransferase and a galactosyltransferase which have been shown to catalyse galactomannan biosynthesis in vitro (M. Edwards et al., 1989, Planta 178, 41–51). There was a close correlation in each case between the levels of the biosynthetic mannosyl- and galactosyltransferases and the deposition of galactomannan. The relative in vitro activities of the mannosyl- and galactosyltransferases in fenugreek and guar were similar, and almost constant throughout the period of galactomannan deposition. In Senna the ratio mannosyltransferase/galactosyltransferase was always higher than in the other two species, and it increased substantially throughout the period of galactomannan deposition. In fenugreek and guar the galactomannans present in the endosperms of seeds at different stages of development had the Man/Gal ratios characteristic of the mature seeds. By contrast the galactomannan present in Senna endosperms at the earliest stages of deposition had a Man/Gal ratio of about 2.3. During late deposition this ratio increased rapidly, stabilising at about 3.3, the ratio characteristic of the mature seed. The levels of -galactosidase in the developing endosperms of fenugreek and guar were low and remained fairly constant throughout the deposition of the galactomannan. In Senna, -galactosidase activity in the endosperm was low during early galactomannan deposition, but increased subsequently, peaking during late galactomannan deposition. The developmental patterns of the -galactosidase activity and of the increase in Man/Gal ratio of the Senna galactomannan were closely similar, indicating a cause-and-effect relationship. The endosperm -galactosidase activity in Senna was capable, in vitro, of removing galactose from guar galactomannan without prior depolymerisation of the molecule. In fenugreek and in guar the genetic control of the Man/Gal ratio in galactomannan is not the result of a post-depositional modification, and must reside in the biosynthetic process. In Senna, the Man/Gal ratio of the primary biosynthetic galactomannan product is controlled by the biosynthetic process. Yet the final Man/Gal ratio of the galactomannan in the mature seed is, to an appreciable extent, the result of galactose removal from the primary biosynthetic product by an -galactosidase activity which is present in the endosperm during late galactomannan deposition.Abbreviations al galactose - Man mannose This work was carried out with the aid of a Cooperative Research Grant (No. CRG 1) awarded by the Agricultural and Food Research Council, UK.     

Morphological aspects of the galactomannan formation in the endosperm ofTrigonella foenum-graecum L. (Leguminosae)

The mode of deposition (secretion) of galactomannan in the cells of the seed endosperm ofTrigonella foenum-graecum has been studied by electron microscopy. In cells which are just beginning to secrete galactomannan there are stacks of rough endoplasmic reticulum (ER). The intracisternal space (containing the enchylema) of the rough ER then swells, becomes vacuolated and forms a voluminous network, with pockets of cytoplasm entrapped within poculiform rough ER. The enchylema contains material which reacts with periodate-thiocarbohydrazidesilver proteinate in a very similar manner to the galactomannan already deposited in the cell wall. It appears that the galactomannan is formed in the intracisternal space of the rough endoplasmic reticulum and then expelled outside the plasmalemma. This mode of deposition contrasts with that of other plant cell wall polysaccharides whose secretion is mediated by Golgi vesicles.Abbreviation ER endoplasmic reticulum This is part six in a series of papers dealing with galactomannan metabolism. Part five: Planta133, 219–222 (1977)     

Water stress and galactomannan breakdown in germinated fenugreek seeds. Stress affects the production and the activities in vivo of galactomannan-hydrolysing enzymes

Imposition of water stress on germinated fenugreek (Trigonella foenum-graecum L.) seeds and isolated fenugreek endosperms after the beginning of galactomannan mobilisation caused a reduction in the rate of breakdown of the polysaccharide relative to unstressed controls. The activities, measured in vitro, of the three hydrolytic enzymes involved in the breakdown process (-d-galactosidase, EC 3.2.1.22;endo--d-mannanase, EC 3.2.1.78;exo--d-mannanase, EC 3.2.1.25) were not decreased. Although there was some accumulation of galactomannan-hydrolysis products in endosperms under stress, there was no clear correlation between sugar levels and the inhibition of galactomannan breakdown. When water stress was applied to fenugreek seeds after germination but before the beginning of galactomannan hydrolysis, both galactomannan breakdown and the development of the hydrolytic enzyme activities were inhibited. Washing of newly germinated seeds for 2 h in water prior to the imposition of stress gave partial relief of the inhibition of galactomannan mobilisation, partial recovery ofendo--d-mannanase levels, and full recovery of -d-galactosidase levels. It is argued: 1) that water stress after germination but before the beginning of galactomannan hydrolysis inhibits the production of hydrolytic enzymes in the endosperm, probably via decreased removal at lowered water content of diffusible inhibitory substances; and 2) that water stress after the beginning of galactomannan hydrolysis decreases the rate of galactomannan breakdown in vivo principally via decreased diffusion at lowered water content of enzymes from the aleurone layer through the storage tissue of the endosperm.Abbreviation PEG polyethyleneglycol     

Cell wall regeneration and galactomannan biosynthesis in protoplasts from carob

Protoplast isolation from endosperms of developing carob (Ceratonia siliqua L.) seeds is reported for the first time. These protoplasts regenerated cell walls within 12 h. In order to assess their potential for galactomannan biosynthesis, the incorporation of radioactivity in the regenerated cell wall polysaccharides (CWP) and extracellular polysaccharides (ECP), after feeding these protoplasts with D-[U-¹⁴C]glucose or D-[U-¹⁴C]mannose was studied. The pattern of the radioactive label distribution in the neutral sugars of the trifluoroacetic acid (TFA) hydrolysate of CWP was different from that of the ECP. In the TFA hydrolysis products of the CWP, immediately after protoplast isolation, the greatest level of radioactivity (approximately 90%) was detected in glucose, galactose and mannose. After 2 days protoplast culture, the label in mannose increased. In contrast, immediately after protoplast isolation, approximately 90% of radioactivity of the ECP was detected in galactose and mannose. However, during culture, the radioactivity incorporation in mannose dropped to one third, while that in galactose and arabinose increased significantly. Hydrolysis of the CWP and ECP with -galactosidase and endo--mannanase confirmed that, at least part of mannose and galactose belonged to galactomannan molecules. These results were compared with those obtained upon feeding developing endosperm tissue with D-[U-¹⁴C]mannose. From our results we concluded that protoplasts from endosperm tissues of developing carob seeds, retained the ability of their original explant to synthesize galactomannan, making protoplasts candidates for the study of galactomannan biosynthesis.     

Characterization of a Schizosaccharomyces pombe morphological mutant altered in the galactomannan content

In a search for Schizosaccharomyces pombe mutants resistant to the antifungal agent papulacandin B, a morphological mutant was isolated. The mutant is round shaped in contrast to the rod shaped parental strain. This morphological defect segregated as a recessive Mendelian character and was not observed in other papulacandin B resistant mutants belonging to the same complementation group. The mutation mapped in the right arm of S. pombe chromosome III very close to pap1 marker. Mutant cell walls were more susceptible to alkali extraction and Novozyme degradation than those from the wild-type. A specific reduction in the cell wall galactomannan fraction was the only significant difference detected as compared to the wild-type strain. Levels of beta (1,3)-glucan and mannan synthases as well as other enzymic periplasmic mannoproteins were very similar in wild type and mutant strains.     

Galactomannan formation and guanosine 5′-diphosphate-mannose: galactomannan mannosyltransferase in developing seeds of fenugreek (Trigonella foenum-graecum L., leguminosae)

The time-course of galactomannan and stachyose (digalactosyl-sucrose) deposition in the fenugreek seed endosperm has been determined, and correlated with standard parameters of seed development. During, and only during, the period of galactomannan deposition, endosperm homogenates are capable of catalysing the transfer of labelled d-mannosyl residues from guanosine 5-diphosphate d-[U-¹⁴C]mannose to a soluble polysaccharide product indistinguishable from galactomannan. The mannosyltransferase activity peaks twice, once at the beginning of galactomannan deposition, and again in the middle of the most rapid phase of galactomannan deposition. The enzyme in the later peak sediments with grossly particulate material (1,000 g pellet), whereas the earlier peak contains a considerable proportion of a particulate enzyme sedimenting at 100,000 g. These observations are discussed in the light of existing information on the ultrastructural aspects of galactomannan deposition. The mannosyltransferase is clearly involved in galactomannan formation in vivo, but the status of an accompanying galactosyltransferase is less clear.Abbreviations GDP guanosine 5-diphosphate - UDP uridine 5-diphosphate     

Structure and biosynthesis of teichoic acids in the cell walls of Staphylococcus xylosus DSM 20266

The simultaneous occurrence of a N-acetylglucosaminyl poly(ribitolphosphate) (-GlcNAc) and a N-acetylglucosaminyl poly(glycerolphosphate) (-GlcNAc) in the cell walls of Staphylococcus xylosus DSM 20266 was demonstrated by different experimental lines:(1) Fractionation of extracted cell wall teichoic acid on DEAE-cellulose, (2) investigation of the composition of cell walls in the growth cycle, (3) in vitro biosynthesis using crude membranes as the source of enzyme.The polymerization of these polymers starts from CDP-ribitol and CDP-glycerol, respectively. In the presence of UDP-N-acetylglucosamine both polymers are substituted with N-acetylglucosamine at a level and with the identical anomeric configuration found in the native cell wall teichoic acids. The in vitro biosynthesis of poly(glycerolphosphate) was unique in that it was highly stimulated by UDP-N-acetylglucosamine and to a lower extent by other UDP-activated sugars. Kinetic studies have provided evidence that this stimulation is due to an increase of V _max while K _m is unchanged. Competition experiments have indicated that poly(ribitolphosphate) and poly(glycerolphosphate) were synthesized in the in vitro system in a close spatial relationship.Abbreviations ADP adenosine 5-diphospho - CDP cytidine 5-diphospho - GDP guanosine 5-diphospho - GalNAc N-acetyl-galactosamine - Glc glucose, glucosyl - GlcNAc N-acetyl-glucosamine - N acetylglucosaminyl - GlcUA glucuronic acid - Gro glycerol - Man mannose, mannosyl - Rit ribitol - SDS sodium dodecyl sulfate - UDP uridine 5-diphospho     

A dual rôle for the endosperm and its galactomannan reserves in the germinative physiology of fenugreek (Trigonella foenum-graecum L.), an endospermic leguminous seed

Some 30% of the reserve material in the fenugreek seed is galactomannan localised in the endosperm; the remainder is mainly protein and lipid in the cotyledons of the embryo. The importance of galactomannan to the germinative physiology of fenugreek has been investigated by comparing intact and endosperm-free seeds. From a purely nutritional point of view the galactomannan's rôle is not qualitatively different from that of the food reserves in the embryo. Nevertheless, due to its spatial location and its hydrophilic properties, the galactomannan is the molecular basis of a mechanism whereby the endosperm imbibes a large quantity of water during seed hydration and is able to buffer the germinating embryo against desiccation during subsequent periods of drought-stress. The galactomannan is clearly a dual-purpose polysaccharide, regulating water-balance during germination and serving as a substrate reserve for the developing seedling following germination. The relative importance of these two rôles is discussed.     

Cell-wall-bound lytic activity in Chlorella fusca: function and characterization of an endo-mannanase

A cell-wall-degrading activity was solubilized from young cells and from mother cell walls of Chlorella fusca by treatment with LiCl. The cytoplasmic enzyme hexokinase was not detectable in these extracts. The LiCl-solubilized activity increased in the cell cycle parallel to the release of autospores. The enzyme was purified on a chromatofocusing column followed by gel filtration. Sodium dodecyl sulfate/polyacryl amide gel electrophoresis of the purified enzyme revealed a molecular weight of 44 kDa, whereas gel filtration indicated a molecular weight of 25 kDa. Cell-wall-lytic activity and -1,4-mannanase activity coeluted in gel filtration and were separated from -d-fucosidase activity. The enzyme degraded isolated cell walls and ivory nut mannan primarily to oligosaccharides with an estimated degree of polymerization 6. The soluble degradation products of the cell wall consisted of 92–96% mannose and 4–8% glucose. It is concluded that the cell-wall-lytic activity is caused by an endo-mannanase. In vivo, this enzyme probably degrades the mother cell wall and, after autospore release, remains bound to it as well as to the surface of the daughter cells by ionic forces. The identity of this bound enzyme with a soluble wall-degrading enzyme previously obtained from mother cells is discussed.     

Pectic polysaccharides in carrot cells growing in suspension culture

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.Abbreviation MW molecular weight     

Changes in cell-wall polysaccharide composition of developingNitella internodes

Changes in the uronide, neutral-polysacharide, and cellulose composition of the cell wall ofNitella axillaris Braun were followed throughout development of the internodes and correlated with changes in growth rate. As the cells increased in length from 4 to 80 mm during development, the relative growth rate decreased. Cell wall thickness, as measured by wall density, increased in direct proportion to diameter, indicating that cell-wall stress did not change during elogation. Cell-wall analyses were adapted to allow determination of the composition of the wall of single cells. The total amounts of uronides, neutral sugars and cellulose all increased during development. However, as the growth rate decreased, the relative proportions of uronides and neutral sugars, expressed as percent of the dry weight of the wall, decreased, while the proportion of cellulose increased. The neutral sugars liberated upon hydrolysis ofNitella walls are qualitatively similar to those found in hydrolysates of higher plant cell walls: glucose, xylose, mannose, galactose, arabinose fucose and rhamnose. Only the percentage of galactose was found to increase in walls of mature cells, while the percentage of all other sugars decreased. The rate of apposition (g of wall material deposited per unit wall surface area per hour) of neutral polysaccharides decreased rapidly with decreasing growth rate during the early stages of development. The rate of apposition of uronides decreased more steadily throughout development, while that of cellulose, after an early decline, remained constant until dropping off at the end of the elongation period. These correlations between decreasing growth rate and decreasing rate of apposition of neutral sugars and uronides indicate that synthesis of these cell-wall components could be involved in the regulation of the rate of cell elongation inNitella.     

Biosynthesis of legume-seed galactomannans in vitro

Particulate enzyme preparations were isolated from developing fenugreek (Trigonella foenum-graecum L.) and guar (Cyamopsis tetragonoloba [L.] Taub.) seed endosperms during the period of galactomannan deposition in vivo. These preparations catalysed the formation of polysacharide products from guanosine 5-diphosphate (GDP)-mannose, from uridine 5-diphosphate (UDP)-galactose and from mixtures of the two nucleotides. The products were analysed by solubility, by complete acid hydrolysis, and by selective enzymatic cleavage using pure enzymes of known specificity. With GDP-[U-¹⁴C]-d-mannose as substrate and a divalent metal cation (Mg⁺², Mn⁺², or Ca⁺²) a highly efficient transfer of labelled d-mannosyl residues was obtained to give a product identified as linear (14)--linked d-mannan. No transfer of galactosyl residues was obtained when GDP-[U-¹⁴C]-d-galactose was the only substrate, although very low and variable amounts of an unidentified product which released labelled glucose on acid hydrolysis were formed. In the presence of UDP-galactose, GDP-mannose and Mn⁺² ions, products were formed which have been characterised as galactomanans — a linear (14)--d-mannan backbone carrying d-galactopyranosyl substituents linked (16)- to mannose. The degree of galactose substitution of the d-mannan backbone was manipulated in vitro by varying GDP-mannose concentrations at constant (saturating) UDP-galactose levels. The transfer of d-galactosyl residues from UDP-galactose to galactomannan was absolutely dependent upon the simultaneous transfer of D-mannosyl residues from GDP-mannose. d-Mannan sequences pre-formed in situ using the mannosyltransferase in the absence of UDP-galactose could not become galactose-substituted in a subsequent incubation either with UDP-galactose alone or with UDP-galactose plus GDP-mannose A model for the interaction of GDP-mannose mannosyltransferase and UDP-galactose galactosyltransferase in galactomannan biosynthesis is proposed.Abbreviations GDP guanosine 5-diphosphate - TLC thinlayer chromatography - UDP uridine 5-diphosphate     

Methanogens with pseudomurein use diaminopimelate aminotransferase in lysine biosynthesis

Methanothermobacter thermautotrophicus uses lysine for both protein synthesis and cross-linking pseudomurein in its cell wall. A diaminopimelate aminotransferase enzyme from this methanogen (MTH0052) converts tetrahydrodipicolinate to l,l-diaminopimelate, a lysine precursor. This gene complemented an Escherichia coli diaminopimelate auxotrophy, and the purified protein catalyzed the transamination of diaminopimelate to tetrahydrodipicolinate. Phylogenetic analysis indicated this gene was recruited from anaerobic Gram-positive bacteria. These results expand the family of diaminopimelate aminotransferases to a diverse set of plant, bacterial and archaeal homologs. In contrast marine methanogens from the Methanococcales, which lack pseudomurein, appear to use a different diaminopimelate pathway for lysine biosynthesis.     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

Further studies on the biosynthesis of the avermectins

Summary The biosynthesis of avermectins was studied further inStreptomyces avermitilis MA5502 by feeding experiments with labeled precursors.¹³C-NMR analysis of the compounds biosynthesized from [2-¹³C]acetate, [1,2-¹³C₂]acetate, [3-¹³C]propionate and [2,3-¹³C₂]propionate confirmed that the aglycone of avermectins is made from seven intact acetate and five propionate units. Feeding experiments with [1-¹³C]2-methylbutyrate and [1-¹³C]isobutyrate have shown that 2-methylbutyrate and isobutyrate are immediate precursors of the starter units of the polyketide chains of avermectin a and b components, respectively. The³H/¹⁴C doublelabeling experiments suggest that the two oleandrose moieties are derived from glucose.     

Molecular weight distribution of cellulose in primary cell walls

The distribution pattern of the degree of polymerization (DP) of cellulose present in the cell walls of mesophyll- and suspension-cultured cells of tobacco was compared to that of newly synthesized ¹⁴C-labeled cellulose from regenerating tobacco protoplasts and suspension-cultured cells. The cellulose was nitrated, and, after fractionation according to differences in solubility in acetone/water, the DP pattern of labeled or unlabeled cellulose nitrate was determined by viscosity measurements. A low (DP<500) and high DP-fraction (DP>2500) of cellulose were predominant in the cell walls of protoplasts, suspension — cultured cells, and mesophyll cells. The average DP of the high molecular weight fraction of cellulose in the cell walls of mesophyll was higher (DP4,000) than in protoplasts or suspension — cultured cells (DP 2,500-3,000). In all cell walls tested, minor amounts of cellulose molecules with a broad spectrum of a medium DP were present. Pulse — chase experiments with either protoplasts or suspension —cultured cells showed that a large proportion of the low and medium DP-cellulose are a separate class of structural components of the cellulose network. The results are discussed in relation to the organization of cellulose in the primary cell wall.Abbreviations DP degree of polymerisation - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indole-3-acetic acid     

Ratio of free to bound polyamines during maturation in mung-bean hypocotyl cells

Free- and bound-polyamine levels were estimated in successive segments of the mung-bean hypocotyl. Three aliphatic polyamines (putrescine, spermidine and spermine) were found in proportions which depended on the state of maturation. In young cells, most of the polyamines were located in the protoplasm whereas in older cells they were mostly bound to the cell walls. Spermidine was always the main bound polyamine, and putrescine, the main free polyamine.Abbreviation EDTA ethylenediaminetetraacetic acid     

Pectins as mediators of wall porosity in soybean cells

The non-invasive technique of fluorescence redistribution after photobleaching was employed on soybean (Glycine max (L.) Merr.) root cells grown in suspension culture to examine macromolecular transport across plant cell walls. Using both fluorescently derivatized dextrans and proteins of graded size, a functional range of diameters for putative trans-wall channels was determined to be 6.6–8.6 nm. A mild treatment with pectinase apparently enlarged the channels, without adversely affecting cell viability, enabling significantly larger molecules to pass through the wall. Treatment of the cells with cellulysin or protease did not have this enlargement effect. It appears that the organization of pectic substances is a major control element in defining the sieving properties of the wall.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Fl-dextran fluorescein-derivatized dextran - FRAP fluorescence redistribution after photobleaching - kDa kilodalton     

An epitope of rice threonine- and hydroxyproline-rich glycoprotein is common to cell wall and hydrophobic plasma-membrane glycoproteins

A monoclonal antibody, LM1, has been derived that has a high affinity for an epitope of hydroxyproline-rich glycoproteins (HRGPs). In suspension-cultured rice (Oryza sativa L.) cells the epitope is carried by three major proteins with different biochemical properties. The most abundant is the 95-kDa extracellular rice extensin, a threonine- and hydroxyproline-rich glycoprotein (THRGP) occurring in the cell wall and secreted into the medium. This THRGP can be selectively oxidatively cross-linked in the presence of hydrogen peroxide and an endogenous peroxidase with the result that it does not enter a protein gel. A second polypeptide with the LM1 epitope (180 kDa), also occurring in the suspension-cultured cells and medium, is not oxidatively cross-linked. Three further polypeptides (52, 65 and 110 kDa) with the characteristics of hydrophobic proteins of the plasma-membrane also carry the LM1 epitope as determined by immuno-blotting of detergent/aqueous partitions of a plasma-membrane preparation and immuno-fluorescence studies with rice protoplasts. At the rice root apex the LM1 epitope is carried by four glycoproteins and is developmentally regulated. The major locations of the epitope are at the surface of cells associated with the developing protoxylem and metaxylem in the stele, the longitudinal radial walls of epidermal cells and a sheath-like structure at the surface of the root apex.Abbreviations AGP arabinogalactan protein - ELISA enzyme-linked immunosorbent assay - HRGP hydroxyproline-rich glycoprotein - THRGP threonine- and hydroxyproline-rich glycoprotein This work was supported by The Leverhulme Trust. We also acknowledge support from The Royal Society and thank Prof. L.A. Staehelin for the carrot extensin, N. Stacey for the rice cell culture and Dr. J. Keen for protein sequencing.     

Pectate distribution and esterification in Dubautia leaves and soybean nodules,studied with a fluorescent hybridization probe

Carbohydrate-hybridization probes (Vreeland and Laetsch, 1989, Planta (177, 423–434) were used to localize the homogalacturonan (pectate) component of pectins in the cell walls of leaves and soybean root nodules. Leaves of two species of the dicotyledon Dubautia were compared; these species contain much pectin but differ in their tissue water relations with respect to their cell-wall properties. Maturation of the primary cell walls in nodules was studied in the Bradyrhizobium japonicum-Glycine max symbiosis. Probe labelling was based on the divalent-cation-mediated association between pectate in tissue sections and fluorescein-conjugated pectate fragments. Pectate was also labelled by mixed-dimer formation with fluorescent polyguluronate derived from alginate. The specificity of the probe for unesterified polygalacturonate was indicated by increased cell-wall labelling after chemical or enzymatic deesterification of tissue sections, in contrast to elimination of labelling by chemical esterification. Postfixation of tissue sections improved retention of soluble pectate. Pectate differences were found in the leaves among cell types, in degree of esterification, and between plant species. The cell walls of soybean nodules were strongly labelled by the pectate probe in nodules one week and three weeks after infection. Pectate was more highly esterified in the central infected zone than in the surrouding cortex. Within the infected zone, walls of uninfected cells and infected cells were similarly labelled by the pectate probe. The results indicate that the pectate molecular probe provides detailed information on pectate distribution at the cellular level for investigations of cell-wall structure, development and physiology.Abbreviations EDTA ethylenedinitrilotetraacetic acid (ethylenediaminetetraacetic acid) - NMR nuclear magnetic resonance spectroscopy - TTB 1,3,5-triazido-2,4,6-trinitrobenene