Galactan Side Chains Research Articles

Down-regulation of an Auxin Response Factor in the tomato induces modification of fine pectin structure and tissue architecture

It has previously been shown that down-regulation of an auxin response factor gene (DR12) results in pleiotropic phenotypes including enhanced fruit firmness in antisense transgenic tomato (AS-DR12). To uncover the nature of the ripening-associated modifications affecting fruit texture, comparative analyses were performed of pectin composition and structure in cell wall pericarp tissue of wild-type and AS-DR12 fruit at mature green (MG) and red-ripe (RR) stages. Throughout ripening, pectin showed a decrease in methyl esterification and in the content of galactan side chains in both genotypes. At mature green stage, pectin content in methyl ester groups was slightly higher in AS-DR12 fruit than in wild type, but this ratio was reversed at the red-ripe stage. The amount of water- and oxalate-soluble pectins increased at the red-ripe stage in the wild type, but decreased in AS-DR12. The distribution of methyl ester groups on the homogalaturonan backbone differed between the two genotypes. There was no evidence of more calcium cross-linked homogalacturan involved in cell-to-cell adhesion in AS-DR12 compared with wild-type fruit. Furthermore, the outer pericarp contains higher proportion of small cells in AS-DR12 fruit than in wild type and higher occurrence of (1-->5) alpha-L-arabinan epitope at the RR stage. It is concluded that the increased firmness of transgenic fruit does not result from a major impairment of ripening-related pectin metabolism, but rather involves differences in pectin fine structure associated with changes in tissue architecture.

Cell wall modifications in chilling-injured plum fruit ( Prunus salicina)

The aim of this study was to analyze the changes in cell wall pectins in normally ripening (juicy) and in chilling-injured plum fruit ( Prunus salicina cv. Fortune) showing mealiness. Total cell wall neutral sugars and uronic acids, solubilization and depolymerization of pectins in water-, CDTA- and Na 2CO 3-soluble fractions of the cell wall (WSF, CSF and NSF, respectively), non-cellulosic neutral sugar compositions of these fractions, and the activities of the cell wall-degrading enzymes polygalacturonase (PG), pectin methylesterase (PME), 1,4-β- d-glucanase/glucosidase and β-galactosidase (β-gal) were determined. No differences in the total content of pectin and neutral sugars between normally ripening and chilling-injured fruit were detected. However, the mealy plums presented a higher level of tightly bound pectin (NSF) and a lower proportion of loosely bound pectin (WSF) than the juicy controls. Lower pectin depolymerization and reduced solubilization of neutral sugars in the WSF and CSF were also detected in the chilling-injured tissues, confirming an alteration in the normal ripening-associated pattern of polyuronide disassembly. While no differences were found in the activities of PG, PME and 1,4-β- d-glucanase/glucosidase between normally ripening and mealy fruit, the latter had reduced β-gal activity. This might have led to differential solubilization of polymers with galactan side chains, but further studies are required to determine if there is a causal relationship between these events. Overall, results indicated that the development of chilling injury symptoms in ‘Fortune’ plums is associated with abnormalities in cell wall metabolism, including a reduction in pectin solubilization and depolymerization and decreased ripening-associated modification of galactose-rich pectin polymers.

Variability in the composition of tissue-specific galactan from flax fibers

Tissue-specific galactan of sclerenchyma fibers, with cell walls of the gelatinous type, was examined in flax plants (Linum usitatissimum L.) of 23 various genotypes. The content and average degree of polymerization of side chains of galactan were estimated before its deposition into the cell wall. The variability of the analyzed parameters of tissue-specific galactan from flax fibers was high; within the same genotype, the scope of paratypic variability between replicates and years of research was comparable to variability between different genotypes. The average length of side chains in the studied samples ranged from 5 to 41 galactose residues. The average degrees of polymerization of galactan side chains in flax fibers was found to be discrete, which could be explained by block assemblage of the polymer in the Golgi apparatus.

Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged

The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called 'natural' composites), rich in pectic neutral sugar side chains and cellulose, as well as 'artificial' composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.

Silicon-induced basal resistance in tomato against Ralstonia solanacearum is related to modification of pectic cell wall polysaccharide structure

Bacterial wilt incidence was reduced by 38.1% and 100% in silicon-treated plants of the moderately resistant tomato genotype King Kong 2 and the resistant genotype Hawaii 7998 grown in peat substrate. At 5 days post inoculation the bacterial population was significantly reduced in stems and roots of genotype Hawaii 7998, and in stems of King Kong 2 in silicon-treated plants compared to non-treated plants, indicating a silicon-induced resistance, since silicon accumulated in roots, but not in stems, while a tolerance effect was observed in the susceptible genotype L390. Characterization of possible molecular mechanisms involved in silicon-mediated resistance by immuno-histochemical analysis of stem cell walls indicated silicon-induced changes in the pectic polysaccharide structure. After infection homogalacturonan with non-blockwise degradation of methyl-esters was increased in vessel walls in non-silicon-treated plants, but not in silicon-treated plants, possibly indicating the action of pathogen pectinmethylesterase. Also the staining of vessel walls for arabinogalactan-protein in infected, non-silicon-treated plants was not observed in silicon-treated plants. In inoculated, silicon-treated plants, staining for arabinan side chains of rhamnogalacturonan I (RG I) was increased in some vessel walls, and fluorescence of antibodies for galactan side chains of RG I overall increased in the xylem parenchyma compared to non-silicon-amended plants. These observations suggest an induced basal resistance on cell wall level after silicon treatment, while the yellow or brown autofluorescence occurring in inoculated, non-silicon-treated plants disappeared.

Chain elongation of pectic β-(1→4)-galactan by a partially purified galactosyltransferase from soybean (Glycine max Merr.) hypocotyls

Pectin is one of the major cell wall polysaccharides found in dicotyledonous plants. We have solubilized and partially purified a beta-(1-->4)-galactosyltransferase (GalT) involved in the synthesis of the beta-(1-->4)-galactan side chains of pectin. The enzyme protein was almost completely solubilized by mixing a crude microsomal preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls with a detergent, Triton X-100 (0.75%, w/v), in buffer. The solubilized enzyme was partially purified by ion-exchange chromatography. The crude membrane-bound GalT transferred Gal from UDP-Gal onto 2-aminobenzamide (AB)-derivatized beta-(1-->4)-galactoheptaose (Gal(7)-AB), leading to the formation of Gal(8-11)-AB by attachment of a series of one to four galactosyl residues; this is similar to what has previously been observed for 2-aminopyridine-derivatized beta-(1-->4)-galactooligomer acceptors (Konishi et al. in Planta 218:833-842, 2004). The partially purified GalT, by contrast, was able to transfer more than 25 galactosyl residues and elongated the chains to about Gal(35)-AB, thus almost reaching the length (43-47 Gal units) of native beta-(1-->4)-galactan side chains found in pectic polysaccharides from soybean cotyledons (Nakamura et al. in Biosci Biotechnol Biochem 66:1301-1313, 2002). Enzyme activity increased with increasing chain length of beta-(1-->4)-galactooligomers and reached maximal activity at heptaose, whereas galactooligomers higher than heptaose showed lower acceptor efficiency.

Pectins from citrus peel cell walls contain homogalacturonans homogenous with respect to molar mass, rhamnogalacturonan I and rhamnogalacturonan II

Pectins of different structures were extracted from citrus peels with water, oxalate, hot dilute hydrochloric acid and cold dilute sodium hydroxide. Homogalacturonans (HGs) were isolated from the four pectins by mild acid hydrolysis after deesterification. The study of their macromolecular properties revealed that they have similar number- and weight-average degrees of polymerisation, in the range of 81–117 galacturonic acid units, irrespective of the initial pectin. It was inferred that citrus peel pectins consist of HG strectches of similar lengths whatever the mode of extraction. Rhamnogalacturonan I (RG I) and rhamnogalacturonan II (RG II) were isolated from the oxalate- and the acid-extracted pectins using an endo-1,4-α-polygalacturonase followed by anion-exchange and size-exclusion chromatographies. RG I was composed mainly of arabinose, galactose, galacturonic acid and rhamnose, suggesting the presence of arabinan and/or (arabino)galactan side chains. Debranched RG I (dRG I) was obtained by treating the RG I arising from acid-extracted pectin with endo-1,5-α- l-arabinanase, endo-1,4-β- d-galactanase, α- l-arabinofuranosidase and β- d-galactosidase in admixture. The molar ratios of galacturonic acid to rhamnose of RGs I and dRG I were very close to 1/1, indicating a strict repeating [GalA–Rha] n pattern in the backbone. It is concluded that citrus pectins consist predominantly of HG, with a few RG I and a minor RG II fraction.

Fast data preprocessing for chromatographic fingerprints of tomato cell wall polysaccharides using chemometric methods

The variability in the chemistry of cell wall polysaccharides in pericarp tissue of red-ripe tomato fruit ( Solanum lycopersicon Mill.) was characterized by chemical methods and enzymatic degradations coupled to high performance anion exchange chromatography (HPAEC) and mass spectrometry analysis. Large fruited line, Levovil (LEV) carrying introgressed chromosome fragments from a cherry tomato line Cervil (CER) on chromosomes 4 (LC4), 9 (LC9), or on chromosomes 1, 2, 4 and 9 (LCX) and containing quantitative trait loci (QTLs) for texture traits, was studied. In order to differentiate cell wall polysaccharide modifications in the tomato fruit collection by multivariate analysis, chromatograms were corrected for baseline drift and shift of the component elution time using an approach derived from image analysis and mathematical morphology. The baseline was first corrected by using a “moving window” approach while the peak-matching method developed was based upon location of peaks as local maxima within a window of a definite size. The fast chromatographic data preprocessing proposed was a prerequisite for the different chemometric treatments, such as variance and principal component analysis applied herein to the analysis. Applied to the tomato collection, the combined enzymatic degradations and HPAEC analyses revealed that the firm LCX and CER genotypes showed a higher proportion of glucuronoxylans and pectic arabinan side chains while the mealy LC9 genotype demonstrated the highest content of pectic galactan side chains. QTLs on tomato chromosomes 1, 2, 4 and 9 contain important genes controlling glucuronoxylan and pectic neutral side chains biosynthesis and/or metabolism.

Immunohistochemical changes in methyl-ester distribution of homogalacturonan and side chain composition of rhamnogalacturonan I as possible components of basal resistance in tomato inoculated with Ralstonia solanacearum

The composition and structure of pectic cell wall polysaccharides of stem sections were investigated in healthy and Ralstonia solanacearum-inoculated tomato genotypes L390 and Hawaii 7996, susceptible and resistant to bacterial wilt, respectively, by immunohistochemical analysis. Constitutive differences between genotypes manifested in methyl-ester distribution of homogalacturonan (HG), arabinan and galactan side chain composition of rhamnogalacturonan I (RG I) and arabinogalactan-protein (AGP) in the xylem parenchyma and in vessel cell walls. After inoculation increased labeling was observed with all the antibodies (JIM5, JIM7, LM2, LM5, LM6, LM7) specific for HG, RG I and AGP epitopes, in the xylem parenchyma and around xylem vessels of stem sections of L390, but not of Hawaii 7996. Also vessel cell walls were stronger stained after inoculation in L390, particularly for the non-blockwise de-esterification of HG, possibly indicating for the first time the non-blockwise action pattern of bacterial pectin methyl esterase. In genotype Hawaii 7996 a reaction to inoculation was observed only in vessel walls, with a significantly increased number of stained vessels—five- and nine-fold for arabinan and galactan epitopes of RG I, respectively. Differences in xylem cell wall structure may play a role as a constitutive resistance mechanism in the multigenic resistance of tomato against bacterial wilt, while changes after inoculation may contribute to induced basal resistance on cell wall level.

Structural analysis and characterization of a mucopolysaccharide isolated from roots of cassava ( Manihot esculenta Crantz L.)

A mucopolysaccharide, referred to as cassava tuber mucilage (CTM), was isolated from the roots of sweet cassava and studied for its characteristic physical, molecular, and structural properties. Sugar composition and molecular properties of purified samples were analyzed using high-performance liquid (HPLC), anion exchange (HPAEC-) and size exclusion (HPSEC- MALLS-RI) chromatographic techniques. Structural properties of crude and purified samples were analyzed and compared using Fourier transformation infrared (FT-IR) and nuclear magnetic resonance spectroscopy. CTM is mainly composed of the major neutral sugars sucrose, fructose, glucose, galactose, and arabinose. It has a molecular average weight of 3.6×10 5, number average molecular mass (Mn) of 2.4×10 6, and a polydispersity index (Mw/Mn) of 1.5. FT-IR spectroscopy revealed high protein content in the crude fractions, whereas purified fractions were mainly carbohydrate. 1H NMR and 13CNMR showed that the mucilage consists of 1, 3-linked glucan chains with 1, 6-linked galactan side chains attached to position 6. The mucilage is purely hemicellulosic and possibly an arabinogalactan, confirmed by SDS gel electrophoresis of crude CTM samples, containing a (1, 3)-linked β-glucan backbone having side chain branching with non-reducing terminal residues mostly of (1, 6)-linked galactose and some arabinose and glucosyl moieties.

MALDI-TOF MS evidence for the linking of flax bast fibre galactan to rhamnogalacturonan backbone

Fibre-specific (1 → 4)-β-galactan extracted from bast fibre peels of developing flax ( Linum usitatissimum L.) stem has been studied to elucidate its structural details. The polysaccharide was characterized by NMR and subjected to partial degradation protocols, including chemical and enzymatic approaches. The oligosaccharide fragments obtained were fractionated by gel permeation chromatography and analyzed for their molecular mass with MALDI-TOF MS. The obtained data show that this flax galactan is a complex RG-I polysaccharide with variable side chain structures. The backbone is composed of the common GalA-Rha repeats with a high degree of branching. These side chains are mainly composed of β-1,4-linked Gal oligomers: (1) short branches of only one or two Gal residue(s); (2) long (linear) branches of up to 26 Gal residues; (3) mixed branches of between 3 and 12 Gal residues (possibly derived from longer linear side chains), that are resistant to galactanase cleavage; (4) side chains of at least 17 Gal residues, decorated with single Ara moieties. The linkage between RG backbone and galactan side chains was confirmed by the presence of fragments with (Rha-GalA) n Hex m structure type. Neither chemical, nor enzymatic hydrolysis yielded oligomeric GalA residues, indicating that RG-I blocks are not interrupted by HGA regions. The polymer can be cleaved only partially by the rhamnogalacturonan hydrolase used, while the remaining part is resistant, probably due to peculiarities of side chain structure. Novel Rha-GalA oligomers were liberated by RG-hydrolase containing two or three Gal attached to Rha near the cleavage site. The native polymer is decorated by acetyl groups, with yet unknown distribution patterns. Treatment with purified and well-characterized galactanase does not change the hydrodynamic volume of flax galactan (despite considerable cleavage of Gal moieties), suggesting a complex “secondary” structure of the polymer.

Alkaline extractability of pectic arabinan and galactan and their mobility in sugar beet and potato cell walls

The organisation of sugar beet and potato cell walls was studied using alkaline extractions following a response surface methodology, simultaneously with solid-state 13C NMR spectroscopy. The influence of two extraction parameters: NaOH concentration (0.05, 0.275, 0.5 M) and temperature (40, 65, 90 °C) on the composition (neutral and acidic sugars) of the residues recovered was established. Treatments of increasing harshness progressively washed off non-cellulosic polysaccharides from the cell walls. Alkaline treatments applied to sugar beet cell wall material (SB-CWM) revealed the presence of diverse pectin populations. The existence of distinct pectin populations in potato cell wall material (P-CWM) was less outstanding. Solid-state 13C NMR applied to SB-CWM and P-CWM and residues after treatment by 0.275 M NaOH at 65 °C revealed two fractions of pectic arabinan and galactan side chains. One fraction was highly mobile, whereas the other one displayed restricted mobility.

Structural changes of homogalacturonan, rhamnogalacturonan I and arabinogalactan protein in xylem cell walls of tomato genotypes in reaction to Ralstonia solanacearum

Healthy and Ralstonia solanacearum-inoculated tomato genotypes susceptible or resistant to bacterial wilt including recombinant inbred lines (RILs) deriving from a cross between the resistant genotype Hawaii7996 and the susceptible Wva700 were compared for symptom and bacterial population development, and for the composition and structure of pectic polysaccharides and arabinogalactan proteins (AGPs) of xylem cell walls by immunological staining of tissue prints. Constitutive differences were observed between resistant and susceptible RILs, with a higher degree of methyl-esterification of homogalacturonan (HG) detected by antibody JIM7 in the resistant plants. After inoculation, decreased methyl-esterification of HG indicated by stronger labeling with antibody JIM5 was observed in all susceptible genotypes and in five of eleven resistant genotypes, with a clear increase in the non-blockwise de-esterification pattern of HG (LM7) only in the susceptible lines, indicating the mode of action of the pectinmethylesterase of R. solanacearum. In the susceptible lines infection generally leads to increased branching of rhamnogalacturonan I indicated by the detection of arabinan (LM6) and galactan (LM5) side chains, and of arabinogalactan protein (LM2), while only few of the resistant genotypes reacted with changes in these epitopes. All the resistant, symptomless genotypes contained relatively high pathogen populations in stems. A clear relation between cell wall composition and degree of latent infection of resistant genotypes was not found.

Cell wall disassembly in ripening fruit.

Fruit softening during ripening involves a coordinated series of modifications to the polysaccharide components of the primary cell wall and middle lamella, resulting in a weakening of the structure. Degradation of polysaccharides and alterations in the bonding between polymers cause an increase in cell separation and a softening and swelling of the wall, which, combined with alterations in turgor, bring about fruit softening and textural changes. A wide range in the extent of cell wall pectic modifications has been observed between species, whereas the depolymerisation of xyloglucan is relatively limited and more consistent. The earliest events to be initiated are usually a loss of pectic galactan side chains and the depolymerisation of matrix glycans, which may begin before ripening, followed by a loss of pectic arabinan side chains and pectin solubilisation. The depolymerisation of pectins may begin during early to mid-ripening, but is usually most pronounced late in ripening. However, some of these events may be absent or occur at very low levels in some species. Cell wall swelling may be related to a loosening of the xyloglucan-cellulose network and to pectin solubilisation, and these processes combined with the loss of pectic side chains increase wall porosity. An increase in wall porosity later in ripening may allow increased access of degradative enzymes to their substrates.

Sugar beet ( Beta vulgaris) pectins are covalently cross-linked through diferulic bridges in the cell wall

Arabinan and galactan side chains of sugar beet pectins are esterified by ferulic acid residues that can undergo in vivo oxidative reactions to form dehydrodiferulates. After acid and enzymatic degradation of sugar beet cell walls and fractionation of the solubilized products by hydrophobic interaction chromatography, three dehydrodiferulate-rich fractions were isolated. The structural identification of the different compounds present in these fractions was performed by electrospray-ion trap-mass spectrometry (before and after 18O labeling) and high-performance anion-exchange chromatography. Several compounds contained solely Ara (terminal or α-1 → 5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was assigned in some cases to the O-2 and in others to the O-5 of non-reducing Ara residues. One compound contained Gal (β-1 → 4-linked-dimer), Ara (α-1 → 5-linked-dimer) and dehydrodiferulate. The location of the dehydrodiferulate was unambiguously assigned to the O-2 of the non-reducing Ara residue and O-6 of the non-reducing Gal residue. These results provide direct evidence that pectic arabinans and galactans are covalently cross-linked (intra- or inter-molecularly) through dehydrodiferulates in sugar beet cell walls. Molecular modeling was used to compute and structurally characterize the low energy conformations of the isolated compounds. Interestingly, the conformations of the dehydrodiferulate-bridged arabinan and galactan fragments selected from an energetic criterion, evidenced very nice agreement with the experimental occurrence of the dehydrodiferulated pectins. The present work combines for the first time intensive mass spectrometry data and molecular modeling to give structural relevance of a molecular cohesion between rhamnogalacturonan fragments.

Composition, structure and solution properties of polysaccharides extracted from leaves of Mesembryanthenum crystallinum

Water and Chelator-soluble polymers were independently isolated from the alcoholic-insoluble substance (AIS) of Mesembryanthenum crystallinum leaves. After precipitation in ethanol and ultrafiltration (40 kD cut-off) of recovered solids dissolved in water, the yield relative of the so-call Lw and LCh polymers to the AIS dry matter was 2.5 ± 0.2 and 7 ± 0.5%. The galacturonic acid contents were 67 ± 3% and 63 ± 5%. The degrees of methylesterification of ca. 45 ± 3% and 50 ± 3%, showed that Lw and LCh belonged to the fairly methylesterified pectin class. From sugar analysis, LCh was shown to contain at least two types of pectic blocks, homogalacturonan (HG: 58%) and rhamnogalacturonan-I (RG-I: 34%). Their structure were deduced after saponification, polygalacturonase treatments, size exclusion chromatography (SEC) onto Sephacryl S-200 and then sugar composition of the collected fractions. The main polymers were methylesterified HG (32%), some of them being linked to non-methylesterified HG (10%), and to polygalacturonase-resistant HG (17%). In addition, there were (1) a highly soluble RG-I with long galactan side chains (RG-I–gal accounting for 9%) and (2) a RG-I with short arabino-galactan side chains (25%), named RG-I–ara/gal that was almost totally lost during SEC analysis, due to its low solubility at room temperature and in absence of chelators. SEC coupled with differential refractive index and light scattering showed that highly methylesterified HG chains exhibited aggregate structures in solution due to intermolecular hydrophobic interactions. These interactions formed hydrophobic clusters, which have been characterized by surface tension measurements and with a polarity probe, the Coomassie Brilliant Blue dye. After alkaline treatment of LCh, the self-assembly of HG disappeared.

In vivo Expression of a Cicer arietinum β-galactosidase in Potato Tubers Leads to a Reduction of the Galactan Side-chains in Cell Wall Pectin

We report the generation of Solanum tuberosum transformants expressing Cicer arietinum betaIII-Gal. betaIII-Gal is a beta-galactosidase able to degrade cell wall pectins during cell wall loosening that occurs prior to cell elongation. cDNA corresponding to the gene encoding this protein was identified among several chickpea beta-galactosidase cDNAs, and named CanBGal-3. CanBGal-3 cDNA was expressed in potato under the control of the granule-bound starch synthase promoter. Three betaIII-Gal transformants with varying levels of expression were chosen for further analysis. The transgenic plants displayed no significant altered phenotype compared to the wild type. However, beta-galactanase and beta-galactosidase activities were increased in the transgenic tuber cell walls and this affected the potato tuber pectins. A reduction in the galactosyl content of up to 50% compared to the wild type was observed in the most extreme transformant, indicating a reduction of 1,4-beta-galactan side-chains, as revealed by analysis with LM5 specific antibodies. Our results confirm the notion that the pectin-degrading activity of chickpea betaIII-Gal reported in vitro also occurs in vivo and in other plants, and confirm the involvement of betaIII-Gal in the cell wall autolysis process. An increase in the homogalacturonan content of transgenic tuber cell walls was also observed by Fourier transform infrared spectroscopy (FTIR) analysis.

Molecular characterisation of soybean polysaccharides: an approach by size exclusion chromatography, dynamic and static light scattering methods

Water soluble polysaccharides from soybean (SSPS) have a pectin-like structure and are used as stabilisers in acidified beverages. Physicochemical properties such as structure, molecular weight and shape or conformation are primary factors controlling their functional properties. Two soybean polysaccharides, a native SSPS and a modified SSPS treated with β-(1→4)- d-galactosidase (GPase/SSPS) were studied by dynamic and static light scattering (DLS, SLS) and size exclusion chromatography (SEC). Consecutive filtrations using a range of membrane pore size removed a small fraction of macromolecular aggregates from dilute polysaccharide solutions with relatively little effect on the major component molecules as monitored by DLS and SEC measurements. Access to aggregate-free dilute solutions of SSPS and GPase/SSPS allowed the direct measurement of molecular characteristics. SLS results showed that SSPS had a weight average molecular weight of (645 ± 11) × 10 3 g/mol and a radius of gyration, R g, of (23.5 ± 2.8) nm. By comparing R g with the hydrodynamic radius, R h (21.1 ± 0.5 nm) obtained from DLS, the structural parameter ρ ( R g/ R h) was found to be 1.1, suggesting that SSPS has an overall globular shape due to a highly branched structure. The modified SSPS had a significantly lower molecular weight (287 ± 18) × 10 3 g/mol but a similar radius of gyration (23.2 ± 1.7 nm). The structure parameter ρ of GPase/SSPS was higher ( ρ = 1.3) because of a smaller hydrodynamic radius (17.7 ± 1.8 nm). This suggests that GPase/SSPS has a much less branched structure yet still differs significantly from a linear random coil conformation ( ρ = 1.7–2.0). The results derived from SLS and DLS are in agreement with the conclusions obtained from a chemical analysis where the reduction of molecular weight of GPase/SSPS was caused by the cleavage of galactan side chains.

Conformation and mobility of the arabinan and galactan side-chains of pectin

The function of the arabinan and galactan side-chains of pectin remains unknown. We describe 13C NMR experiments designed to yield spectra from the most mobile polymer components of hydrated cell walls isolated from a range of plant species. In pectin-rich cell walls, these corresponded to the pectic side-chains. The arabinan side-chains were in general more mobile than the galactans, but the long galactan side-chains of potato pectin showed high mobility. Due to motional line-narrowing effects these arabinan and galactan chains gave 13C NMR spectra of higher resolution than has previously been observed from ‘solid’ biopolymers. These spectra were similar to those reported for the arabinan and galactan polymers in the solution state, implying time-averaged conformations resembling those found in solution. The mobility of the highly esterified galacturonan in citrus cell walls overlapped with the lower end of the mobility range characteristic of the pectic side-chains. The cellulose-rich cell walls of flax phloem fibres gave spectra of low intensity corresponding to mobile type II arabinogalactans. Cell walls from oat coleoptiles appeared to contain no polymers as mobile as the pectic arabinans and galactans in primary cell walls of the other species examined. These properties of the pectic side-chains suggest a role in interacting with water.

Isolation and structural characterization of protopectin from the skin of Opuntia ficus-indica prickly pear fruits

Dilute HCl extraction of Opuntia ficus-indica fruits, previously disencrusted by hot water followed by hot chelating treatments, yielded a series of soluble pectic polysaccharides, which were de-esterified and separated into five fractions by anion exchange chromatography. Three of these fractions were thereafter purified by size exclusion chromatography and characterized by sugar analysis combined with methylation and reduction-methylation. Their study was further supported by 1H and 13C NMR spectroscopy. The results showed that one of these fractions (ASP1) was neutral and consisted of a linear β-(1→4)-galactan. One of the acid fractions (ASP2), with a galacturonic content of about 40%, consisted of a disaccharide repeating unit [→2)-α- l-Rha p-(1→4)-α- d-Gal pA-(1→] backbone, with short linear β-(1→4)-linked galactan side-chains attached to O-4 of the rhamnosyl residues. Another acid fraction (ASP3), with a high galacturonic content (about 62%), consisted of alternating homogalacturonan blocks (1→4) α-linked and rhamnogalacturonan blocks with approximately the same amount of galactopyranosyluronic acid residues in each block. 13C T 1 relaxation times were measured and noticeable differences in molecular motion of the different sugar rings in the repeating units were found.