Alistair J. MacDougall

A new view of pectin structure revealed by acid hydrolysis and atomic force microscopy

Individual pectin polymers and complexes, isolated from the pericarp of unripe tomato (Lycopersicon esculentum var. Rutgers), were subjected to a mild acid hydrolysis and visualised and characterised by atomic force microscopy (AFM). The AFM images confirm earlier studies showing that individual pectic polysaccharides often possess long branches. The AFM data have been used to construct size and molecular weight distributions for the single molecules and complexes, from which the calculated number-average and weight-average molecular weights can then be compared directly with the published literature data on the rheology of bulk samples. Loss of the neutral sugars arabinose, galactose and rhamnose from the pectin samples does not significantly alter either the size or the branching density of the individual polymers, but is reflected in a breakdown of the complexes. Significant loss of galacturonic acid at long hydrolysis times was found to be accompanied by changes in the size and branching of the single polymers and further breakdown of the complexes. The results suggest that rhamnose, arabinose and galactose are not the major components of the individual polymers but are, instead, confined to the complexes. The polysaccharides represent a previously unrecognised branched homogalacturonan with a minimum mean size some three times larger than that previously reported. The complexes consist of homogalacturonans (HGs) held together by rhamnogalacturonan I (RG-I) regions. Comparison of the rate of depolymerisation of the homogalacturonans and complexes with the published data on changes in the intrinsic viscosity of bulk pectin samples, subjected to similar acid hydrolysis, suggests that the different rates of depolymerisation of RG-I and HG contribute separately to the observed changes in intrinsic viscosity during acid hydrolysis. Thus data obtained using a single molecule microscopy technique provides new insights into the behaviour in the bulk.

Calcium binding and swelling behaviour of a high methoxyl pectin gel

Abstract Progress in understanding the structural role of pectic polysaccharides in plant cell walls is currently restricted by a lack of information on the molecular properties of undegraded cell wall pectins. We have examined, in solution, the calcium ion binding behaviour of a high methoxyl cell wall pectin from unripe tomato pericarp, and found it to be comparable to other pectins. After gelation through calcium addition, the affinity of the pectin for calcium ions was increased by at least an order of magnitude, with an estimated stability constant, K , of ∼8000. At pH 6 calcium binding is directly related to crosslink formation and gel stiffness. The swelling of the gel in aqueous salt solution was also examined; the kinetics of swelling were comparable to other polyelectrolyte gel systems. A fraction of the galacturonate residues in the gel does not participate in crosslinking but can contribute to gel swelling through a general polyelectrolyte effect at low ionic strengths. With decreasing concentration of free calcium ions, further marked swelling, and eventually dissolution, occurs as a result of dissociation of calcium crosslinks.

Unexpected branching in pectin observed by atomic force microscopy

Abstract Pectic polysaccharides extracted from unripe tomato plant cell walls have been imaged with an atomic force microscope (AFM). The images obtained reveal for the first time a branched structure for tomato pectins that differs from that proposed for the neutral sugar side chains from enzymatic hydrolysis and sugar analysis. The branches are between 30 and 170 nm long and are relatively linear. This work demonstrates that the AFM is uniquely capable of unambiguously identifying, with minimal sample preparation, individual macromolecules within a heterogeneous population.

Calcium gelation of pectic polysaccharides isolated from unripe tomato fruit

Abstract Cell-wall material was prepared from unripe tomato fruit, and a pectic polysaccharide extracted with cyclohexanediaminetetraacetic acid. The structure of the purified pectic polysaccharide was examined by sugar and methylation analysis, and was typical of a rhamnogalacturonan from the primary cell wall. The physicochemical properties of the isolated polysaccharide were characterised by viscometry and size-exclusion chromatography. The polysaccharide was polydisperse, but of large molecular size as indicated by an intrinsic viscosity of 810 ml. g −1 . At concentrations above ∼ 0.2–0.6% w/w, coil entanglement was observed as an increase in the dependency of viscosity on concentration. For these concentrated solutions, clear elastic gels were formed on addition of calcium ions. At concentrations in the range 0.6–2.8% w/w the shear modulus of the gel showed a c 1.9 dependence on concentration. The modulus of the gel increased linearly with absolute temperature in a rubberlike way, enabling an estimate of cross-link density to be made.

The effect of peptide-pectin interactions on the gelation behaviour of a plant cell wall pectin

The effect of basic peptides on the gelation of a pectin from the cell wall of tomato was examined through the determination of gel stiffness, and swelling behaviour of the gel in water. Poly-L-lysine, poly-L-arginine, and a synthetic peptide, designed to mimic a sequence of basic amino acids found in a plant cell wall extensin, act as crosslinking agents. Circular dichroism studies on the interaction of synthetic extensin peptides with sodium polygalacturonate demonstrated that a conformational change was induced as a result of their complexation. In addition to their effect as crosslinking agents, the polycationic peptides reduced the swelling of the pectin network in water.

Phase Separation of Plant Cell Wall Polysaccharides and Its Implications for Cell Wall Assembly

Concentrated binary mixtures of polymers in solution commonly exhibit immiscibility, resolving into two separate phases each of which is enriched in one polymer. The plant cell wall is a concentrated polymer assembly, and phase separation of the constituent polymers could make an important contribution to its structural organization and functional properties. However, to our knowledge, there have been no published reports of the phase behavior of cell wall polymers, and this phenomenon is not included in current cell wall models. We fractionated cell walls purified from the pericarp of unripe tomatoes (Lycopersicon esculentum) by extraction with cyclohexane diamine tetraacetic acid (CDTA), Na2CO3, and KOH and examined the behavior of concentrated mixtures. Several different combinations of fractions exhibited phase separation. Analysis of coexisting phases demonstrated the immiscibility of the esterified, relatively unbranched pectic polysaccharide extracted by CDTA and a highly branched, de-esterified pectic polysaccharide present in the 0.5 N KOH extract. Some evidence for phase separation of the CDTA extract and hemicellulosic polymers was also found. We believe that phase separation is likely to be a factor in the assembly of pectic polysaccharides in the cell wall and could, for example, provide the basis for explaining the formation of the middle lamella.

Specific degradation of pectins via a carbodiimide-mediated Lossen rearrangement of methyl esterified galacturonic acid residues

A specific, chemical degradation of the methyl esterified galacturonic acid residues of pectins is described. These residues are converted, with hydroxylamine, to hydroxamic acids, and then, with a carbodiimide, to isoureas; the latter undergo a Lossen rearrangement on alkaline hydrolysis. The isocyanates formed are hydrolysed to 5-aminoarabinopyranose derivatives, which spontaneously ring open to give 1,5-dialdehydes. The latter are reduced, in situ, to avoid peeling reactions, with sodium borohydride to give substituted arabitol residues. Thus, overall, partially esterified pectins are specifically cleaved to generate a series of oligogalacturonic acids bearing an arabitol residue as aglycone. Analysis of oligomers so generated discloses the pattern of contiguous nonesterification in a variety of pectins of differing degrees of esterification. Other potential applications are described.

Observations on the crystallization of oligogalacturonates.

Oligogalacturonates were produced by the limited enzymic hydrolysis of polygalacturonic acid and purified by ion-exchange chromatography. The fractions obtained were of limited polydispersity, determined by analytical ion-exchange chromatography. Oligomers with an average degree of polymerization of 10-15 were readily crystallized from aqueous salt solutions at neutral pH as single crystals. Crystal morphology of the salts examined, Na+, K+ and Ca2+ were characteristic of the salt. The wide-angle X-ray diffraction patterns obtained for the sodium salt were consistent with published fibre diffraction data of this salt form.