Pectins

Pectins make up about a quarter to half of the dry mass of typical cell walls from non-woody plant tissues. Some monocots - grasses, cereals, palms and their allies are an exception to this, containing pectins at much lower abundance.

Secondary structure and cross-linking- under suitable conditions the unsubstituted galacturonan chain segments that make most of the length of a pectic molecule can aggregate non-covalently into bundles, and thus connect pectic molecules together. If the galacturonans are strongly anionic, charge repulsion prevents aggregation. The chains aggregate if divalent cations bridge between the anionic carboxyl groups, or if these cease to be anionic due to esterification and protonation.

It is often stated that the pectic aggregates within the cell wall are of the "egg-box" structure, where two pectic chains in a twofold helical conformation retain calcium ions between them like eggs in a egg-box. Using solid-state NMR we can probe the pectic conformations in situ. We have shown that both twofold and threefold helices are present and that there is a balance between them. We have proposed a more complex ‘cable’ model for chain aggregation in vivo that includes both helical anionic forms complexing calcium, and may also incorporate high-ester chain regions in the threefold helical conformation.

Is calcium the only possible cation? We have shown by EELS that calcium is certainly present in the same places as anionic pectins and presumably much of the galacturonan in the cell wall has calcium as its counterion, but it is quite possible that other cations are present too. Some, like copper and lead, bind to aggregated galacturonans much more strongly than calcium does. Sodium and magnesium may be present at sufficient levels to contribute to the charge balance and we cannot rule out the presence of aluminium, ammonium, titanium and organic cations such as polyamines.

A large unknown area in pectin chemistry concerns the possible presence in vivo of covalent cross-links between pectic molecules. Certainly pectins are much harder to extract from cell walls than their non-covalent chemistry suggests, and the old term ‘protopectin’ reflects that fact that the molecule must be altered to extract it. Strictly, pectin is whatever has been extracted and it may well lack covalent bonds present in ‘protopectin’. Such inter-polymer bonds might connect pectins with other, insoluble polymers such as xyloglucans, but alternatively the pectic molecules could simply be cross-linked to one another. Flory theory shows that an average of two cross-links per macromolecule will produce an infinite, insoluble network.

There have been a small number of reports that the percentage of galacturonoyl esters in cell walls exceeds the number of methyl esters. We have confirmed this for potato cell walls by two relatively simple methods, a copper binding and a delicate titration protocol. So what are the non-methyl esters? We do not know. They could be intermolecular cross-links, or at least some of them could be. Alternatively they could be intramolecular. We have done simple modelling experiments to show that an ester linkage is sterically possible from the carboxyl of one galacturonic acid unit to O-2 of the preceding one. Technically this would be a lactone, but it would be much less strained and thus potentially more stable than the known (2,6)-lactones formed by uronic acids. Sterically it would resemble the O-2 –H- O-6 hydrogen bond that stabilises the structure of cellulose I.

Borate diesters are another likely way in which pectins are cross-linked in vivo, and there are enticing signs that these linkages may be broken and re-formed during growth. One of the borate-esterified apiosyl residues in each RGII unit is capable of cross-linking to another RGII unit, and this mechanism may account for the rapid and drastic effects of boron deficiency in growing plant cells. We are trying to image boron in cell walls by EELS to see if its distribution corresponds to this hypothesis. The levels present are very low, but we have encouraging signs for potato and flax cell walls.

Mechanical function - there is fairly clear evidence that pectins are the main constituents of the middle lamella which attaches one dicot cell to the next. Obviously, whatever kind of bonding links pectic molecules originating from one cell with those from its neighbour, the cross-links must be formed after these pectins are in place and not during their synthesis within the cell. Some of the cross-links are mediated by calcium. Our EELS studies, and related work by a number of other groups, have shown that calcium is concentrated at the points of maximum intercellular stress, consistent with a role in intercellular adhesion. However the removal of calcium ions with chelating agents is not usually sufficient to separate cells and other mechanisms of cross-linking must also be present.

We have shown that disruption of the pectic gel in the primary cell wall can induce swelling normal to the plane of the wall. We suggest that the layers of the primary wall, defined by sheets of microfibrils, are attached to one another by pectins in the same way as the primary wall of one cell is attached to the next. Do pectins also have a role in the mechanical properties of the cell wall within its own plane, those properties that control growth of the cell? The evidence is conflicting but the answer is probably yes - in complex ways that remain to be elucidated.