Some pectins gain more and more interest for their health modulating activities. Endogenous as well as exogenous enzymes play an important role in determining the pectic structures present in plant tissue, food products, or ingredients at a given time.
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In this paper functional and structural characteristics of pectin are described with special emphasis on the structural elements making up the pectin molecule, their interconnections and present models which envisage the accommodation of all structural elements in a macromolecule. Attention is also given to analytical methods to study the pectin structure including the use of enzymes as analytical tools. Pectin is one of the major plant cell wall components and probably the most complex macromolecule in nature, as it can be composed out of as many as 17 different monosaccharides containing more than 20 different linkages [ 1 , 2 , 3 ].
In a plant, pectin is present in the middle lamella, primary cell and secondary walls and is deposited in the early stages of growth during cell expansion [ 4 ]. Its functionality to a plant is quite divers. First, pectin plays an important role in the formation of higher plant cell walls [ 5 ], which lend strength and support to a plant and yet are very dynamic structures [ 4 ]. In cell walls of some fruits and vegetables, the pectin content can be substantially higher and the protein content lower [ 6 ].
Second, pectin influences various cell wall properties such as porosity, surface charge, pH, and ion balance and therefore is of importance to the ion transport in the cell wall [ 7 ]. Furthermore, pectin oligosaccharides are known to activate plant defense responses: they elicit the accumulation of phytoalexin which has a wide spectrum of anti-microbial activity [ 8 , 9 , 10 ]. Finally, pectin oligosaccharides induce lignification [ 11 ] and accumulation of protease inhibitors [ 12 ] in plant tissues. Pectin is used in foods mainly as gelling, stabilizing, or thickening agent in products such as jam, yoghurt drinks, fruity milk drinks, and ice cream [ 13 ].
Most of the pectin used by food industry originates from citrus or apple peel from which it is extracted at low pH and high temperature and is primarily a homogalacturonan [ 14 ]. In products that naturally contain pectin, e. Native or added pectic enzymes can play an important role in these changes [ 15 ].
Plant products, fresh, extracted or processed, constitute a large part of the human diet. As a fiber naturally present in these food products, pectic substances fulfill a nutritional function [ 16 , 17 ]. Next to its nutritional status, pectin increasingly gains interest as a possible health promoting polysaccharide and several studies have been conducted to prove its health promoting function. One study showed the beneficial influence of vegetable pectin-chamomile extract on shortening the course of unspecific diarrhea and relieving associated symptoms [ 18 ].
Another study revealed that carrot soup contains pectin derived oligosaccharides that block the adherence of various pathogenic micro-organisms to the intestinal mucosa in vitro, which is an important initial step in the pathogenesis of gastrointestinal infections [ 19 , 20 ]. Furthermore, pectins were shown to have immuno-regulatory effects in the intestine, to change the ileal microbial activity, to change the morphology of the small intestinal wall [ 21 , 22 ], to lower the blood cholesterol level [ 23 , 24 , 25 ], and to slow down the absorption of glucose in the serum of diabetic and obese patients [ 25 , 26 , 27 ].
To better understand the bio-functionality of pectic polysaccharides scientific elucidation of the structures responsible for the beneficial effect is very important [ 28 ]. Schematic representation of pectin structural elements [ ]. The minimum estimated length of this backbone is, for citrus, sugar beet, and apple pectin 72— GalA residues [ 36 ]. The methyl-esterification in particular has gained a lot of attention in pectin research, because it strongly determines the physical properties of pectin.
However, not only the amount of methyl-esterification is important, but also the distribution of these esters is. The suggestion made by Rees and Wight [ 42 ] that HG elements could be interspersed with single L-rhamnose residues, resulting in a kink of the molecule, was convincingly argued against by Zhan et al. These authors could not isolate this internal rhamnose Rha from an endo-polygalacturase digest of citrus pectin, indicating a scarcity or complete lack of interspersing single rhamnose residues.
Furthermore, based on molecular modeling, the presence of a kink in the molecule caused by interspersing Rha is further undermined [ 44 ]. Part of the GalA residues in XGA is methyl-esterified and the methyl esters are found to be equally distributed among the substituted and unsubstituted GalA residues [ 44 , 45 ].
Although XGA has been mainly identified in reproductive tissues such as fruits and seeds [ 42 , 44 ], Zandleven et al. Sycamore cells that are cultured in suspension can have as many as repeats of this disaccharide [ 42 , 47 ]. In contrast, in sugar beet pectin oligosaccharides with a maximum length of only 20 residues of alternating Rha and GalA units were isolated. However, it is unclear whether the acid hydrolysis extraction might have caused backbone breakdown, thus underestimating the RGI backbone length [ 48 ].
The rhamnosyl residues of RGI can be substituted at O-4 with neutral sugars side chains [ 49 , 50 , 47 ]. The ratio between these alternative substituted oligosaccharides suggests that hairy regions are composed, in part, of different repeating units [ 49 ]. Rhamnogalacturonan II RGII is a highly conserved structure in the plant kingdom and can be released by endo-polygalacturonase action. The structure is characterized as a distinct region within HG, containing clusters of four different side chains with very peculiar sugar residues, such as apiose, aceric acid, 3deoxy-lyxoheptulosaric acid DHA , and 3-deoxy-mannooctulosonic acid KDO.
These side chains are attached to a HG fragment of approximately nine GalA residues, of which some are methyl-esterified [ 3 , 59 , 60 ]. The structure of RGII seems to be highly conserved in the plant kingdom. Only the apiofuranosyl residues of the 2-O-methyl- d -xylose-containing side chains in each of the subunits of the dimer participate in the cross-linking [ 61 ].
Pectin and AGII often co-extract and are subsequently difficult to separate from each other [ 67 ]. It has even been demonstrated that a small fraction of carrot tap root cell wall AGPs is linked to pectin [ 68 ]. Mode of action of pectinases involved in the degradation of homogalacturonan, rhamnogalacturonan I and xylogalacturonan see text for abbreviations.
Pectins and Pectinases
Terminal end of rhamnogalacturonan I is represented in gray to stress that indicated exo-activity only exists with a single sugar moiety. Figure has been adapted from Hilz et al. The enzyme randomly attacks its substrate and produces a number of GalA oligosaccharides [ 74 ]. Within the products formed, the Rha residues can be substituted with single galactose units [ 49 ].
The enzyme is intolerant for acetyl-esterification of the RGI backbone [ 70 , 77 ]. Removal of arabinan side chains from saponified hairy regions of pectin resulted in an increased catalytic efficiency of Aspergillus aculeatus RGL, whereas the removal of galactan side chains decreases the enzyme efficiency [ 80 ]. The RGL activity increased after removal of acetyl groups [ 80 ]. The enzyme is intolerant for galactose substitutions and has not yet been assigned to a glycosyl hydrolase family since no sequence information is available.
Rhamnogalacturonan galacturono hydrolase is able to release a GalA moiety connected to a rhamnose residue from the non-reducing side of RGI chains but is unable to liberate GalA from HG [ 82 ]. XGH has a requirement for xylosyl side chains and is therefore believed to cleave between two xylosidated GalpA residues [ 83 ]. Removal of ester linkages of galacturonan by saponification increases the enzyme activity [ 84 ]. Pectin cross links as described in literature. Figure adapted from Hilz [ ]. Low methyl-esterified pectins are thought to gel according to the egg box model [ 85 ], first suggested for alginates [ 86 ].
Sections of two pectic chains, which must be free of ester groups, are held together by a number of calcium ions. It is reported that blocks of 7—20 free GalA residues are required for association with calcium [ 87 , 88 , 89 ]. Pectins originating from spinach and sugar beet contain ferulic acid residues in the arabinan side chains. The demonstration that RGII exists in primary walls as a dimer that is covalently crosslinked by a borate diester [ 61 ] was a major advance in the understanding of the structure and function of this pectic polysaccharide.
RGII is covalently linked to HG and as a consequence dimer formation results in the cross-linking of two HG chains, which could lead to the formation of a three-dimensional pectic network in muro [ 60 ]. This network contributes to the mechanical properties of the primary wall and is required for normal plant growth and development.
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Changes in wall properties resulting from decreased borate cross-linking of pectin lead to many of the symptoms associated with boron deficiency in plants [ 2 , 60 , 61 ]. Lamport [ 95 ] suggested that HG could be linked to relatively non-polar putative alcohols by uronyl esters. These observations have been revisited, with the additional hypothesis that particular pectin methylesterase s PME could catalyze a trans-esterification reaction [ 97 ].
The energy imparted in the methyl ester bond is used in the wall by PME to synthesize cross-links between HG chains; the methanol is released and the carboxylgroup of the galacturonosyl moiety is attached to a —OH group of a galacturonosyl moiety of another HG chain. As HG is mainly deposited in the cell wall in a methylesterified form, it is evident that these molecules hold an enormous potential for cross-linking.
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Interestingly, within the Arabidopsis genome, about 60 PME genes have been found that await further characterization [ 98 ]. It is possible that PMEs specialized in catalyzing the formation of uronyl esters can be found among these. More work is needed to further substantiate the abundance, formation, and role of this cross-link.
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It has become clear that pectin is a very complex macromolecule and that it is a big challenge to accommodate all available information in a model structure. Some of the most cited hypothetical models are summarized below. A proposed structure for the rhamnogalacturonan. Reprinted from Talmadge et al.
De Vries et al. The degradable unsubstituted part was defined as the smooth region HG. The observed neutral sugar distribution curves, obtained by anion-exchange and size-exclusion chromatography, indicated a specific ratio of smooth versus hairy regions within the different eluted populations. In fractions of extracts obtained with various extractants from ripe and unripe and fractionation by anion-exchange chromatography three main types of pectin molecules were identified, having one, two, or three hairy regions, respectively.
Characterization of CRISPR Mutants Targeting Genes Modulating Pectin Degradation in Ripening Tomato
These types were identified by grouping subfractions with equal amounts of anhydrogalacturonic acid according to the ratio of moles neutral sugars per mole anhydrogalacturonic acid present. Pectin model based upon the sugar composition and molecular weights of the different pectin extracts B, C, and D. Type A and Type E are considered to be degraded pectins.
Horizontal lines: rhamnogalacturonan backbone of the pectin molecule. Branched areas: blocks of neutral sugar side chains number and length are arbitrary. Reprinted from de Vries et al. Hypothetical structure of apple pectin and of the prevailing population of MHR isolated here from.