Work

If Homogalacturonan Were a Side Chain of Rhamnogalacturonan I. Implications for Cell Wall Architecture

Year: 2003

Type: review

Abstract: Pectin, an important cell wall component of dicotyledonous plants, is probably the most complex macromolecule in nature. Here, we critically summarize the large amount of data on pectin structure. An alternative model for the macromolecular structure of pectin is put forward, together with ideas on how pectins are integrated into the plant cell wall. Pectin is composed of as many as 17 different monosaccharides (Fig. 1; for review, see Ridley et al., 2001; Voragen et al., 2003). These monosaccharides are organized in a number of distinct polysaccharides, the structures of which are schematically shown in Figure 1. Together, these polymers form the pectin network (Visser and Voragen, 1996; Ridley et al., 2001; Voragen et al., 2003). Schematic representative structures of the constituent polysaccharides of pectin. The symbols for the various monosaccharide building units are explained in the accompanying legend. The predominant linkage types are indicated in the text. Underneath the diagrams with the monosaccharide building units, the symbol for the polymeric structure of that polysaccharide is indicated. These symbols will be used in the rest of the paper. The first structural elements of pectin to be discussed have a backbone of 1,4-linked α-d-GalpA residues and are referred to as galacturonans. Three types of galacturonan can be distinguished in the plant cell wall: the unsubstituted homogalacturonan (HG), rhamnogalacturonan II (RG-II; see below), and xylogalacturonan (XGA). The GalA residues of HG can be methyl-esterified at C-6 and carry acetyl groups on O-2 and O-3. XGA is a branched galacturonan with β-d-Xylp-(1→3) side chains (Schols et al., 1995; Visser and Voragen, 1996). The degree of xylosylation can vary between 25% (watermelon [Citrullis vulgaris]) and 75% (apple [Malus domestica]; Schols et al., 1995; Visser and Voragen, 1996). The GalA residues of XGA can be methyl-esterified as in HG (Schols et al., 1995). The methyl-esterification of HG, in particular, has drawn the attention of many research groups, because it determines to a large extent the industrial applicability of pectin. Not only the amount of methyl-esterification is important, but also the distribution of methyl groups on the HG backbone. Blocks of more than 10 unesterified GalA residues generally yield pectin molecules, which are sensitive to Ca2 +-cross-linking (Daas et al., 2001). HGs can contain clusters of four different (heterooligomeric) side chains with very peculiar sugar residues (such as Api, AceA, Dha, and Kdo). These side chains, together with the approximately nine galacturonyl residues to which they are connected, are referred to as RG-II (O'Neill et al., 2001; Ridley et al., 2001). The name RG-II is somewhat misleading, because it suggests that this structure contains a rhamnogalacturonan backbone just like rhamnogalacturonan I (RG-I). However, the Rha residues are much less abundant in RG-II than in RG-I, and they are present in the side chains of RG-II instead of in the backbone. Although the structure of RG-II is highly conserved in plants, new structural details are continuously being added to this complex element (Ridley et al., 2001). The distribution of the four side chains in RG-II, as well as the distribution of RG-II in HG, still remain to be established. It is not known whether XGA can harbor RG-II elements. Another constituent polysaccharide of pectin, RG-I, is composed of a repeating disaccharide unit [→2)-α-l-Rhap-(1→ 4)-α-d-GalpA-(1→]n, where n can be larger than 100 (McNeil et al., 1980; Visser and Voragen, 1996). The galacturonyl residues can carry acetyl groups on O-2 and O-3. The rhamnosyl residues can be substituted at O-4 with neutral sugars (McNeil et al., 1980; Lau et al., 1987). The proportion of branched Rha residues generally varies from approximately 20% to approximately 80% depending on the source of the polysaccharide (Visser and Voragen, 1996), although essentially unbranched RG-I molecules have also been reported in the literature (Penfield et al., 2001). The side chains can be single unit [β-d-Galp-(1→4)] but also polymeric, such as arabinogalactan I (AG-I) and arabinan (50 glycosyl residues or more). AG-I is composed of a 1,4-linked β-d-Galp backbone; α-l-Araf residues can be attached to the O-3 of the galactosyl residues (Ridley et al., 2001). The arabinans consist of a 1,5-linked α-l-Araf backbone, which can be substituted with α-l-Araf-(1→2)-, α-l-Araf-(1→3)-, and/or α-l-Araf-(1→3)-α-l-Araf-(1→3)-side chains (Ridley et al., 2001). Complexes of RG-I, AG-I, and arabinan are often referred to as pectic hairy regions (HR), in which AG-I and arabinan are the “hairs.” The abundance of HR and the amount/nature of the hairs can differ considerably from species to species. Arabinogalactan II (AG-II), is mainly associated with proteins (arabinogalactan proteins or AGPs), and it is still unclear whether this polysaccharide is part of the pectin complex. Pectin and AG-II often seem to co-extract and are subsequently difficult to separate from each other, suggesting that they can be covalently linked. AG-II is composed of 1,3-linked β-d-Galp backbone, containing short side chains of α-l-Araf-(1→6)-[β-d-Galp-(1→6)]n (n = 1, 2, or 3; Ridley et al., 2001). The galactosyl residues of the side chains can be substituted with α-l-Araf-(1→3) residues. The major moiety of AGPs (>90%) consists of polysaccharides. The protein moiety is rich in Pro/Hyp, Ala, Ser, and Thr (Gaspar et al., 2001, and refs. therein). In Arabidopsis, mature AGPs are between 10 and 369 amino acids long. At the C terminus of these proteins, a glycosylphosphatidylinositol anchor signal sequence is present. This signal can be replaced by a glycosylphosphatidylinositol anchor, which allows the protein to be inserted in the cell membrane. Although the fine structure of the various pectic constituents is largely known, it is still unclear how these structural elements are combined into a macromolecular structure. It is likely that these constituents are covalently linked to each other (Ridley et al., 2001; Voragen et al., 2003). For a long time, pectin was either depicted as is shown in Figure 2A, in which HG and RG-I form one continuous backbone (Visser and Voragen, 1996), or the connection between HG and RG-I was left undiscussed. The most important feature of the model in Figure 2A is that the “pectic backbone” is an extended chain comprising RG-I (including the neutral hairs [not shown]), HG (or XGA [not shown]), and (isolated) Rha residues interspersing the HG regions. This model was consistent with the observed length periodicity of HG (approximately 70–100 GalA residues; Thibault et al., 1993), endo-polygalacturonase-resistant rhamnogalacturonan regions with a GalA to Rha ratio of >1, and the typical RG-I structure (McNeil et al., 1980; Visser and Voragen, 1996). Recently, we have summarized evidence for an alternative macromolecular structure of pectin (Fig. 2, B–D) that is more consistent with the most recent results in pectin research. The main difference between the alternative models and that in Figure 2A is that the HG constituents are depicted as side chains of RG-I. The most important motivations for proposing this alternative structure are briefly described below (for a detailed discussion, see Voragen et al., 2003). (a) It seems unlikely that the biosynthetic machinery inserts rhamnosyl residues in the nascent pectic backbone in an irregular pattern. (b) The length periodicity of HG is explained in a different way. Upon treatment of pectin with dilute acid, the α-l-Rhap-(1→4)-α-d-GalpA linkages in rhamnogalacturonans are (selectively) split. It seems likely that similarly sized HG fragments would be obtained with both models. (c) A thorough study in which pectin was exhaustively treated with endo-polygalacturonase, followed by a careful fractionation of the resulting fragments, has not provided evidence for the existence of isolated Rha residues interspersing two HG structural elements (Zhan et al., 1998). (d) Studies in which pectin preparations were treated with exoPG or endo-XGA hydrolase suggest that XGA is not an integral part of the pectic backbone (J.-P. Vincken, G. Beldman, and H.A. Schols, unpublished data). Because XGA and HG have the same backbone structure, it is not unreasonable to hypothesize that this holds also for HG. Recently, Oechslin et al. (2003) have also suggested that XGA might be a side chain of RG-I, based on a detailed structural investigation of pectic substances that were strongly associated with cellulose. (5) Atomic force microscopy (AFM) of citrus pectin indicated that HG branches might be connected to a pectic backbone (Round et al., 2001). Although the interpretation of the micrographs is rather complex, we think that the AFM results could indicate that HG side chains are attached to an RG-I backbone. Schematic representation of macromolecular structures of pectin, which are in accordance with the observed length periodicity of HG. A, In this model, the pectic backbone is an extended chain with HG and RG-I regions (adapted from Visser and Voragen, 1996). HG regions can be interspersed with rhamnosyl residues. The “smooth” regions are mainly composed of HG. The “hairy” regions consist of RG-I, XGA, arabinan, and AG-I. For simplicity, XGA, arabinan, and AG-I are not shown. AI, AII, and AIII illustrate the transition of HG to RG-I, rhamnogalacturonan regions with a GalA to Rha ratio of >1, and a Rha residue interspersing two HG constituents, respectively. R is reducing end; NR is nonreducing end. B through D, Models for pectin in which RG-I is decorated with neutral (AG-I, arabinan, and possibly AG-II) and HG/XGA side chains (adapted from Voragen et al., 2003). It is unknown how HG and XGA are attached to RG-I. It is possible that unique pectin molecules exist, in which only one type of side chain is present (B), or in which various side chains are distributed at random in RG-I polymer (C), or in which the side chains are arranged in a cluster-like fashion (D). The abundance of the various polysaccharide units and their length is arbitrary, as is the position of RG-II in the HG polymer. For simplicity, all hairs are drawn to one side of the RG-I backbone. The exact orientation of hairs with respect to each other remains to be established. It is very possible that the hairs point out in various directions, away from the RG-I backbone. For symbols, see Figure 1. AFM will be a powerful tool to elucidate the macromolecular structure of pectin in this is combined with of pectin and/or dilute combined with of the is to elucidate the exact of the point of of HG to RG-I. will be to how the various HG, are distributed in the RG-I backbone. In an number of is RG-I carry only one of side chain (Fig. are a random distribution of hairs (Fig. cluster-like of hairs of the same (Fig. and cluster-like of different of hairs It be that it is unlikely that all hairs point into one It is probably more to the hairs in various directions, to the RG-I backbone. However, in Figure this has not been because it would the The that can be by treatment with hydrolase or to the existence of cluster-like The HG, arabinan, and AG-I AG-II) hairs are very different in and In different be the wall by different of this an important in wall at various and in different A new model for the pectin network in the and the cell wall. A, of two cell by a two 1, The is from the pectin molecules in the cell which are Ca2 to HG hairs in subsequently of pectin of Pectin methyl can the methyl groups from HG, HG sensitive to Ca2 These pectic probably the pectins that can be from the wall 2, RG-II of of pectin by of pectin can be inserted in the the of the mature cell wall has been The RG-I backbone is a from the same RG-I will both of the in and the pectic molecules from from the wall. of the from the cell to the RG-II can between two of pectin but also between pectic molecules the same AG-I and arabinans the in the and have an important in wall for simplicity, are not shown in this methyl-esterification in HG chains are indicated with cell cell For other symbols, see Figure 1. In the we the large structural of pectin In the plant cell these in much larger Three different of for pectic molecules will be discussed HGs can different in of which the and seem to be most in of et al., unesterified HG chains can in a complex referred to as in which the groups of two GalA residues form a that can a Ca2 et al., et al., At 10 unesterified galacturonyl residues are to a between chains et al., In this Ca2 can be Pectin molecules can be in different A, through of Ca2 between the unesterified groups of the residues of two HG than nine are to a connection et al., HG molecules can also be by which can be between the residues of the side chains of It has been suggested that can be as a of a in which a methyl-esterified GalA residue is the In wall polysaccharide can as an molecules of RG-II can complex with a (Fig. et al., the residues of the side chains in each of the of the in the et al., Because RG-II is an integral part of HG and can two HG and in in a and et al., The of RG-II in the plant and structural indicate a distinct in wall for this constituent of pectin. it was suggested that HG could be to other by (Fig. and to approximately of the GalA residues could be in this way. Recently, these have been et al., 2001). It was suggested that pectin can a methyl-esterified HG as a and other HG molecules as an Because HG is mainly in the cell wall in a methyl-esterified it is that these molecules an for in Arabidopsis, have been that et al., 2001). It is possible that in the of can be more is to the and of this the a number of have been that different pectic structures in the wall. The of these have been et al., of with various have shown that pectin to the of the plant cell wall. This was by the of et al. in which and different of were that the wall one cell can contain each a type of HG. polysaccharides are also in a For the distribution of pectic in the of from that of the (for review, see Voragen et al., and refs. therein). but not different pectic are at different of For in the of in than of The suggest that the wall a of pectic molecules to or at a In very the (a) various HG can one cell with that of Ca2 to the cell and containing (b) arabinan is generally present the wall of is mainly present to the of (c) RG-II seems to be present the in the (d) AG-II to be to the or in of cell for elements (Ridley et al., 2001, refs. et al., Voragen et al., 2003). The of pectic polysaccharides suggests that the various constituents of pectin each have their in the of the wall and At this of whether or how pectic to of the wall is very The main for this is that on the of cell wall polysaccharides, pectin in particular, is still in (Ridley et al., 2001). The first in the backbone of a pectic structural element remains to be although it seems as important has been very The first in one of the pectic has been only a et al., Because of have been to with pectin a A number of with an pectin have been (Penfield et al., 2001; et al., et al., for review, see Voragen et al., of which the is probably (O'Neill et al., 2001). These contain of RG-II, but the two residues present in RG-II are replaced by residues. a of the of RG-II and the of this are The that plant on pectic polysaccharide The can be by of or This is in with in which or plant were et al., et al., 2001). It was observed that results in with This is of to and The of the could not be by the of et al., The to be in a RG-II and the attached in the side and the to form the is to of that observed with in have also been a to as has been described et al., in this have a galacturonyl in their and cell is The exact of this still to be but a in pectin be The of the to have less cell wall and also have cell et al., The galacturonyl of the of these was more or less to of However, the to regions of unesterified HG, by and It has been suggested that the are in a An alternative for the of the various structural elements of pectin is to in have been with from et al., et al., and et al., The two are of hairs from the RG-I backbone, the can this backbone. and were in the cell wall of the I to their This was not possible with because the and not this was in signal to the arabinans they were in the wall. of the isolated cell wall of the that the amount of and both and was in the and with the The and cell were also microscopy in with The was used to the of hairs and as a tool for RG-I. with the by in their that the the from the cell wall. In to the of in the of a of the to the was observed in The in of the RG-I backbone with the of the suggests that the hairs not with other cell wall cell seem to a of either or arabinan hairs the because both the and the of the of the and et al., et al., In to of the RG-I backbone to with and large in the et al., This strongly suggests that RG-I a more in cell wall than the In the we have that pectin a large of pectic molecules, which can differ considerably in their In the these molecules are in different a larger the of various pectic molecules and the of different can be in the which are to for various This will with how the pectic network might be in the wall. In wall a network with a pectin network and The is to be the structure of the the determines possible models for the plant cell wall: (a) the (b) the and (c) the The first model is by in one and between In the model, as a of which and subsequently pectin are is of The model of and one In all of these pectin the in the and is to the of and the of the new ideas pectin macromolecular structure, in which pectic molecules are as with different (Fig. 2, have for the of the plant cell wall. a new model (Fig. for (a) the of the and (b) of and (c) of wall that the are as such in and that they are not the cell by the of to the models discussed pectin has a much more important than the of the In the wall consists of a which is in a of This will be for a more detailed of the we to this it is not which polysaccharide is first in the cell pectin, or cellulose. for of pectin and in the cell and for pectin first et al., For model, we have that pectin is cellulose. the is as a separate between a Ca2 HG with a degree of The of these polymers and whether these HGs are connected to other wall polymers have not been that arabinan or are first in the cell of two of pectin molecules are HG hairs can be by which are suggested to in a or The unesterified regions of HGs different sensitive to Ca2 and can form a with in with have shown that and Ca2 can to of such as the et al., 1998). The pectin the HG, which can be from the wall of many plant species In we hypothesize that the is a Ca2 by the first of pectin to of the cell wall of two The point at which the is not the of the cell wall. In it has been observed that the with the cell wall and This model is consistent with by that the of unesterified HG with cell et al., and with the that RG-II is from the (Ridley et al., 2001). the first of pectin has been two are to in to the of the cell wall and the (a) cell wall polymers but also are continuously being added on both of the Ca2 (or which is away from the cell membrane. the is (b) are at the cell and into the where they can be by This is also a continuous which can be as a The are from the by of in the that different of are in the wall. In Figure the of two is but it be that more can be inserted in a way. For simplicity, other are not indicated in the hypothesize that the pectin molecules in the are in the wall by two predominant is the in which the RG-I backbone of the pectic molecules are the with in see also because new is continuously being added to the wall at the The hairs between the with in the of of the pectic is evidence that mainly the pectin molecules highly substituted with side chains in one or with (or because with a were to these polysaccharides et al., Oechslin et al., 2003). it has been shown that the pectin molecules with of a are in the wall et al., 2001). These results indicate a for arabinan in pectin in the although this has not been in with a arabinan et al., a more predominant in the of the wall (for review, see Voragen et al., 2003). the various pectic in the wall are connected the of the wall. It seems likely that the RG-II a predominant in this with in which is consistent with that RG-II the cell wall but not in the This is also in with that have which can in et al., et al., 2001). between pectic one pectin also be in that the of the pectic molecules in the The RG-II is in the but is present the wall. AG-I is probably more abundant to the cell arabinan is more abundant in the cell wall the In we that the pectin network is in a The RG-I backbone probably with the cell the hairs have a more orientation with respect to the RG-I backbone. The HG hairs are than the neutral and are to shown in or with of the pectic to we the observed length periodicity of HG of approximately to 100 GalA residues et al., 1993), it can be that each pectin has a of to HG hairs of and a of approximately HG the generally monosaccharide of In model, the of one pectin on the number of RG-II elements HG as well as on the exact of the RG-II element the HG These are in with the model of the plant cell wall by and suggested that the wall was of various and that the between of different was approximately The of the was approximately which is also consistent with the length periodicity of HG. The model in Figure is consistent with the of in the wall. In the various pectic molecules are in the wall they molecules with which are in would be to in the pectin to the cell membrane. the model can that pectin can a wall of a network is et al., et al., 2001). The of such is the is to that of et al., In these seem to for the of by pectic molecules with more HG et al., et al., et al., 2001). detailed are to whether the wall in these is by Ca2 or other of are also of protein and in the of which could to et al., In this we have provided for the first time, to a detailed on how the pectin network be present in the plant cell wall. model is in a structure in which HG, XGA, and arabinan as side chains to RG-I, a of different hairs in various a large number of can be The pectin be in the wall in a the plant cell to different in the wall and to to and at a point or It is that a number of to be the between RG-I and HG to be which will not be an the distribution of hairs in the RG-I backbone will be an the hairs in an at random or in fashion in a number of RG-II remain to be many RG-II elements HG hairs are the RG-II elements distributed in the HG structural these are to on the of the molecules for the of the various In a more detailed of will a on the of different polysaccharide structures that can be the of the first to the pectin biosynthetic in pectin research seem to be of at the of these in will in the of the various polysaccharides. These will important to to pectin in different

Source: PLANT PHYSIOLOGY

DOI: 10.1104/pp.103.022350