Biosynthesis of Pectin

The plant cell wall is composed of polysaccharides and proteins. In addition, some cells have walls impregnated with lignin. In all cases, the polysaccharides constitute the major part of the wall. The wall polysaccharides are often classified into cellulose, hemicelluloses, and pectin, and these three types are represented in almost all cell walls in varying proportions. Cellulose is the main load-bearing structure and is composed of β-1,4-linked glucan chains organized in more or less crystalline microfibrils. Hemicelluloses include several different polymers, chiefly xylans, xyloglucans, and (gluco)mannans, which are characterized by having a backbone of β-1,4-linked sugars with an equatorial linkage configuration (Scheller and Ulvskov, 2010). Pectin is the third group of polysaccharides, characterized by relatively high extractability using acid or chelators and a high content of GalUA. Together, the hemicelluloses and pectins constitute the matrix in which cellulose microfibrils are embedded. The interactions between the different polysaccharides ensure the strong yet dynamic and flexible properties of the cell wall. Various pectic polysaccharides can be detected in the cell wall, including homogalacturonan (HG), xylogalacturonan (XGA), apiogalacturonan, rhamnogalacturonan I (RGI), and rhamnogalacturonan II (RGII; Fig. 1). In Arabidopsis (Arabidopsis thaliana) leaves, the cell wall contains approximately 50% (w/w) pectin but the content varies depending on environment, tissue, and species (Zablackis et al., 1995). The ratio between HG, XGA, RGI, and RGII is also variable, but typically HG is the most abundant polysaccharide, constituting about 65% of the pectin, while RGI constitutes 20% to 35% (Mohnen, 2008). XGA and RGII are minor components, each constituting less than 10% (Zandleven et al., 2007; Mohnen, 2008). The different pectic polysaccharides are not separate molecules but covalently linked domains. Unbranched homopolymer chains of α-1,4-linked d-GalUA are described as HG. The backbone of GalUA residues can be substituted at various positions with other sugar moieties, including Xyl (XGA) and apiofuranose (apiogalacturonan). In XGA, a single Xyl is attached to the O-3 position of some GalUA residues. Additional Xyl residues can be attached to the first Xyl with β-1,4 linkage (Zandleven et al., 2006). XGA is particularly abundant in reproductive tissues but is also present in other tissues, such as Arabidopsis leaves (Zandleven et al., 2007). In aquatic angiosperms such as Lemna and Spirodela, the galacturonan backbone is decorated with d-apiofuranose at O-2 or O-3 (Hart and Kindel, 1970; Ovodov et al., 1971; Longland et al., 1989). Schematic structure of pectin. Pectin consists of four different types of polysaccharides, and their structures are shown. Kdo, 3-Deoxy-d-manno-2-octulosonic acid; DHA, 3-deoxy-d-lyxo-2-heptulosaric acid. HG and RGI are much more abundant than the other components (see text). Besides the simple substitutions in XGA and apiogalacturonan, clusters of complex side chains are also attached onto the O-2 or O-3 position in the galacturonan backbone to form RGII (Fig. 1). These side chains are composed of 12 types of glycosyl residues linked together by at least 22 different glycosidic bonds. Some of the glycosyl residues and glycosidic linkages found in RGII side chains are rare and considered unique in plant polysaccharides (e.g. 2-O-methyl-l-Fuc, l-aceric acid, and α-1,3- xylofuranose). Despite its complexity, the structure of RGII is highly conserved among vascular plants (Matsunaga et al., 2004; O’Neill et al., 2004). Frequently, RGII exists as a dimer mediated by borate ion attached to the A branch. Boron is an essential micronutrient for plants, most likely due to an important role of RGII dimerization in ensuring the integrity of the cell wall (O’Neill et al., 2001). Thus, although RGII is a minor component of pectin, it seems to have an essential role, and that may be the main reason why very few mutants with altered RGII have been identified, although many genes must be involved in RGII biosynthesis. RGI is the only type of pectin not built upon pure galacturonan backbones. Instead, it is a branched polymer with a backbone of disaccharide (α-1,4-d-GalA-α-1,2-l-Rha) repeats. The Rha residues in the backbone can be substituted with β-1,4-galactan, branched arabinan, and/or arabinogalactan side chains. Unlike RGII, the structure of the side chains of RGI can vary greatly among plants. Even a model suggesting that HG and its substituted variants are side chains of RGI was proposed (Vincken et al., 2003). However, the discovery of RGI backbone oligomers with HG and XGA as nonreducing end extensions suggests otherwise (Nakamura et al., 2002; Coenen et al., 2008). Galacturonic acid in pure galacturonan backbones is further modified by methyl esterification at the C-6 carboxyl position and/or by O-acetylation at the O-2 or O-3 position, whereas, in RGI, only the O-3 acetylation has been unambiguously reported (Komalavilas and Mort, 1989; Ishii, 1995, 1997). The degree of methyl and acetyl esterification is variable and affects the physicochemical properties of the pectin, especially the formation of calcium-mediated interactions between HG chains. For example, the dimerization of homopolygalacturonan chains mediated by calcium ions was found to require stretches of at least nine unmodified GalUA residues and could be disrupted by methyl esterification (Liners et al., 1992). The primary roles of cell walls are to give physical strength to the plant and to provide a barrier against the outside environment. The main role of pectin is to participate in these two functions together with the other polymers. Especially HG and RGII are well known to be involved in strengthening the wall. The mechanical properties of HG and RGII have been reviewed and described thoroughly in other publications (Ryden et al., 2003; Caffall and Mohnen, 2009) and will not be described further here. HG plays additional roles besides pure mechanical support. Plant pathogens cause degradation of pectin, and oligogalacturonides (i.e. α-1,4-linked oligomers of GalUA) are well established to be part of a signaling cascade that senses wall degradation upon pathogen attack (Ridley et al., 2001; D’Ovidio et al., 2004; Kohorn et al., 2009). Compared with the many studies of HG and RGII function, much less is known about the function of RGI, and it has not been summarized in recent reviews. Treatment of leaf epidermis with arabinanase resulted in hindering of the opening and closing of the stomata (Jones et al., 2003). The proposed hypothesis is that the arabinans work as spatial regulators of the proximity of HG domains. This regulation may prevent the formation of Ca2+-mediated interactions between HG regions; hence, the removal of arabinan may induce stiffening of the cell wall. This hypothesis of the RGI side chains as plasticizers in cell walls that undergo large physical remodeling is in agreement with observations of cell walls functioning under extreme water deficient conditions. Arabinan and galactan are highly mobile polymers compared with the other components of pectin, and this mobility is influenced by the hydration of the cell wall. Therefore, arabinan and galactan are the first polymers to be mobilized upon the hydration of dry cell walls (Tang et al., 1999). Likewise, arabinan and galactan retain mobility relatively longer than other cell wall polymers upon desiccation and may fill the cavities created upon physical rearrangement of the cell wall. Interestingly, an unusually high Ara content (approximately 38 mol %) was observed in the resurrection plant Myrothamnus flabellifolius and inferred to represent pectic arabinan (Moore et al., 2006). Seeds also undergo extreme desiccation and may have a high content of arabinans (Shiga and Lajolo, 2006; Gomez et al., 2009). Marama bean (Tylosema esculentum), a native bean from southern Africa with a water content down to 5%, has as much as 60% (w/w) Ara in the noncellulosic fraction of the cell wall, practically all from a highly branched arabinan (M. Mosele and J. Harholt, unpublished data). Further evidence for the role of RGI side chains in the physical properties of the wall comes from the investigation of transgenic plants. Ulvskov et al. (2005) analyzed the mechanical properties of the cell wall of potato (Solanum tuberosum) tubers from wild-type and transformant plants with decreased contents of galactan or arabinan. The force needed to induce failure of the cell wall decreased in both types of transgenic tubers. The elastic properties of the tubers were also altered, with a stiffening of the cell wall, as observed in stomatal cells (Jones et al., 2003; Ulvskov et al., 2005). Overall, the to that the components of RGI in the wall and a role in wall cell wall polymers function as polymers that are mobilized hemicelluloses have this role, components of pectin may also as is highly abundant in the of species et al., and is and it was that polymers arabinan, are Arabidopsis and et al., 2009). Arabidopsis to mol Ara in the noncellulosic cell wall The Ara was by to be the arabinan in the may have a function, but it also has a more role in as it has been that on arabinan has an important role in of the upon hydration et al., 2009). polysaccharides are to be in it be that some in the or that some in the wall. the evidence for is pectic have been to with and and Mohnen, et al., 2001; and 2003; et al., and pectic can be found in the but not in et al., the few pectic that have been have all been to be in the the of pectin it is that a large of are to these the of the et al. have that different and are will be only three of these have been unambiguously Pectin has been for many and HG in was in et al., of arabinan and galactan was also et al., and and Thus, a is known about in plant tissues, but it is the of this to give a of these will on the studies that have to of the genes and involved in the of pectin. For other recent of pectin Caffall and and et al. HG in was reported in of a of work by different it very of the pectin were A major was the of the HG from and the of an for and Mohnen, The in that residues to the nonreducing end of the HG (Scheller et al., 1999). The work of and the first of a pectin the HG in Arabidopsis by et al. The was in cells and to acid in Interestingly, the fraction from Arabidopsis that also a has not been to have but the two with each other and form a complex in (Mohnen, 2008). is to be a type II (i.e. a with a single and the main the and other are in the et al., 2009). has of which are represented in The group of to and has in Arabidopsis (Fig. of the was in pectin to the of et al., The is highly deficient in HG, suggesting an of in pectin biosynthesis. However, the of has not been and although the decreased of HG it also decreased et al., 2005). functions have been proposed for of the known as in in decreased content in the wall, especially a in the end present in of most plants et al., 2007). This to the that is involved in the end et al., 2007). The contains a GalUA linked to Xyl with an and it seems that could this an is not present in Fig. an hypothesis was proposed that plays an important role in that is to et al., 2007; Caffall et al., 2009). The end that contains has not been in the two are not the of is it be that the on is although it is that all are it is many are involved in pectin biosynthesis. A recent by Caffall et al. an of the functions of the different in Arabidopsis on and of However, between the and in the mutants the of to each with the at to on the of the of The contains from and The was using in the In pectic was reported as as and the were using and of the by degradation and for the in HG described the of a of the and 2003). This was further by et al. very with and in of Ara to both arabinan and galactan In these studies by Ishii, and was as and Ara was into the in the However, the Ara in pectic arabinan and galactan is almost in the is not but it was as a by et al. were to in of onto that plants have that to The was and the et al., 2007). the to but was found to be a known as due to its with a of et al., 2002; et al., 2006). is a relatively abundant on the may have additional its as a is not in the et al., 2010). In of galactan was reported in et al., The have been further with of the and the of et al., 2004; et al., 2001; et al., 2004). were found in et al., and potato et al., that could from onto RGI in that could be with but not with The of et al. only with of RGI backbone with galactan side but not with RGI backbone or with In et al. in bean using Despite the to galactan of the genes involved in the have been plants with a decreased content of galactan have been by of that the of plants is not et al., et al., 2009). constitutes a major part of in primary and mutants be in cell wall In the of galactan mutants both in the that the residues to the RGI backbone and in the that the galactan In of the of studies of arabinan and galactan the of the involved in the of RGI and substituted is two have been and as involved in RGI and et al., 2006; et al., 2008). and are in and of (Fig. to the of et al. are type II on studies of are in the et al., 2006; et al., et al., 2010). is a of the mutants a of Ara in the cell wall et al., 2006). Further that the arabinan side chains to be of but than in the of in and and to by in or in have been in of a Ishii, and unpublished data). of and The from and The were using in the of besides Arabidopsis (Fig. The to is and are not of under the of the in not the arabinan of J. Harholt, and unpublished data). However, of and in and using the arabinan different from the type in all three mutants J. Harholt, and unpublished data). a from in and The of are wall of mutants of some of the has not to the discovery of cell wall and unpublished data). involved in the of pectin and hemicelluloses have been to function in by several (Scheller et al., 2007; Mohnen, et al., 2008). seems that such could but to this are The only strong evidence is the described that and are together in a complex involved in the of HG (Mohnen, 2008). for the of is the that of into polysaccharides the et al., 2006). The form of has been found in plants and is suggesting that is (i.e. the that to likely the to the the is a but on the side of suggesting that it with in the These that the are present in a complex or interactions between sugar and have not been unambiguously but an of was to be with of but not of pectic galactan et al., 2006). This of the of the hypothesis that sugar could with in and are both involved in arabinan and also are for the of complex The conserved suggesting that could a role in complex and/or were in cells and analyzed by under and nonreducing conditions. nonreducing the of or to be than J. Harholt, and unpublished data). and were by this and by J. Harholt, and unpublished data). The XGA is in (Fig. and linkage of mutants a decreased content of XGA in the mutants et al., 2008). with a XGA the XGA of in cells and in using of Xyl from to GalUA the function of as a XGA of mutants using the two not the major XGA present in tissues but only in the and the XGA was not in the This that are to by the other of RGII has not been much A group of found to be involved in RGII is the Arabidopsis in of of this with characterized functions include and et al., 2008). These have and can Xyl from onto This glycosidic linkage is only present in RGII, suggesting the role of this in pectin biosynthesis. The of and has been in using from various including cells et al., et al., and in leaves and and unpublished data). Besides and but not function well as Compared with other for which has been the are and high and can also Xyl onto RGII from and plants but not from the type et al., 2006). This the in RGII from mutants that to be detected in sugar of cell walls and of the the in Arabidopsis has four with three to have function in it is likely that the of is due to are for the only of this Arabidopsis that has not been However, the of in as well as the proximity of and prevent the of mutants to the in the RGII, with its complex structure and unique sugar must require a large of for its However, has to the of of This may be due to for or the of A cell in was reported to be deficient in residues of RGII, suggesting that the was involved in RGII et al., However, genes in and are to be involved in and the Arabidopsis mutants have in RGII structure et al., 2009). et al. evidence suggesting that and are This suggests that the on RGII in the was a and that is not involved in RGII biosynthesis. esterification of pectin using as methyl has been in and Mohnen, but have been unambiguously A was to be deficient in pectin, and the was identified, it to be a et al., 2007). The is a of a large group of but their has not been and it is not known many of the are involved in pectin biosynthesis. a large group of pectin not be that the Arabidopsis less than pectin the degree of pectin must be very important for plants. Pectin acetylation with as the has also been in and for the to in two Arabidopsis mutants have been that have decreased of pectin acetylation in leaves and unpublished data). The wall have of the The is to a involved in acetylation of the in the et al., 2001). Arabidopsis has three additional of the and these are most likely involved in acetylation in different it is the are or have function (e.g. as The are with many is also not the are for pectin, but most likely this is not the of the in Arabidopsis and a in are highly in tissues with wall which pectin. Therefore, the in these tissues may be involved in acetylation than pectin In species (e.g. and sugar RGI side chains are by of acid to O-3 of Ara in arabinans or to of in et al. (2005) have that acid and arabinanase stomatal function in plants outside and it may be that pectic is but of genes involved in of have been in et al., 2007; et al., 2010). These genes to a large the of this are the most likely for pectin but evidence for or against this hypothesis has been The recent of a and the of the of the give into the of pectin biosynthesis. The of HG and also the RGII backbone is by the group of genes in as described has and in with the of HG in both species et al., 2007). described in Arabidopsis a complex with and is involved in HG as well (Mohnen, 2008). However, is in or is also involved in HG the of has not been established and have been observed et al., 2002; et al., 2005). not to a of HG with complex formation in angiosperms as the most recent is a of Arabidopsis but could be found (Fig. arabinan is present in cell other of the could be to be involved in arabinan biosynthesis. The Arabidopsis in both and Interestingly, the most a separate which also has a The or of XGA in and has not been has genes to Arabidopsis has three or four et al., in the and have a single RGII is present in has not been Some of the rare sugars of RGII have been found in but in than compared with plants (Matsunaga et al., 2004). The of an in suggests the of at least part of RGII in The of pectin plant for a at cell walls to the of The and are known to HG in their cell walls et al., et al., 2007; et al., 2008). RGI and its side chains of arabinan and galactan or RGII, to have not been in with and which and has been observed in but the were found in the et al., 2008). are for and several species (e.g. and However, the wall polysaccharides found in these species are the is to much evidence for functions of In the known in cell walls between and species and the plants that a be very and could give on functions of and many other of cell wall biosynthesis. Pectin many the large of which have not been of a very large of has not to the discovery of many involved in pectin biosynthesis. this is due to that in more than are to in wall or This the of will be most for the A of the of may be a However, is a of for it may well be that many of the pectin function in and that the have or These that it may be to the on the most and has to be a very to including involved in wall et al., et al., 2005). The for pectin are to be less such may be to that are likely to be involved in pectin biosynthesis. or of tissues with of a is a to involved in that Cellulose and were in this et al., et al., 2004). Some plant are highly in pectin (e.g. Seeds of some (e.g. are highly in pectic which as a of genes in such plant tissues and likely to for further of has been in to for involved in galactan biosynthesis. A of to galactan was the high of of the group of and Ulvskov, unpublished data). with its could galactan and a at and mutants be et al. to the of cells with an of cellulose to of noncellulosic polysaccharides, including pectin, pectic be identified, but were In to the of functions to found in by or it is also that are of not found in on work to the discovery of outside have been et al., 2004; et al., 2004; et al., 2009). these a has been to involved in cell wall et al., 2009). The is not part of and it that some of the major in of pectin could outside Pectin has many and to minor side chains of arabinans and not be observed by the in the to pectin biosynthesis. However, it may that for the major galactan or the involved in the RGI backbone have The RGI backbone is also a major component of pectin, yet in a has been The of the backbone has not been the needed for such studies is not and is not to The be but have Interestingly, the end of contains a Rha hence, must be a involved in its Arabidopsis and are deficient in the but for the has from studies (Scheller and Ulvskov, 2010). is more likely to be a and the other and are to be than as for the The linkage in the end of the linkage found in hence, of the involved in may provide for other including involved in RGI biosynthesis. RGII contains Rha all linked in different other are needed for RGII yet only has been seems that not more of these have been that RGII mutants are to a For the reason for the is that are several genes this also be the for all the other seems that are not by the of Arabidopsis each have three to four have proposed that the of may form with different and different (Scheller et al., 2007). This could both the and the of with could also the in the functions of The that can be by the of other is by the of which its sugar from to in the of et al., the the of and the for can be that in of pectin can be Pectin is an important especially as an in the of the of pectin of pectins for and could the of that are not of their For example, potato and sugar are in large but are not as for pectin Pectin is not a major component of for which of cell walls with a pectin the structure of pectin has an important on et al., 2010). is essential to a of the and other that the pectin polysaccharides, and this must be an important of However, also be that this is only the of the important roles in the polymers are and plants have an of and than of the polysaccharides are into the wall by that are not is an important of wall but may a role as the wall is by and that are only to et al., 2007; Kohorn et al., 2009). all these work together is the