Wheat arabinoxylans: Exploiting variation in amount and composition to develop enhanced varieties

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Journal of Cereal Science 46 (2007) 261–281 www.elsevier.com/locate/jcs

Wheat arabinoxylans: Exploiting variation in amount and composition to develop enhanced varieties Luc Saulniera,, Pierre-Etienne Sadoa, Ge´rard Branlardb, Gilles Charmetb, Fabienne Guillona a

UR1268 Biopolyme`res, Interactions, Assemblages, INRA, F-44316 Nantes, France UMR1095 Ame´lioration et Sante´ des Plantes, INRA, F-63100 Clermont-Ferrand, France

b

Received 24 January 2007; received in revised form 25 May 2007; accepted 14 June 2007

Abstract Arabinoxylans (AX) are the major polymers of wheat grain cell walls. The content and the structure of AX polymers show large differences between tissues and between wheat cultivars that affect the end-use properties and nutritional quality of the grain. The development of new wheat cultivars with enhanced quality, therefore, requires methods to exploit this variation and it is essential to understand and modulate the mechanisms controlling the key events of cell-wall polymer synthesis. This paper summarises recent knowledge on the structure and physicochemical properties of AX including variation between cultivars and tissues, methods for analysis and screening, biosynthetic mechanisms and approaches to identifying key genes. This knowledge is essential to understand AX properties and defined possible targets for plant breeding. r 2007 Elsevier Ltd. All rights reserved. Keywords: Biosynthesis; Cell wall; Cereal; Ferulic acid; Grain; Pentosans; Viscosity; QTL; Plant breeding

1. Introduction Cereals are staple foods for human nutrition and their incorporation into a wide range of products is of great economic importance. The major components of the grain are starch (approximately 60–70% of grain, 70–80% of flour) and proteins (approximately 10–15%) with nonstarch polysaccharides derived from the cell walls only accounting for about 3–8% of the total. Nevertheless, these components have major effects on the use of cereal grain (including milling, baking, and animal feed) due to their viscosity in aqueous solution but also to their hydration properties. As dietary fibre, they also have a major impact on the nutritional quality of cereal foods (Fincher and Stone, 1986; Saulnier et al., 2007). In wheat grain, arabinoxylans (AX) are the major polymers of cell walls. The effects of AX on the end use properties and nutritional quality of cereal grains have been discussed elsewhere (Fincher and Stone, 1986; Izydorczyk Corresponding author. Tel.: +33 2 40 67 50 62; fax: +33 2 40 67 50 66.

E-mail address: [email protected] (L. Saulnier). 0733-5210/$ - see front matter r 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2007.06.014

and Biliaderis, 1995; Saulnier et al., 2007) and are not presented here. The amount and the structure of AX polymers show large differences according to grain tissue but also between wheat cultivars. However, the different effects of AX are not fully explained in terms of structure/function relationships and the development of new wheat cultivars with enhanced nutritional and technological qualities requires a better control of variation in AX. It is therefore necessary to understand the mechanism controlling the key events in cell-wall polymer synthesis and possibly to modulate them. In this context, this paper summarises our recent knowledge of the structure and physicochemical properties of AX. This knowledge is essential to understand the properties of AX and to define possible targets for plant breeding. Variability in AX structure, among wheat cultivars and during grain development, but also due to environmental conditions, is then presented and discussed in relation to developing new cultivars with enhanced properties. Knowledge of AX variability is also required to understand AX biosynthesis and to identify candidate genes. In this respect, methods to determine the variability of AX and

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new tools to screen AX variability are presented. Finally, our current knowledge of AX biosynthesis is reviewed including forward and reverse genetic approaches that have been used to study the control of AX biosynthesis in wheat grain. 2. Structure and physicochemical properties of AX Numerous papers have been devoted to the composition, structure and properties of AX in cereal grains. For reviews, see Fincher and Stone (1986), Izydorczyk and Biliaderis (1995), Saulnier et al. (2007), and Vinkx and Delcour (1996). 2.1. General structure and chemical heterogeneity The cereal grain is a complex organ composed of different tissues (see Fig. 1), which have cells walls with different properties and compositions. Tissues from maternal origin form the outer part of the kernel and have primarily a role of protection. Cell walls in these tissues are thick, hydrophobic and essentially formed of cellulose and complex xylans but also generally contain significant amount of lignin. In endosperm tissues, cell walls represent 2–7% of the tissue, they are thin and hydrophilic and essentially formed of the two polymers: AX and (1,3)(1,4)linked b-D-glucans, the proportions of which can vary significantly according to the cereal species. For example, rye, wheat and sorghum are rich in AX, whereas barley and oats exhibit high levels of b-glucans. In addition, low amounts of other polymers such as glucomannan (2–7%), cellulose (2–4%) and structural proteins are also reported in these walls. Arabinogalactan peptides are also present in wheat endosperm cell walls (Van den Bulck et al., 2002, 2005).

Hence, AX are the major polymer in the cell wall of wheat grain. They are formed of a linear backbone of (1-44)-linked b-D-xylopyranosyl units. The xylose generally represents more than 50% of the constitutive sugars and a great diversity of side chains are present on the main chain on the O-2 or O-3 positions or both. Single units of a-L-arabinofuranose and a-D-glucuronic acid (and its methyl ether, 4-O-methyl-glucuronic acid) are the most frequent side-chains, although xylopyranosyl and galactopyranosyl residues associated with arabinofuranosyl residues are also found as short side-chains of 2–3 sugar units. In addition, acetic acid and hydroxycinnamic acids, ferulic and p-coumaric acids, are found as esters. Ferulic and p-coumaric esters are linked to the O-5 of the arabinofuranosyl units. This general description actually masks a wide diversity in composition and structure associated with the different cellular types present in the various tissues of wheat grain. From a structural as well as a botanical point of view, it is essential to distinguish the AX from endosperm tissues (starchy endosperm and aleurone layer) from those found in the outer part of the kernel. Furthermore, although the histological origin of wheat milling fractions is not strictly defined and may vary to a large extent, endosperm tissues are essentially recovered in the flour whereas the outer parts of the kernel are associated with the bran fraction. 2.2. Composition and structure of AX from starchy endosperm 2.2.1. General features The AX in the endosperm of wheat are only composed of arabinose and xylose and for this reason are often referred to as pentosans. They are found as water-extractable (WE-AX)

Fig. 1. Wheat grain, showing component tissues. (Reprinted from Surget and Barron, 2005.)

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and water-unextractable (WU-AX) fractions. In wheat flours, the average amounts are 0.5% and 1.7% (g/100 g flour) for WE-AX and WU-AX, respectively (Dervilly-Pinel et al., 2001b; Ordaz-Ortiz and Saulnier, 2005; Saulnier et al., 1995a). Thus, total AX content in wheat flour is about 2.2% and approximately 1/4 of total AX are WE-AX (Table 1). The arabinose to xylose ratio (A/X) is often used to characterise the structure of AX, and the average value of A/X ratio is 0.5 for WE-AX. However, the A/X ratio is an approximate characterisation of AX structure, which is better described by the substitution pattern of the xylose backbone by arabinose residues. The main structural features of wheat flour AX are depicted in Fig. 2. Arabinose residues are mainly present as single side-chain units as mono-substitutions on position O-3 (mXyl3; 21%), or di-substitutions on position O-2 and O-3 (dXyl; 13%) of the xylosyl residues of the backbone. Mono-substitution on O-2 (mXyl2) is rare in wheat. On average 66% of xylosyl residues of the backbone are unsubstituted (uXyl)

Table 1 AX content in wheat flour and grain of 20 French cultivars (table adapted from Ordaz et al., 2005) Flour

Mean Min. Max. Std. dev. c.v. (%)

(Table 2). In addition to these major structural features, the presence of small amounts of short arabinose side-chains have been suggested by methylation analysis (Gruppen et al., 1992b; Izydorczyk and Biliaderis, 1993). The structural features of WE-AX are well documented, but less information is available for WU-AX, which represent the major part of AX in cell walls of the endosperm. WU-AX have been studied after alkaline extraction (AE-AX) (Gruppen et al., 1991, 1992a, b; Gruppen et al., 1993b) or xylanase extraction (XE-AX) (Ordaz-Ortiz and Saulnier, 2005). The structure of WUAX is very close to that of WE-AX but the average molecular weight and A/X ratio are slightly higher for WU-AX than for WE-AX (Izydorczyk and Biliaderis, 1995). The higher A/X ratio corresponds to a higher proportion of mXyl and a lower proportion of uXyl in WU-AX (AE-AX or XE-AX) compared with WE-AX whereas the proportions of dXyl are similar in WE-AX and WU-AX. Table 2 AX contents and structural features of WE-AX from 90 wheat lines (Synthetic  Opata cross)

Grain a

b

c

WE-AX g/100 g

Total AX g/100 g

Total AX g/100 g

0.51 0.26 0.75 0.16 30.9

2.18 1.66 2.87 0.34 15.4

5.76 4.79 6.92 0.63 11.00

Mean Min. Max. Std. dev. c.v. (%)

Calculated as the sum of Ara and Xyl extracted by water in g/100 g of flour. Ara is corrected for the presence of arabinogalactans. b Calculated as the sum of Ara and Xyl in g/100 g of flour. c Calculated as the sum of Ara and Xyl in g/100 g of grain.

uX

Total AXa

WE-AXb

g/100 flour

g/100 g flour

2.35 1.65 3.14 0.30 12.7

0.51 0.30 0.80 0.11 22.3

WU-AXc

Xylose in WE-AX (%)d

A/X

g/100 g flour

mXyl

dXyl

uXyl

0.47 0.39 0.57 0.04 8.5

1.84 1.33 2.57 0.26 14.3

20.8 16.1 27.3 2.3 11.1

13.0 7.7 19.3 2.6 20.2

66.2 60.0 70.3 1.9 2.9

a

a

A

263

Calculated as the sum of Ara and Xyl in g/100 g of flour. Calculated as the sum of Ara and Xyl extracted by water in g/100 g of flour. Ara is corrected for the presence of arabinogalactans. c Calculated as total AXWE-AX d Calculated from 1H NMR. b

A

dX

mX2

A

A

mX3

A

F

Ga

Ac

X

A

A

F

X

A

G

X

Fig. 2. Main structural features of AX from endosperm (A) and outer tissues (B) of cereal grains A: arabinose; X: xylose; G: galactose; Ga: glucuronic acid; F: ferulic acid; uX: unsubstituted xylose; dX: di-substituted xylose; mX3: O-3 mono-substituted xylose; mX2: O-2 mono-substituted xylose (rare in wheat endosperm AX). (Reprinted from Saulnier, L., Guillon, F., Sado, P.-E., Rouau, X. (2007). Plant cell wall polysaccharides in storage organs: xylans (food applications), Vol. Section ) I. Plant Polysaccharides *. Elsevier BV, Amsterdam, The Netherlands.)

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Although ferulic acid is an important structural element of AX, the amount linked to AX is very low and represents 0.2–0.4% of WE-AX (w/w) and 0.6–0.9% of WU-AX in wheat (Bonnin et al., 1999). This corresponds to about 2–4 ferulic acid residues per 1000 xylose residues in WE-AX (6–10 for WU-AX). Dehydrodiferulic acids were also detected in low amount (10–15 times less than ferulic acid) in WE-AX from wheat (Dervilly-Pinel et al., 2001b). For WU-AX, the amount of dehydrodiferulic acids is only 4 times lower than ferulic acid (Lempereur et al., 1998). 2.3. Chemical heterogeneity of WE-AX and WU-AX Numerous studies have revealed the chemical heterogeneity of WE-AX and WU-AX from wheat endosperm. Using ethanol fractionation, sequential ammonium sulphate precipitation or DEAE-cellulose chromatography, different authors have demonstrated the presence of a range of polymers exhibiting different A/X ratios in AX populations initially extracted by water from flours (Fincher and Stone, 1986; Izydorczyk and Biliaderis, 1995; Vinkx and Delcour, 1996). The mechanisms of the fractionation are generally not fully understood and comparison of the fractions obtained by the different methods is therefore not straightforward. Among the different methods used, the heterogeneity of WE-AX was demonstrated using ethanol precipitation and size exclusion chromatography (Dervilly et al., 2000; Dervilly-Pinel et al., 2001a); isolated sub-populations exhibited A/X ratios ranging from 0.31 up to 1.06, whereas the starting WE-AX had a ratio of 0.56. Linear relationships have been found between the A/X ratio and mXyl, dXyl and uXyl in WE-AX populations obtained by graded fractionation (Dervilly et al., 2000; Izydorczyk and Biliaderis, 1995). The A/X ratio is strongly positively correlated to the proportion of dXyl and negatively correlated to the proportion of uXyl, whereas the proportion of mXyl is independent of the A/X ratio (Delcour et al., 1999; Dervilly et al., 2000). In other words, the A/X ratio of WE-AX is essentially determined by the level of dXyl. The structural heterogeneity of WE-AX is not limited to arabinose substitution and is also observed for ferulic acid (Izydorczyk and Biliaderis, 1995). WE-AX with low A/X ratios contain higher amounts of ferulic acid (Dervilly et al., 2000), which suggest that ferulic acid residues might be esterified only to arabinose substituents located at position O-3 of mono-substituted xylose residues (mXyl), as observed for the structure of feruloylated oligosaccharides by different authors (Ishii, 1997; Lequart et al., 1999; Rhodes et al., 2002).

A random distribution of the arabinosyl units on the xylan backbone has been calculated and compared with the experimentally determined distribution (Dervilly-Pinel et al., 2004). For a given A/X ratio, a simulated random distribution gave a higher weight to mono-substitution than was experimentally observed, which suggests that regulation mechanisms favouring di-substitution occur during the biosynthesis of the polymer. Different experimental approaches, based on specific chemical degradation (periodate oxidation and Smith’s degradation) and degradation with purified xylanase, have been used to obtain insight into the distribution of arabinose residues. Contiguous blocks of three (Gruppen et al., 1993a), four (Izydorczyk and Biliaderis, 1994) and up to six (Dervilly-Pinel et al., 2004) substituted xylosyl residues have been observed in wheat flour WE-AX. Some authors have described WE-AX and WU-AX as being built of different regions with high or low substitution, the proportions of which could vary in the AX molecule (Gruppen et al., 1993b; Izydorczyk and Biliaderis, 1994). However, AX with low and high degrees of substitution have been isolated by various means, which suggests that a range of polymer structures exists, in contrast with the suggestions of distinct regions in AX. In fact, structural studies on WE-AX or WU-AX have been carried out on bulk populations isolated from flours and the range of polymer structures observed might therefore reflect structural heterogeneity within wheat endosperm. In conclusion, the different models proposed for AX from wheat flour support an irregular distribution of arabinosyl residues along the chain, but biosynthetic mechanisms favour di-substitution. 2.5. Composition and structure of AX from aleurone layer The aleurone layer is a specific tissue of cereal endosperm, which due to milling procedures and tissue properties is mainly associated with bran fractions. In wheat, the AX of the aleurone layer, although structurally closely related to the starchy endosperm AX, exhibits specific features. Firstly, the aleurone AX are not WE-AX and have a lower A/X ratio (0.3–0.4) than the starchy endosperm AX (Table 3; Antoine et al., 2003). Furthermore, aleurone AX are heavily esterified compared with those of the starchy endosperm, and ferulic acid and dehydrodiferulic acids represent about 3.2% and 0.45% of the WU-AX, respectively (Antoine et al., 2003; Parker et al., 2005). Some p-coumaric acid is also associated with aleurone cell walls (Antoine et al., 2004; Rhodes et al., 2002). In addition, acetyl groups which are probably esterified to the xylose backbone of AX were also detected in aleurone cell walls (Rhodes et al., 2002).

2.4. Models for AX structure in starchy endosperm 2.6. AX from maternal tissues Variation in the structure of cereal endosperm AX is well documented, but information on the distribution of arabinose residues on the xylan backbone is scarcer, and discussion is still open.

Numerous studies have been dedicated to AX from the bran of wheat and other cereals. However, bran is a multilayered composite isolated during the milling process,

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Table 3 Composition of the outer tissues of wheat grain

Outer pericarpc Inner pericarp+testa+NEc Inner pericarpd Testa+NEd Aleuronec

AX (g/100 g)a

Glc (g/100 g)

Uronic A. (g/100 g)

A/X (mg/g)b

Ferulic A. (mg/g)

DHD (mg/g)

44.1 38.0 49.0 55.0 20.8

29.3 17.6 15.8 3.8 15.0

nd nd 9.5 3.2 nd

1.14 0.36 1.06 0.13 0.39

3.7 5.0 E1 E5 6.7

4.1 0.91 E0.5 E0.3 0.95

AX: sum of arabinose and xylose; Glc: glucose, mainly present as mixed linked beta-glucan in aleurone, and cellulose in other tissues; A/X: arabinose/ xylose ratio; DHD sum of ferulic acid dehydrodimers, NE: nucellar epidermis. a In g/100 g of tissue. b In mg/g of tissue. c Adapted from Antoine et al. (2003). d Adapted from Parker et al. (2005).

which is composed of the different tissues of maternal origin found in the outer part of cereal grains. Furthermore, the aleurone layer that is part of the endosperm is generally associated with the bran fraction. Therefore, bran comprises a diversity of tissues and associated polymers, especially in the case of wheat (Antoine et al., 2003; OrdazOrtiz et al., 2005; Parker et al., 2005). Table 3 clearly shows that the nucellar epidermis and testa are characterised by a very low A/X ratio (0.13) whereas the cross-cells and pericarp (also known as beeswing bran) are characterised by a very high A/X ratio (1.14). In addition, glucuronic acid (or its 4-omethyl ester) and galactose are also present in bran AX, which are also described as glucuronoarabinoxylans or heteroxylans (AX; see Fig. 2). The diversity of the structure is also well illustrated by the degradation of isolated layers or total bran fractions with endoxylanases (Beaugrand et al., 2004; Benamrouche et al., 2002; Ordaz-Ortiz et al., 2005). The nucellar layer is completely degraded by endoxylanase whereas the pericarp is resistant. The heterogeneity of bran tissues also explains the different AX fractions isolated after AE-AX. Wheat fractions with low A/X ratio (Schooneveld-Bergmans et al., 1999a; Shiiba et al., 1993) that precipitate upon neutralisation clearly originate from the nucellar epidermis tissues. However, the main part of AX from the bran is derived from the pericarp. These polymers are soluble at neutral pH after AE-AX and are characterised by a highly branched and complex structure (Brillouet and Joseleau, 1987; Schooneveld-Bergmans et al., 1999a; Shiiba et al., 1993). The level of substitution of the xylan backbone of AX from the outer pericarp is very high (80%), with a high proportion of dXyl (40%). The A/X ratio is close to 1 and arabinose residues are mainly found as terminal side chains, but also associated with xylose and galactose in short side-chains. In addition, glucuronic acid and xylose residues are also found as terminal side-chains (Brillouet and Joseleau, 1987). A high level of hydroxycinnamic acids characterises outer tissues of the kernel. Feruloylated oligosaccharides derived from AX (Ishii, 1997) and feruloylated AX (Ng

et al., 1997; Schooneveld-Bergmans et al., 1999c) have been isolated from bran tissues. Large amounts of dehydrodiferulic acid are also found in the outer tissues (Antoine et al., 2003; Parker et al., 2005; Saulnier and Thibault, 1999). Ferulic acid accounts for 0.9% (w/w) of AX in the pericarp from wheat and very similar amounts are observed for dehydrodimers (Antoine et al., 2003; Saulnier and Thibault, 1999), which corresponds to about 30 ferulic acid residues per 1000 xylose residues. In addition, acetic acid is present in wheat bran (Kabel et al., 2002; Mandalari et al., 2005; Saulnier et al., 1995b) and is probably linked to the xylan backbone in the cell walls. 3. Conformation and physico-chemical properties of AX AX exhibit different physico-chemical characteristics such as water solubility, viscosity, gelling and hydration properties, which are the basis of their functional properties in different processes and food systems. The physicochemical properties of macromolecules depend basically on the conformations of the macromolecules that drive chain–chain interactions with other polymer chains and with the solvent. The conformation and interaction properties can be modulated by fine structural changes of the macromolecules. 3.1. Molecular weight The chain length of polymers is an important determinant of their physico-chemical properties (Saulnier et al., 2007). The Mw of WE-AX of wheat is in the range of 200–300 kD with a high polydispersity index (I) of 1.7–2 (I ¼ Mw/Mn) (Dervilly et al., 2000; Dervilly-Pinel et al., 2001a, b). This high polydispersity actually reflects a range of polymers exhibiting different masses and structures (polymolecularity). For example, starting with a WE-AX population (Mw ¼ 280 kD; I ¼ 1.8), a range of polymers was isolated with Mw varying from 70 kD up to 655 kD and A/X ratios from 0.4 up to 1.2; no simple relationship was observed between the Mw and the structure of the polymers (Dervilly et al., 2000; Dervilly-Pinel et al., 2001a).

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Determination of the Mw of water-insoluble AX requires an extraction procedure, which can lead to possible degradation of the polymer chain. Nevertheless, WU-AX solubilised with BaOH2 from wheat endosperm exhibit higher Mw than WE-AX and still have a high polydispersity index (Gruppen et al., 1991). Similarly, highly substituted AX can be isolated from bran after AE-AX. A Mw of 293 kD (I ¼ 3) has been determined for wheat bran AX (Schooneveld-Bergmans et al., 1999b), which is very close to the value determined for maize bran AX (Chanliaud et al., 1996). Low substituted AX isolated from the outer part of the grain have a strong tendency to form aggregates so that it is almost impossible to determine the Mw of individual chains in aqueous systems (Ebringerova et al., 1994; SchooneveldBergmans et al., 1999b).

calculated from the radius of gyration–molecular weight relationship (RgEM nw ). This depends on chain flexibility (n ¼ 1 for a fully extended rigid chain, 1/3 for a compact sphere and 0.5 for Gaussian chains) and a value of 0.47 has been found for WE-AX (Dervilly-Pinel et al., 2001a; Picout and Ross-Murphy, 2002) indicating that they behave as random coils which is contrary to their usually reported extended rigid rod character (Andrewartha et al., 1979; Izydorczyk and Biliaderis, 1995). In addition, the presence of arabinose side-chains has no significant influence on the conformation (rigidity) of the xylan backbone as indicated by molecular modelling (Dervilly-Pinel, 2001; Ordaz-Ortiz et al., 2004). Furthermore, the persistence length is not significantly affected by A/X ratio so that it can be concluded that arabinose substitution has no real influence on the conformational behaviour of AX in solution.

3.2. Conformation of xylan chains

3.3. Water solubility

According to X-ray fibre diffraction studies, xylans are described as extended chains forming twisted ribbon-like strands, with three-fold symmetry (Nieduszynski and Marchessault, 1972). This extended conformation is often compared with that of b-(1-44) linked polysaccharides such as cellulose and mannan chains, but actually the three-fold screw symmetry of xylans differs considerably from the two-fold symmetry of cellulose and is not favourable for association with it (Almond and Sheehan, 2003; Yui et al., 1995). Furthermore, the b-(1-44)-xylan chain is more flexible than the conformationally restricted b-(1-44)-cellulose or mannan chains (Almond and Sheehan, 2003). Despite the intrinsic flexibility of the xylan chain, AX are generally described as rigid molecules because the extended conformation observed in the crystalline state is generally wrongly assigned to the rigidity of the polymer chain. Actually, the polymer chain rigidity is directly measured by the persistence length (Lp), which represents an estimate of the length through which the propagation of the chain in a given direction is preserved. Lp values of about 3–5 nm (6–10 xylose residues) are predicted for xylan chains from molecular modelling (Mazeau et al., 2005). Lp has been experimentally determined for AX in solution (DervillyPinel et al., 2001a; Picout and Ross-Murphy, 2002). Depending on the method used for calculation, different absolute values for Lp were obtained: 7.871.4 nm (Dervilly-Pinel et al., 2001b) or 3.170.6 nm (Picout and RossMurphy, 2002). These values, when compared with those of very flexible 1-46 linked polymers such as pullulans (Lp ¼ 2 nm) (Roger and Colonna, 1992) or very stiff polysaccharides present in solution as double or triple helix complex such as xanthan (100–150 nm) (Berth et al., 1996; Milas et al., 1996) or schizophillan (145 nm) (Burchard, 2005), are typical of semi-flexible polysaccharides such as galactomannans (Lp ¼ 4.5–9) nm that behave as random coils (Burchard, 2005). The random coil behaviour of AX is further confirmed by the hydrodynamic parameter n

The water solubility of polysaccharide depends on the delicate balance between chain–chain and chain–solvent interactions. Structural factors such as chain length, the presence of side-chain groups and their distribution will modify this balance and the solubility behaviour of the polymers. Generally, the presence of side chains that prevent chain–chain interactions favours water solubility of the polymers. In the case of AX, water solubility is not only related to structural features of the polymer chain but also to covalent linkage to other cell-wall polymers. For example, in endosperms WU-AX have a higher A/X ratio than WE-AX which should lead to higher water solubility, but the presence of a high proportion of chain–chain crosslinking through covalent ‘‘diferulic bridges’’ render WUAX WU-AX. Similar conclusions are applicable to the highly branched AX found in pericarp tissues. However, arabinose side-chains may affect AX solubility and different studies have shown that removal of arabinose residues by controlled acid hydrolysis (Dea and Rees, 1973) or using arabinofuranosidase (Andrewartha et al., 1979) give rise to aggregation and precipitation of the polymer. The mechanism of the aggregation is not clear, although generally described as an interaction between ‘‘unsubstituted’’ regions of the polymer chain. 3.4. Viscosity The viscosity of a polymer solution is directly related to the fundamental molecular properties (molecular conformation, molecular weight, and molecular weight distribution) and concentration of the polymer. According to the concentration, dilute, semi-dilute and concentrated regimes are observed for polysaccharides (Doublier and Cuvelier, 2006). All polysaccharides exhibit the same behaviour that depends on the reduced concentration c[Z] were c is the concentration and [Z] the intrinsic viscosity. Intrinsic viscosity is actually a measure of the hydrodynamic volume of the isolated polymer in

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solution and must not be confused with the viscosity of the solution. For c[Z]o1, the dilute regime is observed and under these conditions the behaviour is Newtonian (viscosity increase linearly with shear rate). Above this concentration, the shear rate dependence of the solution also has to be taken into account, and polysaccharides have a shear thinning behaviour (Doublier and Cuvelier, 2006). WE-AX exhibit this general behaviour (Izydorczyk and Biliaderis, 1995). Therefore, intrinsic viscosity is the best parameter to compare the thickening potential of different polysaccharides. Intrinsic viscosities reported for WE-AX in wheat are in the range 200–600 ml/g with an average value of 400 ml/g (Rattan et al., 1994; Saulnier et al., 1995a). Although some differences between studies can result from different extraction procedures (and especially in the inactivation of endogenous enzymes), for the same extraction procedure variation is cultivar related, and may depend on variation in the molecular weight or in the conformation of the polymer. The relation between the intrinsic viscosity and molecular mass of macromolecules is usually expressed in the form of the empirical Mark-Houwink–Sakurada equation: [Z] ¼ KM aw , where K and a are empirical parameters which depend on the polymer–solvent pair and on the temperature, and are both related to chain stiffness. The value of the exponent ‘‘a’’ gives information on the general conformation of the polymer. For flexible linear chains 0.5p a p1, whereas stiff chains display larger values of a (values as high as 1.8 are reported for rod-like chain conformations) (Lefebvre and Doublier, 2005). The values of the coefficient ‘‘a’’ determined for wheat (a ¼ 0.74) WE-AX (Dervilly-Pinel et al., 2001a) are well within the range for a flexible chain. The conformational behaviour of AX in solution is in fact remarkably similar to that of galactomannans and is characteristic of semiflexible polysaccharides (Dervilly-Pinel et al., 2001a; Picout and Ross-Murphy, 2002). As pointed out earlier, the conformation of the AX chain is not affected by the degree of branching of the xylan backbone. The viscosity of AX solution is therefore mainly dependant on changes in the concentration and Mw of the polymer, and contrary to the generally accepted assumption (Izydorczyk and Biliaderis, 1995), structural features such as A/X ratio have probably very limited effects on the viscosity of AX solution. However, the formation of dehydrodiferulic bridges that have been detected in WE-AX (Dervilly-Pinel et al., 2001b) might strongly affect the viscosity of AX solutions by increasing dramatically the Mw. The viscosity-generating potential of WE-AX has a strong influence on the functional, technological, and nutritional properties of AX. As early as 1956, Udy (1956) showed that 95% of the intrinsic viscosity of wheat flour water extract was due to WE-AX, and pointed out that the average size of the soluble molecules was a cultivar-specific characteristic as well as the amount of water-soluble polysaccharide.

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3.5. Hydration properties The hydration properties (water absorption or retention properties) of insoluble AX have a strong impact on the functional properties of AX, as they can modify the distribution of water among the different components of food, which is very critical in cereal products and especially during bread making (Saulnier et al., 2007). The literature on the hydration properties of AX is rather confusing and the results reported as ‘‘water-binding capacity’’ do not actually reflect the same measurements. The water-binding capacity e.g. the amount of water retained by a known weight of fibre under centrifugation, was determined for water-unextractable solids (WUS) containing about 50% (w/w) of WU-AX: the value was 7.6 g/g (Wang et al., 2003). Furthermore, the swelling properties (the volume occupied by a known weight of fibre) of WUS with similar composition were shown to be 26 ml/g (Gruppen et al., 1993b). A ‘‘water-binding capacity’’ has been determined for WE-AX, which is actually the amount of non-freezable water. This has been determined by differential scanning calorimetry (Girhammar and Nair, 1992) or NMR (Andrewartha et al., 1979) and a value of about 0.40g/g was being measured irrespective of the origin of WE-AX (wheat, rye, and barley) or the A/X ratio. 3.6. Gelation Solution of feruloylated AX can thicken and gel with free radical-generating agents such as chemicals (ferric chloride, ammonium persulphate) or enzymatic systems (hydrogen peroxide/peroxidase, linoleic acid/lipoxygenase, laccase/oxygen) (Figueroa-Espinoza et al., 1998; Geissman and Neukom, 1973; Georget et al., 1999; Hoseney and Faubion, 1981; Izydorczyk et al., 1990; Ng et al., 1997). The gelation primarily results from the formation of threedimensional networks of AX chains anchored by dehydrodimers of ferulic acids. The free radical mechanism of ferulic acid coupling gives rise to different dehydrodimers (Ralph et al., 1994), and the 8-50 (normal and benzofuran forms), 8-O-40 , 5-50 and 8-80 dehydrodiferulates have been detected in AX gels, the 8-O-40 being the most abundant. Higher oligomers of ferulic acids such as the 4-O-80 , 5-500 dehydrotrimer found in maize pericarp cell walls (Bunzel et al., 2005; Rouau et al., 2003) were also detected in gelled wheat AX but in lower concentrations than dehydrodimers (Carvajal-Millan et al., 2005b). After a few days of storage, enzymatically induced AX gels tend to be weaker. This effect is attributed to radical reactions catalysed by the still active enzymatic systems that result in a loss of ferulic acid coupling products and a depolymerisation of AX chains. It can be overcome by thermal inactivation of the enzyme (Carvajal-Millan et al., 2005a). The structural characteristics of AX play a determinant role in their gelation ability. Comparison of AX from

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different sources has shown that a high content of ferulic acid, a high molecular weight/hydrodynamic volume and a low substituted xylan backbone are favourable features to form strong gels (Dervilly-Pinel et al., 2001; Izydorczyk et al., 1991; Izydorczyk and Biliaderis, 1995b; Rattan et al., 1994). A positive correlation has thus been established between gel rigidity and intrinsic viscosity of AX. During the gelation process, the monomers of ferulic acid esters disappear since they are converted into dimers or higher oligomers. The concentrations of dimers and trimers are, however, insufficient to account for the decrease in monomers. The formation of higher oligomers has been suggested although they have not been detected so far. Although the formation of covalent cross-links is the major mechanism of gel formation, gel stiffness was shown to increase whereas no new ferulate cross-links were detected (Carvajal-Millan et al., 2005b). This suggested that additional weak linkages such as hydrogen bonding, van der Waals interactions or chain entanglements could play a role in the gel structure. 4. Interactions of AX with other cell-wall components The cell wall of cereal grains mainly consist of AX and mixed-linked b-glucans in the endosperm and AX, cellulose and variable amount of lignin in the outer tissues. The tight association of the different polymers in the wall is illustrated in Fig. 4. The aleurone cell wall was specifically labelled with antibodies directed against mixed-linked bglucans (green in Fig. 4) (Meikle et al., 1994) and AX (red in Fig. 4) (Guillon et al., 2004; Ordaz-Ortiz et al., 2004). The wall appeared as a multilayered system, AX are more abundant at the interface between cells and cells corners, whereas mixed linked b-glucans are concentrated close to the plasma membrane. However, both polymers are distributed across the wall giving a yellow colour and suggesting close interactions. Similar co-localisation of the polymers is also observed in the central endosperm cell walls. Interactions of AX with the different polymers of the wall are likely to occur through hydrogen bonds, covalent bonds or mechanical entanglement. 4.1. Interaction with mixed-linked b-glucans and cellulose Although both AX and b-glucans are co-localised in the walls of cereal grain and in cereals in general (Buckeridge et al., 2004), their possible interactions had never been studied in detail. However, a xylan/b-glucan complex with an unusual low substitution of AX and a high proportion of b(1-44) linkages for b-glucan was recovered after AE-AX of cell-wall material from barley grist (Izydorczyk and MacGregor, 2000) suggesting possible chain–chain interactions. Cellulose–hemicellulose interactions through hydrogen bonding are well documented in the case of xyloglucan (Vincken et al., 1995), but such interactions are less clear for AX. As a matter of fact, hydrogen bonding is restricted

by the presence of arabinose side-chains that hinder chain–chain interactions of the highly substituted AX found in the endosperm or outer pericarp cell walls of cereal grains. The coating of AX to cellulose micro-fibrils is actually very low when compared with xyloglucan (Dervilly-Pinel, 2001; Saulnier et al., 2007). Furthermore, few differences are found between WE-AX exhibiting degrees of substitution varying from 25% (A/X:0.4) up to 50% (A/X:0.8), showing that large blocks of unsubstituted xylose which are suggested to facilitate chain–chain association are not likely to occur even in low substituted WE-AX. Very highly substituted AX isolated from maize pericarp, that are structurally related to those in wheat pericarp, did not bind at all to cellulose under the same conditions, indicating that hydrogen bonding of AX to cellulose is not likely to occur in pericarp tissue (Saulnier et al., 1995b). 4.2. AX–AX interactions through phenolic acid bridge Because they can act as cross-linking agents between polysaccharides, or between polysaccharides and lignin, ferulic acids contribute to wall assembly, promoting tissue cohesion and restricting cell expansion (Iiyama et al., 1994), and also control the mechanical properties of mature tissues (Antoine et al., 2003). The different dehydrodimers (Ralph et al., 1994) and trimers (Bunzel et al., 2003; Funk et al., 2005; Rouau et al., 2003) found in plant tissues (see Figs. 3 and 4) indicate that the crosslinking reaction is an oxidative mechanism probably mediated by peroxidase in vivo (Fry et al., 2000). Feruloylation of AX is therefore an essential aspect of cell-wall development and tissue properties. The amount and type of hydroxycinnamic acids vary greatly according to the different tissues in cereal grains. Clearly, the outer tissues of the kernel and the aleurone layer are very rich in ferulic acid and dehydrodimers (Antoine et al., 2003; Parker et al., 2005). Irrespective of the tissue, 8-O-40 and 5-80 benzofuran dehydrodimers were mainly found but the 5-50 , 8-50 , and 8-80 forms were also detected in durum and bread wheats (Beaugrand et al., 2004; Peyron et al., 2001). Coumaric acid is mainly located in the aleurone while the dehydrotrimer has only been detected in the pericarp tissue of wheat (Antoine et al., 2004; Funk et al., 2005; Rouau et al., 2003). 4.3. Other covalent interactions Dehydrodiferulic bridges are likely to be the major parameter that explains differences between WE-AX and WU-AX in endosperm and aleurone cell walls of cereal grains. Rhodes et al. (2002) suggested that in addition to dehydrodiferulic bridges, protein-polysaccharide crosslinking through tyrosine–hydroxycinnamic acid dimerisation could also occur in aleurone cell wall (Rhodes and Stone, 2002). Dehydro-diferulic acid–tyrosine complexes have been isolated from rye and wheat flour doughs (Piber

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269 HO

HO

OH HO

O

O

OH

O

O OMe

O

OMe

HO H

O

H

O OMe MeO OH

OMe

HO

O

OMe

OH

OH

8-5'

8-O-4' O

OH

OH HO

O

MeO HO

O

OH MeO

OH O

O

OMe OH

OH

O HO HO

O OMe

OH

OMe

OMe

OH

5-5'

8-8'

MeO OH

4-O-8', 5'-5"

O

Fig. 3. Structure of dehydrodimers and dehydrotrimer of ferulic acid identified in cereal grains. (Reprinted from Saulnier, L., Guillon, F., Sado, P.-E., Rouau, X. (2007). Plant cell wall polysaccharides in storage organs: xylans (food applications), Vol Section ) I. Plant Polysaccharides *. Elsevier BV, Amsterdam, The Netherlands.)

and Koehler, 2005), but this cross-link between AX and proteins may be the result of the processing conditions. In addition, the outer tissues of the grain generally contain lignin. Different cross-links of AX to lignin have been suggested (Iiyama et al., 1994). Firstly, hydroxycinnamic acids are known to be directly esterified or etherified to the lignin surface (Lam et al., 1994, 2001) and it is likely that all ferulic acid etherified to lignin is also esterified to AX (Iiyama et al., 1994). Direct ester link between uronic acid on glucuronoxylans and the hydroxyl groups of lignin surfaces and direct ether linkages between AX and lignin involving, for example, the primary hydroxyl of arabinose side-chains have also been proposed (Iiyama et al., 1994), but to date no such linkages have been demonstrated. 5. Variation in AX content and structure 5.1. Genetic variability among wheat cultivars

Fig. 4. Double labelling of AX and beta-glucans in aleurone cell walls from wheat grain. A polyclonal anti-xylan and a monoclonal anti-betaglucan antibodies were used with a second stage goat anti-rabbit (orangered fluorescence) and a second stage goat anti-mouse (green fluorescence) antibodies, respectively. Yellow fluorescence indicates the presence of both beta-glucans and AX. (Reprinted from Saulnier, L., Guillon, F., Sado, P.-E., Rouau, X. (2007). Plant cell wall polysaccharides in storage organs: xylans (food applications), Vol. Section ) I. Plant Polysaccharides *. Elsevier BV, Amsterdam, The Netherlands.)

Variation in the amount of WE-AX and in their fine structure has been demonstrated among wheat cultivars. In Table 2, the amount and structural features of flour WEAX isolated from the ITMI population ‘‘Synthetic  Opata’’ grown in Clermont-Ferrand (France) are reported. Although, the range of variation observed in this population (90 lines) cannot be considered as representative of the variability present in the species; this survey illustrates very well the wide diversity in terms of structure and amount of

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endosperm AX in wheat. WE-AX represented from 0.3% to 0.8% of flour (dry weight basis) and the A/X ratio varied from 0.39 to 0.57 for the population, as generally observed for WE-AX in wheat flours (Courtin and Delcour, 2002; Izydorczyk and Biliaderis, 1995). The average proportions of uXyl, mXyl, and dXyl were 66.2%, 20.8%, and 13%, respectively, but wide variation in the structure of flour AX also exists among the Synthetic  Opata population as reported in a different survey of wheat varieties (Andersson et al., 1994; Dervilly et al., 2000). A strong correlation between the A/X ratio and the proportion of dXyl (r ¼ 0.90, po0.001), and uXyl (r ¼ 0.83, po0.001) was observed in the Synthetic  Opata population (Fig. 5), whereas the correlation was very small with mXyl (r ¼ 0.34, p ¼ 0.001). Similar relationships between the A/X ratio and the proportions of dXyl and uXyl were previously reported in WE-AX fractions obtained by graded fractionation (Dervilly et al., 2000; Izydorczyk and Biliaderis, 1995), originating from different part of the kernel (Delcour et al., 1999) or isolated from different varieties (Ordaz-Ortiz and Saulnier, 2005). Variation in the structural features of WU-AX among wheat cultivars is less well documented, but a recent study used XE-AX of flours from 20 wheat cultivars (OrdazOrtiz and Saulnier, 2005). The A/X ratios were statistically different for WE-AX and XE-AX and varied from 0.47 to 0.58 and from 0.51 to 0.67, respectively. The average proportions of uXyl and mXyl for both populations were significantly different; XE-AX had more mXyl and consequently less uXyl than WE-AX; but the proportions of dXyl were similar. Furthermore, the A/X ratios of WE-AX and XE-AX were correlated (r:0.78), showing that although WE-AX and WU-AX had different structures, the variation in structure observed among wheat varieties for WE-AX was also observed for WU-AX. Environmental conditions are likely to affect the amount and the structure of AX in cereal endosperm, but variation

was mainly genetically determined. In fact, WE-AX content, A/X ratio and viscosity of flour water extract, were compared in 19 cultivars grown in three French locations. Broad sense heritability (h2 ¼ s2g/s2g+s2e) estimates were 0.7570.10 for WE-AX, 0.83 70.05 for A/ X and 0.8070.05 for viscosity (Martinant et al., 1999). These values are very high, compared with those observed for complex traits such as yield or protein content, which have heritability values ranging from 0.1 to 0.4. Such high heritabilities are confirmed by very high correlations among evaluation sites (see Fig. 6). We have recently developed two recombinant doubled haploid populations from crosses between parental lines exhibiting extreme values (high and low) for the viscosity of wheat flour water extract (Fig. 6), which is an indirect measure of the content of WE-AX (see tools for screening 4.5 4.0 Viscosity Location 2

270

3.5 3.0 2.5 2.0 1.5 2.0

2.5

3.0 3.5 4.0 Viscosity Location 1

4.5

5.0

Fig. 6. Correlation between 2 growing locations for the viscosity of flour water-extract. The extreme lines RE 99006 and CF 99007 have been used as parent for developing a recombinant Double Haploı¨ d population. Trials were carried out in 1999 on 33 lines—location 1: INRA-Site de Croue¨l—63100, Clermont-Ferrand, France—location 2: INRA—Ferme du Moulon—91190 Gif sur Yvette, France. Viscosity is the relative viscosity of a flour water extract compared with pure water.

30

20

10

CF99007

RE99006

Number of lines

40

0 1

Fig. 5. Relative amount of the structural element of the xylan backbone as function of A/X ratio in WE-AX isolated from 90 wheat flours (Synthetic–Opata descendants).

2 3 Viscosity of flour water extract

4

Fig. 7. Distribution of the viscosity of flour water-extract in the 300 DH progeny from the cross between the two extreme lines RE 99006 and CF 99007. Harvest 2003—Clermont-Ferrand, France. Viscosity is the relative viscosity of a flour water extract compared with pure water.

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AX variability). The distribution of the viscosity of flour water extracts (WE-AX content) from one of these crosses is shown in Fig. 7. The main feature of these distributions (the second one is not shown) is their continuousness. In the case of a single major gene, for a trait with such a high heritability, a bimodal distribution would have been expected (as observed, for example, with grain hardness or carotenoid content). This clearly shows that the genetic control of WE-AX content and composition is oligo- or polygenic. Molecular maps are being developed for these new populations and will allow QTL identification (see Section 7). In addition, the specific effects of the 1BL/1RS translocation in wheat on AX structure have been studied (Biliaderis et al., 1992; Selanere and Andersson, 2002). In these lines, the short arm of chromosome 1R of rye has replaced the short arm of wheat 1B. In one study, the authors found that the amounts of WE-AX and total AX in flours were not significantly different between normal wheat and 1Bl/1RS lines although rye contains significantly higher levels of AX (Biliaderis et al., 1992). However, a recent investigation of a limited number of lines indicated that 1BL/1RS translocation wheats contained higher proportions of total AX in flour (Selanere and Andersson, 2002). Both studies showed that WE-AX from 1BL/1RS translocation wheats exhibited significantly lower molecular weights than those from normal wheat. 5.2. Effect of the environment Although, the amount and the structure of AX in wheat endosperm are mainly genetically determined, some influence of environmental conditions has been reported (Andersson et al., 1993; Coles et al., 1997). A study of 49 varieties grown in 1987 and 1988 in Sweden, including winter and spring wheat varieties (Andersson et al., 1992), showed that the amount of WE-AX in flour was higher in 1987 compared with 1988, and that the non-starch polysaccharides content was different in winter and spring wheat flours. A positive relationship was also observed between the amount of AX in wheat grain and drought (Coles et al., 1997). However, in this latter case, the AX content was measured in the whole grain and did not give the variation in the endosperm (flour). Further studies are needed to determine the impact of environmental factors on the AX in wheat endosperm, effects on their amount, their physicochemical properties and their ratio of WE-AX to WU-AX. 5.3. Spatial and temporal changes A combination of immunocytochemistry techniques using specific antibodies against the structural features of AX and infra-red and Raman micro-spectroscopy techniques that determine the spectral fingerprint of AX has revealed that changes occur in the structure of AX during grain development (Mills et al., 2005; Barron et al., 2005; Philippe et al., 2006a–d). During the very early stage of

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endosperm development, (1–3)-linked b-glucans are transiently detected in the forming cell wall. During the first stage of cell division, mixed-linked b-glucans appear and are uniformly distributed. AX are detected later, at the beginning of endosperm cell differentiation, when the grain has reached its final length. Variation in the structural features of AX is subsequently observed, depending on developmental stage and location in the grain. At the beginning of cell differentiation, the degree of substitution of AX with arabinose is higher in the cell walls of prismatic cells than in those of central cells, but these differences then disappear and the AX are less substituted in the endosperm of mature grains. In the walls of the aleurone cells, AX appear to be little substituted and highly esterified with phenolic acids. The walls of the modified aleurone and modified sub-aleurone cells in the crease region are spectrally different from those of the rest of endosperm (Philippe et al., 2006a). This specific region of the wheat kernel exhibits different cellular types (an absence of peripheral and prismatic cells (Mares and Stone, 1973)), and different patterns of gene expression as shown for barley (Doan et al., 1996) and for wheat (Drea et al., 2005). The modified aleurone and sub-aleurone are also thought to be important in setting up the developmental patterning as the change from division to differentiation in the endosperm spreads from this region to the rest of endosperm. Altogether, these results indicate a fine spatial and temporal tuning of the biosynthesis of cell-wall polymers. Further studies are needed to fully understand the heterogeneity of wall structure in wheat endosperm and possibly to relate this spatial heterogeneity to the variation observed in the structure of WE-AX and WU-AX between wheat cultivars. 5.4. Tools for screening AX variability Several methods have been developed for the screening and identification of plants with altered cell-wall polysaccharide structure. Fourier-transform infra-red (FT-IR) micro-spectroscopy (Chen et al., 1998; Mouille et al., 2003) has been developed for the selection of cell-wall mutants and can be used for wheat endosperm cell walls. Other methods based on the quantification of neutral monosaccharides have also been used to screen cell-wall mutants (Reiter et al., 1997), and have been used in a number of studies to estimate the AX content of wheat flours or grains (Bell, 1985; Izydorczyk and Biliaderis, 1995). However, the use of such methods for whole grain does not give information on the proportions of AX in the endosperm and the outer parts of the kernel. The WE-AX content can be determined after extraction with water and determination of the neutral sugar content, based either on chromatographic or colorimetric analysis (Rouau and Surget, 1994). Alternatively, viscosity measurements of the water extract from flour or grain provide a functional way to determine the amount of WE-AX (Bhatty et al., 1991; Saulnier et al., 1995b; Grosjean et al., 1999),

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although endogenous variation in molecular weight or the effects of endoxylanases (endogenous or microbial contamination) may affect the validity of the results. The measurement of the A/X ratio alone is not sufficient to describe the complex structure of AX and the variation in this. However, the characterisation of alterations in the wall polysaccharide composition usually requires purification using differential extraction procedures and additional structural characterisation. Furthermore, 1H NMR, the reference method used to investigate the structure of AX (Izydorczyk and Biliaderis, 1995), requires quite large amounts of sample and is rather tedious to run on large number of samples. Enzyme treatment and subsequent chromatographic analysis (i.e. enzymatic fingerprinting) is an alternative method to study structural variation in plant polysaccharides (e.g. the proportions of mono- and disubstituted residues in the case of WE-AX and WU-AX). Endoxylanases hydrolyse (1,4) linkages between b-Dxylopyranosyl residues and the specific bonds cleaved depend on the side-chain residues (Biely et al., 1997; Kulkarni et al., 1999). Therefore, endoxylanase action is affected by the structure of AX, and in turn the degradation products of AX by endoxylanases give information on the structure of the polymers. Xylanase fingerprinting of wheat grain AX was carried out on various grain tissues: the endosperm, aleurone layer, intermediate layer and pericarp isolated by dissection of grains, as well as on flours and whole grains (Ordaz-Ortiz et al., 2005). High structural heterogeneity of AX according to tissues was observed with some peaks being excellent markers for endosperm tissues and for mono- and disubstituted forms of AX (Ordaz-Ortiz et al., 2005). Xylanase fingerprinting can also be applied to large wheat populations in order to identify the chromosome regions controlling the structural features of AX. The method can also be used in combination with FT-IR micro-spectroscopy to screen for variation in AX structure within the endosperm. 6. Manipulating AX in wheat grain 6.1. Candidate genes for the biosynthesis of AX The biosynthesis of the cell wall remains a major unsolved problem in plant biology. Biochemical approaches to identify the enzymes and genes involved have been hindered by the liability of the enzymes and by our ignorance of their biosynthetic mechanisms. Although substantial progress has recently been made in the identification and functional characterisation of cellulose synthases (CesA) and the corresponding genes and of several transferases such as xylosyl, galactosyl, and fucosyl transferases, little is known about the genes and enzymes involved in synthesis of the backbones of xylans (Hazen et al., 2003; Burton et al., 2006; Lerouxel et al., 2006). The biosynthesis of AX in cereal cell-wall grain requires the orchestration in a defined order of several enzymes and the

spatial and temporal heterogeneity of AX probably imply heterogeneity in the biosynthetic machinery (i.e. different sets of enzymes could be implicated in the synthesis of low substituted AX and highly substituted AX). 6.2. The synthesis of the building blocks of AX The presence of the right building blocks is a prerequisite for the biosynthesis of AX. These are specific nucleotide sugars and ferulic acid. These molecules must be synthesised and channelled to the right location within the cell for polymer assembly. Two pathways are required, part of the nucleotide sugar interconversion pathway (for review, see Seifert (2004)), and part of the lignin pathway that leads to ferulic acid (for review, see Barriere and Ralph, 2004). The two major nucleotide sugars are namely UDP-Dxylose (pyran form) and UDP-L-arabinose (furan form). The UDP-D-xylose units maybe made from UDP-Dglucuronic acid by two types of decarboxylase, the UDPxylose-synthase (UXS) or UDP-apiose/UDP-xylosesynthase (AXS). The UXS gene family (six members in Arabidopsis) encodes both soluble and membrane-bound enzymes with the synthesis of UDP-xylose occurring both in the cytosol and in the endomembrane system (Harper and Bar-Peled, 2002). Two Arabidopsis genes distantly related to the UXS genes encode AXS (Seifert, 2004). UDP-L-arabinose is made from UDP-D-xylose by a UDP-D-xylose-4 epimerase in the endomembrane system (Seifert, 2004). An epimerase could also be necessary for the synthesis of the furan form (less stable than the pyran). The mur4 mutant of Arabidopsis has facilitated the identification of a small gene family of three other potential xylose-epimerase genes (Burget et al., 2003). Several other nucleotide sugars may be required for AX biosynthesis but in small amounts, such as UDP-Dglucuronic acid (UDP-D-GlcA), and UDP-D-Galactose (UDP-D-Gal) (Fincher and Stone, 1986). The lignin pathway has been mostly studied in maize and in Arabidopsis (Goujon et al., 2003; Halpin, 2004). The part of the pathway that leads to ferulic acid within the cell is still controversial, but the biosynthesis of ferulic acid in wheat seedlings seems to be correlated with the activity of O-methyltransferases (Lam et al., 1996). Studies of maize mutants (Barriere and Ralph, 2004) suggests the presence of a CCoAOMT hub that leads to feruloyl CoA (Fig. 8). The transfer of ferulic acid from feruloyl-CoA to AX is also supported by work on rice suspension cells (YoshidaShimokawa et al., 2001). However, other groups suggested that feruloyl-glucose (1-O-feruloyl-b-glucose) was the precursor for the intracellular feruloylation of AX in wheat suspension culture, and that feruloyl CoA was required for feruloylation of proteins (Obel et al., 2003). Another pathway described using an Arabidopsis mutant suggest that the accumulation of soluble ferulic acid could result from the oxidation of coniferaldehyde by coniferaldehydedehydrogenase (Nair et al., 2004).

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UGE

UDP-D-Gal

UDP-D-Gal GAE

UGDU

UDP-D-Glc

DP-D-GlcA UXS

273

UDP-D-GalA

UDP-D-GlcA

AXS

UXS UDP-D-Api UDP-D-Xyl

UDP-D-Xyl Phenylalanine or tyrosine

Caffeoyl CoA Lignin pathway

XS

UXE

CCoAOMT

UDP-L-Ara ?

Feruloyl CoA

Xylan

AT

Feruloyl Glc

AX

? FCoApSFt CCR ? Coniferaldehyde

Peroxidases ME

Coniferaldehyde Dehydrogenase

Ferulic acid

F-AX

Golgi

F-AX Cell wall

Cytoplasm

Fig. 8. Schematic representation of AX biosynthesis. The building blocks are synthesised or channelled in the Golgi apparatus to be assembled. The xylan backbone is synthesised by a xylan synthase (XS) then subtituted by arabinose residues by possibly several arabinosyltransferases (AT). Others transferases add in minor amounts residues and side chains made of galactose (Gal) and glucuronic acid (GlcA). Ferulic acid molecules are linked to some arabinosyl residue to form feruloylated AX (F-AX), which are then exported to the wall. The ferulic acid is further reticulated in the wall by peroxidases. Other modifying enzymes (ME) are possibly involved to obtain the final AX found in muro. The precursors and the different enzymes (circular boxes) involved in arabinoxylan biosynthesis are indicated. Sugar and ferulic acid transferases are indicated in red. The hypothesis for ferulic acid synthesis and incorporation in AX are also shown. The sugar building blocks: UDP-D-Glc (glucose); UDP-D-GlcA (glucuronic acid); UDP-D-GalA (galacturonic acid); UDP-D-Xyl (xylose); UDP-D-Api (apiose); UDP-L-Ara (arabinose); and UDP-D-Gal (galactose). The enzymes: UGD : UDP-D-glucose dehydrogenase; UXS : UDP-D-xylose synthase; AXS : UDP-D-apiose/UDP-D-xylose synthase; UXE : UDP-D-xylose 4-epimerase; GAE : UDP-D-glucuronic acid 4-epimerase; UGE : UDP-D-glucose 4-epimerase; FCoApSFt: feruloyl-CoA:polysaccharide feruloyltransferase; CCR: cinnamoyl CoA reductase; CCoAOMT: caffeoyl Coenzyme A O-methyltransferase; XS: xylan synthase; AT: arabinosyltransferases; and ?: unknown enzymes. (Adapted from Saulnier et al., 2007.)

6.3. The formation of the linkages found in feruloylated AX The range of linkages found in AX (Fig. 2) requires at least six different enzyme activities to be formed. Four classes of enzyme are needed for AX assembly: glycosyltransferases (for the glycosidic bonds), feruloyltransferase (feryloylation), oxidative enzymes (ferulic acid dimerisation), and acetylesterases (xylose acetylation). In plants, glycosyltransferases represent a very large multigene family with several hundreds of members found in monocots and dicots (Coutinho et al., 2003). The presence of motifs and the conservation of these genes allow their classification into subfamilies, some of which have been extensively studied and had functions assigned (Hamann et al., 2004). Although most glycosyltransferases have been identified by motifs and sequence homology or other bioinformatics tools (Egelund et al., 2004) and inserted in the CAZY database (Coutinho and

Henrissat, 1999), relatively few of them have been assigned a particular function, with little known about the glycosyltransferases involved in cell-wall polysaccharide biosynthesis. Different glycosyltransferase activities are required for the formation of the three main glycosidic bonds found in AX, namely the b-1,4-linked xylose backbone and the a-1,2- and a-1,3-linked arabinose side-chains. 6.4. b-1,4-Xylosyltransferase or xylan synthase EC 2.4.2.24 The biosynthesis of the b(1,4)-xylan backbone of AX is catalysed by UDP-D-xylose:1,4-b-D-xylan 4-b-D-xylosyltransferase (EC 2.4.2.24 ), commonly called xylan synthase, using uridine 50 -diphosphoxylose (UDP-Xyl) as the donor substrate (Urahara et al., 2004). The evidence that the xylan backbone of AX is made first comes from the fact that arabinosyltransferase activity is dependent on the

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synthesis of unsubstituted xylan as an acceptor molecule (Porchia et al., 2002), but the size of these acceptor molecule is not known. Thus, it is possible that small xylan molecules are linked end to end to form the final xylan backbone that could be up to several thousand residues long (Fincher and Stone, 1986). The enzyme activity for xylan biosynthesis has been detected in tissues undergoing secondary wall formation in various plants (Gregory et al., 2002), but also in wheat seedlings (Kuroyama and Tsumuraya, 2001; Porchia and Scheller, 2000) and most interestingly in barley endosperm (Urahara et al., 2004). Two potential xylosyltransferases were partially purified and characterised (Mr ¼ 38,000 and 40,000) from French bean (Rodgers and Bolwell, 1992). The presence of one of these enzymes seems to correlate with a peak of activity during the period of maximum secondary wall synthesis (Rodgers and Bolwell, 1992). However, the structural features of the synthesised xylan have not been characterised in detail. Studies of wheat seedlings indicate that b-1,4-xylosyltransferase activity can be detected with exogenous acceptor substrates such as xylooligosaccharides (Kuroyama and Tsumuraya, 2001) or with endogenous acceptors in which case xylan molecules (4500 kDa or more than 3300 residues) are synthesised (Porchia and Scheller, 2000). With the latter, UDP-arabinose had no effect on the incorporation of xylose into xylan (in wheat seedling) which suggests that xylosyltransferase is independent of the availability of UDP-arabinose (Porchia and Scheller, 2000). Recent work on developing barley endosperms showed that the maximal activity of a b(1,4)-xylosyltransferase occurred between 13 and 14 days after flowering (Urahara et al., 2004), a stage at which the deposition of AX is very active. However, increased deposition of AX continued after xylosyltransferase activity ceased (after 25 days), suggesting the presence of other b-1,4-xylosyltransferases which were not detected in the assay (Urahara et al., 2004). As yet, only a a-xylosyltransferase activity has been identified at the molecular level. A small gene family of glycosyltransferase encode this enzyme (GT34, seven members in Arabidopsis) and some members may be implicated in xyloglucan biosynthesis (Faik et al., 2002). We can speculate that xylansynthase belongs to a multigene family with several isoforms implicated in the biosynthesis of different type of xylans (AX, heteroxylans, and xylan) and in the different walls (primary, secondary, type I, type II). This multigene family may share features with other backbone synthesising enzymes such as cellulose synthase (chain of b-1,4-glucosyl residue, CesA, GT2), mannose synthase (chain of b-1,4-mannosyl residues, CslA, GT2), or callose synthase (chain of b-1,3-glucosyl residues, GT48). These enzymes have high Mw (4 500 amino acid) with several predicted membrane spanning domains (4–17). However, neither the enzyme nor its gene has been characterised at the molecular level. The catalytic mechanism for b-1,4-xylosyltransferase is presumably, by analogy with glycosidases, the inverting type (as opposed to

retaining) and most likely follows a single displacement mechanism where the acceptor performs a nucleophilic attack at carbon C-1 of the sugar donor (Gibson et al., 2002). Xylan synthase is likely to be a processive enzyme meaning that it remains attached to its substrate and performs multiple rounds of catalysis before dissociating, and it may form a complex with arabinosyltransferase (Porchia et al., 2002). 6.5. Arabinosyltransferase (a-1,2 and/or a-1,3) The xylan backbone of cereal AX is mainly decorated with mono- and di-substitutions of a-L-arabinosyl residues attached through (1-3) and (1-2) linkages. As for xylan activity, arabinosyltransferase activity has been detected in secondary wall forming tissues of several plants (Rodgers and Bolwell, 1992) and in monocot seedlings (Porchia et al., 2002). A putative arabinosyltransferase of Mw ¼ 70,000 (corresponding to about 630 amino acids) was partially purified from the Golgi-bound fraction in French bean (Rodgers and Bolwell, 1992). Arabinosyltransferase activity identified in the microsomal fraction of wheat seedlings has been described, based on the product characterisation, as an AX arabinosyltransferase (Porchia et al., 2002). Interestingly an Arabidopsis mutant (ARAD1) that has a reduced amount of arabinose in the walls of leaves and stems has been recently characterised (Harholt et al., 2006). This mutation could affect the arabinosyltransferase gene that is part of a large glycosyltransferase family in plants (GT47), with 39 and 25 members in Arabidopsis and rice, respectively. Three members of this family involved in cellwall biosynthesis have been previously identified. These are two galactosyltransferases for xyloglucan biosynthesis (Li et al., 2004) and glucuronosyltransferase for pectic rhamnogalacturonan II biosynthesis (Iwai et al., 2002). 6.6. Other glycosyltransferases Other enzymes are required for adding the minor components of AX (short side-chains, galactose and glucuronic acid). Several other glycosyltransferase activities are probably implicated in the minor substitutions found on the xylan backbone. These activities have not been studied for AX biosynthesis but galactosyltransferase, for example, has been identified for xyloglucan biosynthesis (Li et al., 2004) and could be used to identify related enzymes. 6.7. AX feruloylation The feruloylation of arabinosyl residues of AX has been described in monocot (wheat and rice) cell suspension cultures (Obel et al., 2003; Yoshida-Shimokawa et al., 2001). An enzyme (feruloyl-CoA: arabinoxylan-trisaccharide O-hydroxcinnamoyl transferase) activity has been isolated from rice that indicates that feruloyl-CoA is

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a substrate (Yoshida-Shimokawa et al., 2001). The enzyme implicated in the feruloylation and its gene remains to be identified. The oxidative coupling to form diferulate could be catalysed by peroxidases, oxidases, or non-enzymic system involving active oxygen species. Results obtained using maize suspension cells suggested that the coupling of feruloyl residues was catalysed by peroxidases rather than oxidases (Encina and Fry, 2005). 6.8. The location of AX biosynthesis Like other non-cellulosic polysaccharides, AX biosynthesis takes place in the Golgi apparatus and the polymer is then exported to the wall by vesicular transport. Direct evidence for this comes from immunolocalisation of AX in Golgi vesicles (Philippe et al., 2006) and the localisation of xylan synthase (Bolwell and Northcote, 1983; Gregory et al., 2002; Porchia and Scheller, 2000) and arabinosyl transferase (Porchia et al., 2002) activites in the Golgi. The feruloylation of AX has not been localised at such a level but seems to occurs inside the cell (Fry et al., 2000; Obel et al., 2003; Yoshida-Shimokawa et al., 2001) and therefore most likely also occurs in the Golgi apparatus. Experiments using labelled 3H arabinose and 14C ferulic acid have indicated an intracellular location for AX feruloylation in cell-suspension cultures of Festuca, maize and wheat (Fry et al., 2000; Obel et al., 2003; Myton and Fry, 1994). The dimerisation of ferulic acid has long been believed to only take place in the cell wall but the most recent studies (Fry et al., 2000; Obel et al., 2003) indicate that dimerisation also take place intracellularly, at least for some of the 8-50 dehydrodimers (Obel et al., 2003). One limitation to the study of AX biosynthesis results from the fact that the structure of the AX prepared from the wall might not precisely reflect its biosynthesis inside the cell. Indeed, important structural modifications of the polymer could occur in muro and also form part of its biosynthesis. For example, the reticulation by laccases and peroxidases of the ferulic acid residues and the removal of certain arabinose residue by an arabinosylfuranosidase. The presence in the Golgi or in the wall of glycosylhydrolases (CAZY, GH) implicated in the biosynthesis of AX is not excluded. For example, an endoglucanase (Korrigan) has been found to be involved in cellulose biosynthesis, together with the glycosyltransferases known as CesA from the GT2 family (Lane et al., 2001). Biochemical characterisation has only been carried out on a small number of enzymes implicated in cell-wall biosynthesis, of the hundreds of plant glycosyltransferases. To date, less than 20 genes identified in mutant screens are likely to play a direct role in polysaccharides synthesis. The number of enzymes identified by mutant screens compared with biochemical data shows the importance of genetics for unravelling complex biosynthetic pathways such as polysaccharide biosynthesis. It is also not surprising that all of the enzymes implicated in AX biosynthesis remains to be

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identified and characterised at the molecular level. A synthetic view of the biosynthetic pathways for AX is shown in Fig. 8. 7. Forward genetic approaches 7.1. Quantitative genetic: the QTL approach Traits such as fruit size, flowering time, morphology, and light responsiveness differ among lines, ecotypes, or accessions of a particular plant species, and a number of these have been successfully analysed genetically. Because they are typically inherited in a quantitative manner, they are more challenging to analyse than single gene traits, and isolation of the responsible genes is more difficult. Contrary to some expectations, the QTLs underlying natural variation have proved to be variant alleles of genes that play a central role in the trait under study, and not minor, secondary genes with an indirect role (Hazen et al., 2003). Thus, the identification of genes controlling natural quantitative differences in cell-wall properties may lead to the identification of important genes involved in AX biosynthesis (Hazen et al., 2003). The inheritance of cell-wall composition has been studied in several species including wheat, barley and maize (Lempereur et al., 1997; Lundvall et al., 1994; Powell et al., 1985). In general, genotype  environment interactions are observed, with complex patterns of inheritance indicative of control by many genes. QTLs affecting mixed-linked glucan content in oats and barley, dietary fibre content and sugar composition of pericarp in maize, dietary fibre and AX content in rye and the ratio of Ara to Xyl in wheat flour have been identified (Boros et al., 2002; Han et al., 1995; Hazen et al., 2003; Kianian et al., 2000; Lu¨bberstedt et al., 1997; Martinant et al., 1998; Mechin et al., 2000). However, the specific genes underlying the QTLs were not identified in any of these studies, due to the complexity of the plant genomes, the lack of adequate genomic resources, or both. Genetic analyses of recombinant progenies from crosses have allowed us to identify quantitative trait loci (QTL) for either WE-AX or A/X ratio. QTLs for WE-AX, often measured as viscosity of flour extract, have been located on chromosomes 1BL and chromosome 7A in the progeny from Re´cital  Renan (Groos et al., 2004), each of which explain 18% of the phenotypic variation. The QTL on chromosome 1B was also found in two other non-pedigreerelated progenies (the ITMI population ‘‘Synthetic  Opata and Courtot  Chinese Spring) (Martinant et al., 1999, 1998) with major effects on both the relative viscosity of flour extracts (r2 ¼ 32–37%) and the A/X ratio (r2 ¼ 35–42%). Other QTL analyses were performed on the ITMI progeny grown at Clermont Ferrand a third year studying different features of flour AX (Table 4). The contents of WE-AX, WU-AX and ferulic acid in flour water extracts as well as structural features such as the A/X ratio and the proportions of uXyl, mXyl, and dXyl were

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Table 4 QTL analysis of the ITMI progeny for different features of wheat flour AX

QTL analyses revealed the importance of long arm of chromosome IB1 in controlling AX characteristics.

Features

Chromosomes

Markers

R2a

8. Reverse genetic approach: tilling and eco-tilling

WE-AX contentb

1AS 1BL

ksue18D bcd508b

17.5 24.7

WU-AX contentc

1BL 7AS

cdo346b fbb264

16.1 19.7

Total AX contentd

1BL

bcd508b-cdo346b

29.8

A/X

1AS 1BL 6AS

ksued18D ksue11d-bcd508b psr167b

11.0 36.4 14.3

dXylf MXyl UXyl

1BL 7DL 1AS 1BL 3BL

bcd508b bcd129 ksue18D bcd508b bg131

35.7 – 14.5 17.7 10.2

Ferulic A. in WE-AXg G/100g of flour

1As 1BL 5DL

ksue18D bcd508b cdo1508

12.6 18.2 10.6

3Bs

abg471b-bcd1418

12.6

1BL 6AL

bcd508b cdo388a-fbb164a

21.1 15.1

Direct genetic approaches require the evaluation of a huge number of genotypes, either recombinant progenies or mutant collections, to identify chromosome regions or genes associated with variation in traits. This approach may be tedious and costly, particularly for complex traits such as cell-wall composition and structure. An alternative approach, which can be used when the biosynthesis pathway is at least partially known, is to look for allelic variation in candidate genes (usually those coding for key enzymes of the pathway) in collections of genotypes. Such allelic variation may include either the insertion or deletion of sequences (varying in size from 1 to some dozen of base pairs), or single base substitution, the so-called single nucleotide polymorphisms (SNPs). The former type of variation is readily detected by any system able to discriminate amplified fragments according to their size (e.g. by agarose gel electrophoresis and capillary electrophoresis). SNPs are more abundant and are less easy to detect. An obvious method would be to amplify and sequence the full length of the gene to reveal every difference. This approach is still expensive, but may be more attractive in the future as the cost of automatic sequencing decreases. An elegant method to detect SNPs without sequencing is targeted induced local lesions IN genomes (TILLING). Although initially proposed to detect chemically induced single base substitutions, the method can also be applied to natural variation and is called ECO-TILLING. The method is based on the ability of some specific endonucleases to specifically cut at mismatch positions. Therefore, in a mixture of two DNAs (one reference and one being tested), the occurrence of a SNP in the second DNA strand will result in two fragments on digestion, which are then easily detected by any fragment size analyser (Fig. 9). Moreover, the detection is often possible in DNA pools from eight or even more individuals. In chemically mutagenised populations of

e

Ferulic A. in WE-AXg G/100 g of WE-AX Relative viscosityh a

Only QTLs with LOD score higher than 2 were recorded. Calculated as the sum of Ara and Xyl extracted by water in g/100 g of flour. Ara is corrected for the presence of arabinogalactans. c Calculated as total AXWE-AX. d Calculated as the sum of Ara and Xyl in g/100 g of flour. e Ratio of arabinose to xylose calculated from the content of arabinose and xylose in total AX. f uXyl: unsubstituted xylose residue, mXyl: mono-substituted xylose residue, dXyl: di-substituted xylose residue, % determined in WE-AX from 1H NMR and neutral sugar composition. g Ferulic acid content esterified to WE-AX. Content were expressed as g/100 g of flour or in g/100 g of WE-AX. h Relative viscosity measured on flour water extract as described in Martinant et al. (1998). b

determined. QTL analyses of this densely mapped population showed that in addition to chromosome 1BL, the short arm of chromosome 1A also had an important effect on AX characteristics. A total of five characteristics were significantly associated with the bcd508b marker on chromosome 1BL (WE-AX content, the proportions of dXyl and uXyl, the ferulic acid content in flour water extracts and the relative viscosity of flour water extract). Three other characteristics were also significantly associated with at least one locus in the vicinity of the bcd508b marker. Interestingly, four AX characteristics were significantly associated with a QTL close to the ksue18D marker on chromosome 1AS (WE-AX content, A/X ratio of total AX, proportion of uXyl, and the ferulic acid content in flour water extract). In addition to these two major QTLs some other markers were significantly associated with AX characteristics but with smaller effects.

Fig. 9. Schematic representation of the TILLING strategy.

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bread wheat, one may expect one SNP every 25 kb. In other word, 100 novel alleles could be expected in a population of 1000 plants for a gene of 2.5 kb. In contrast, natural variation occurs at a much lower rate. SNP frequency estimates are usually given for whole sample studies. Ravel et al. (2006a) have reported an average of one SNP every 300 bp in a collection of 200 bread wheats from worldwide origins, but this rate varied considerably from one gene to another. ECO-TILLING is therefore less likely to identify a large number of new alleles, but those, which are detected, have been retained by natural or human selection, and thus are more likely to have positive effects. One major difficulty in SNP detection is to ensure that the same sequence is being studied in all individuals (orthologous sequences). In a diploid species, a first difficulty may arise from the presence of gene duplications or gene families (paralogous sequences). This is the case for xylan synthases or epimerases, which are members of gene families. In polyploid species such as bread wheat, several copies may represent even ‘‘single genes’’, one on each ancestral genome (homoeologous sequences). It is therefore necessary to design genome specific primers prior to using TILLING or ECO-TILLING, which is not a trivial task in many cases. If all homoeologous and paralogous sequences are present in databases, a clustering approach may enable all gene copies to be identified, then sequence comparison may allow the desired specific primers to be designed. If not, the amplification from well-designed BAC (Bacterial Artificial Chromosomes) pools offers the opportunity to isolate the homoeologous sequences (Ravel et al., 2006b). Once SNP variation has been detected in mutagenised or natural collections, the role of the candidate gene can be predicted by establishing statistical associations between gene variation and trait variation. This approach is relatively straightforward, although some caution should be taken to avoid spurious associations caused by longrange linkage disequilibrium (Remington et al., 2001; Rafalski et al., 2002). Because linkage disequilibrium usually declines rapidly in natural collections, a statistical association between a given SNP and a phenotypic trait may be due to a closely related gene, but probably not a gene elsewhere in the genome. Therefore, this method is very efficient for selection between closely mapped candidate genes (e.g. Ravel et al., 2006b), and can be used to save effort in functional genomic studies.

9. Conclusion AX are the major polymers of wheat grain cell walls and their structure varies between tissues. The impact of WEAX originating from the endosperm on the viscosity of aqueous solution is without doubt one of the major factors that determine the impacts of AX on different uses of cereal grain, as it modifies the rheology of solutions and its behaviour in dough and in the gastrointestinal tract. The

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hydration properties of WU-AX can also affect water distribution in food systems. The structural diversity of AX has been extensively studied with the aim of relating the structure to specific functional properties. Clearly, the largest differences in AX structure relate to the tissue of origin, especially in the outer parts of the grain. The endosperm also exhibits relatively large variation in the amount and the structure of AX, both within the tissue but also between different varieties. The relationship between this structural variation and the so-called ‘‘functional properties’’ of AX is not straightforward, especially for the level of substitution by arabinose side-chains. The presence of ferulic acid is more clearly related to the formation of firmer gels upon oxidative conditions and to the modification of the mechanical properties of the outer layers, but variation in AX feruloylation among wheat cultivars, especially in the endosperm or aleurone tissues, is little documented. The biological significance of the structural diversity of AX is not yet understood. AX and cell walls are involved in water transport or diffusion during different physiological stages: grain development, desiccation, and germination. It is therefore possible that structural variation is involved in modulating the hydration properties of the cells walls to regulate the water content of the grain. Similarly, arabinose substitution may play a role in controlling interactions with other cell-wall components, but up to now the mechanisms of xylan aggregation are poorly understood and need further study. Although the structural diversity of AX observed in the endosperm of wheat varieties is not fully understood nor explained in term of functional properties, the genetic control of this variability is now well established suggesting that plant breeding could be used to produce new types of wheat with improved nutritional quality and processing properties. Elucidation of the mechanisms that control the synthesis of AX in the endosperm is a challenge for the future and will require studies of their deposition during grain development, their spatial diversity within the endosperm and the identification of the critical enzymes and genes involved in their biosynthesis.

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