The low-molecular-weight glutenin subunits of wheat gluten

Journal of Cereal Science 39 (2004) 321–339 www.elsevier.com/locate/jnlabr/yjcrs Review The low-molecular-weight glutenin subunits of wheat gluten Renato D’Ovidio*, Stefania Masci Dipartimento di Agrobiologia e Agrochimica, Università degli Studi della Tuscia, Via San Camillo de Lellis, s.n.c., 01100 Viterbo, Italy Received 26 September 2003; revised 19 December 2003; accepted 19 December 2003 Abstract Low-molecular-weight glutenin subunits (LMW-GS) are polymeric protein components of wheat endosperm and like all seed storage proteins, are digested to provide nutrients for the embryo during seed germination and seedling growth. Due to their structural characteristics, they exhibit features important for the technological properties of wheat flour. Their ability to form inter-molecular disulphide bonds with each other and/or with high-molecular-weight glutenin subunits (HMW-GS), is important for the formation of the glutenin polymers, which are among the biggest macromolecules present in nature, and determine the processing properties of wheat dough. Explanation of the structural basis for these correlations continues to intrigue researchers and, while earlier emphasis had been on HMW-GS, considerable attention is now being focused on the LMW-GS. LMW-GS are a highly polymorphic protein complex, including proteins with gliadin-type sequences. Difficulty in separating single components, arising from the complexity of the group, has limited the characterisation of the individual proteins and the establishment of clear-cut relationships with quality parameters. Here we review results concerning different aspects of LMW-GS, including their structural characteristics, genetic control, and relationships with quality parameters. In addition, we emphasise the distinction between the components with sequences unique to the LMWGS fraction and those behaving like glutenin subunits (incorporated into polymers), but with sequences corresponding to gliadins. q 2004 Elsevier Ltd. All rights reserved. Keywords: Triticum aestivum L; Low-molecular-weight glutenin subunits; Gluten polymer; Wheat quality 1. Introduction Low-molecular-weight glutenin subunits (LMW-GS) are among the major components of wheat storage proteins, collectively known as prolamins because of their high content of the amino acids proline and glutamine. Prolamins are used as nutrients by the embryo, during the early phase of germination and seedling growth, before photosynthesis is established. Wheat prolamins are subdivided into gliadins and glutenins, according to their polymerisation properties: gliadins are monomeric proteins that form only intramolecular disulphide bonds, if present, whereas glutenins are polymeric proteins whose subunits are held together by inter-molecular disulphide bonds, although intra-chain bonds are also present. Gliadins can be divided into four Abbreviations: HMW-GS, high-molecular-weight glutenin subunits; LMW-GS, low-molecular-weight glutenin subunits. * Corresponding author. Tel.: þ 39-761-357323; fax: þ 39-761-357238. E-mail addresses: dovidio@unitus.it (R. D’Ovidio), masci@unitus.it (S. Masci). 0733-5210/$ - see front matter q 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2003.12.002 groups, named a-, b- (although these two groups have similar structural characteristics), g- and v-gliadins. When glutenins are reduced, two types of subunits are released, based on molecular weight: the high-molecular-weight glutenin subunits (HMW-GS) (in the molecular weight range 70,000 – 90,000) and the LMW-GS (in the molecular weight range 20,000 – 45,000). HMW-GS and LMW-GS are cross-linked to form the so-called glutenin polymers, which are amongst the largest molecules in nature, with molecular weights exceeding one million (Wrigley, 1996). HMW-GS comprise only a few components and have been widely studied, whereas LMW-GS include a large number of polypeptides and their structure, organisation and relationship to grain processing quality have not yet been investigated to the same degree as for the HMW-GS. 2. History and classification LMW-GS were first identified by gel filtration of extracts of wheat flour as high-molecular-weight gliadins linked by 322 R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 Table 1 Summary of different classification systems for LMW-GS Classification based on SDS-PAGE mobility LMW-GS group Predominant sequence type B LMW-s; LMW-m C a-type; g-type D v-type N-terminal amino acid LMW-GS type sequence LMW-s LMW-m LMW-I a-type g-type v-type N-terminal AA sequence SHIPGLMETSH(R/C)IISQQQQVRVPVPNMQVDPKELQSPARQLNP- disulphide bonds, distinguishing them from monomeric gliadins (Beckwith et al., 1966). Starch gel electrophoresis gave further evidence for their existence, but technical difficulty in separating them from co-migrating gliadins (Elton and Ewart, 1966), did not allow a better definition. Later, Nielsen et al. (1968) designated this particular fraction as low-molecular-weight glutenin, because their viscosity and electrophoretic mobility differed from those of the gliadin fraction. A further insight into the fraction came from analysis of the reduced glutenin components by SDSPAGE (Payne and Corfield, 1979). Pioneering work from these authors showed that the glutenin subunits could be subdivided into A, B, and C groups, according to their mobility, the A group corresponding to HMW-GS and the B and C groups to LMW-GS. They also demonstrated that high-molecular-weight gliadins, once reduced and separated by SDS-PAGE, had mobilities similar to those of B and C subunits of LMW-GS. Shewry et al. (1983) characterised an aggregated gliadin fraction, which in retrospect was probably similar to the high-molecular-weight gliadins and low-molecular-weight glutenins described previously, and reported a major unique N-terminal sequence for the fraction, along with minor sequences corresponding to a-, b-, and g-gliadins. A further subdivision of LMW-GS was reported by Jackson et al. (1983) who recognized three groups of LMWGS on the basis of electrophoretic mobility in SDS-PAGE previously described, namely the B and C groups (Payne and Corfield, 1979), and the additional D group (Table 1 and Fig. 1). Unfortunately, this new group of proteins did not have electrophoretic mobilities in accord with the previous divisions, which assigned alphabetical letters in order of increasing electrophoretic mobility for the groupings in SDS-PAGE. The mobilities of D subunits fall approximately between those of the A and B subunits, near where the v-gliadins migrate in gliadin fractions. A further deficiency of the scheme is that the A, B, C, and D nomenclature sometimes causes confusion with the A, B, and D designations for the genomes of bread wheat. Unfortunately, a better nomenclature system has not yet been proposed. Jackson et al. (1983) described three D subunit components in the bread wheat cultivar Chinese Spring, but additional ones have been identified recently in other genotypes (Egorov et al., 2000; Gianibelli et al., 2002a,b; Johansson, 1996; Nieto-Taladriz et al., 1998; Odintsova et al., 2000). Subsequent analyses showed that the D group is actually composed of modified v-gliadin components that have acquired a cysteine residue. The identification of cysteine residues in v-gliadins, which typically lack this amino acid residue, was the first evidence that gliadin-like subunits were present in the glutenin polymers (Masci et al., 1993, 1999). This had been previously suggested by Tao and Kasarda (1989) who found a- and g-type N-terminal sequences in glutenin preparations from the bread wheat cultivar Chinese Spring. LMW-GS with a- and g-type N-terminal sequences are the most abundant proteins in the so-called C group, with at least thirty components being detected by two-dimensional analyses (Masci et al., 2002). As for the D subunits, it is probable that they form part of the glutenin fraction because the numbers of cysteine residues is different from that in Fig. 1. Two-dimensional electrophoresis (IEF £ SDS-PAGE) of glutenin subunits of the bread wheat cultivar Chinese Spring. The HMW-GS and the B-, C-, and D-type groups of LMW-GS are indicated. R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 the a- and g-gliadins. Although the only experimental evidence for this hypothesis is the demonstration that LMW-GS were linked by disulphide bonds to components with g-type sequences (Keck et al., 1995; Köhler et al., 1993), this is supported by comparisons of gene and deduced protein sequences. g-Gliadin clones with nine cysteine codons instead of the typical eight have been identified in durum and bread wheats (D’Ovidio et al., 1995; Scheets and Hedgcoth, 1988) while a-gliadin genomic clones with numbers of cysteine residues other than the typical six have been identified by Anderson et al. (1997). 3. The typical LMW-GS Most of the typical LMW-GS are present in the B group, although a smaller proportion of components with gliadinlike sequences are also present within this group (Masci et al., 2002; Tao and Kasarda, 1989). On the basis of Nterminal amino acid sequences, three subgroups of typical LMW-GS can be recognized (Table 1), called LMW-s, LMW-m, and LMW-i types, according to the first amino acid residue of the mature protein: serine, methionine, or isoleucine, respectively. LMW-s type subunits are the most abundant in all genotypes analysed and their average molecular mass (35,000 – 45,000) is higher than that of LMW-m type subunits (30,000 –40,000) (Tao and Kasarda, 1989; Lew et al., 1992; Masci et al., 1995). The N-terminal amino acid sequence of LMW-s type subunits is SHIPGL-, whereas the N-terminal sequences of LMW-m type subunits are more variable and include METSHIGPL-, METSRIPGL-, and METSCIPGL- (Kasarda et al., 1988; Lew et al., 1992; Masci et al., 1995; Tao and Kasarda, 1989). However, both LMW-s and LMW-m type subunits contain eight cysteine residues, two of which are involved in intermolecular disulphide bonds (see following paragraphs). Comparisons of the N-terminal sequences of LMW-s and LMW-m type subunits show that the former differ from the latter in the absence of the first three amino acid residues, although LMW-s type sequences starting with SRIPGL- or SCIPGL- have not so far been identified. The isolation of a LMW-GS gene from the bread wheat cultivar Yecora Rojo and analysis of the corresponding LMW-s type polypeptide product, named ‘42 K LMW-GS’, suggested that the SHIPGL- sequence might originate through differential gene processing, possibly at the translational level (Masci et al., 1998). The deduced mature amino acid sequence of the gene encoding the 42K LMW-GS is MENSHIPGL and it is possible that the presence of an asparagine residue (N) instead of the characteristic threonine (T), present in most of the LMW-GS genes isolated, may account for differential processing giving rise to the sequence variation observed. The findings of Dupont et al. (2003) suggesting that a single v-gliadin gives rise to two different N-terminal sequences due to post-translational cleavage by an asparaginyl peptidase, and the finding in bread wheat of a LMW-GS 323 polypeptide with the N-terminal sequence MENSHIPGL (Ikeda, personal communication) support this hypothesis. Thus, when considering the nucleotide sequences of typical LMW-GS, no distinction between LMW-s and LMW-m types can be made (see below). Among typical LMW-GS, the LMW-i type (Cloutier et al., 2001), which was first identified by Pitts et al. (1988), can be considered as a variant form. These LMW-GS lack the N-terminal region, starting directly with the repetitive domain after the signal sequence, with ISQQQQ- being the deduced N-terminal sequence of all LMW-i type genes isolated so far (Table 2). Although the N-terminal region is missing, the typical eight cysteine residues are all present in the C-terminal domain (Fig. 2). Cloutier et al. (2001) and Ikeda et al. (2002) presented evidence that they are expressed in the endosperm. A glutenin polypeptide in the durum wheat cultivar Langdon, with the N-terminal sequence IXQQQQP-, has recently been identified, thus confirming that LMW-i genes are actually expressed in wheat endosperm. We have also obtained evidence that they are incorporated into the glutenin polymers (Masci et al., 2003). At the same time, Ikeda (personal communication) has confirmed the expression of LMW-i type subunits by Nterminal amino acid sequencing of spots isolated from 2D gels. On the basis of the structural characteristics of the B, C and D groups, Kasarda (1989) suggested the existence of two functional groups of LMW-GS. One group, which includes the majority of the B-type sub-units, acts as chain extenders of the growing polymers because of their ability to form two inter-molecular disulphide bonds. The second group, which includes most of the C and D-type LMW subunits, act as chain terminators of the growing polymer, having only one cysteine available to form an intermolecular disulphide bond. New classes of low molecular mass proteins are likely to be discovered in the future, thus Clarke et al. (2000) and Anderson et al. (2001) have identified genes encoding new types of wheat endosperm proteins, with sequence similarity to gliadins, LMW-GS, and to 1-hordeins. The encoded proteins are characterised by distinctive N-terminal sequences, a smaller central repetitive domain than in typical LMW glutenin subunits, and the presence of more cysteine residues. However, whether these proteins are present in monomeric or polymeric form is not yet known. 4. Chromosomal localisation of LMW glutenin subunit genes The application of different one and two-dimensional separation systems to nullisomic-tetrasomic, ditelocentric and inter-varietal chromosome substitution lines of the bread wheat cultivar Chinese Spring, allowed Payne and colleagues (Jackson et al., 1983; Payne et al., 1984, 1985) to establish that most LMW-GS (B, C, and D groups) are 324 R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 Table 2 Sequences of cloned LMW-GS genes deposited in data banks N-terminal Genotype Accession number Chr. METSHIPGLEKPSMETSHIPSLEKPL- T. aestivum cv Norin 61 T. aestivum cv Norin 61 and cv Chinese Spring; T. durum cv Langdon T. aestivum cv Norin 61, Chinese Spring and subsp. tibeticum T. aestivum cv Norin 61, cv Chinese Spring, cv Cheyenne and subsp. tibeticum; T. durum cv Mexicali T. aestivum cv Norin 61, cv Cheyenne and cv Chuannong 16 T. aestivum cv Norin 61; T. durum cv Langdon T. aestivum cv Norin 61 T. aestivum cv Norin 61, cv Chinese Spring, cv Cheyenne and cv Chuannong 16 T. aestivum cv Norin 61 and cv Yecora Rojo; T. durum cv Lira 42 T. aestivum cv Norin 61 T. aestivum/Thinopyrum intermedium addition lines, Thinopyrum intermedium Thinopyrum intermedium T. aestivum cv Norin 61 T. aestivum cv Norin 61, Cheyenne, Yamhill and line 1CW, T. durum cv Langdon, T. aestivum/Thinopyrum intermedium addition lines, T. monococcum T. aestivum/Thinopyrum intermedium addition lines, Thinopyrum intermedium T. monococcum T. aestivum cv Chinese Spring; T. durum line 21 T. aestivum cv Chinese Spring T. aestivum cv Cheyenne, cv Neepawa and line 1CW AB062851 AB062852, Y14104, X84887 1D 1B AB062865-67, AY214450, AY299457 1D AB062873-74, X84959, X51759, U86026, AY299458 1A, 1D AB062872, U86028, M11077, AY296753 1D AB062868-70, X62588, AJ293099 AB062871 AB062875, X13306, U86027, U86029, AJ 519835 1A 1A 1D AB062853-60, AB062862, Y18159, Y17845 1B AB062861 AY214458, AY214451-52 1B – AY214454 AB062863-64 AB062876-78, AB008497, AJ293097, U86030, X07747, AY214456, AY146587-88 – – 1A AY214457, AY214455, AY214453 – AF072898 X84960, AJ007746 X84961 AB007763-64, U86025, M11335-36, AJ519837, AJ519838 AF073525, AJ293098 AY299459, AY296752, AJ519836 1A 1B 1D – METSRVPGLEKPWMETSCIPGLERPW- METSCISGLERPWMDTSCIPGLERPWMDTSCIPGLERPRMETRCIPGLERPWMENSHIPGLERPSMENSHIPGLERLSMESNIIISFLKPWMESNIIISFLEPW IENSHIPGLEKPSISQQQ- QQQQ Unknown Unknown Unknown Unknown Pseudogene Pseudogene T. monococcum, T. durum cv Langdon T. aestivum cv Neepawa, cv Chuannong 16 and subsp tibeticum 1A – N-terminal amino acid sequences are deduced from gene sequences containing a partial or complete signal sequence. Underlined N-terminal sequences are found at protein level (Lew et al., 1992; Masci et al., 1998). In bold are sequences with known chromosomal localization, specified in the Chr. column. N-terminal amino acid sequences found at protein level but not yet at gene level: METSCIPGLERPS, METSHIPGLERPS, METSRIPGLE, METSHIPGLEKPL (Lew et al., 1992). Fig. 2. Diagrams representing the structures of typical LMW-GS as deduced from their encoding genes. (A) LMW-m and LMW-s type (B) LMW-i type. S: Signal peptide; N: N-terminal region; REP: repetitive domain (small boxes indicate repeat motifs); C-TER I, II, III: C-terminal regions (see text for details). Cysteine residues are indicated by asterisks with sequential numbers, or the lettering system according to Köhler et al. (1993). The asterisks encircled in (A) indicate the cysteines that are most likely to be involved in inter-molecular disulphide bonds. Their position is also variable (as indicated by the brackets). Boxed asterisks in (B) indicate the two extra cysteine residues reported in the C-terminal domain of LMW-i type LMW-GS. R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 encoded by genes on the short arms of the group 1 chromosomes, at the complex Gli-1 loci that also encode gand v-gliadins. They also found some components in the same mobility range as C-type subunits that were encoded by genes on the short arms of the group 6 chromosomes, and suggested that they were contaminating monomeric gliadins. We now know that some of the C-type LMW-GS are actually encoded on the group 6 chromosomes, and correspond to a-type LMW-GS (Masci et al., 2002). Most attention has been paid to B-type LMW-GS, because they are the most abundant and have the greatest impact on technological properties. The development of a two-step SDS-PAGE procedure, which gives a more effective one-dimensional separation of B-type LMW-GS, allowed Singh and Shepherd (1988) to detect a low level of recombination between genes coding for LMW-GS and gliadins at the Gli-B1 locus, resulting in the designation of a separate locus for 1B-coded LMW-GS, called Glu-B3. However, they did not find any recombination between LMW-GS and gliadins coded at the Gli-A1 and Gli-D1 loci. The Glu-B3 locus was mapped on the short arm of chromosome 1B, between Gli-B1 (at 2 cM) and the centromere (Pogna et al., 1990). Subsequently, Ruiz and Carrillo (1993) found recombination between Gli-A1 and Glu-A3 in durum wheat, and estimated a distance of 1.3 cM. Liu and Shepherd (1995) confirmed the occurrence of recombination between Gli-A1 and Glu-A3 in durum wheat, and also found recombination (3.07 cM) within the Glu-B3 locus, designating the new locus as Glu-B4. They also detected a B-type LMW-GS in the durum wheat cultivar Edmore, encoded by a locus located about 20.9 cM from Glu-B3 (Liu, 1995). They suggested that this locus could be the same as the Glu-B2 locus previously described by Jackson et al. (1985) as encoding a D-type subunit. These analyses were complicated by the difficulty in distinguishing, at the protein level, the typical LMW-GS from modified gliadins (see below). However, restriction fragment length polymorphism (RFLP) analysis, using probes specific for typical LMW-GS and g-gliadins, allowed a clear distinction between these two types of genes. Segregation analysis showed genetic distances between Glu-3 and Gli-1 similar to those reported above based on protein analyses (Dubcovsky et al., 1997). Moreover, RFLP analysis also identified a locus encoding typical LMW-GS on the short arm of chromosome 1B which was different to Glu-B3. This locus probably corresponds to the Glu-B2 locus identified previously by Liu and Shepherd (1995), based on protein analysis, and was designated XGlu-B2. Two novel LMW-GS present in accessions of Indian bread wheat have been mapped to loci on chromosome 1D (Glu-D4) and chromosome 7D (Glu-D5) (Sreeramulu and Singh, 1997). Although their amino acid compositions differ from those of typical LMW-GS and other gluten proteins, they have been defined as LMW-GS because of their polymeric behaviour, and because polyclonal antibodies raised against them cross-reacted strongly with typical 325 LMW-GS, but not with HMW-GS or gliadins. Moreover, their N-terminal amino acid sequences resembled those of B- and C-type LMW-GS, being KETXXI- for the Glu-D4 encoded subunit (similar to the METSHI- sequence), and VXVPV- for Glu-D5 encoded subunit (similar to VRVPVpeculiar of a-type gliadins). The possibility that typical LMW-GS are encoded by loci in addition to Glu-3, is also suggested by the identification by RFLP analysis, of a new locus (Xucd1) encoding these subunits on the long arm of chromosome 7Am of T. monococcum (Dubcovsky et al., 1997). Further, Vaccino et al. (2002) suggested the existence of other loci encoding typical LMW-GS based on a high recombination frequency between the Gli-B1 locus and two DNA fragments which hybridised with a probe specific for typical LMW-GS, and were associated with two LMW-GS bands. D subunits are encoded by loci on the short arm of chromosomes 1 (Payne et al., 1985). However, until their structural characteristics were defined, there was confusion over their encoding loci. Jackson et al. (1983) suggested that the Glu-B2 locus, located on the satellite of the short arm at 17 cM from Glu-B1 and 22 cM from Gli-B1, encoded for a D-type subunit, although the same locus was later found to encode a B-type subunit (Liu, 1995). This observation might be due to the fact that Glu-B2 is a complex locus, or result from the use of different electrophoretic systems to identify the subunit. Most recently, Nieto-Taladriz et al. (1998) have shown that a D-type LMW-GS is encoded by the same GluB3 locus as typical B-type LMW-GS, thus confirming that it is a complex locus. Finally, it has been confirmed that the majority of C subunits are encoded on the same chromosomes as ggliadins, a-gliadins and typical LMW-GS, namely on the short arms of chromosomes 1 and 6 (Masci et al., 2002). A summary of the different loci encoding for B-, C-, and D-type subunits is shown in Fig. 3. 5. LMW-GS in wild wheats and other cereals LMW-GS are not only present in cultivated wheats, but also in wild relatives and in cereals related to wheat. LMWGS or proteins that are clearly similar to LMW-GS have been reported in the genera Elymus (Obukhova et al., 1997), Dasypyrum (Blanco et al., 1991), Elytrigia (Gupta and Shephard, 1990) and Hordeum (barley) (Atienza et al., 2002; Ladogina et al., 1989). LMW-GS have been extensively studied in T. tauschii, the D genome donor of bread wheat (Gianibelli et al., 2000, 2002a,b; Hsam et al., 2001; Pfluger et al., 2001; Vensel et al., 1997), and show a much wider variation in pattern than in the D-genome encoded LMW-GS present in bread wheat (Masci et al., 1991; Lafiandra et al., 2000). Other wheat relatives, including T. monococcum (Tranquilli et al., 2002; Corbellini et al., 1999) and other A-genome species (Lee et al., 1999), T. turgidum var. dicoccoides (AABB) 326 R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 Fig. 3. Chromosomal localization of genes encoding LMW-GS. Relative positions of loci are not proportional to their actual genetic distances, but are only indicative. The question mark indicates that the positions of the Glu-D4 and Glu-D5 loci are not known. (Ciaffi et al., 1991, 1993; Pagnotta et al., 1995), T. dicoccum (AABB) (Galterio et al., 2001) and T. polonicum (AABB) (Liu and Shepherd, 1996) also show large variation in LMW-GS, as do obsolete cultivars and landraces (Jaroslava et al., 2001; Ovesna et al., 2001). The extensive variation in LMW-GS present in relatives of wheat has been proposed as a source of genes for wheat breeding to widen or improve the properties of flour and semolina for different end-uses. 6. Structure and organisation of typical LMW-GS genes The typical LMW-GS are encoded by gene families at each of the orthologous Glu-3 loci, whose copy numbers are not known. However, estimates of the total gene copy number, based on Southern blot analyses, varied from 10 –15 (Harberd et al., 1985) to 35 –40 (Cassidy et al., 1998; Sabelli and Shewry, 1991) in hexaploid wheat. Recent sequence analyses of bacterial artificial chromosomes (BAC) clones revealed that two LMW-GS genes in T. monococcum may be separated from each other more than 150 kbp (Wicker et al., 2003). Information on the structure of genes encoding LMWGS derives from the characterisation of more than 70 DNA clones reported in data banks. These are isolated from 15 different genotypes belonging mainly to T. aestivum and T. durum, but also including some sequences from T. monococcum, Thinopyrum intermedium and T. aestivum/T. intermedium addition lines (Table 2). Additional LMW-GS clones (whose sequences are not present in data banks) from T. aestivum and a few from T. boeoticum and T. tauschii have been also isolated and their deduced amino acid sequences reported (Benmoussa et al., 2000; Ciaffi et al., 1999; Lee et al., 1999). Based on these data, the general structure of a typical LMW-GS (Figs. 2 and 4) can be proposed. This shows four main structural regions including a signal peptide of 20 amino acids, a short Nterminal region (13 amino acids) that usually contains the first cysteine residue, a repetitive domain rich in glutamine codons and a C-terminal region. As suggested by Cassidy et al. (1998), this latter region can be further subdivided into three distinctive regions: a cysteine-rich region containing five cysteine residues, a glutamine-rich region containing a cysteine residue and stretches of glutamine residues, and a C-terminal conserved sequence containing the last cysteine residue (Fig. 4). Most of the full-length genes vary from 909 bp (as in the sequence U86028) to 1167 bp (as in the sequence AB062878) in size with the molecular masses of the encoded mature proteins ranging from about 32,000 to 42,800. The number of repeats present in the repetitive domain is mainly responsible for this length variation, ranging between about 12 and 25. Comparison of allelic genes suggests that this variation can result from deletion and/or insertion of repeat units (D’Ovidio et al., 1999), most probably caused by unequal crossing-over and/or slippage during replication as suggested for the evolution of other prolamins (Shewry et al., 1989). The length of each repeat unit can vary between 15 and 27 bp with the following consensus sequence: CCA1 – 2 TTT T/C CA/G CAG/A CAA1 – 4 (Fig. 5). The repetitive domain is also mainly responsible for the general hydrophilic character of LMW-GS. The locations of the cysteine residues in the sequence distinguishes between those LMW-GS that form intermolecular disulphide bonds (first and seventh) from those forming intra-molecular disulphide bonds (Fig. 2). Based on the distribution of cysteine residues, the LMW-GS proteins can be classified into three main different types: (i) those with one cysteine in the short N-terminal domain; (ii) those R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 327 Fig. 4. Multiple alignment of sequences of typical LMW-GS. Sequences are derived from gene sequences in data banks and are representative of all the different types of LMW-GS genes so far reported in durum and bread wheats. A complete list of sequences deposited in data banks is reported in Table 2. Sig, signal peptide; Nter, the short N-terminal region present in some LMW-GS; Rep, repetitive domain that has been excluded from the figure. Three glutamines usually flank the first and the last repeat within the repetitive domain; CterI, CterII and CterIII represent sub-regions of the C-terminal part of the protein and indicate a cysteine-rich region, a glutamine-rich region, and the final conserved part of the protein, respectively. Cysteine residues are highlighted in bold. Asterisks indicate conserved amino acid positions. with a cysteine residue in the repetitive domain replacing that in the N-terminus; and (iii) those with eight cysteines in the C-terminal part of the protein (Fig. 2). This different cysteine distribution could lead to functional differences, especially regarding the third group. However, this has not yet been demonstrated. Glutamine and proline account for almost 50% of the total amino acids, being about 30 and 15 mol %, respectively. A characteristic of LMW-GS genes is the preferential use of the CAA codon (about 70%) in place of the CAG triplet to encode glutamine residues, which is also typical of other endosperm proteins such as gliadins and HMW-GS. The deduced sequences of the mature LMW-GS also show different N-terminal sequences, which have been used to divide the different gene sequences into groups (Table 2). Comparison of these N-termini with those detected at the protein level show that some correspond to proteins, although no protein sequences corresponding to others have been reported (Table 2). Comparisons between the coding regions of LMW-GS genes show that they share more than 80% identity at both the nucleotide and deduced protein levels. These sequences can be grouped into five clusters containing members that are encoded by different genomes, but share more than 90% identity. Among the five clusters, one containing sequences encoding LMW-GS with the N-terminus ISQQQ- (LMW-i type) is clearly differentiated from the other four groups (Fig. 6). Inclusion of a-, g- and v-gliadin sequences in the same analysis clearly shows that they fall into distinct groups (Fig. 6), but also shows that they have some sequence similarities which suggest a common evolutionary origin. The sequence similarity is mainly evident in the parts that encode the C-terminal and repetitive domains of the protein, with the latter encoding the basic PF(P/S)Q motif, where serine is characteristic of LMW-GS (Table 3). The sequences of the proteins deduced from the LMW-GS, a-, and g-gliadin genes also show a conserved distribution of some cysteine residues. 328 R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 Fig. 5. Repeat motif present in the repetitive region of LMW-GS. The three examples include a LMW-GS with a short (12 repeats) and two LMW-GS with long repetitive (22 and 25) regions. LMW-GS with a long repetitive region often contains repeat motifs with polyglutamine stretches or a cysteine residue at the beginning of the repetitive region (underlined). c, consensus sequence. R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 329 7. Synthesis, processing and trafficking of LMW-GS The initiation of LMW-GS synthesis has been reported to occur at the same time as that of HMW-GS, between 5 and 13 days after anthesis, depending on the genotype and growing conditions (Ng et al., 1991; Gupta et al., 1996; Grimwade et al., 1996) and continues until maturity, when the desiccation process starts. In contrast, Panozzo et al. (2001) reported that LMW-GS are only synthesised in significant amounts at about 14 days after anthesis, after the HMW-GS. Using a semi-quantitative PCR approach, it has been demonstrated that no significant differences in transcript accumulation occur between different LMW-GS genes in the cultivar Cheyenne at 15 days post-anthesis (Altenbach, 1998). Like all prolamins, LMW-GS are secretory proteins that are synthesised on the rough endoplasmic reticulum (ER) and then pass into the lumen, where they fold and form aggregates together with the other prolamin proteins, but it has not yet been shown if the process is assisted by molecular chaperones (Shewry, 1999). LMW-GS, together with other gluten proteins, are deposited in protein bodies in the developing endosperm cells. It has been proposed that there are two different routes for protein body formation, one predominating for gliadins, that are deposited into the vacuole via the Golgi apparatus, and one mainly for glutenins, that accumulate directly into the lumen of the ER (Rubin et al., 1992). It is therefore possible that at least typical LMW-GS (B subunits) follow this latter route. It seems reasonable that the choice of one route or the other depends on the properties of the protein that influences solubility or stability, so that monomeric proteins, such as gliadins, might be easily transportable via the Golgi, whereas polymers are not (Shewry, 1999). However, sequence variation in the C-terminal region has also been suggested to influence the secretion route (Altschuler and Galili, 1994). There are no specific reports on the trafficking of C-type and D-type LMW-GS but, based on the solubility hypothesis, it is possible that smaller aggregates such as dimers and trimers follow the same route as gliadins, whereas those incorporated into higher molecular weight polymers accumulate into the ER lumen. 8. Regulation of LMW-GS genes Fig. 6. Dendrogram showing the relationship between the coding regions of genes for typical LMW-GS and gliadins. Nucleotide sequences are specified by their accession number. A, B, or D at the end of the accession number indicates the chromosomal assignment of the sequence. Similar groups are given using protein sequences. Grey boxes indicate Met- or Sertype LMW-GS (LMW-m or LMW-s, respectively), whereas the slashed box groups the Ile-type LMW-GS (LMW-i). The gliadin sequences group separately from the LMW-GS sequences. Little is known about the regulation of wheat gluten protein synthesis. Like other prolamin genes, the expression of LMW-GS genes is primarily controlled at the transcriptional level (Bartels and Thompson, 1986) by trans-acting factors binding to sequences in cis-acting elements called ‘the 2 300 element’, ‘endosperm box’, or ‘prolamin box’ (Colot et al., 1987; Forde et al., 1985). In fact, it has been shown that sequences present between 2 326 and 2 160 bp upstream of the transcription starting point, which contain 330 R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 Table 3 Consensus sequences of the repeat motifs found in LMW-GS and gliadins LMW-GS g-gliadin From Anderson et al., 2001 a-gliadin From Anderson and Greene, 1997 v-gliadin From Hsia and Anderson, 2001 p Some LMW-GS genes can contain 1 or 2 repeat with up to 11 Q. The consensus sequences of LMW-GS, g-, a- and v-gliadins are deduced the comparison of 26, 21, 27, and 1 sequences, respectively. Some LMW-GS genes can contain one or two repeat with up to 11 Q. the endosperm box, confer endosperm-specificity to a typical LMW-GS (Colot et al., 1987). The endosperm box contains two conserved sequences, the 50 TGTAAAGT (the endosperm or E motif), and the 30 G(A/G)TGAGTCAT (GCN4-like motif or GLM) (Hammond-Kosack et al., 1993). The E and GLM motifs are both required for endosperm-specific gene expression. These two conserved sequences are sequentially occupied by nuclear factors during endosperm development and control transcription of the LMWG1D1 gene (Hammond-Kosack et al., 1993). In vivo foot-printing experiments showed that during the early stages of seed development, when the transcription rate is low, only the endosperm motif becomes occupied by a trans-acting factor named ESBF-I, whereas the GCN4 motif becomes occupied by a second trans-acting factor, named ESBF-II, before the stage of maximum gene expression. Similar results have been obtained with an ‘endosperm box’ from a gene encoding a C hordein (the barley homologue of v-gliadin). They also showed that the GCN4 motif is a key element in the nitrogen response, and, for this reason, is also called nitrogen element or N-motif. Under a low nitrogen regime, the GCN4 motif acts as negative regulatory element, whereas under optimal nitrogen regime this element acts as positive element interacting with the endosperm motif (Muller and Knudsen, 1993). It has been shown that the maize endosperm-specific transcription factor Opaque-2 (O2), that influences the accumulation of some maize storage proteins (zeins), can also activate transcription of the LMW-G1D1 gene in plant protoplasts and in yeast cells (Holdsworth et al., 1995). A seed-specific basic leucine zipper transcriptional activator from wheat (storage protein activator, SPA), similar in sequence to the O2 factor, has been found to bind to the GCN4 motif in both maize and tobacco leaf protoplasts. Both SPA and O2 activate transcription to similar levels (Albani et al., 1997). Even less is known about post-translation modifications of LMW-GS. Laurière et al. (1996) have presented indications that LMW-GS are N-glycosylated with xylose and suggested also that LMW-GS sorting occurs in the Golgi apparatus, although there was no direct evidence for such occurrence. Environmental conditions also influence gluten protein synthesis, including the synthesis of LMW-GS with effects being genotype dependent (Andrews et al., 1994; Bonfil et al., 1997; Luo et al., 2000; Marchylo et al., 1990; Panozzo and Eagles, 2000; Triboi et al., 2000). The most important environmental factors considered include fertilisation (i.e. nutritional) conditions (reviewed by Shewry et al., 2001) and temperature (Blumenthal et al., 1990; Perrotta et al., 1998). It is well established that an increased supply of nitrogen causes a general increase in grain protein content, although there are no specific increases in LMW-GS with respect to the other prolamins (Altenbach et al., 2002; Luo et al., 2000; Triboi et al., 2000). In contrast to nitrogen, sulphur availability has different effects on the various classes of wheat storage proteins (Shewry et al., 2001). Because LMW-GS are sulphur-rich proteins, their synthesis is negatively affected by sulphur deficiency (Shewry et al., 2001; Zhao et al., 1999), and this effect is even more marked if high nitrogen is supplied (Wooding et al., 1994). Shewry et al. (2001) speculate that S-rich and S-poor prolamins may present minor differences in the sequences of their GCN4like motifs, and that this might account for a differential ability to bind possible signalling factors released in conditions of sulphur deficiency. Considering the effects of temperature on wheat storage protein accumulation, Perrotta et al. (1998) and Altenbach et al. (2002) both showed that changes in temperature did not affect the proportions of mRNAs for any prolamins, including LMW-GS, although the initiation and termination of transcription were slightly earlier when temperatures were increased. Similar results were also obtained at the protein level by determining the protein composition of heat-stressed samples, although differences in the aggregation behaviour were found (Ciaffi et al., 1995, 1996) R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 9. Structural characteristics of LMW-GS and their role in the formation of the glutenin polymer Very little is known about the structures of LMW-GS. Tatham et al. (1987) proposed that the N-terminal domain forms irregularly distributed b-turns, whereas a-helices are predominant in the C-terminal domain. These results were supported by Thompson et al. (1994) who studied heterologously expressed LMW-GS and subunits extracted from wheat. Attempts to predict the secondary structure of the Nterminal part of the repetitive domain did not attribute any regular structure to a 42 K LMW-GS, whereas the Cterminal portion of the domain was predicted to be more compact, with the probable presence of a-helices (Masci et al., 1998). Application of flexibility modelling to the same 42K LMW-GS indicated that the repetitive domain was highly flexible, particularly where stretches of glutamines were present. This subunit contains two cysteine residues that are likely to be involved in inter-molecular disulphide bonds, namely the first (Cys-43 according to Masci et al. (1998) or Cbp according to Köhler et al. (1993)) and the seventh (Cys-295 or Cx). These residues are predicted to be located or surrounded by regions of high flexibility, which might be a mechanism that facilitates polymerisation. Structural prediction of the 42 K LMW-GS also suggests that the different numbers of glutamine residues comprising each repeat might have evolved to avoid the formation of regular intra- or inter-molecular interactions that might give rise to strong interacting aggregates, which are resistant to degradation by enzymes during germination. The remaining six cysteine residues in the 42 K LMWGS are likely to be involved in intra-molecular disulphide bonds, as suggested by direct amino acid sequencing of cystine-containing peptides, similarity with the closely related g-gliadins, and site-directed mutagenesis of cysteine residues (Keck et al., 1995; Köhler et al., 1993; Müller et al., 1998; Muller and Wieser, 1997; Orsi et al., 2001; Shewry and Tatham, 1997; Thompson et al., 1993, 1994). Although most of the identification of disulphide bonds has been performed on gluten, so that it is not possible to ascertain if some cystine-containing peptides were derived from the same or different polypetides (Keck et al., 1995; Köhler et al., 1993; Müller et al., 1998), this organisation of intramolecular disulphide bonds in LMW-GS is generally accepted. There is little doubt that the first (Cbp) and seventh (Cx) cysteines are involved in inter-molecular disulphide bonds,; this latter residue has in fact been found to be linked to several different polypeptides, namely HMW-GS and a modified g-gliadin (probably a C-type LMW-GS) (Keck et al., 1995; Köhler et al., 1993) (Fig. 2). Site-specific mutagenesis of cysteine residues coupled with the use of an in vitro system composed of wheat germ extract and bean microsomes has allowed Orsi et al. (2001) to determine which intra-molecular disulphide bonds are essential for the correct folding of a LMW-GS. This study also supported the involvement of the first and seventh 331 cysteines in inter-molecular disulphide bonding. According to these authors, the three intra-molecular disulphide bonds play different roles in the structural maturation of the LMWGS encoded by the B11-33 clone (Okita et al., 1985). The most important linkage is between the second (Cys-134 according to Orsi et al. (2001) or Cc according to Köhler et al. (1993)) and the fifth (Cys-169 or Cf1) cysteines, since its elimination causes extensive protein aggregation. The formation of this bond may be a critical step to reach the appropriate folding: in its absence, adhesive sites might become exposed, leading to the formation of inappropriate intra- and inter-molecular disulphide bonds that result in insoluble aggregates. In contrast, the elimination of either of the two other intra-molecular disulphide bonds, Cys-142/ Cys-162 (Cd/Ce) or Cys-170/Cys-280 (Cf2/Cy) did not have instead any significant effects, thus suggesting that they have roles in the stabilisation of the local structure. Because folding occurs co-translationally in prolamins, and disulphide bond formation also occurs very rapidly as soon as the protein is transported into the ER lumen, it can be speculated that this mechanism has been favoured because it ensures proper folding and aggregation behaviour. The role of enzymes in the formation of disulphide bonds in prolamins is also unresolved. The use of protein disulphide isomerase (PDI) and cysteine-containing synthetic peptides based on LMW-GS indicated that it did have a strong oxidising effect, but this effect was inversely correlated to protein molecular weight, inferring that the enzyme does not play an important role in vivo (Bauer and Schieberle, 2000). However, in vivo studies indicate that PDI might be involved in the assembly of wheat storage proteins within the ER, based mainly on its abundance in developing endosperm (Dupont et al., 1998; Grimwade et al., 1996; Shimoni et al., 1995). Moreover, the in vitro synthesis of a g-gliadin in disulphide-deficient microsomes resulted in the formation of incorrect disulphide bonds, and this was reversed when microsomes were reconstituted with PDI (Bulleid and Freedman, 1988). The dynamics of formation of both intra- and intermolecular disulphide bonds is a matter of debate. LMW-GS are major components of the glutenin polymers, being about 5 –6 times more abundant than HMW-GS (Clarke et al., 2000; Kasarda, 1989). However, it is not known whether they are randomly incorporated into the glutenin polymer, or their incorporation is subject to particular constraints due to their structure, timing of synthesis, or the involvement of specific enzymes. Lindsay and Skerritt (1998) proposed that the pattern of release of glutenin subunits from glutenin polymers by stepwise reduction indicates that this structure has a precise organisation. They found that LMW-GS were initially released as dimers, with monomers appearing after increasing the concentration of the reducing agent (DTT). B subunits were released at a low concentration of DTT, whereas C subunits were released over a wider concentration of reducing agent. This has been interpreted to indicate that B subunits are present in the biggest polymers, 332 R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 since inter-molecular bonds are broken first, whereas C subunits are involved in polymers with a wider range of molecular weight. On the basis of the results reported by these authors, B subunits may behave like a type of adhesive to stabilise the glutenin polymers, since once they are released, complete depolymerisation occurs. The same authors suggested, based on transmission electron microscopy (TEM) images, that LMW-GS form discrete clustered structures, although it was not possible to determine whether these associations were covalent or noncovalent (Lindsay and Skerritt, 2000). There are indications that the structural characteristics of LMW-GS have an influence on polymerisation behaviour. We have recently shown that a LMW-GS with an extended deletion in its repetitive domain, polymerised more rapidly during in vitro re-oxidation that the wild-type subunit, suggesting that the length of the repetitive region may influence the accessibility of cysteine residues involved in inter-molecular disulphide bonds (Patacchini, 2003; Patacchini et al., 2003). Moreover, the role of the first cysteine residue in polymerisation was confirmed, since a mutant LMW-GS, in which this cysteine residue was replaced by an arginine, formed a lower amount of polymers than the corresponding wild-type LMW-GS. The correlation between the presence of cysteine residues available for inter-molecular disulphide bonds and the polymerisation capacity can also be inferred from the results of Veraverbeke et al. (2000) who found that B subunits formed more polymers than C subunits after in vitro re-oxidation, with the latter mostly remaining in the monomeric form. This difference may occur because B subunits have two cysteine available for inter-molecular bonds, whereas C subunits may only possess one cysteine available for inter-molecular disulphide bonds. 10. Influence of LMW-GS on quality properties LMW-GS are important for the end-use quality of durum wheat, in particular subunits encoded by loci present on chromosome 1B (Josephides et al., 1987). The best pastamaking characteristics are associated with the presence of a specific allelic form of typical LMW-GS, named LMW-2 (Payne et al., 1984). This allele also seems to be important for determining breadmaking properties (Peňa et al., 1994). LMW-2 comprises a group of polypeptides, encoded by the Glu-B3 locus, which is genetically linked to the Gli-B1 locus, which contains genes encoding g- and v-gliadins, designated 45 and 35, respectively. Most commonly grown durum wheat cultivars have either the LMW-2/g-45 (plus vgliadin 35) or the LMW-1/g-42 (plus v-gliadins 33, 35 and 38) allelic forms, the latter being associated with poor quality pasta-making properties (Fig. 7). Because of the close association between g-42 and g-45 with LMW-1 and LMW-2, respectively, it was initially believed that quality characteristics were dependent on the presence of Fig. 7. SDS-PAGE of glutenins (A) and acid polyacrylamide gel electrophoresis (A-PAGE) of gliadins (B) in biotypes 42 (1) and 45 (2) of the durum wheat cv. Lira. LMW-GS comprising the LMW-1 and LMW2 groups are indicated. Arrows indicate the g-42 and the g-45 gliadins. the specific g-gliadins rather than the associated LMW-GS. However, many more recent studies have demonstrated the importance of LMW-GS, and it is now commonly accepted that g-42 and g-45 are only genetic markers for quality (Boggini and Pogna, 1989; Pogna et al., 1988). LMW-2 also exert a positive effect on gluten strength when present in hexaploid tritordeum, an amphiploid of Triticum durum £ Hordeum chilense (Alvarez et al., 1999). There are indications that the better quality associated with the presence of LMW-2 in durum wheat is mainly due to the fact that the subunits are more abundant than the LMW-1 subunits (Autran et al., 1987; D’Ovidio et al., 1992; Masci et al., 1995) and that structural differences may play only a minor role (D’Ovidio et al., 1999; Masci et al., 1998). In support of this, D’Ovidio et al. (1999) showed that allelic genes encoding major components of the LMW-1 and LMW-2 groups differed only by 15 amino acid substitutions within the repetitive domain. Although it is not possible to exclude the possibility that other LMW-GS belonging to the LMW-1 and 2 groups are responsible for their different effect on end-use properties, this result supports the hypothesis that quantitative differences in LMW-GS are important. Further support comes from the poor processing properties of the durum wheat cultivar Demetra that possesses the LMW-2 group, but has a low amount of LMW-GS, caused either by a lower number of subunits composing the LMW-2 and to a lower level of expression of those LMW-GS present (Masci et al., 2000a). The main difference between the LMW-1 and LMW-2 protein groups is the presence of a slow moving Glu-B3 R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 coded LMW-GS in the latter (D’Ovidio et al., 1999; Masci et al., 1995; Nieto-Taladriz et al., 1997; Ruiz and Carrillo, 1995, 1996). This slow moving LMW-GS corresponds to the 42 K LMW-GS (Masci et al., 1998) in most genotypes and it is consistently the most abundant LMW-GS polypeptide (Masci et al., 1995). Allelic variants of the LMW-2 group have also been described and in all cases these are associated with good technological properties (Brites and Carrillo, 2001; Ruiz and Carrillo, 1995, 1996; Vàzquez et al., 1996). Although HMW-GS are the major group of gluten proteins that determine the bread-making characteristics of dough, LMW-GS also play an important role. In general, the LMW-GS are associated with dough resistance and extensibility (Metakovskii et al., 1990; Andrews et al., 1994; Cornish et al., 2001), and some allelic forms of LMW-GS show even greater effects on these properties than HMW-GS (Payne et al., 1987; Gupta et al., 1989, 1994). Similarly, null alleles of LMW-GS have detrimental effects on these parameters (Benedettelli et al., 1992). Differences in the total amount of LMW-GS, associated with specific allelic forms, have also been reported to be an important cause of quality differences in bread wheat (Gupta and MacRitchie, 1994). In support of this hypothesis, Wieser and Kieffer (2001) found that twice the amount of LMW-GS was necessary to obtain the same dough resistance as they achieved with HMW-GS, as determined by rheological measurements and baking tests on a micro-scale. The 42 K LMW-GS may also be present in good quality bread wheat (Masci et al., 2000b), but it is not associated with the LMW-2 group, which does not appear to occur in hexaploid wheat. Different allelic forms of LMW-GS seem to play different roles in determining different quality parameters (Luo et al., 2001). Thus the Glu-A3 alleles influence protein content, SDS sedimentation volume, and mixograph midline peak value of New Zealand wheat cultivars, whereas the Glu-D3 alleles do not have any influence on the SDSsedimentation value. Reports of correlations between particular allelic forms of LMW-GS and quality parameters in bread wheat are often contradictory, a possible explanation being that gene interactions and environmental effects may play a fundamental role (Benedettelli et al., 1992; Gupta et al., 1994; Killermann and Zimmermann, 2000; Nieto-Taladriz et al., 1994). Although quantitative effects (i.e. differences in amount of LMW-GS) seem to be predominant in both durum and bread wheat, structural differences in the proteins may also play a role, as indicated by the results of incorporation experiments using the 2 g Mixograph. In these experiments, similar amounts of single LMW-GS are used, and the differences observed should therefore to be attributable to structural differences only. The ability to express single polypeptides in heterologous systems, such as bacterial systems, has been of great value for the analysis of LMWGS, as single components are very difficult to purify from 333 wheat. Lee et al. (1999) have incorporated four different LMW-GS polypeptides into bread wheat dough using proteins expressed in E. coli using genes isolated from wild relatives of wheat. These polypeptides differed in structural characteristics with amino acid substitutions affecting polarity, charge and side-chain structures, and minor deletions/insertions. The differences observed in the Mixograph parameters were explained in terms of cysteine accessibility and non-covalent interactions deriving from structural differences between the heterologously expressed LMW-GS. Patacchini (2003) has expressed three LMW-GS genes from durum wheat, differing either in the number of cysteine residues and in the length of the repetitive domains, in E. coli, and incorporated the purified proteins into durum wheat dough. Mixographic analyses showed that a wildtype LMW-GS increased the mixing-time (MT) (Patacchini, 2003), a parameter that is positively correlated with glutenin polymer size (Hoseney, 1985). In contrast, a mutated subunit lacking the first cysteine residue (involved in inter-molecular disulphide bonds) and another with a truncated repetitive domain decreased the MT, this effect being greater with the first mutant which presumably acted as a chain terminator of glutenin polymers. Most qualitative evaluations of LMW-GS have been performed on B-type subunits, as they are the most abundant and easiest to detect. Very little is known about the role of C-type subunits, probably because a procedure allowing their detailed study has only recently been developed (Masci et al., 2002). A few studies have dealt with the role of D-type subunits on quality characteristics with contrasting results: their reported presence in smaller polymers or in the propanolsoluble fraction suggests that they should be negatively correlated with quality. Similarly, the presence of a single cysteine residue that terminates growth of the glutenin polymer may indicate a role as chain terminator (Masci et al., 1993; Tao and Kasarda, 1989; Egorov et al. 2000; Gianibelli et al., 2002a,b). SDS-sedimentation tests on two biotypes of the bread wheat cultivar Newton differing at the Gli-D1/Glu-D3 loci showed lower values for the biotype that possessed D-type subunits, providing further support for their negative contribution to visco-elastic properties (Masci et al., 1991). However, Nieto-Taladriz et al. (1998) suggested that the presence of a 1B-coded D-type subunit might be responsible for the higher SDS sedimentation test value of the bread wheat cultivar Prinqual. The apparently contrasting effects reported by these authors may be due to the different genetic backgrounds analysed. 11. Manipulation of LMW-GS composition through classical and advanced breeding for wheat quality improvement The limited variation in LMW-GS in cultivated wheats, which may result from the intense selection over recent 334 R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 decades, forces breeders to look for new allelic variants that can be found in landraces or wild wheat relatives. One way to modify the technological properties of durum wheat is to introduce the D genome, which is present in bread wheat. Pogna et al. (1996) have introduced a naturally occurring translocation of a segment carrying the Gli-D1/Glu-D3 loci on chromosome 1A of the bread wheat cultivar Perzivan-1, into two durum wheat cultivars and have compared the alveographic parameters of the wild-type durum wheats with those carrying the translocated segment. Their results showed that the genotypes with the Gli-D1/Glu-D3 translocation showed increases in dough strength and extensibility and decreases in tenacity, although effects of the Gli-A1/Glu-A3 alleles of cv. Perzivan-1, could not be excluded. In order to exclude the effects of associated genes, the recent development of reliable genetic transformation protocols for wheat allows the effects of single LMW-GS genes on different structural and qualitative characteristics to be determined. Genetic transformation allows new allelic variants or additional copies of LMW-GS genes to be introduced into wheat, in order to increase the amount of the encoded proteins. The effect of the introduction of multiple copies of a LMW-GS gene into bread wheat by means of particle bombardment, on glutenin polymer organisation and quality predictive tests has been evaluated (Masci et al., 2003). Although the rationale of this work was to improve gluten strength by increasing the amount of LMW-GS, the very high expression of the transgenic polypeptide (up to 16-fold with respect to an average LMW-GS) resulted in a lower SDS sedimentation volume of the transgenic wheat with respect to untransformed lines. Such behaviour has been interpreted in terms of an optimum size for glutenin polymers that, when exceeded, negatively affects gluten strength. In support of this hypothesis, expression of a lower amount of a transgenic LMW-GS in pasta wheat increased dough strength, although a second line expressing the same gene showed reduced dough strength due to partial suppression of endogenous LMW-GS genes (Tosi, 2002; Tosi et al., 2004). Additional contributions of biotechnology to breeding for wheat quality are the development of PCR and immunological assays for specific quality-related LMWGS alleles. They not only facilitate selection, but also allow the purity of wheat grain and flour to be tested. PCR assays specific for components of the LMW-2 group have proved to be very efficient even when using a very limited amount of endosperm tissue. Their effectiveness and ease of use, as compared to SDS-PAGE analysis, can speed up the selection of genotypes possessing the good quality LMW2 components. Moreover, since these markers are co-dominant, homozygous can be easily distinguished from heterozygous genotypes (D’Ovidio, 1993; D’Ovidio and Porceddu, 1996; D’Ovidio et al., 1999). Monoclonal antibodies specific for the LMW-2 allele (Kovacs et al., 1995) or subunits encoded by the GluB3 locus (Denery-Papini et al., 1995) have been shown to provide efficient identification of these components in total glutenin preparation. Moreover, antibodies able to identify LMW-GS encoded by the Glu-D3 locus (Brett et al., 1993) or to discriminate between alleles at the GluA3 and GluB3 loci (Andrews and Skerritt, 1996; Partridge et al., 2001) have been used to correlate the presence of specific LMWGS with dough properties. 12. Conclusions LMW-GS are a complex group of proteins that share the ability to form inter-molecular disulphide bonds that allow their incorporation into glutenin polymers. Comparison of data at the protein and gene levels demonstrates heterogeneity with the presence of sequences related to a-, g-, and v-gliadins, as well as a distinct group of components that can be defined as the typical LMW-GS. Sequence comparison, biochemical analyses and molecular modelling show that these typical LMW-GS contain a specific number and distribution of cysteine residues that confer the ability to form two inter-molecular disulphide bonds with other LMW-GS or with HMW-GS, thus contributing to the formation of the glutenin polymers. Conversely, the number and distribution of cysteine residues in gliadins allows them to form only intra-molecular disulphide bonds, with the result that they are monomeric components of the gluten. However, mutational events that affect the number of cysteine residues in gliadins may enable them to form inter-molecular disulphide bonds, thus participating in glutenin polymer formation. Consequently, the biochemical distinction between the B-, C-, and D-type LMW-GS does not reflect common structural characteristics, but represents an initial attempt to classify the different components within this complex group of proteins. In fact, a pioneering proteomic study reported by Kasarda and colleagues (Lew et al., 1992; Tao and Kasarda, 1989) showed that most of the C- and D-type LMW-GS and some of the B-type are a-, g-, and v-gliadin that have acquired the ability to participate in the formation of the glutenin polymers. Consequently, they can also been called modified gliadins, gliadin-like, or a-, g-, and v-glutenins (D’Ovidio et al., 2000; Southan and MacRitchie, 1999; Masci et al., 2002). Although a number of typical LMW-GS sequences have been reported, little is known about the composition and organisation of the gene family. Furthermore, more information is required to define the specific contributions of single components to gluten properties, especially in bread wheat, in order to perform a more rational selection of quality-related components in breeding programs. It is probable that proteomic and genomic approaches will contribute greatly to this understanding, including also the discovery of additional polymeric endosperm components. R. D’Ovidio, S. Masci / Journal of Cereal Science 39 (2004) 321–339 Acknowledgements The authors are supported by the Italian Minister for University and Research (MIUR), projects ‘Aspetti biochimici, genetici e molecolari delle proteine della cariosside dei frumenti in relazione alle caratteristiche nutrizionali e tecnologiche dei prodotti derivati’ (PRIN (2002)) and ‘Espressione genica ed accumulo di proteine d’interesse agronomico nella cellula vegetale: meccanismi trascrizionali e post-trascrizionali’ (FIRB RBNE01TYZF). The authors wish to dedicate this paper to Dr Donald D. Kasarda. References Albani, D., Hammond-Kosack, M.C.U., Smith, C., Conlan, S., Colot, V., Holdsworth, M., Bevan, M.W., 1997. 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