J Appl Genetics (2014) 55:27–42 DOI 10.1007/s13353-013-0187-4
PLANT GENETICS • ORIGINAL PAPER
Molecular characterisation and evolution of HMW glutenin subunit genes in Brachypodium distachyon L. Saminathan Subburaj & Guanxing Chen & Caixia Han & Dongwen Lv & Xiaohui Li & Friedrich J. Zeller & Sai L. K. Hsam & Yueming Yan
Received: 15 August 2013 / Revised: 10 November 2013 / Accepted: 19 November 2013 / Published online: 4 December 2013 # Institute of Plant Genetics, Polish Academy of Sciences, Poznan 2013
Abstract Brachypodium distachyon, a small wild grass within the Pooideae family, is a new model organism for exploring the functional genomics of cereal crops. It was shown to have close relationships to wheat, barley and rice. Here, we describe the molecular characterisation and evolutionary relationships of high molecular weight glutenin subunits (HMWGS) genes from B. distachyon. Sodium dodecyl sulphate– polyacrylamide gel electrophoresis (SDS-PAGE), high performance capillary electrophoresis (HPCE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses demonstrated that there was no HMW-GS expression in the Brachypodium grains due to the silencing of their encoding genes. Through allele-specific polymerase chain reaction (ASPCR) amplification and cloning, a total of 13 HMW-GS encoding genes from diploid, tetraploid and hexaploid Brachypodium species were obtained, and all of them had typical structural features of y-type HMW-GS genes from common wheat and related species, particularly more similar to the 1Dy12 gene. However, the presence of an in-frame premature stop codon (TAG) at position 1521 in the coding region resulted in the conversion of all the genes to pseudogenes. Further, quantitative real-time PCR (qRT-
Saminathan Subburaj and Guanxing Chen contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13353-013-0187-4) contains supplementary material, which is available to authorized users. S. Subburaj : G. Chen : C. Han : D. Lv : X. Li (*) : Y. Yan (*) College of Life Science, Capital Normal University, 100048 Beijing, China e-mail: [email protected] e-mail: [email protected] F. J. Zeller : S. L. K. Hsam Division of Plant Breeding and Applied Genetics, Technical University of Munich, 85354 Freising, Germany
PCR) analysis revealed that HMW-GS genes in B. distachyon displayed a similar trend, but with a low transcriptional expression profile during grain development due to the occurrence of the stop codon. Phylogenetic analysis showed that the highly conserved Glu-1-2 loci were presented in B. distachyon, which displayed close phylogenetic evolutionary relationships with Triticum and related species. Keywords Brachypodium distachyon . HMW glutenin subunits . AS-PCR . qRT-PCR . Phylogeny and evolution
Introduction The Poaceae grass family includes some major temperate cereal crops, such as wheat, rice, corn, barley, rye and sorghum and forage grasses. These cereals contain 7–15 % protein in mature grains and serve as important protein sources for human consumption and animal feed. Brachypodium distachyon, a small wild grass within the Pooideae subfamily, has emerged as a new model system for the study of these temperate cereals (Draper et al. 2001; Hasterok et al. 2004; Vogel et al. 2006a, b), and its whole genome sequencing has been completed recently (International Brachypodium Initiative 2010). Because of its short life cycle (Garvin et al. 2008; Mur et al. 2011), very small genome in monocots (Wolny and Hasterok 2009) and low nutritional requirements (Vogel et al. 2009), B. distachyon has become more suitable for functional and comparative genomics studies. Prolamins are important endosperm storage proteins in cereal grains. Based on their molecular weights and sulphur contents, they can be further classified into three subgroups: high molecular weight (HMW) prolamins, S-rich prolamins and S-poor prolamins (Shewry et al. 1999). Gluten, the most important grain prolamin in common wheat (Triticum aestivum L.), comprises HMW prolamins (HMW-GS), S-
28
rich prolamin [α/β, γ-gliadins and B/C-type low molecular weight (LMW)-GS] and S-poor prolamins (ω-gliadins and Dtype LMW-GS). It is known that HMW-GS mainly affect the dough elasticity, while LMW-GS and gliadins determine dough viscosity and extensibility, respectively. These dough properties are essential for bread-making quality (Payne 1987; Shewry and Halford 2002). Genes coding for HMW-GS are located at the Glu-1 loci on the long arm of the group 1 homeologous chromosomes of the A, B and D genomes in hexaploid bread wheat. Glu-1 loci include two tightly linked genes Glu-1-1 and Glu-1-2, which encode a larger x-type (80–88 kDa) and a smaller y-type (67– 73 kDa) HMW-GS, respectively (Mackie et al. 1996). However, only 3–5 subunits generally express in the individual hexaploid cultivars due to the silencing of the genes coding for y-type HMW-GS at the Glu-A1 and Glu-B1 loci (Payne 1987). Orthologous HMW-GS are present in Triticum and related species such as Aegilops and rye (De Bustos and Jouve 2003; Yan et al. 2003a, 2004; Zhang et al. 2008; Jin et al. 2012). Apart from the critical role for wheat end products, HMW-GS genes are also able to provide useful information for understanding the evolutionary relationships among Triticeae and its related species (Yan et al. 2003b). B. distachyon is the only annual member of the tribe Brachypoideae (Khan and Stace 1999) and has a strong physiological and genetic similarity to its “core pooids”, a subgroup that includes wheat, barley and rye (Huo et al. 2009). Originally, B. distachyon acts as a connecting bridge between wheat and rice, but phylogenetic analysis indicated that B. distachyon has higher sequence similarities with the wheat genome (Ozdemir et al. 2008). Also, the divergence time between wheat and B. distachyon is closer than that between wheat and rice (Vogel et al. 2006b; Mur et al. 2011). Multiple alignment of several grass genomes showed that B. distachyon contains prolamin-like genes named as Brachypodins-III , which were under the class of HMW glutenins (Xu and Messing 2009). Moreover, an inactive copy of HMW glutenin-like gene was found in B. distachyon ’s Glu-1 loci by orthologous comparison between different grass genomes (Gu et al. 2010). The grain characteristics of Brachypodium showed more resemblance to that of the wheat species (Ozdemir et al. 2008; Opanowicz et al. 2011) than to rice. However, B. distachyon is similar to rice by the presence of glutelin-like protein bodies in the endosperm (Larré et al. 2010). Furthermore, proteomic analysis of some hexaploid B. distachyon accessions showed that their major seed storage proteins were 11S-globulins (Laudencia-Chingcuanco and Vensel 2008), whereas, in the mature stages of grains, an unstoppable proteinaceous matrix made B. distachyon grains similar to wheat with significant similarities in the grain endosperm development (Hands and Drea 2012). Apart from the presence of globulins, there were some prolamin proteins displaying homology to HMW glutenins in the grains of
J Appl Genetics (2014) 55:27–42
diploid Bd21 (Larré et al. 2010). Also, Wang et al. (2010) and Hammami et al. (2011) found that there were enormous amounts of LMW-like glutenins with similar electrophoretic patterns to wheat in Brachypodium seeds. More recently, a highly conserved Glu-3 locus was confirmed to be present in Brachypodium as those in Triticum and its related species (Wang et al. 2012a). Until now, limited information on the structural features and evolution of HMW-GS genes in B. distachyon has been available. In the current study, we have conducted further investigation on the molecular characterisation of HMW-GS genes from diploid, tetraploid and hexaploid B. distachyon accessions and their phylogenetic revolutionary relationships with Triticum and other Poaceae species. Our results reveal a highly conserved Glu-1 locus that is present in B. distachyon and close evolutionary relationships with Aegilops and Triticum species.
Materials and methods Plant materials The seeds of B. distachyon accessions of Bd2 (PI185133), Bd3 (PI185134), Bd4 (PI208216), Bd10 (PI226452), Bd12 (PI227011), Bd13 (PI233228), Bd14 (PI239713), Bd16 (PI239715), Bd17 (PI239716), Bd21 (PI254867), Bd21-3, BdISKP1 and Bd347 were kindly provided by Dr. John Vogel, United States Department of Agriculture—Agriculture Research Service (USDA-ARS), USA, and Dr. Chengtao Lin, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (CAAS), China. Accessions Bd2, Bd3, Bd21-3 and Bd21 were diploid (2n=10), while other genotypes are hexaploid, except the tetraploid Bd347 (Vogel et al. 2006b). The hexaploid common wheat cultivar Chinese Spring (Triticum aestivum L., 2n=6x=42, AABBDD) with HMW-GS compositions of Null, 1Bx7+1By8 and 1Dx2+1Dy12, and spelt wheat cv. Renval (Triticum spelta L., 2n=6x=42, AABBDD) with 1Ax1, 1Bx13*+1By19* and 1Dx2+1Dy12 (Yan et al. 2003b) were used as standards for glutenin identification as well as the controls for polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR) amplifications. Apart from this, T. durum (Null, 1Bx7+1By8), T. monococcum (1Ax+1Ay), Ae. tauschii (1Dx5t +1Dy12.5t), Oryza sativa and Setaria italica were also used as controls in the PCR amplification reactions. SDS-PAGE and HPCE Brachypodium matured seeds (about 50 mg) were ground to fine powder with liquid nitrogen. HMW-GS were extracted and separated on 10 % sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) according to Yan et al.
J Appl Genetics (2014) 55:27–42
(2003a), with minor modifications. Chinese Spring (CS) was used as the control and a protein marker (14–100 kDa) was used to measure the molecular mass of HMW-GS. Extracted HMW-GS were precipitated with cold acetone for high performance capillary electrophoresis (HPCE) analysis. Palletised HMW-GS samples were resolved in 0.2 ml of 25 % (v/v) acetonitrile (ACN)+0.1 % (v/v) trifluoroacetic acid (TFA) and centrifuged at 13,000 rpm for 10 min. A Beckman P/ACE 800 system with Gold Software for system control and data acquisition was used to separate glutenin subunits based on Yan et al. (2003c) and Li et al. (2012). HPCE was carried out with an acidic buffer containing 0.1 M phosphate-glycine (pH 2.5), 20 % (v/v) ACN and 0.05 % (w/v) hydroxypropyl methylcellulose (HPMC). Uncoated fused-silica capillaries of 30 cm in length (25 cm to the detector) and 50-μm inner diameters were used at 12.5 kV and 38 °C. The sample was injected at 11 kV for 8 s and UV absorbance at 200 nm was used to detect HMW-GS.
29
1Dx2 (X03346), 1Bx7 (X13927), 1Bx14 (AY367771), 1By8 (AY245797), 1Sy (AF513640) and 1By16 (EF540765) deposited in GenBank, four pairs of allele-specific (AS) PCR primers PF3a+PR3a, PF3b+PR3b, PF3c+PR3c and PF3d+ PR3d (+1 bp to +1977 bp) were designed and used to amplify HMW-GS encoding genes in Brachypodium. All the primers used and their sequence details are listed in Table 1. PCR amplification was performed on a Thermal Cycler with a 30-μl reaction mixture containing 100 ng DNA as a template, 2× GC buffer (MgCl2 +Plus), LA-Taq polymerase (Takara), 0.4 mM dNTP and 0.5 μM of each oligonucleotide primer and DD H2O to a 30-μL volume. The PCR reaction system was 95 °C for 4 min initial denaturation, followed by 34 cycles of 45 s at 95 °C, 1 min at 60 °C, 2.10 min at 72 °C and 10 min at 72 °C as the final extension step. The PCR products were separated in 1.0 % agarose gels. Molecular cloning, sequencing and comparison of HMW-GS genes
LC-MS/MS The protein bands on SDS-PAGE gel were excised and subjected to trypsin digestion (Jin et al. 2012). The digested peptide samples were separated and identified by HPLCESI-MS/MS (Thermo Fisher Scientific). The identified proteins were obtained by using the MASCOT software through searching with the raw file for the NCBInr protein sequence database. The following search parameters were used in all MASCOT searches: peptide false discovery rate (FDR)
PLANT GENETICS • ORIGINAL PAPER
Molecular characterisation and evolution of HMW glutenin subunit genes in Brachypodium distachyon L. Saminathan Subburaj & Guanxing Chen & Caixia Han & Dongwen Lv & Xiaohui Li & Friedrich J. Zeller & Sai L. K. Hsam & Yueming Yan
Received: 15 August 2013 / Revised: 10 November 2013 / Accepted: 19 November 2013 / Published online: 4 December 2013 # Institute of Plant Genetics, Polish Academy of Sciences, Poznan 2013
Abstract Brachypodium distachyon, a small wild grass within the Pooideae family, is a new model organism for exploring the functional genomics of cereal crops. It was shown to have close relationships to wheat, barley and rice. Here, we describe the molecular characterisation and evolutionary relationships of high molecular weight glutenin subunits (HMWGS) genes from B. distachyon. Sodium dodecyl sulphate– polyacrylamide gel electrophoresis (SDS-PAGE), high performance capillary electrophoresis (HPCE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses demonstrated that there was no HMW-GS expression in the Brachypodium grains due to the silencing of their encoding genes. Through allele-specific polymerase chain reaction (ASPCR) amplification and cloning, a total of 13 HMW-GS encoding genes from diploid, tetraploid and hexaploid Brachypodium species were obtained, and all of them had typical structural features of y-type HMW-GS genes from common wheat and related species, particularly more similar to the 1Dy12 gene. However, the presence of an in-frame premature stop codon (TAG) at position 1521 in the coding region resulted in the conversion of all the genes to pseudogenes. Further, quantitative real-time PCR (qRT-
Saminathan Subburaj and Guanxing Chen contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s13353-013-0187-4) contains supplementary material, which is available to authorized users. S. Subburaj : G. Chen : C. Han : D. Lv : X. Li (*) : Y. Yan (*) College of Life Science, Capital Normal University, 100048 Beijing, China e-mail: [email protected] e-mail: [email protected] F. J. Zeller : S. L. K. Hsam Division of Plant Breeding and Applied Genetics, Technical University of Munich, 85354 Freising, Germany
PCR) analysis revealed that HMW-GS genes in B. distachyon displayed a similar trend, but with a low transcriptional expression profile during grain development due to the occurrence of the stop codon. Phylogenetic analysis showed that the highly conserved Glu-1-2 loci were presented in B. distachyon, which displayed close phylogenetic evolutionary relationships with Triticum and related species. Keywords Brachypodium distachyon . HMW glutenin subunits . AS-PCR . qRT-PCR . Phylogeny and evolution
Introduction The Poaceae grass family includes some major temperate cereal crops, such as wheat, rice, corn, barley, rye and sorghum and forage grasses. These cereals contain 7–15 % protein in mature grains and serve as important protein sources for human consumption and animal feed. Brachypodium distachyon, a small wild grass within the Pooideae subfamily, has emerged as a new model system for the study of these temperate cereals (Draper et al. 2001; Hasterok et al. 2004; Vogel et al. 2006a, b), and its whole genome sequencing has been completed recently (International Brachypodium Initiative 2010). Because of its short life cycle (Garvin et al. 2008; Mur et al. 2011), very small genome in monocots (Wolny and Hasterok 2009) and low nutritional requirements (Vogel et al. 2009), B. distachyon has become more suitable for functional and comparative genomics studies. Prolamins are important endosperm storage proteins in cereal grains. Based on their molecular weights and sulphur contents, they can be further classified into three subgroups: high molecular weight (HMW) prolamins, S-rich prolamins and S-poor prolamins (Shewry et al. 1999). Gluten, the most important grain prolamin in common wheat (Triticum aestivum L.), comprises HMW prolamins (HMW-GS), S-
28
rich prolamin [α/β, γ-gliadins and B/C-type low molecular weight (LMW)-GS] and S-poor prolamins (ω-gliadins and Dtype LMW-GS). It is known that HMW-GS mainly affect the dough elasticity, while LMW-GS and gliadins determine dough viscosity and extensibility, respectively. These dough properties are essential for bread-making quality (Payne 1987; Shewry and Halford 2002). Genes coding for HMW-GS are located at the Glu-1 loci on the long arm of the group 1 homeologous chromosomes of the A, B and D genomes in hexaploid bread wheat. Glu-1 loci include two tightly linked genes Glu-1-1 and Glu-1-2, which encode a larger x-type (80–88 kDa) and a smaller y-type (67– 73 kDa) HMW-GS, respectively (Mackie et al. 1996). However, only 3–5 subunits generally express in the individual hexaploid cultivars due to the silencing of the genes coding for y-type HMW-GS at the Glu-A1 and Glu-B1 loci (Payne 1987). Orthologous HMW-GS are present in Triticum and related species such as Aegilops and rye (De Bustos and Jouve 2003; Yan et al. 2003a, 2004; Zhang et al. 2008; Jin et al. 2012). Apart from the critical role for wheat end products, HMW-GS genes are also able to provide useful information for understanding the evolutionary relationships among Triticeae and its related species (Yan et al. 2003b). B. distachyon is the only annual member of the tribe Brachypoideae (Khan and Stace 1999) and has a strong physiological and genetic similarity to its “core pooids”, a subgroup that includes wheat, barley and rye (Huo et al. 2009). Originally, B. distachyon acts as a connecting bridge between wheat and rice, but phylogenetic analysis indicated that B. distachyon has higher sequence similarities with the wheat genome (Ozdemir et al. 2008). Also, the divergence time between wheat and B. distachyon is closer than that between wheat and rice (Vogel et al. 2006b; Mur et al. 2011). Multiple alignment of several grass genomes showed that B. distachyon contains prolamin-like genes named as Brachypodins-III , which were under the class of HMW glutenins (Xu and Messing 2009). Moreover, an inactive copy of HMW glutenin-like gene was found in B. distachyon ’s Glu-1 loci by orthologous comparison between different grass genomes (Gu et al. 2010). The grain characteristics of Brachypodium showed more resemblance to that of the wheat species (Ozdemir et al. 2008; Opanowicz et al. 2011) than to rice. However, B. distachyon is similar to rice by the presence of glutelin-like protein bodies in the endosperm (Larré et al. 2010). Furthermore, proteomic analysis of some hexaploid B. distachyon accessions showed that their major seed storage proteins were 11S-globulins (Laudencia-Chingcuanco and Vensel 2008), whereas, in the mature stages of grains, an unstoppable proteinaceous matrix made B. distachyon grains similar to wheat with significant similarities in the grain endosperm development (Hands and Drea 2012). Apart from the presence of globulins, there were some prolamin proteins displaying homology to HMW glutenins in the grains of
J Appl Genetics (2014) 55:27–42
diploid Bd21 (Larré et al. 2010). Also, Wang et al. (2010) and Hammami et al. (2011) found that there were enormous amounts of LMW-like glutenins with similar electrophoretic patterns to wheat in Brachypodium seeds. More recently, a highly conserved Glu-3 locus was confirmed to be present in Brachypodium as those in Triticum and its related species (Wang et al. 2012a). Until now, limited information on the structural features and evolution of HMW-GS genes in B. distachyon has been available. In the current study, we have conducted further investigation on the molecular characterisation of HMW-GS genes from diploid, tetraploid and hexaploid B. distachyon accessions and their phylogenetic revolutionary relationships with Triticum and other Poaceae species. Our results reveal a highly conserved Glu-1 locus that is present in B. distachyon and close evolutionary relationships with Aegilops and Triticum species.
Materials and methods Plant materials The seeds of B. distachyon accessions of Bd2 (PI185133), Bd3 (PI185134), Bd4 (PI208216), Bd10 (PI226452), Bd12 (PI227011), Bd13 (PI233228), Bd14 (PI239713), Bd16 (PI239715), Bd17 (PI239716), Bd21 (PI254867), Bd21-3, BdISKP1 and Bd347 were kindly provided by Dr. John Vogel, United States Department of Agriculture—Agriculture Research Service (USDA-ARS), USA, and Dr. Chengtao Lin, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (CAAS), China. Accessions Bd2, Bd3, Bd21-3 and Bd21 were diploid (2n=10), while other genotypes are hexaploid, except the tetraploid Bd347 (Vogel et al. 2006b). The hexaploid common wheat cultivar Chinese Spring (Triticum aestivum L., 2n=6x=42, AABBDD) with HMW-GS compositions of Null, 1Bx7+1By8 and 1Dx2+1Dy12, and spelt wheat cv. Renval (Triticum spelta L., 2n=6x=42, AABBDD) with 1Ax1, 1Bx13*+1By19* and 1Dx2+1Dy12 (Yan et al. 2003b) were used as standards for glutenin identification as well as the controls for polymerase chain reaction (PCR) and quantitative real-time PCR (qRT-PCR) amplifications. Apart from this, T. durum (Null, 1Bx7+1By8), T. monococcum (1Ax+1Ay), Ae. tauschii (1Dx5t +1Dy12.5t), Oryza sativa and Setaria italica were also used as controls in the PCR amplification reactions. SDS-PAGE and HPCE Brachypodium matured seeds (about 50 mg) were ground to fine powder with liquid nitrogen. HMW-GS were extracted and separated on 10 % sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) according to Yan et al.
J Appl Genetics (2014) 55:27–42
(2003a), with minor modifications. Chinese Spring (CS) was used as the control and a protein marker (14–100 kDa) was used to measure the molecular mass of HMW-GS. Extracted HMW-GS were precipitated with cold acetone for high performance capillary electrophoresis (HPCE) analysis. Palletised HMW-GS samples were resolved in 0.2 ml of 25 % (v/v) acetonitrile (ACN)+0.1 % (v/v) trifluoroacetic acid (TFA) and centrifuged at 13,000 rpm for 10 min. A Beckman P/ACE 800 system with Gold Software for system control and data acquisition was used to separate glutenin subunits based on Yan et al. (2003c) and Li et al. (2012). HPCE was carried out with an acidic buffer containing 0.1 M phosphate-glycine (pH 2.5), 20 % (v/v) ACN and 0.05 % (w/v) hydroxypropyl methylcellulose (HPMC). Uncoated fused-silica capillaries of 30 cm in length (25 cm to the detector) and 50-μm inner diameters were used at 12.5 kV and 38 °C. The sample was injected at 11 kV for 8 s and UV absorbance at 200 nm was used to detect HMW-GS.
29
1Dx2 (X03346), 1Bx7 (X13927), 1Bx14 (AY367771), 1By8 (AY245797), 1Sy (AF513640) and 1By16 (EF540765) deposited in GenBank, four pairs of allele-specific (AS) PCR primers PF3a+PR3a, PF3b+PR3b, PF3c+PR3c and PF3d+ PR3d (+1 bp to +1977 bp) were designed and used to amplify HMW-GS encoding genes in Brachypodium. All the primers used and their sequence details are listed in Table 1. PCR amplification was performed on a Thermal Cycler with a 30-μl reaction mixture containing 100 ng DNA as a template, 2× GC buffer (MgCl2 +Plus), LA-Taq polymerase (Takara), 0.4 mM dNTP and 0.5 μM of each oligonucleotide primer and DD H2O to a 30-μL volume. The PCR reaction system was 95 °C for 4 min initial denaturation, followed by 34 cycles of 45 s at 95 °C, 1 min at 60 °C, 2.10 min at 72 °C and 10 min at 72 °C as the final extension step. The PCR products were separated in 1.0 % agarose gels. Molecular cloning, sequencing and comparison of HMW-GS genes
LC-MS/MS The protein bands on SDS-PAGE gel were excised and subjected to trypsin digestion (Jin et al. 2012). The digested peptide samples were separated and identified by HPLCESI-MS/MS (Thermo Fisher Scientific). The identified proteins were obtained by using the MASCOT software through searching with the raw file for the NCBInr protein sequence database. The following search parameters were used in all MASCOT searches: peptide false discovery rate (FDR)
