Identification of novel glutelin subunits and a comparison of glutelin composition between japonica and indica rice (Oryza sativa L.)

Abstract

Glutelin, a major protein in rice grains, is encoded by a multigene family. However, its protein composition is not well characterised. Here, we identified and characterised two novel glutelin subunits, GluBX and GluC. The individual glutelin subunits of japonica cv. Nipponbare and indica cv. 93-11 rice were analysed using 2-dimensional gel electrophoresis, LC–MS/MS, and Western blotting. Comparison of the glutelin profiles between three japonica and three indica cultivars indicated two distinct subunits (GluA-1 and GluA-3 isomers) and a distinction in the subunit composition (notably GluA-3 and Lys-rich GluB-1 components) of these two subspecies. Sequence alignment revealed different nutritional (Lys residues) and functional (Cys residues) characteristics between the type-A and type-B glutelin subfamilies. We also analysed amino acid and total protein contents of the grains in thirty-five cultivars, and we demonstrated that the Lys-rich glutelin composition of indica cultivars is superior to that of japonica cultivars. The Lys-rich and Cys-poor GluBX subunit is a native protein and is a high nutritional protein in grains. Our combined approaches for the identification of glutelin subunits have revealed the nutritional characteristics of individual subunits in rice, and this knowledge will provide new insights for improving grain quality during rice breeding.

Introduction

The nutritional quality of rice (Oryza sativa L.) is incredibly important because rice is one of the most important crops in the world, providing 21% of the calories and 15% of the protein for the global human population. The protein content and amino acid composition are regarded as key determinants for the nutritional quality of the crop (Ufaz and Galili, 2008). The protein content of rice grains accounts for 9% of the seed dry weight. The cereal seed storage proteins include four components: 1) water-soluble albumin, 2) dilute saline-soluble globulin, 3) alcohol-soluble prolamin, and 4) acid- or alkali-soluble glutelin. Among these protein components, glutelin is much safer for humans than albumin and globulin (Ito et al., 2005), and glutelin is more digestible than prolamin (Chandi and Sogi, 2007; Van Der Borght et al., 2006). With respect to rice, glutelin accounts for approximately 80% of the seed storage proteins. The high nutritive value of glutelin is attributed to its amino acid composition, as most cereals are generally depleted of Lys (Ufaz and Galili, 2008). Thus, the seed protein composition, composed of mostly glutelin, is an attractive target for enhancing the nutritional value of rice grains.

Mature rice glutelin is derived from the cleavage of proglutelin (57 kDa) to form the 35 kDa acidic (α-polypeptide) and 22 kDa basic (β-polypeptide) polypeptides, which are linked by a disulfide bond (Adachi et al., 2003; Shewry and Halford, 2002; Wang et al., 2009). Glutelins are encoded by a multigene family that includes four subfamilies: 1) type-A, 2) type-B, 3) type-C, and 4) type-D (Kawakatsu et al., 2008). Type-A and type-B are the major subfamilies, consisting of four (GluA-1, GluA-2, GluA-3, and the GluA-4 pseudogene) and five members (GluB-1, GluB-2, GluB-4, GluB-5, and the GluB-3 pseudogene) (Qu et al., 2008; Takaiwa et al., 1991), respectively. The corresponding glutelin subunits share more than 80% sequence similarity within each subfamily (type-A or type-B), and share 60% similarity between subfamilies type-A and type-B (Katsube-Tanaka et al., 2004). Lys is the first limiting essential amino acid for most cereals (Ufaz and Galili, 2008). Glutelin type-B proteins contain more Lys than type-A proteins; thus, it has been speculated that rice grains containing greater Lys-rich type-B components have a higher nutritive value than grains with less type-B components (Katsube-Tanaka et al., 2004; Takaiwa et al., 1991; Van Der Borght et al., 2006). Modern methods for rice grain nutrition enhancement include optimising the seed protein composition with related mutants (Kawakatsu et al., 2010; Motoyama et al., 2009), which requires prerequisite knowledge of the native seed storage proteins (in particular rice glutelin). Therefore, not only would one have to isolate and identify certain Lys-rich glutelin subunits, but they would also have to determine the glutelin composition needed to improve the rice grain nutrition.

The published rice genomic sequences facilitated the discovery of glutelin family members (Ohyanagi et al., 2006). Presently, fifteen glutelin genes have been reported, nine of which have been well identified and characterised in japonica rice (Kawakatsu et al., 2008; Qu et al., 2008). Nevertheless, some of the glutelin subunits have not been identified in rice genomic sequences via experimental approaches. All glutelin members have similar molecular weights (MWs) and a high degree of sequence homology. Two-dimensional and/or sodium dodecyl sulphate polyacrylamide gel electrophoresis (2D/SDS-PAGE) and high performance liquid chromatography (HPLC) are widely used techniques for separating the glutelin subunits. Additionally, Western blotting and/or peptide mass spectrometry (MS) are used to identify the glutelin subunits. Currently, the major glutelin subunits (GluA-1, GluA-2, GluA-3, GluB-1, GluB-2, GluB-4, and GluB-5) and two major subfamilies (type-A and type-B) have been successfully discriminated using these methods (Katsube-Tanaka et al., 2004; Khan et al., 2008a, 2008b; Van Der Borght et al., 2006). However, it is still important to discover new family members and to identify glutelin subunits in rice; this knowledge will further advance the usage of glutelins as nutritional proteins.

In this study, individual glutelin subunits of indica cv. 93-11 and japonica cv. Nipponbare were isolated using 2D-PAGE, and further identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS) and database searching. Finally, Western blot analysis using subunit-specific antibodies was employed. Based on our subunit identification results, we discovered two novel glutelin subunits, which we named GluBX and GluC, in the UniProtKB database using a bioinformatics approach. The two novel subunits were further characterised via molecular cloning, prokaryotic expression, and Western blot analysis. Following glutelin subunit identification, the glutelin composition and the nutritive value (specifically Lys) of the glutelin members in three japonica and three indica cultivars were assessed by 2D-gel analysis. Furthermore, the amino acid composition and the total protein content of thirty-five cultivars were analysed to assess the nutritive value of the grain. Together, our results further clarify the relationship between grain nutritional quality, which includes the grain protein content and amino acid composition, and the nutritional glutelin composition (Lys-rich glutelin subunits). These findings will provide new insights for improving the quality of grain in rice breeding.