A single amino acid substitution in puroindoline b impacts its self-assembly and the formation of heteromeric assemblies with puroindoline a
Introduction
The grain composition determines the end-uses properties of wheat such as milling and baking properties. These technological features are closely related to the supramolecular assemblies between endosperm constituents, namely starch, proteins and lipids. More precisely, the milling properties of wheat grain are closely related to grain hardness, a major physical parameter describing grain texture.
Grain hardness is related to the compactness of the endosperm starch-protein matrix and is genetically controlled by allelic variations of a major locus (Hardness locus, Ha) located on the short arm of the chromosome 5D. The Ha-locus, that determines the ‘Hard’ or ‘Soft’ character of wheat grain, encodes the puroindoline genes (Morris et al., 2001, Morris, 2002). Two major proteins were expressed, puroindoline (PINA) and puroindoline b (PINB). Puroindolines (PINs) are small cationic proteins (approximately 13 kDa) containing five disulfide bonds and a tryptophan-rich domain. Their predicted 3D structure is homologous to the structure of 2S albumins of dicot seeds (Lesage et al., 2011). Allelic variations due to point mutations, null mutations or deletions of puroindoline genes (Pina-D1 and Pinb-D1) are associated with increased grain hardness while the presence of both wild type (WT) genes, i.e. Pina-D1a and Pinb-D1a are necessary for the expression of softness (Morris, 2002, Bhave and Morris, 2008b). A single nucleotide mutation in Pinb-D1 leading to a glycine-to-serine change at position 46 in PINB (G46S; PINB-D1b) was the first hardness-related mutation reported (Giroux and Morris, 1998). Other PINB mutations were further highlighted that all confirmed a relationship to the ‘Hard’ character of wheat grain (e.g. Pinb-D1c, L60P and Pinb-D1d, W44R) (Lillemo and Morris, 2000). Finally, it was shown that the over-expression of the soft alleles in Hard cultivars led to a significant decrease of grain hardness (Bhave and Morris, 2008b) and that a cooperative interaction between WT PINA and PINB is associated to grain softness (Wanjugi et al., 2007, Alfred et al., 2014).
While PINs are undoubtedly involved in grain hardness, the question of their precise role in the cohesion of the starch-protein matrix remains totally open. It has been suggested that the interaction of PINs with lipids may control the association between the starch granules and proteins, via membrane remnants found at this interface (Douliez et al., 2000). Indeed, both PINA and PINB interact with lipids in vitro, via their respective tryptophan-rich domain. It has been suggested that the observed differences in the lipid binding properties of PINs are linked to small changes in the conformation of the tryptophan-rich domain (Dubreil et al., 1997, Le Guernevé et al., 1998, Clifton et al., 2007a, Clifton et al., 2007b, Clifton et al., 2011b). Furthermore, in the case of G46S and W44R PINB mutants, it has been shown that these single mutations can affect, in vitro, the penetration into an anionic phospholipid (DPPG) monolayers (Clifton et al., 2007b, Clifton et al., 2011b). Besides grain hardness, the lipid binding properties are related to two other important functional properties of these unique plant proteins, i.e. their antimicrobial and foaming properties (Douliez et al., 2000, Bhave and Morris, 2008a).
Many works have focused on the interactions of PINs with lipids, while only few data are available concerning their interactions with other components, such as proteins. Like prolamins, the major storage proteins of wheat endosperm, PINs contents increase during endosperm development, slow down during maturation and PINs are degraded upon seed germination (Dubreil et al., 1998, Pauly et al., 2013). Using high resolution electron microscopy, it was shown that PINs were located in storage protein bodies of developing endosperm (Lesage et al., 2011) to be finally found, after desiccation, in both the protein matrix and at the starch-protein interface (Dubreil et al., 1998). A relationship between endosperm texture and the size of storage protein polymers was evidenced by asymmetrical flow field-flow fractionation (AF4). These studies suggested that PINs could also interact with proteins, impacting both polymer sizes and consequently endosperm texture (Lesage et al., 2012).
Beyond PIN-lipid interaction, different studies have highlighted the aggregative properties of PINA in vitro (Dubreil et al., 2003, Clifton et al., 2011a). Consequently, we decided to pinpoint the self-association of PINA and especially PINB in relation with mutations describing endosperm texture in soft/hard wheat, i.e. wild-type PINA (PINA-D1a) and PINB (PINB-D1a), and mutants PINB (PINB-D1b) and PINB (PINB-D1d) containing the Gly-46 to Ser-46 and the Trp-44 to Arg-44 mutations respectively. We also studied the aggregative properties of PINA and PINB mixtures in relation with the observed interdependence of both proteins on grain hardness (Wanjugi et al., 2007).
Section snippets
Chemicals
All chemicals were sourced from Sigma–Aldrich or Merck and were of the highest purity available. Protein solutions (0.5 g L−1) were prepared in phosphate buffer solution Na2HPO4 and NaH2PO4 at ionic strength 0.02 and 0.1 M at pH 7.2, pH 5.5 or pH 9.0.
Wheat material
PINA and PINB were purified from three wheat Triticum aestivum varieties: the soft variety Paledor (Pina-D1a and Pinb-D1a alleles), the hard varieties, Recital (Pina-D1a and Pinb-D1b alleles-G46S mutation) and Courtot (Pina-D1a and Pinb-D1d alleles
Characterization of purified puroindolines
PIN proteins were isolated from three wheats cultivars displaying contrasted endosperm textures: soft Paledor (Pina-D1a and Pinb-D1a alleles), hard variety Recital (Pina-D1a and the mutant Pinb-D1b alleles) and hard variety Courtot (Pina-D1a and Pinb-D1d alleles).
In a preliminary step, the purity and primary structure of isolated PINs was checked by using different methods, including SDS and acid-PAGE, reversed-phase HPLC and mass spectrometry. In SDS-PAGE, PINA and PINB migrated as a single
Conclusion
In solution, PINs form aggregates that are polydisperse in size. A significant proportion of PINA self-associates into dimers while different aggregative properties are displayed by PINB proteins. Indeed, wild type PINB, i.e. PINB-D1a, tends to form monomers while PINBs from hard varieties, i.e. PINB-D1b and PINB-D1d, form large aggregates. The aggregative properties of mixed protein dispersions show that PINB proteins interfere with PINA aggregation in good agreement with the independence of
Acknowledgments
We are grateful to André Lelion for his expert technical assistance in purification of proteins. We wish to thank Sophie Guilois for her technical assistance with AF4.
ESI-MS experiments were performed using the facilities of the Biopolymers, Structural Biology platform, INRA Nantes, France.
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