Relationships between puroindoline A-prolamin interactions and wheat grain hardness.

Nathalie Geneix,Isabelle Nadaud,Didier Marion,Khalil Elmorjani,Michèle Dalgalarrondo,Caroline Tassy,Bénédicte Bakan,Pierre Barret,Ajay Kumar

doi:10.1371/journal.pone.0225293

Nathalie Geneix, Isabelle Nadaud + Show 7 more

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https://doi.org/10.1371/journal.pone.0225293

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Abstract

Grain hardness is an important quality trait of cereal crops. In wheat, it is mainly determined by the Hardness locus that harbors genes encoding puroindoline A (PINA) and puroindoline B (PINB). Any deletion or mutation of these genes leading to the absence of PINA or to single amino acid changes in PINB leads to hard endosperms. Although it is generally acknowledged that hardness is controlled by adhesion strength between the protein matrix and starch granules, the physicochemical mechanisms connecting puroindolines and the starch-protein interactions are unknown as of this time. To explore these mechanisms, we focused on PINA. The overexpression in a hard wheat cultivar (cv. Courtot with the Pina-D1a and Pinb-D1d alleles) decreased grain hardness in a dose-related effect, suggesting an interactive process. When PINA was added to gliadins in solution, large aggregates of up to 13 μm in diameter were formed. Turbidimetry measurements showed that the PINA-gliadin interaction displayed a high cooperativity that increased with a decrease in pH from neutral to acid (pH 4) media, mimicking the pH change during endosperm development. No turbidity was observed in the presence of isolated α– and γ-gliadins, but non-cooperative interactions of PINA with these proteins could be confirmed by surface plasmon resonance. A significant higher interaction of PINA with γ-gliadins than with α–gliadins was observed. Similar binding behavior was observed with a recombinant repeated polypeptide that mimics the repeat domain of gliadins, i.e., (Pro-Gln-Gln-Pro-Tyr)8. Taken together, these results suggest that the interaction of PINA with a monomeric gliadin creates a nucleation point leading to the aggregation of other gliadins, a phenomenon that could prevent further interaction of the storage prolamins with starch granules. Consequently, the role of puroindoline-prolamin interactions on grain hardness should be addressed on the basis of previous observations that highlight the similar subcellular routing of storage prolamins and puroindolines.

Highlights

Grain hardness is a major quality trait that determines the milling properties of wheat and subsequent end-uses of flour
Wheat hardness and puroindoline A-prolamin interactions position 44, i.e., in the tryptophan-rich domain (TRD) that determines the physicochemical properties of PIN [1]
In our experiments, hardness changes are due only to the overexpressed puroindoline A (PINA), a result strengthened by the absence of modifications in the contents of the major putative molecular targets of puroindolines, i.e., lipids and proteins, which control the interactions of the protein matrix with starch granules

Summary

Introduction

Grain hardness is a major quality trait that determines the milling properties of wheat and subsequent end-uses of flour. Two major proteins are expressed, puroindoline A (PINA) and puroindoline B (PINB). PINA and PINB are small (approximately 13 kDa based on their amino acid sequence) cationic proteins (pI around 10), containing five disulfide bonds and a tryptophan-rich domain (TRD). Deletion of the Pina-D1 gene or single mutation of the Pinb-D1 gene is associated with a hard phenotype, whereas the presence of both Pina-D1a and Pinb-D1a genes is associated with the soft phenotype [1]. Single mutations can affect the TRD (Pinb-D1b, Pinb-D1d) or small hydrophobic domains (PinbD1c). Null mutations of the Pinb-D1 gene, which is associated with a hard phenotype, are due to the insertion of a stop codon in the coding sequence leading to the absence of PINB [1]

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