Antifungal activity of puroindoline protein from soft wheat against grain molds and its potential as a biocontrol agent.

Mold growth reduces the quality of stored grains, besides producing toxins that pose a potential threat to human health. Therefore, prevention of grain mold growth during storage is important to ensure a safe and high-quality product, preferably using an eco-friendly antifungal agent. The puroindoline (PIN) protein was extracted by Triton X-114 and identified by QE mass spectrometry. Aspergillus flavus has attracted much attention because of its toxic secondary metabolites, and PIN protein showed a significant inhibition on A. flavus growth. Scanning electron microscopy revealed altered spore morphology of A. flavus following PIN protein treatment, and propidium iodide staining showed incomplete spore cell membranes. The disruption and deformation of A. flavus spores suggest that the cell walls and cell membranes were compromised. Decreased mitochondrial membrane potential and increased levels of intracellular reactive oxygen specieswere detected using JC-1 and 2,7-dichlorodihydrofluorescein diacetate staining, respectively. PIN protein could effectively inhibit the growth and aflatoxins B1 production of A. flavus in stored grains, such as wheat and rice. PIN proteins can inhibit the growth of many common grain storage molds, including Penicillium, Aspergillus spp. (A. flavus, A. glaucus, A. kawachii, A. ochraceus and A. niger), Alternaria and Fusarium graminearum, in a dose-dependent manner. PIN protein has a significant inhibitory effect on the growth of grain molds, with a stronger inhibitory effect noted in wheat and rice. Our study provides a novel and simple theoretical basis for the selection and storage of mold resistance in grains and food during storage.

Genomics-Enabled Analysis of Puroindoline b2 Genes Identifies New Alleles in Wheat and Related Triticeae Species.

Kernel hardness is a key trait of wheat seeds, largely controlled by two tightly linked genes Puroindoline a and b (Pina and Pinb). Genes homologous to Pinb, namely Pinb2, have been studied. Whether these genes contribute to kernel hardness and other important seed traits remains inconclusive. Using the high-quality bread wheat reference genome, we show that PINB2 are encoded by three homoeologous loci Pinb2 not syntenic to the Hardness locus, with Pinb2-7A locus containing three tandem copies. PINB2 proteins have several features conserved for the Pin/Pinb2 phylogenetic cluster but lack a structural basis of significant impact on kernel hardness. Pinb2 are seed-specifically expressed with varied expression levels between the homoeologous copies and among wheat varieties. Using the high-quality genome information, we developed new Pinb2 allele specific markers and demonstrated their usefulness by 1) identifying new Pinb2 alleles in Triticeae species; and 2) performing an association analysis of Pinb2 with kernel hardness. The association result suggests that Pinb2 genes may have no substantial contribution to kernel hardness. Our results provide new insights into Pinb2 evolution and expression and the new allele-specific markers are useful to further explore Pinb2's contribution to seed traits in wheat.

Effects of Rationally Designed Physico-Chemical Variants of the Peptide PuroA on Biocidal Activity towards Bacterial and Mammalian Cells.

Antimicrobial peptides (AMPs) often exhibit wide-spectrum activities and are considered ideal candidates for effectively controlling persistent and multidrug-resistant wound infections. PuroA, a synthetic peptide based on the tryptophan (Trp)-rich domain of the wheat protein puroindoline A, displays strong antimicrobial activities. In this work, a number of peptides were designed based on PuroA, varying in physico-chemical parameters of length, number of Trp residues, net charge, hydrophobicity or amphipathicity, D-versus L-isomers of amino acids, cyclization or dimerization, and were tested for antimicrobial potency and salt and protease tolerance. Selected peptides were assessed for effects on biofilms of methicillin-resistant Staphylococcus aureus (MRSA) and selected mammalian cells. Peptide P1, with the highest amphipathicity, six Trp and a net charge of +7, showed strong antimicrobial activity and salt stability. Peptides W7, W8 and WW (seven to eight residues) were generally more active than PuroA and all diastereomers were protease-resistant. PuroA and certain variants significantly inhibited initial biomass attachment and eradicated preformed biofilms of MRSA. Further, P1 and dimeric PuroA were cytotoxic to HeLa cells. The work has led to peptides with biocidal effects on common human pathogens and/or anticancer potential, also offering great insights into the relationship between physico-chemical parameters and bioactivities, accelerating progress towards rational design of AMPs for therapeutics.

Co-expression of high-molecular-weight glutenin subunit 1Ax1 and Puroindoline a (Pina) genes in transgenic durum wheat (Triticum turgidum ssp. durum) improves milling and pasting quality.

BACKGROUND: Durum wheat is considered not suitable for making many food products that bread wheat can. This limitation is largely due to: (i) lack of grain-hardness controlling genes (Puroindoline a and b) and consequently extremely-hard kernel; (ii) lack of high- and low-molecular-weight glutenin subunit loci (Glu-D1 and Glu-D3) that contribute to gluten strength. To improve food processing quality of durum wheat, we stacked transgenic Pina and HMW-glutenin subunit 1Ax1 in durum wheat and developed lines with medium-hard kernel texture. RESULTS: Here, we demonstrated that co-expression of Pina + 1Ax1 in durum wheat did not affect the milling performance that was enhanced by Pina expression. While stacking of Pina + 1Ax1 led to increased flour yield, finer flour particles and decreased starch damage compared to the control lines. Interestingly, Pina and 1Ax1 co-expression showed synergistic effects on the pasting attribute peak viscosity. Moreover, Pina and 1Ax1 co-expression suggests that PINA impacts gluten aggregation via interaction with gluten protein matrix. CONCLUSIONS: The results herein may fill the gap of grain hardness between extremely-hard durum wheat and the soft kernel durum wheat, the latter of which has been developed recently. Our results may also serve as a proof of concept that stacking Puroindolines and other genes contributing to wheat end-use quality from the A and/or D genomes could improve the above-mentioned bottleneck traits of durum wheat and help to expand its culinary uses.

Diversity, distribution of Puroindoline genes and their effect on kernel hardness in a diverse panel of Chinese wheat germplasm.

BACKGROUND: Kernel hardness, which has great influence on the end-use properties of common wheat, is mainly controlled by Puroindoline genes, Pina and Pinb. Using EcoTILLING platform, we herein investigated the allelic variations of Pina and Pinb genes and their association with the Single Kernel Characterization System (SKCS) hardness index in a diverse panel of wheat germplasm. RESULTS: The kernel hardness varied from 1.4 to 102.7, displaying a wide range of hardness index. In total, six Pina and nine Pinb alleles resulting in 15 genotypes were detected in 1787 accessions. The most common alleles are the wild type Pina-D1a (90.4%) and Pina-D1b (7.4%) for Pina, and Pinb-D1b (43.6%), Pinb-D1a (41.1%) and Pinb-D1p (12.8%) for Pinb. All the genotypes have hard type kernel hardness of SKCS index (>60.0), except the wild types of Pina and Pinb combination (Pina-D1a/Pinb-D1a). The most frequent genotypes in Chinese and foreign cultivars was Pina-D1a/Pinb-D1b (46.3 and 39.0%, respectively) and in Chinese landraces was Pina-D1a/Pinb-D1a (54.2%). The frequencies of hard type accessions are increasing from 35.5% in the region IV, to 40.6 and 61.4% in the regions III and II, and then to 77.0% in the region I, while those of soft type are accordingly decreasing along with the increase of latitude. Varieties released after 2000 in Beijing, Hebei, Shandong and Henan have higher average kernel hardness index than that released before 2000. CONCLUSION: The kernel hardness in a diverse panel of Chinese wheat germplasm revealed an increasing of kernel hardness generally along with the latitude across China. The wild type Pina-D1a and Pinb-D1a, and one Pinb mutant (Pinb-D1b) are the most common alleles of six Pina and nine Pinb alleles, and a new double null genotype (Pina-D1x/Pinb-D1ah) possessed relatively high SKCS hardness index. More hard type varieties were released in recent years with different prevalence of Pin-D1 combinations in different regions. This work would benefit the understanding of the selection and molecular processes of kernel hardness across China and different breeding stages, and provide useful information for the improvement of wheat quality in China.

The grain Hardness locus characterized in a diverse wheat panel (Triticum aestivum L.) adapted to the central part of the Fertile Crescent: genetic diversity, haplotype structure, and phylogeny.

Wheat belongs to the most important crops domesticated in the Fertile Crescent. In this region, fortunately, locally adapted wheat landraces are still present in farmers' fields. This material might be of immense value for future breeding programs. However, especially wheat germplasm adapted to the central part of the Fertile Crescent has been poorly characterized for allelic variation at key loci of agricultural importance. Grain hardness is an important trait influencing milling and baking quality of wheat. This trait is mainly determined by three tightly linked genes, namely, Puroindoline a (Pina), Puroindoline b (Pinb), and Grain softness protein-1 (Gsp-1), at the Hardness (Ha-D) locus on chromosome 5DS. To investigate genetic diversity and haplotype structure, we resequenced 96 diverse wheat lines at Pina-D1, Pinb-D1, Gsp-A1, Gsp-B1, and Gsp-D1. Three types of null alleles were identified using diagnostic primers: the first type was a multiple deletion of Pina-D1, Pinb-D1, and Gsp-D1 (Pina-D1k), the second was a Pina-D1 deletion (Pina-D1b); and the third type was a deletion of Gsp-D1, representing a novel null allele designated here as Gsp-D1k. Sequence analysis resulted in four allelic variants at Pinb-D1 and five at Gsp-A1, among them Gsp-A1-V was novel. Pina-D1, Gsp-B1 and Gsp-D1 sequences were monomorphic. Haplotype and phylogenetic analysis suggested that (1) bread wheat inherited its 5DS telomeric region probably from wild diploid Ae. tauschii subsp. tauschii found within an area from Transcaucasia to Caspian Iran; and that (2) the Ha-A and Ha-B homoeoloci were most closely related to sequences of wild tetraploid T. dicocco ides. This study provides a good overview of available genetic diversity at Pina-D1, Pinb-D1, and Gsp-1, which can be exploited to extend the range of grain texture traits in wheat.

Molecular characterization of the puroindoline-a and b alleles in synthetic hexaploid wheats and in silico functional and structural insights into Pina-D1.

Kernel hardness determined by two tightly linked Puroindoline genes, Pina-D1 and Pinb-D1, located on chromosome 5DS define commercially important characteristics, uses, major grades and export markets of wheat. This study was conducted to characterize Pina-D1 and Pinb-D1 alleles, in fifteen synthetic hexaploid wheats (SHWs) and its relation with grain hardness. Additionally, in silico functional analyses of puroindoline-a protein was conducted for better understanding of their putative importance in grain quality. Six different Pina-D1 alleles were identified in the SHWs, of which three i.e. Pina-D1a, Pina-D1c and Pina-D1d were already known whereas the other three had new sequence polymorphisms and were designated as Pina-D1w, Pina-D1x and Pina-D1y. Three different Pinb-D1 alleles were identified which have been reported earlier and no novel sequence polymorphism was detected. It was concluded that despite some primary, secondary and 3D structure variations, ligand binding sites and disulfide bonds discrepancies, the main features of PINA, i.e. the tryptophan-rich domain, the cysteine backbone, the signal peptide and basic identity of the proteins were all conserved. In silico analysis showed that puroindolines having binding capacity with small parts of prolamins causing celiac disease of human, however their potential role is not obvious. Conclusively, the new Pina-D1 alleles with modest effect on grain hardness, and insight into their functional and structural characteristics are important findings and their putative role in celiac disease require further studies to validate.

Dissection of a novel major stable QTL on chromosome 7D for grain hardness and its breeding value estimation in bread wheat.

Grain hardness (Gh) is important for wheat processing and end-product quality. Puroindolines polymorphism explains over 60% of Gh variation and the novel genetic factors remain to be exploited. In this study, a total of 153 quantitative trait loci (QTLs), clustered into 12 genomic intervals (C1-C12), for 13 quality-related traits were identified using a recombinant inbred line population derived from the cross of Zhongkemai138 (ZKM138) and Chuanmai44 (CM44). Among them, C7 (harboring eight QTLs for different quality-related traits) and C8 (mainly harboring QGh.cib-5D.1 for Gh) were attributed to the famous genes, Rht-D1 and Pina, respectively, indicating that the correlation of involved traits was supported by the pleotropic or linked genes. Notably, a novel major stable QTL for Gh was detected in the C12, QGh.cib-7D, with ZKM138-derived allele increasing grain hardness, which was simultaneously mapped by the BSE-Seq method. The geographic pattern and transmissibility of this locus revealed that the increasing-Gh allele is highly frequently present in 85.79% of 373 worldwide wheat varieties and presented 99.31% transmissibility in 144 ZKM138-derivatives, indicating the non-negative effect on yield performance and that its indirect passive selection has happened during the actual breeding process. Thus, the contribution of this new Gh-related locus was highlighted in consideration of improving the efficiency and accuracy of the soft/hard material selection in the molecular marker-assisted process. Further, TraesCS7D02G099400, TraesCS7D02G098000, and TraesCS7D02G099500 were initially deduced to be the most potential candidate genes of QGh.cib-7D. Collectively, this study provided valuable information of elucidating the genetic architecture of Gh for wheat quality improvement.

Characterization and Differentiation of Grain Proteomes from Wild-Type Puroindoline and Variants in Wheat.

Premium wheat with a high end-use quality is generally lacking in China, especially high-quality hard and soft wheat. Pina-D1 and Pinb-D1 (puroindoline genes) influence wheat grain hardness (i.e., important wheat quality-related parameter) and are among the main targets in wheat breeding programs. However, the mechanism by which puroindoline genes control grain hardness remains unclear. In this study, three hard wheat puroindoline variants (MY26, GX3, and ZM1) were compared with a soft wheat variety (CM605) containing the wild-type puroindoline genotype. Specifically, proteomic methods were used to screen for differentially abundant proteins (DAPs). In total, 6253 proteins were identified and quantified via a high-throughput tandem mass tag quantitative proteomic analysis. Of the 208 DAPs, 115, 116, and 99 proteins were differentially expressed between MY26, GX3, and ZM1 (hard wheat varieties) and CM605, respectively. The cluster analysis of protein relative abundances divided the proteins into six clusters. Of these proteins, 67 and 41 proteins were, respectively, more and less abundant in CM605 than in MY26, GX3, and ZM1. Enrichment analyses detected six GO terms, five KEGG pathways, and five IPR terms that were shared by all three comparisons. Furthermore, 12 proteins associated with these terms or pathways were found to be differentially expressed in each comparison. These proteins, which included cysteine proteinase inhibitors, invertases, low-molecular-weight glutenin subunits, and alpha amylase inhibitors, may be involved in the regulation of grain hardness. The candidate genes identified in this study may be relevant for future analyses of the regulatory mechanism underlying grain hardness.

The relationship between grain hardness, dough mixing parameters and bread-making quality in winter wheat.

The influence of grain hardness, determined by using molecular markers and physical methods (near-infrared (NIR) technique and particle size index-PSI) on dough characteristics, which in turn were determined with the use of a farinograph and reomixer, as well as bread-making properties were studied. The material covered 24 winter wheat genotypes differing in grain hardness. The field experiment was conducted at standard and increased levels of nitrogen fertilization. Results of molecular analyses were in agreement with those obtained by the use of physical methods for soft-grained lines. Some lines classified as hard (by physical methods) appeared to have the wild-type Pina and Pinb alleles, similar to soft lines. Differences in dough and bread-making properties between lines classified as hard and soft on the basis of molecular data appeared to be of less significance than the differences between lines classified as hard and soft on the basis of physical analyses of grain texture. Values of relative grain hardness at the increased nitrogen fertilization level were significantly higher. At both fertilization levels the NIR parameter determining grain hardness was significantly positively correlated with the wet gluten and sedimentation values, with most of the rheological parameters and bread yield. Values of this parameter correlated with quality characteristics in a higher degree than values of particle size index.

Features and Possible Applications of Plant Lipid-Binding and Transfer Proteins.

In plants, lipid trafficking within and inside the cell is carried out by lipid-binding and transfer proteins. Ligands for these proteins are building and signaling lipid molecules, secondary metabolites with different biological activities due to which they perform diverse functions in plants. Many different classes of such lipid-binding and transfer proteins have been found, but the most common and represented in plants are lipid transfer proteins (LTPs), pathogenesis-related class 10 (PR-10) proteins, acyl-CoA-binding proteins (ACBPs), and puroindolines (PINs). A low degree of amino acid sequence homology but similar spatial structures containing an internal hydrophobic cavity are common features of these classes of proteins. In this review, we summarize the latest known data on the features of these protein classes with particular focus on their ability to bind and transfer lipid ligands. We analyzed the structural features of these proteins, the diversity of their possible ligands, the key amino acids participating in ligand binding, the currently known mechanisms of ligand binding and transferring, as well as prospects for possible application.

Antifungal Effects of Fusion Puroindoline B on the Surface and Intracellular Environment of Aspergillus flavus.

Aspergillus flavus infection is a major issue for safe food storage. In this study, we constructed an efficient prokaryotic expression system for puroindoline B (PINB) protein to detect its antifungal activity. The Puroindoline b gene was cloned into pET-28a (+) vector and expressed in Escherichia coli. Treatment with fusion PINB revealed that it inhibits mycelial growth of A. flavus, a common grain mold. Moreover, fusion PINB-treated A. flavus mycelium withered and exhibited a sunken spore head. As fusion PINB concentration increased, electrical conductivity in mycelium also increased, indicative of cell membrane damage. Furthermore, intracellular malate dehydrogenase and succinate dehydrogenase activity decreased, revealing a disruption in the tricarboxylic acid cycle. Moreover, the dampened activity of the ion pump Na+K+-ATPase negatively affected the intracellular regulation of both ions. Catalase and superoxide dismutase activity decreased, thus reducing antioxidant capacity, a result confirmed with an increase in malondialdehyde content. Changes to the GSH/GSSG ratio indicated a shift to an intracellular oxidative state. At the same time, laser scanning confocal microscopy assay showed the accumulation of reactive oxygen species and nuclear damage. Therefore, the PINB fusion protein may have the potential to control A. flavus in grain storage and food preservation.

Molecular and physical characterization of grain hardness in European spring common wheat (Triticum aestivum L.).

Grain hardness is the single most important trait in determining technological properties and end-use quality of wheat product. This trait is controlled by two genes (Pina-D1 and Pinb-D1) at the Hardness (Ha) locus. Soft endosperm kernels are characterized by the presence of alleles 'a' in both genes (Pina-D1a and Pinb-D1a), while the medium hard and hard grain is the result of deletion in the Pina-D1 gene or single mutation of the Pinb-D1 gene. The aim of the current study was to determine the relationship between common wheat grain hardness and the presence of puroindoline genes. Eighty-one spring common wheat cultivars from Europe were analysed for grain hardness by SKCS (Single Kernel Characterization System) and Pin-D1 genes. The analysed genotypes were divided into three hardness classes: hard, medium and soft and they showed four allelic combinations in Pin-D1 genes. The SKCS results showed that hard wheat was the major type in European cultivars, whereas molecular analysis showed differential allelic combinations of puroindoline genes among these classes. The conducted analyses suggest that another major gene or other factors were influencing kernel texture. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02897-3.

Toward the Genetic Basis and Multiple QTLs of Kernel Hardness in Wheat.

Kernel hardness is one of the most important single traits of wheat seed. It classifies wheat cultivars, determines milling quality and affects many end-use qualities. Starch granule surfaces, polar lipids, storage protein matrices and Puroindolines potentially form a four-way interaction that controls wheat kernel hardness. As a genetic factor, Puroindoline polymorphism explains over 60% of the variation in kernel hardness. However, genetic factors other than Puroindolines remain to be exploited. Over the past two decades, efforts using population genetics have been increasing, and numerous kernel hardness-associated quantitative trait loci (QTLs) have been identified on almost every chromosome in wheat. Here, we summarize the state of the art for mapping kernel hardness. We emphasize that these steps in progress have benefitted from (1) the standardized methods for measuring kernel hardness, (2) the use of the appropriate germplasm and mapping population, and (3) the improvements in genotyping methods. Recently, abundant genomic resources have become available in wheat and related Triticeae species, including the high-quality reference genomes and advanced genotyping technologies. Finally, we provide perspectives on future research directions that will enhance our understanding of kernel hardness through the identification of multiple QTLs and will address challenges involved in fine-tuning kernel hardness and, consequently, food properties.

Plant Antimicrobial Peptides: State of the Art, In Silico Prediction and Perspectives in the Omics Era.

Even before the perception or interaction with pathogens, plants rely on constitutively guardian molecules, often specific to tissue or stage, with further expression after contact with the pathogen. These guardians include small molecules as antimicrobial peptides (AMPs), generally cysteine-rich, functioning to prevent pathogen establishment. Some of these AMPs are shared among eukaryotes (eg, defensins and cyclotides), others are plant specific (eg, snakins), while some are specific to certain plant families (such as heveins). When compared with other organisms, plants tend to present a higher amount of AMP isoforms due to gene duplications or polyploidy, an occurrence possibly also associated with the sessile habit of plants, which prevents them from evading biotic and environmental stresses. Therefore, plants arise as a rich resource for new AMPs. As these molecules are difficult to retrieve from databases using simple sequence alignments, a description of their characteristics and in silico (bioinformatics) approaches used to retrieve them is provided, considering resources and databases available. The possibilities and applications based on tools versus database approaches are considerable and have been so far underestimated.

Binary Colloidal Crystal Layers as Platforms for Surface Patterning of Puroindoline-Based Antimicrobial Peptides.

The ability of bacteria to form biofilms and the emergence of antibiotic-resistant strains have prompted the need to develop the next generation of antibacterial coatings. Antimicrobial peptides (AMPs) are showing promise as molecules that can address these issues, especially if used when immobilized as a surface coating. We present a method that explores how surface patterns together with the selective immobilization of an AMP called PuroA (FPVTWRWWKWWKG-NH2) can be used to both kill bacteria and also as a tool to study bacterial attachment mechanisms. Surface patterning is achieved using stabilized self-assembled binary colloidal crystal (BCC) layers, allowing selective PuroA immobilization to carboxylated particles using N-(3-dimethylaminopropyl)-N'-ethyl carbodiimide (EDC) hydrochloride/N-hydroxysuccinimide (NHS) coupling chemistry. Covalent immobilization of PuroA was compared with physical adsorption (i.e., without the addition of EDC/NHS). The AMP-functionalized colloids and BCC layers were characterized by X-ray photoelectron spectroscopy, ζ potentials, and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Surface antimicrobial activity was assessed by viability assays using Escherichia coli. MALDI-TOF MS analysis revealed that although not all of PuroA was successfully covalently immobilized, a relatively low density of PuroA (1.93 × 1013 molecules/cm2 and 7.14 × 1012 molecules/cm2 for covalent and physical immobilization, respectively) was found to be sufficient at significantly decreasing the viability of E. coli by 70% when compared to that of control samples. The findings provide a proof of concept that BCC layers are a suitable platform for the patterned immobilization of AMPs and the importance of ascertaining the success of small-molecule grafting reactions using surface-MALDI, something that is often assumed to be successful in the field.

Structural consequences of the interaction of puroindolines with gluten proteins.

The effect of puroindolines (PINs) on structural characteristics of wheat proteins was investigated in Triticum turgidum ssp. durum (cv. Svevo) and Triticum aestivum (cv. Alpowa) and in their respective derivatives in which PIN genes were expressed (Soft Svevo) or the distal end of the short arm of chromosome 5D was deleted and PINs were not expressed (Hard Alpowa). The presence of PINs decreased the amount of cold-SDS extractable proteins and the accessibility of protein thiols to specific reagents, but resulted in facilitated solvation of gluten proteins, as detected by tryptophan fluorescence measurements carried out on minimally mixed flour/water mixtures. We propose that PINs and gluten proteins are interacting in the grain or flour prior to mixing. Hydrophobic interactions between PINs and some of the gluten proteins modify the pattern of interactions among gluten proteins, thus providing an additional mechanistic rationale for the effects of PINs on kernel hardness.

Changes in the starch-protein interface depending on common wheat grain hardness revealed using atomic force microscopy.

The atomic force microscope tip was used to progressively abrade the surface of non-cut starch granules embedded in the endosperm protein matrix in grain sections from wheat near-isogenic lines differing in the puroindoline b gene and thus, hardness. In the hard near-isogenic wheat lines, starch granules exhibited two distinct profiles corresponding either to abrasion in the surrounding protein layer or the starch granule. An additional profile, only identified in soft lines, revealed a marked stop in the abrasion at the protein-starch transition similar to a lipid interface playing a lubricant role. It was related to the presence of both wild-type puroindolines, already suggested to act at the starch-protein interface through their association with polar lipids. This study revealed, for the first time, in situ differences in the nano-mechanical properties at the starch-protein interface in the endosperm of wheat grains depending on the puroindoline allelic status.