Puroindoline Proteins Research Articles

Recent developments on the contribution of glutenin and puroindoline proteins to improve wheat grain quality

AbstractBackground and ObjectivesWheat is most staple food crop for making different end products. The major factors determining the wheat flour end‐use are glutenins and puroindolines. It is very important to identify the composition, variation, and functional characteristics of these key factors.FindingsThe glutenin subunits comprise two subgroups—high molecular weight glutenin subunits (HMW‐GS), and low molecular weight glutenin subunits (LMW‐GS). Puroindolines genes depict a wide diversity of both PINA and PINB allelic forms. The prevalence and relative proportions of these types may vary depending on several genetic and environmental factors.ConclusionCurrent review summarizes recently published research on the impact of glutenin genes governing gluten strength and puroindolines genes related to grain softness. It encompasses recent reports on the development of DNA‐based molecular markers utilized to identify novel alleles associated with HMW‐GS, LMW‐GS, and puroindoline genes in diverse wheat genotypes.Significance and NoveltySubstantial advancements have been made to identify associations between gene expression and grain quality and to develop improved wheat grain quality lines. Nevertheless, a novel marker system such as multiplex polymerase chain reaction and high throughput analysis is needed to screen large numbers of markers at low cost for rapid and precise analysis of germplasm, mapping, and marker‐assisted selection.

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.

Production and phenotypic characterization of nascent synthetic decaploids derived from interspecific crosses between a durum wheat cultivar and hexaploid Aegilops species

Wheat and its relatives include some allopolyploid species such as Aegilops juvenalis (Thell.) Eig (DDMMUU) and Aegilops vavilovii (Zhuk.) Chenn. (DDMMSS). Here, we successfully produced for the first time three allodecaploid lines with the AABBDDMMUU and AABBDDMMSS genomes through interspecific crosses between a tetraploid wheat cultivar and the two Aegilops species. Spike architecture of the synthetic lines showing a brittle rachis phenotype strongly resembled that of the parental hexaploid Aegilops species, but not that of the parental tetraploid wheat. Phenotypic differences of the spike and spikelet morphology were caused by one of the parental Aegilops species. Glumes of the synthetic decaploids were extremely hard, and all three synthetic lines had a soft texture with a smooth starch surface in endosperm cells. The soft-textured grains of the synthetic decaploids were due to accumulation of the puroindoline proteins derived from the genomes of parental allohexaploid Aegilops species. Therefore, the nascent synthetic decaploid lines highly reflect the phenotypic characteristics of the pollen parents. With further wheat breeding, the synthetic decaploids could have great potential to improve various traits.

Transfer of grain softness from 5U-5A wheat-Aegilops triuncialis substitution line to bread wheat through induced homeologous pairing

Bread wheat sustains genes for grain softness on “Ha” locus of short arm of 5D chromosome. Pina-D1 and Pinb-D1 alleles of “Ha” locus together are responsible for soft grain texture of wheat. The wheat cultivar WL711 had wild type Pinb-D1a and mutated Pina allele, imparting hard kernel texture. A 5U-5A substitution line of Aegilops triuncialis had extra soft grains. Efforts were made to transfer puroindolines from Aegilops triuncialis 5U as 5U-5A substitution line BTC17 to its homeologous chromosome 5A of bread wheat. A total of 367 5U-5A substitution recombinants developed through induced homeologous pairing by ph1b deletion of Ph1 locus were screened for the presence of Pina and Pinb. Only 23 lines were found to have both Pina and Pinb. Grain hardness index of recombinants with functional puroindolines was reduced from 6 to 67%. Deletion mapping of wheat 5A chromosome specific SSR markers revealed the maintenance of synteny in 5U and 5A homeologous chromosomes and puroindolines were found to be translocated at the telomeric end of 5A short arm. Translocation of puroindolines from 5U was confirmed by sequencing of Pina and Pinb genes. The Pina nucleotide sequence was found 100% similar to Aegilops markgrafii (CC). Puroindoline proteins extracted from the selected softer lines exhibited significant antimicrobial activity against gram positive and gram negative bacteria. Chromosome 5U puroindolines transferred to 5A may be pyramided with 5D of soft wheat to develop extra soft varieties for superior biscuit making quality.

Genetic variability of Hin genes in selected barley germplasm and potential implications for grain texture

Grain hardness (texture) in barley is a commercially important trait, associated with water-uptake during the steeping phase of malting, quantified by measuring endosperm hardness. Hordoindolines are seed proteins of barley, related to the wheat puroindoline proteins. The Hordoindoline genes Hina, Hinb-1 and Hinb-2 are orthologs of wheat Pin genes. The information on contributions of Hin genes to barley grain hardness is limited and somewhat ambiguous. Hence this study aimed to investigate the genetic diversity of Hin genes in 12 Australian barley cultivars and 14 landraces originating from geographic centers of diversity. The grain hardness was determined by the Single Kernel Characterisation System (SKCS) and ranged between 24.2–78.5 SKCS units for cultivars and 48.7–94.4 SKCS units for landraces. Hin gene amplifications and DNA sequencing indicated notable genetic diversity, with seven Hina, six Hinb-1 alleles and eight Hinb-2 alleles detected. Some of the SNPs led to substitutions at the potentially functionally important tryptophan rich domain and certain basic and hydrophobic residues in the putative proteins. A novel Hinb-1 allele with an insertion at nucleotide position 210, leading to a frame-shift mutation, was discovered in landrace L400211 from Ethiopia that had a high SKCS hardness value, indicating harder grain. The study suggests that Hin gene variations may contribute to barley grain hardness, and selection of certain alleles may be useful for breeding for varying barley grain textures.

Sequence Diversity and Identification of Novel Puroindoline and Grain Softness Protein Alleles in Elymus, Agropyron and Related Species

The puroindoline proteins, PINA and PINB, which are encoded by the Pina and Pinb genes located at the Ha locus on chromosome 5D of bread wheat, are considered to be the most important determinants of grain hardness. However, the recent identification of Pinb-2 genes on group 7 chromosomes has stressed the importance of considering the effects of related genes and proteins. Several species related to wheat (two diploid Agropyron spp., four tetraploid Elymus spp. and five hexaploid Elymus and Agropyron spp.) were therefore analyzed to identify novel variation in Pina, Pinb and Pinb-2 genes which could be exploited for the improvement of cultivated wheat. A novel sequence for the Pina gene was detected in Elymus burchan-buddae, Elymus dahuricus subsp. excelsus and Elymus nutans and novel PINB sequences in Elymus burchan-buddae, Elymus dahuricus subsp. excelsus, and Elymus nutans. A novel PINB-2 variant was also detected in Agropyron repens and Elymus repens. The encoded proteins detected all showed changes in the tryptophan-rich domain as well as changes in and/or deletions of basic and hydrophobic residues. In addition, two new AGP sequences were identified in Elymus nutans and Elymus wawawaiensis. The data presented therefore highlight the sequence diversity in this important gene family and the potential to exploit this diversity to modify grain texture and end-use quality in wheat.

Wheat puroindolines tether to starch granule surfaces in puroindoline-null (Pin-null) plants

The interaction of wheat puroindolines (pin) with starch granules from a pin-null cereal crop, rice, was investigated using an in vitro immunofluorescent method. Rice seed cross-sections were incubated with soft (Augusta) and hard (Langdon) durum wheat extracts. The in vitro localization of puroindolines on rice seed cross sections were compared to the in vivo localization of puroindolines from transgenic Pin+ rice seeds using the Durotest anti-Pin monoclonal antibody. Wheat puroindolines were observed to localize with high affinity for rice starch granule surfaces both in vitro and in vivo. The strong interactions of puroindolines with starch granules plays important roles in both plant defense and grain texture. The obtained results confirmed that puroindolines interact with starch granule surfaces in rice, a pin-null cereal, and that an in vitro localization method can be used to predict the in vivo targeting and deposition of puroindoline proteins.

Genotypic Variation in Wheat Flour Lysophospholipids.

Lysophospholipids (LPLs) are the most abundant polar lipids in wheat endosperm and naturally complex with amylose, affecting starch physicochemical properties. We analyzed LPLs in wheat flour from 58 cultivars which differ by grain hardness using liquid chromatography mass spectrometry (LCMS). There were significant differences in LPL content between cultivars, demonstrating that genotype rather than environment contributes most to the total variance in wheat endosperm LPLs. Polar lipids such as LPLs may play a role in grain hardness through their interaction with puroindoline proteins, however, no strong correlation between kernel hardness and LPLs was detected. This may reflect the location of LPLs within the starch granule as opposed to the puroindoline proteins outside starch granules. LPLs may have an indirect relationship with kernel hardness as they could share the same origin as polar lipids that interact with puroindoline on the starch granule surface.

Kernel softness in wheat is determined by starch granule bound Puroindoline proteins

Wheat has a vital position in agriculture because it is a staple food for masses and variation in grain hardness governs its applications. Soft wheats have softer endosperm texture that mills easily, so needs less energy to mill, produces smaller particles, and small amount of starch is damaged after milling as compared to hard wheat. Soft texture results from higher level of friabilin whereas hard texture results from low level of friabilin on starch granule surface. Friabilin, a marker of kernel texture is primarily composed of Puroindolines (PINs) and its genes (Pins) are located on the Hardness (Ha) locus. The Pins are the molecular-genetic basis of kernel softness in wheat. When both Pins are in their ‘wild state’ (Pina-D1a and Pinb-D1a), wheat kernel is soft. Absence or mutation in one of the Pins results in hard grain texture with different effects on end use and milling qualities. Pina-D1b genotypes gave harder grain texture, higher protein content, water absorption of flour, damaged starch granules and greater flour yield than hard wheat. Recently, other Pins like genes, Pin b variant genes located on the long arm of chromosome 7A were reported in bread wheat with more than 70% similarity to Pinb (Pinb-D1a) at the DNA level. Other genes located on chromosomes 1A, 2A, 5A, 7A, 5B, 2D and 6D also affect kernel texture. However the main determinants are the variants in the allelic diversity of Puroindoline family genes. Contemporary studies show that Pins are multifunctional family of genes having a range of functions from grain hardness to natural defense against insects and pathogens such as viruses, bacteria and fungi.

Surface Lipids Play a Role in the Interaction of Puroindolines with Wheat Starch and Kernel Hardness

Kernel hardness is an important quality characteristic of common wheat. In this study, we investigated the role of starch surface lipids on the interaction of puroindoline proteins and starch granules through in vitro starch–protein binding experiments and flour reconstitution. SDS‐PAGE showed that there were no puroindoline proteins on the starch granule surface when surface lipids were removed or when defatted starch was incubated with puroindoline proteins. However, the puroindoline protein bands were present when defatted starch was incubated with lipids followed by purified puroindoline proteins, which indicated that starch surface lipids play a role in the binding of puroindolines to starch granules. The hardness of flour tablets and dough sheets made from reconstituted flour, which combined defatted starch incubated with lipids and puroindolines with gluten, was lower than for the control reconstituted flour, which was made from defatted starch and gluten. The results of scanning electron microscopy also showed that starch granules were embedded in the gluten in the gluten + defatted starch + lipids + puroindolines treatment. These results confirmed that starch surface lipids are involved in the interaction of puroindolines with wheat starch and kernel hardness.

Empirical rheology and pasting properties of soft-textured durum wheat (Triticum turgidum ssp. durum) and hard-textured common wheat (T. aestivum)

Puroindoline (PIN) proteins are the molecular basis for wheat kernel texture classification and affect flour milling performance. This study investigated the effect of PINs on empirical rheology and pasting properties in Triticum turgidum ssp. durum and Triticum aestivum. Soft wheat (cv. Alpowa), durum wheat (cv. Svevo) and their derivatives in which PINs were deleted (Hard Alpowa) or expressed (cv. Soft Svevo). Presence of PINs affected flour particle size and damaged starch. PINs increased the pasting temperature and breakdown viscosity, while the effect on peak viscosity and setback were not consistent. Presence of PINs was negatively associated with GlutoPeak gluten aggregation energy and farinograph dough stability, suggesting a weakening of the gluten matrix. As regards dough extensibility, the role of PINs was evident only in common wheat: 5DS distal end deletion increased the resistance to extension, without affecting the dough extensibility. This study showed PINs to have different impact on pasting and rheological properties of T. aestivum and T. turgidum ssp. durum flours.

Role of Lipid Composition on the Interaction between a Tryptophan-Rich Protein and Model Bacterial Membranes.

The interaction between tryptophan-rich puroindoline proteins and model bacterial membranes at the air-liquid interface has been investigated by FTIR spectroscopy, surface pressure measurements, and Brewster angle microscopy. The role of different lipid constituents on the interactions between lipid membrane and protein was studied using wild type (Pin-b) and mutant (Trp44 to Arg44 mutant, Pin-bs) puroindoline proteins. The results show differences in the lipid selectivity of the two proteins in terms of preferential binding to specific lipid head groups in mixed lipid systems. Pin-b wild type was able to penetrate mixed layers of phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) head groups more deeply compared to the mutant Pin-bs. Increasing saturation of the lipid tails increased penetration and adsorption of Pin-b wild type, but again the response of the mutant form differed. The results provide insight as to the role of membrane architecture, lipid composition, and fluidity on antimicrobial activity of proteins. Data show distinct differences in the lipid binding behavior of Pin-b as a result of a single residue mutation, highlighting the importance of hydrophobic and charged amino acids in antimicrobial protein and peptide activity.

Production of bioactive wheat puroindoline proteins in Nicotiana benthamiana using a virus-based transient expression system

The emergence of antibiotic resistant pathogenic strains of bacteria has necessitated the development of novel antimicrobial agents. The puroindoline A and B (PINA and PINB) proteins of wheat, well-known for their roles in determining the important phenotype of grain texture, are also antimicrobial, making them attractive as natural bio-control agents. However, the biochemical basis of PIN functionality remains unclear due to limitations in expressing them at the required yield and purity and lack of accurate tertiary structure. This study focussed on rapid transient expression of PINs targeted to different subcellular compartments (chloroplast, apoplast, endoplasmic reticulum and cytosol) of Nicotiana benthamiana leaf cells using the deconstructed tobacco mosaic virus-based ‘magnICON®’ system. The expressed recombinant PINs were characterised by Western blot using the Durotest anti-friabilin antibody, enzyme-linked immunosorbent assays (ELISA) and antimicrobial activity tests. Maximum yield of the His-tagged PINs occurred when targeted to the chloroplast. Both PINs exhibited oligomeric and monomeric forms on gels, but Western blots with the widely used Durotest anti-friabilin antibody identified only oligomeric forms. Only the PINs purified by a hydrophobic interaction method exhibited monomeric forms with the anti-His tag antibody, indicating correct folding. Interestingly, the Durotest antibody did not bind to monomers, suggesting their epitope may be obscured. PINs purified by His-tag affinity purification under native conditions or by the hydrophobic method exhibited antimicrobial activities. The successful in planta expression and optimisation of purification will enable future studies to examine the detailed structure of the PINs and explore novel bio-control applications in health, food and/or agriculture.

Diversity and distribution of puroindoline-D1 genes in Aegilops tauschii

Puroindoline-D1 genes are the key genes that decide kernel texture in bread wheat, but limited genetic resources have impeded the improvement of wheat quality. In this study, the puroindoline-D1 genes from 110 accessions of Aegilops tauschii Coss. (the D genomic ancestor of bread wheat) were isolated and sequenced to identify new alleles and discover their genetic diversities. Three new alleles: Pina-D1o, Pinb-D1dt and Pinb-D1it, were discovered, and they were only one nucleotide different to Pina-D1a, Pinb-D1a and Pinb-D1i, respectively. Two new puroindoline proteins were translated from the Pina-D1o and Pinb-D1dt. Pinb-D1it coded the same protein as Pinb-D1o, Pinb-D1m, Pinb-D1h, Pinb-D1n, AJ302108, Pinb-D1i and Pinb-d1it, which are common phenotypes found at the Pina-D1 and Pinb-D1 A. tauschii locus; Pina-D1h, Pina-D1f, Pina-D1e, Pina-D1d, Pina-D1c and AY608595 also coded the same amino acid at the Pina-D1 locus. Total eight Pina-D1 alleles, seven Pinb-D1 alleles and 20 haplotypes were found in these accessions. The synonymous mutations produced two kinds of synonymous haplotypes in our experiment. The first kind contained Pina-D1d/Pinb-D1i, Pina-D1c/Pinb-D1h, Pina-D1c/Pinb-D1i, Pina-D1d/pinb-D1h, Pina-D1j/pinb-D1i and Pina-D1e/Pinb-D1i, and the second kind contained Pina-D1a/Pinb-D1i and Pina-D1a/Pina-D1it. The eight synonymous haplotypes made up the majority of haplotypes, and covered also the majority of the geographical distribution. The large numbers of synonymous haplotypes and their wide distribution suggested that the puroindoline-D1 genes play important roles in A. tauschii survival. New puroindoline alleles and the haplotypes in A. tauschii will improve our understanding of puroindolines evolution and lead to increasing of wheat quality.

Molecular characterization of diverse wheat germplasm for puroindolineproteins and their antimicrobial activity

Thirty different wheat genotypes, including landraces, cultivars, and related wild species, were analyzed for puroindoline genes and proteins. Full-length Pina and Pinb genes were amplified using gene-specific markers where 13 genotypes amplified both genes, while 17 genotypes had either one or none of the puroindoline genes. None of the puroindoline genes were detected in any of the Triticum durum cultivars. Genotypes with both puroindoline genes had a soft grain texture, while other genotypes had hard grains. Related nonprogenitor species with A, C, M, U, and S genomes also showed the presence of either one or both the puroindoline genes, indicating that these species can also be utilized for development of extra-soft wheat. SDS-PAGE analysis of selected wheat genotypes revealed that puroindoline proteins are approximately 13-14 kDa and the protein results were in agreement with the PCR results. Puroindoline proteins of selected 3 soft wheat genotypes HPW 89, Naphal, and IITR-9 also exhibited antimicrobial activity against gram-positive and gram-negative bacteria. Both genes of a soft wheat cultivar, HPW 89, were successfully cloned in the Escherichia coli DH-5 $\alpha$ strain. Introgression and molecular characterization of diverse Pina and Pinb genes to develop extra-soft wheat is in progress.

Puroindoline allelic diversity in Indian wheat germplasm and identification of new allelic variants.

Grain hardness is an important quality trait that influences product development in wheat. This trait is governed by variation in puroindoline proteins (PINA and PINB). Our study evaluated 551 Indian wheat germplasm lines for diversity in Pina and Pinb genes. Eighty-two lines were shortlisted for full length sequencing and grain hardness studies. Sequencing studies identified six unknown alleles: two for the Pina gene and four for the Pinb gene. Five of them were novel with non-synonymous changes in the corresponding amino acid sequences. Identified mutations in the deduced mature proteins and their pre- and pro-peptides influenced the hardness characteristics of the grain. We classified these 82 varieties into different hardness categories with reference to international and Indian systems of classification. The majority of Indian wheat varieties were categorized as hard. This study revealed that unexplored Indian wheat germplasm can be a good source of genetic variability for both Pina and Pinb genes, helping in marker-assisted breeding and in obtaining wheat with different textural properties.

The antimicrobial domains of wheat puroindolines are cell-penetrating peptides with possible intracellular mechanisms of action.

The puroindoline proteins (PINA and PINB) of wheat display lipid-binding properties which affect the grain texture, a critical parameter for wheat quality. Interestingly, the same proteins also display antibacterial and antifungal properties, attributed mainly to their Tryptophan-rich domain (TRD). Synthetic peptides based on this domain also display selectivity towards bacterial and fungal cells and do not cause haemolysis of mammalian cells. However, the mechanisms of these activities are unclear, thus limiting our understanding of the in vivo roles of PINs and development of novel applications. This study investigated the mechanisms of antimicrobial activities of synthetic peptides based on the TRD of the PINA and PINB proteins. Calcein dye leakage tests and transmission electron microscopy showed that the peptides PuroA, Pina-M and Pina-W→F selectively permeabilised the large unilamellar vesicles (LUVs) made with negatively charged phospholipids mimicking bacterial membranes, but were ineffective against LUVs made with zwitterionic phospholipids mimicking eukaryotic membranes. Propidium iodide fluorescence tests of yeast (Saccharomyces cerevisiae) cells showed the peptides were able to cause loss of membrane integrity, PuroA and Pina-M being more efficient. Scanning electron micrographs of PINA-based peptide treated yeast cells showed the formation of pits or pores in cell membranes and release of cellular contents. Gel retardation assays indicated the peptides were able to bind to DNA in vitro, and the induction of filamental growth of E. coli cells indicated in vivo inhibition of DNA synthesis. Together, the results strongly suggest that the PIN-based peptides exert their antimicrobial effects by pore formation in the cell membrane, likely by a carpet-like mechanism, followed by intracellular mechanisms of activity.

Expression, purification and antimicrobial activity of puroindoline A protein and its mutants

Wheat puroindoline proteins, PINA and PINB, play key roles in determining wheat grain hardness as well as in defending the plant against pathogens. PINA has much greater membrane-binding property and antimicrobial activity because it contains more tryptophan residues in the unique tryptophan-rich domain (TRD). In order to obtain proteins with higher antimicrobial activity, mutants of PINA containing two or three copies of TRD, designated ABBC and ABBBC, respectively, were constructed and expressed in E. coli Rosetta-gami (DE3). Metal affinity chromatography was used to purify the soluble affinity-tagged recombinant proteins. The secondary structures of the recombinant proteins were predicted by the online program Protein Homology/analog Y Recognition Engine v2.0 and experimentally assessed using circular dichroism. Minimum inhibition concentration tests and fluorescence microscope analyses were employed to evaluate the antimicrobial activities of the mutants. The results showed that the purified recombinant ABBC was correctly folded and presented significantly higher antimicrobial activities against E. coli and S. aureus than wild-type PINA, suggesting its potential use as an antimicrobial agent. The results also confirmed that TRD is a determinant of the antimicrobial activity of PINA and demonstrated that it is feasible to enhance the antimicrobial activity of PINA by adding one copy of TRD.

The RNA-mediated silencing of one of the Pin genes in allohexaploid wheat simultaneously decreases the expression of the other, and increases grain hardness

The RNAi-mediated silencing of Pina and Pinb, the two genes responsible for the grain texture of allohexaploid wheat, was induced and analysed in two wheat cultivars, Kontesa and Torka. A characterization of the two genes in non-transgenic plants revealed that Pinb carries a point mutation, designated Pinb-D1c in both cultivars. This mutation does not influence transcript abundance or protein content. Two silencing cassettes of the hpRNA type were constructed and used for stable transformation via Agrobacterium. In total, 43 transgenic lines representing the two cultivars were obtained, transformed with the silencing cassettes for Pina or for Pinb or co-transformed with both cassettes. The relative transcript levels of the two genes in the same progeny plant were found to be similar, independent of the silencing cassette used. The reduction in the Pina and Pinb transcript levels in the segregating T(1) progeny of Kontesa and Torka transformed with one of the silencing cassettes exceeded 80%. Co-transformation with the silencing cassettes for both genes resulted in a reduction of over 91% of Pina and Pinb transcripts in some segregating T(1) progeny of Kontesa. The silencing was transmitted to the T(4) kernel generation of the T(3) lines. A significant reduction or lack of both puroindoline proteins in the silenced lines correlated with an essential increase in grain hardness. The discussion covers some new insights into the function of the Pin genes, including the simultaneous silencing of both, independent of the siRNA signal.

Puroindolines, Pin alleles, hordoindolines and grain softness proteins are sources of bactericidal and fungicidal peptides

The puroindoline (PIN) proteins in the wheat grain are considered the primary genetic basis of texture. Their lipid-binding nature that enables an effect on texture is also likely related to their antimicrobial properties. A number of mutations leading to hard texture are reported, but their collateral effects on any antimicrobial activities are unknown. To evaluate whether antimicrobial activity is conserved at the tryptophan rich domain (TRD) and affected by point mutations, we designed a series of peptides based on the TRD of proteins encoded by the alleles Pina-D1a, Pina-D1m, Pinb-D1a, Pinb-D1b, Pinb-D1l and Pinb-D1q, as well as by the grain softness protein-1, hordoindoline-a and hordoindoline-b genes. PuroA, Pina-M (Pro-35 to Ser), Hina and GSP-1 peptides displayed measurable antibacterial activity, Pina-M higher than PuroA. Replacement of Arg-39 with Gly decreased the activity considerably, and the five Trp residues in PuroA could not be substituted with Phe without major loss of activity. Except for Pinb-L (Lys-45 to Glu) and Hinb1a, all displayed antifungal activity and none displayed haemolytic activity. The work demonstrates a potential role for this protein family in pathogen protection, stresses the importance of Trp and basic residues, and suggests potential applications in food and health industries.