Anti‐biofilm and sporicidal activity of peptides based on wheat puroindoline and barley hordoindoline proteins

The broad‐spectrum activity of antimicrobial peptides (AMPs) and low probability of development of host resistance make them excellent candidates as novel bio‐control agents. A number of AMPs are found to be cationic, and a small proportion of these are tryptophan‐rich. The puroindolines (PIN) are small, basic proteins found in wheat grains with proposed roles in biotic defence of seeds and seedlings. Synthetic peptides based on their unique tryptophan‐rich domain (TRD) display antimicrobial properties. Bacterial endospores and biofilms are highly resistant cells, with significant implications in both medical and food industries. In this study, the cationic PIN TRD‐based peptides PuroA (FPVTWRWWKWWKG‐NH₂) and Pina‐M (FSVTWRWWKWWKG‐NH₂) and the related barley hordoindoline (HIN) based Hina (FPVTWRWWTWWKG‐NH₂) were tested for effects on planktonic cells and biofilms of the common human pathogens including Pseudomonas aeruginosa, Listeria monocytogenes and the non‐pathogenic Listeria innocua. All peptides showed significant bactericidal activity. Further, PuroA and Pina‐M at 2 × MIC prevented initial biomass attachment by 85–90% and inhibited >90% of 6‐h preformed biofilms of all three organisms. However Hina, with a substitution of Lys‐9 with uncharged Thr, particularly inhibited Listeria biofilms. The PIN based peptides were also tested against vegetative cells and endospores of Bacillus subtilis. The results provided evidence that these tryptophan‐rich peptides could kill B. subtilis even in sporulated state, reducing the number of viable spores by 4 log units. The treated spores appeared withered under scanning electron microscopy. The results establish the potential of these tryptophan‐rich peptides in controlling persistent pathogens of relevance to food industries and human health. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Phenotypic effects of alleles of the common wheat puroindoline genes

Eighty-five common winter wheat cultivars and lines mostly selected in Ukraine were studied with the use of the NIR method to determine the relative index of hardness and protein content. Actual data concerning hardness were compared with data obtained from previous genetic studies and published sources. A significantly varying hardness index for genotypes with the same alleles of the puroindoline genes confirms the presence of extra genes that have effect on this trait.     

Stability of puroindoline peptides and effects on wheat rust

Peptides modelled on the tryptophan rich domain of puroindolines and the related grain softness protein-1 have a broad range of antibacterial and antifungal activities. With the aims of further investigating the activities of these antimicrobial peptides we studied their activity against wheat rust diseases and environmental stability. PINA-based peptides were found to have high pH and thermal stability in addition to being stable over long periods at room temperature. These properties could make them excellent candidates as preservatives in food. PuroA, Pina-R39G and PuroB peptides adversely affected the morphology of the stripe rust spores (Puccinia striiformis f. sp. tritici), while PuroA and PuroB showed moderate inhibition of their germination. Additionally, GSP-5D reduced the germination of leaf rust spores (P. triticina). PuroA and PuroB sprayed onto stripe rust infected plants effected a moderate reduction in the number of stripe rust uredinia on wheat seedlings, as did PuroB sprayed onto the seedlings and allowed to coat the leaves for 5 day prior to spore infection. The results suggest that the presence of the PIN-based peptides may lower frequency of initial infection foci.     

Real Time RT-PCR and flow cytometry to investigate wheat kernel hardness: role of puroindoline genes and proteins

Developing seeds from Triticum aestivum (wheat) cultivars were collected after flowering and analysed for puroindoline a and b gene expression by Real Time RT-PCR. Mature seeds were investigated for the presence and the amount of starch-associated puroindoline a and b proteins by flow cytometry. Puroindoline a gene and protein were found to have a predominant role in controlling wheat kernel hardness.     

Characterization of HSP-70 cognate proteins from wheat

Summary Animal and plant cells contain a family of constitutively expressed HSP-70 cognate proteins that are localized in different subcellular locations and are presumed to play a role in protein folding and transport. Utilizing antibodies raised against the yeast endoplasmicreticulum-localized HSP-70 cognate termed BiP/GRP-78, as well as antibodies raised against the Escherichia coli HSP-70 protein DnaK, we have identified and characterized a large family of closely related proteins in wheat. One protein band of 78 kDa that is apparently closely related to yeast BiP was localized in the endoplasmic reticulum. This band cross-reacted with the yeast BiP but not with the DnaK-specific antibodies. The yeast BiP antibodies also recognized a cytoplasmic protein of 70 kDa that is probably related to the HSC-70 cognate proteins. These two proteins were further confirmed as HSP-70 cognates by their ability to bind to an ATP-agarose column. Probing of proteins from purified wheat mitochondrial preparations with the yeast BiP and DnaK-specific antibodies showed that this organelle contained a family of HSP-70-related proteins. The yeast BiP antibodies recognized two mitochondrial proteins of 60 and 58 kDa, but failed to detect any protein in the size rang of 70 to 80 kDa. However, the presence of immunologically distinct proteins of 90 and 78 kDa, as well as of lower molecular weight from this family in the mitochondria, was shown by probing with the DnaK-specific antibodies. A new protein of 30 kDa, cross-reacting with anti-yeast BiP antibodies, was detected only in developing seeds, close to their maturity. The evolution of HSP-70 cognate proteins in wheat as shown in this study is discussed.     

Genetic and biochemical analysis of common wheat cultivars lacking puroindoline a

Puroindoline a (Pin-a) and puroindoline b (Pin-b), two basic isoforms encoded by the Pina-D1 and Pinb-D1 loci respectively, involved in controlling grain texture in wheat, were isolated from starch granules of soft wheat cultivars using three different extraction procedures, and fractionated by acidic polyacrylamide gel electrophoresis (A-PAGE). Tris buffer containing 1% Triton X-114 extracted Pin-a and small amounts of Pin-b, whereas 1% SDS preferably extracted Pin-b. Large amounts of both puroindolines were isolated by a solution containing 50% propan-2-ol and 50 mM NaCl. This solution extracted reduced amounts of Pin-b and no traces of Pin-a from starch granules of 20 hard common wheats containing the null allele Pina-D1b. The absence of Pin-a was confirmed by immunostaining with an anti-Pin-a antiserum. With the exception of two cultivars, null Pin-a cultivars gave no PCR fragment with three primer pairs specific to either the coding region or the promoter region of Pina-D1a, suggesting that major changes had occurred at the Pina-D1 locus in these genotypes. Cultivars Fortuna and Glenman were unique in giving size-specific PCR fragments with all primer pairs for the allele Pina-D1a and showed a cytosine deletion at position 267 in the coding region of the Pin-a gene, which resulted in a TGA stop codon at position 361. However, there was no evidence of a mutated protein in the A-PAGE or SDS-PAGE patterns of Fortuna and Glenman. The novel gene, provisionally named Pina-D1c, is the first null allele due to a point mutation that has been identified at the Pina-D1 locus.     

Expression of puroindoline a enhances leaf rust resistance in transgenic tetraploid wheat

The purouindoline gene (pin) coding for puroindoline proteins (PINs) is located on chromosome 5D, controls grain hardness, and the PINs have in vitro antimicrobial activity against gram-positive (G+) bacteria, gram-negative (G-) bacteria and fungi. Wheat leaf rust caused by Puccinia triticina is one of the most important fungal diseases for common wheat with AABBDD genomes. Tetraploid wheat (AABB genome) varieties Luna and Venusia were transformed with the purouindoline a (pinA) gene by bombardment, express PINA consititutively. Transgenic plants showed enhanced response to leaf rust in greenhouse and field. Comparative study of harvesting parameters showed significant differences between transgenic and control plants. These indexes were significantly lower (P < 0.05) in control plants than that in transgenic plants, which suggests that they are significantly affected by pinA gene and that the puroindoline a protein (PINA) can effectively inhibit in vivo the growth of fungal, and the transgenic tetraploid wheat can grow well in Hubei Province, Central China, where the tetraploid wheat varieties Luna and Venusia have poor yield due to their disease-sensitivity.     

Expression of wheat puroindoline genes in transgenic rice enhances grain softness

The puroindoline genes (pinA and pinB) are believed to play critical roles in wheat (Triticum aestivum L.) grain texture. Mutations in either gene are associated with hard wheat. No direct evidence exists for the ability of puroindolines to modify cereal grain texture. Interestingly, puroindolines appear to be absent in cereal species outside of the tribe Triticeae, in which the dominant form of grain texture is hard. To assess the ability of the puroindolines to modify cereal grain texture, the puroindolines were introduced into rice (Oryzae sativa L.) under the control of the maize ubiquitin promoter. Textural analysis of transgenic rice seeds indicated that expression of PINA and/or PINB reduced rice grain hardness. After milling, flour prepared from these softer seeds had reduced starch damage and an increased percentage of fine flour particles. Our data support the hypothesis that puroindolines play important roles in controlling wheat grain texture and may be useful in modifying grain texture of other cereals.     

Seed-specific expression of the wheat puroindoline genes improves maize wet milling yields

The texture of maize ( Zea mays L.) seeds is important to seed processing properties, and soft dent maize is preferred for both wet-milling and livestock feed applications. The puroindoline genes ( Pina and Pinb ) are the functional components of the wheat ( Triticum aestivum L.) Hardness locus and together function to create soft grain texture in wheat. The PINs (PINA and PINB) are believed to act by binding to lipids on the surface of starch granules, preventing tight adhesion between starch granules and the surrounding protein matrix during seed maturation. Here, maize kernel structure and wet milling properties were successfully modified by the endosperm-specific expression of wheat Pins ( Pina and Pinb ). Pins were introduced into maize under the control of a maize γ- Zein promoter. Three Pina/Pinb expression positive transgenic lines were evaluated over two growing seasons. Textural analysis of the maize seeds indicated that the expression of PINs decreased adhesion between starch and protein matrix and reduced maize grain hardness significantly. Reduction in pressure required to fracture kernels ranged from 15.65% to 36.86% compared with control seeds. Further, the PINs transgenic maize seeds had increased levels of extractable starch as characterized by a small scale wet milling method. Starch yield was increased by 4.86% on average without negatively impacting starch purity. The development of softer maize hybrids with higher starch extractability would be of value to maize processors.     

Characterization and antifungal properties of wheat nonspecific lipid transfer proteins

This study simultaneously considered the phylogeny, fatty acid binding ability, and fungal toxicity of a large number of monocot nonspecific lipid transfer proteins (ns-LTP). Nine novel full-length wheat ns-LTP1 clones, all possessing coding sequences of 348 bp, isolated from abiotic- and biotic-stressed cDNA libraries from aerial tissues, exhibited highly conserved coding regions with 78 to 99 and 71 to 100% identity at the nucleotide and amino acid levels, respectively. Phylogenetic analyses revealed two major ns-LTP families in wheat. Eight wheat ns-LTP genes from different clades were cloned into the expression vector pPICZalpha and transformed into Pichia pastoris. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, Western blotting, and in vitro lipid binding activity assay confirmed that the eight ns-LTP were all successfully expressed and capable of in vitro binding fatty acid molecules. A comparative in vitro study on the toxicity of eight wheat ns-LTP to mycelium growth or spore germination of eight wheat pathogens and three nonwheat pathogens revealed differential toxicities among different ns-LTP. Values indicating 50% inhibition of fungal growth or spore germination of three selected ns-LTP against six fungi ranged from 1 to 7 microM. In vitro lipid-binding activity of ns-LTP was not correlated with their antifungal activity. Using the fluorescent probe SYTOX Green as an indicator of fungal membrane integrity, the in vitro toxicity of wheat ns-LTP was associated with alteration in permeability of fungal membranes.     

Determination and evaluation of the sequence and textural effects of the puroindoline a and puroindoline b genes in a population of synthetic hexaploid wheat

Aegilops tauschii (2n=2 x=14, DD) is a rich source of genetic variability for hexaploid wheat (Triticum aestivum, 2n=6 x=42, AABBDD) improvement. This variability can be accessed through utilizing synthetic hexaploid wheat lines, which contain genomes from Ae. tauschii and T. turgidum (2n=4 x=28, AABB). Numerous desirable characteristics can and have been introgressed into common hexaploid wheat with this germplasm. In this work, the genetic variability in the two puroindoline genes (a and b) contained on the D genome, and the relationship that sequence polymorphisms in these genes have on endosperm texture among a population of 75 CIMMYT synthetic hexaploid accessions is described. Kernel texture was evaluated using the single kernel characterization system (SKCS). Kernel texture differed significantly (P0.0001) among the synthetic hexaploid accessions (range 2.6–40.9) and the parent types, durum or Ae. tauschii. The interaction term between parent types was also a significant effect (P0.0001). In addition to the wild-type protein sequences of the puroindoline genes (those present in Chinese Spring and all other soft wheats), three other translated sequences were identified in puroindoline a and two others in puroindoline b. These protein sequences were associated with significantly (P0.0001) softer endosperm textures than the wild-type protein sequences. As the softer alleles are expressed in a hexaploid background, they are immediately available to wheat breeding programs.     

Evidence of physical interactions of puroindoline proteins using the yeast two-hybrid system

The puroindoline proteins PINA and PINB play key roles in determining wheat grain texture and also have potential antimicrobial roles. Many recent studies show that their roles in grain texture involve some interaction or interdependence, and their antimicrobial activity may also involve formation of protein complexes. The issue of whether any homo- and/or heteromeric associations occur amongst the PIN proteins is thus critical for understanding their biological functions and exploiting them for grain texture modifications or antimicrobial applications, but is as yet unresolved. This work has utilised the well-established yeast two-hybrid system to directly address this issue. The results confirm occurrence of in vivo interactions between the two PIN proteins for the first time, and show that PINB interacts with itself and also interacts, although somewhat weakly, with PINA, while PINA is a weaker interactor. The results explain the many reported observations suggesting a co-operative interaction between the two proteins and provide a rapid and efficient tool for testing the effects of various alleles/mutations on the interactions and lipid binding properties of these proteins, which are of functional significance to grain texture and antimicrobial defence functions.     

Distribution of puroindoline alleles in bread wheat cultivars of the Yellow and Huai valley of China and discovery of a novel puroindoline a allele without PINA protein

Kernel hardness is one of the most important factors determining the milling and processing quality of bread wheat (Triticum aestivum L.). In the present study, 267 wheat cultivars and advanced lines from the Yellow and Huai Valley of China, CIMMYT, Russia and Ukraine were used for identification of SKCS (Single Kernel Characterization System) hardness and puroindoline alleles. Results indicated that Pinb-D1b is the most popular genotype in wheat cultivars from the Yellow and Huai Valley, Russia and Ukraine, whereas PINA null is a predominant genotype in wheat cultivars and advanced lines from CIMMYT. Molecular characterization of PINA-null alleles indicated that one Chinese landrace Chiyacao had the allele Pina-D1l with a single nucleotide C deletion at position 265 in Pina coding region based on sequencing results, and 35 of 39 PINA-null alleles belonged to Pina-D1b according to PCR amplification with the sequence-tagged site (STS) marker Pina-N developed previously. The remaining three cultivars (Jiangdongmen, Heshangtou and Hongquanmang from China) with PINA-null alleles were characterized at the DNA level by a primer walking strategy, and the results showed that all three cultivars with PINA-null alleles possessed a uniform 10,415-bp deletion from −5,117 bp to +5,298 bp (ATG codon references zero), designated as Pina-D1r. Correspondingly, an STS marker Pina-N2 with an expected fragment size of 436-bp spanning the 10,415-bp deletion was developed for detection of the Pina-D1r allele. This study provided a useful molecular marker for straightforward detection of one of the PINA-null alleles and would also be helpful to further understand the molecular and genetic basis of kernel hardness in bread wheat.     

Recurrent deletions of puroindoline genes at the grain hardness locus in four independent lineages of polyploid wheat

Polyploidy is known to induce numerous genetic and epigenetic changes but little is known about their physiological bases. In wheat, grain texture is mainly determined by the Hardness (Ha) locus consisting of genes Puroindoline a (Pina) and b (Pinb). These genes are conserved in diploid progenitors but were deleted from the A and B genomes of tetraploid Triticum turgidum (AB). We now report the recurrent deletions of Pina-Pinb in other lineages of polyploid wheat. We analyzed the Ha haplotype structure in 90 diploid and 300 polyploid accessions of Triticum and Aegilops spp. Pin genes were conserved in all diploid species and deletion haplotypes were detected in all polyploid Triticum and most of the polyploid Aegilops spp. Two Pina-Pinb deletion haplotypes were found in hexaploid wheat (Triticum aestivum; ABD). Pina and Pinb were eliminated from the G genome, but maintained in the A genome of tetraploid Triticum timopheevii (AG). Subsequently, Pina and Pinb were deleted from the A genome but retained in the A(m) genome of hexaploid Triticum zhukovskyi (A(m)AG). Comparison of deletion breakpoints demonstrated that the Pina-Pinb deletion occurred independently and recurrently in the four polyploid wheat species. The implications of Pina-Pinb deletions for polyploid-driven evolution of gene and genome and its possible physiological significance are discussed.     

Mapping QTLs for grain hardness and puroindoline content in wheat ( Triticum aestivum L.)

Genes for puroindoline-a (Pin-a), puroindoline-b (Pin-b) and grain-softness proteins (GSP) have been shown to be linked to the dominant Ha locus responsible for the soft texture of the grain. Though linkage has been demonstrated of the puroindoline genes to the Ha locus, there is no clear evidence that puroindoline content is the product of the gene Ha. A segregating population of 115 recombinant inbred lines (RILs) originating from a cross between the hexaploid Synthetic wheat ( Triticum durum x Aegilops tauschii, W 7984) and the cultivar 'Opata' (M 85) was studied in two different experimental years to detect Quantitative Trait Loci (QTLs) for three traits: grain hardness (Hard), puroindoline-a (Pin-a) and puroindoline-b (Pin-b) contents. The detection of QTLs was performed using marker linear regression. Negative correlation coefficients (-0.86 and -0.80) were identified between grain hardness and puroindoline content (a and b, respectively) on data obtained in 1996. Results obtained in 1999 confirmed the negative correlation between Hard and Pin-a (-0.73); however a positive correlation coefficient was found with Pin-b content (0.41). Total phenotypic variation explained by each QTL was calculated (R2). For each of the Hard, Pin-a and Pin-b traits one major QTL was detected on the short arm of chromosome 5D, located close to the mta9 allele (puroindoline-a). For the first year (1996) the QTL in this region explained around 63% of the phenotypic variability in grain hardness, 77% in Pin-a and 45% in Pin-b contents. These values were confirmed in trials carried out in 1999 with a R2 value of 0.71, 0.72 and 0.25 for Hard, Pin-a and Pin-b, respectively. In 1996 and 1999 a second major QTL was detected for grain hardness on the long arm of the same chromosome. Present results indicate that it cannot be definitely concluded that puroindoline content represents a linear explanation for variations in grain hardness.     

Characterization of wheat gliadin proteins by combined two-dimensional gel electrophoresis and tandem mass spectrometry

A proteomics-based approach was used for characterizing wheat gliadins from an Italian common wheat (Triticum aestivum) cultivar. A two-dimensional gel electrophoresis (2-DE) map of roughly 40 spots was obtained by submitting the 70% alcohol-soluble crude protein extract to isoelectric focusing on immobilized pH gradient strips across two pH gradient ranges, i.e., 3-10 or pH 6-11, and to sodium dodecyl sulfate-polyacrylamide electrophoresis in the second dimension. The chymotryptic digest of each spot was characterized by matrix-assisted laser desorption/ionization-time of flight mass spectrometry and nano electrospray ionization-tandem mass spectrometry (MS/MS) analysis, providing a "peptide map" for each digest. The measured masses were subsequently sought in databases for sequences. For accurate identification of the parent protein, it was necessary to determine de novo sequences by MS/MS experiments on the peptides. By partial mass fingerprinting, we identified protein molecules such as alpha/beta-, gamma-, omega-gliadin, and high molecular weight-glutenin. The single spots along the 2-DE map were discriminated on the basis of their amino acid sequence traits. alpha-Gliadin, the most represented wheat protein in databases, was highly conserved as the relative N-terminal sequence of the components from the 2-DE map contained only a few silent amino acid substitutions. The other closely related gliadins were identified by sequencing internal peptide chains. The results gave insight into the complex nature of gliadin heterogeneity. This approach has provided us with sound reference data for differentiating gliadins amongst wheat varieties.     

Characterization of wheat germ lipase

Some properties of wheat germ lipase were determined with a fluorometric assay of enzymatic cleavage converting the nonfluorescent 4-methyl umbelliferone butyrate (4-MUB) to the highly fluorescent 4-methyl umbelliferone (4-MU). Optimum reaction conditions were attained at buffer pH 7·5 and temperature 30°. Lineweaver-Burk plots were linear. Relative cation combination effectiveness as reaction activators was Ca + Mg + K > Ca + Mg + K + Na > Ca + Mg + Na > Ca + Mg > Mg > Ca, with no reaction effects of K, Na, and K + Na without Ca or Mg. Highly significant inhibitors of lipase reaction were CN⁻, aflatoxin, Cu²⁺, Fe³⁺, S²⁻, and EDTA.     

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.     

Inheritance of grain proteins in wheat

Summary Diallel crosses between five divergent vulgare wheat cultivars were made in order to evaluate the mode of inheritance and combining ability of grain proteins. Significant differences in grain protein content were found between cultivars and their hybrids. It was established that the inheritance of seed protein in the F₁ generation included both additive and non-additive gene action.