The genes encoding granule-bound starch synthases at the waxy loci of the A, B, and D progenitors of common wheat.

Three genes encoding granule-bound starch synthase (wx-TmA, wx-TsB, and wx-TtD) have been isolated from Triticum monococcum (AA), and Triticum speltoides (BB), by the polymerase chain reaction (PCR) approach, and from Triticum tauschii (DD), by screening a genomic DNA library. Multiple sequence alignment indicated that the wx-TmA, wx-TsB, and wx-TtD genes had the same extron and (or) intron structure as the previously reported waxy gene from barley. The lengths of the three wx-TmA, wx-TsB, and wx-TtD genes were 2834 bp, 2826 bp, and 2893 bp, respectively, each covering 31 bp in the untranslated leader and the entire coding region consisting of 11 exons and 10 introns. The three genes had identical lengths of exons, except exonl, and shared over 95% identity with each other within the exon regions. The majority of introns were significantly variable in length and sequence, differing mainly in length (1-57 bp) as a result of insertion and (or) deletion events. The deduced amino acid sequence from these three genes indicated that the mature WX-TMA, -TSB, and -TTD proteins contained the same number of amino acids, but differed in predicted molecular weight and isoelectric point (pI) due to amino acid substitutions (13-18). The predicted physical characteristics of the WX proteins matched the respective proteins in wheat very closely, but the match was not perfect. Furthermore the exon5 sequences of the wx-TmA, wx-TsB, and wx-TtD genes were different from a cDNA encoding a waxy gene of common wheat previously reported. The striking difference was that an insertion of 11 amino acids occurred in the cDNA sequence that could not be observed in the exons of the A, B, and D genes. It was noted, however, that the 3' end of intron4 of these genes could account for the additional 11 amino acids. The sequence information from the available waxy genes identified the intron4-exon5-intron5 region as being diagnostic for sequence variation in waxy. The sequence variation in the waxy genes provides the basis for primer design to distinguish the respective genes in common wheat, and its progenitors, using PCR.     

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.     

Use of DNA Markers in Prediction of Hybrid Performance and Heterosis for a Three-Line Hybrid System in Rice

Two Cytoplasmic Male Sterile lines were crossed with fourteen restorer lines of rice widely grown in the western regions of Maharashtra, India, to produce 28 F₁ hybrids which were evaluated for eight agronomically important traits, contributing to yield potential, in replicated field trials. The hybrid performance was recorded along with heterosis and heterobeltiosis. All the rice lines under investigation were subjected to marker-based variability analysis. An attempt was made to correlate genetic distance based on specific markers for each trait individually, as well as average genetic distance based on all specific markers, with hybrid performance and heterosis, by regression analysis. Specific markers could cluster the parental lines in different groups and showed significant correlation with hybrid performance. The data also supports the proposition that epistasis is the basis of heterosis. The analysis, however, revealed a lack of significant predictive values for field application.     

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.     

OrthoRBH: A streamlined pipeline for mining large gene family sequences in related species

Plant and animal genomes are replete with large gene families, making the task of ortholog identification difficult and labor intensive. OrthoRBH is an automated reciprocal blast pipeline tool enabling the rapid identification of specific gene families of interest in related species, streamlining the collection of homologs prior to downstream molecular evolutionary analysis. The efficacy of OrthoRBH is demonstrated with the identification of the 13-member PYR/PYL/RCAR gene family in Hordeum vulgare using Oryza sativa query sequences. OrthoRBH runs on the Linux command line and is freely available at SourceForge.

Availability

http://sourceforge.net/projects/ orthorbh/     

TOO MANY MOUTHS promotes cell fate progression in stomatal development of <Emphasis Type="Italic">Arabidopsis</Emphasis> stems

Mutations in TOO MANY MOUTHS (TMM), which encodes a receptor-like protein, cause stomatal patterning defects in Arabidopsis leaves but eliminate stomatal formation in stems. Stomatal development in wild-type and tmm stems was analyzed to define TMM function. Epidermal cells in young tmm stems underwent many asymmetric divisions characteristic of entry into the stomatal pathway. The resulting precursor cells, meristemoids, appropriately expressed cell fate markers such as pTMM:GFP. However, instead of progressing developmentally by forming a guard mother cell, the meristemoids arrested, dedifferentiated, and enlarged. Thus asymmetric divisions are necessary but not sufficient for stomatal formation in stems, and TMM promotes the fate and developmental progression of early precursor cells. Comparable developmental and mature stomatal phenotypes were also found in tmm hypocotyls and in the proximal flower stalk. TMM is also a positive regulator of meristemoid division in leaves suggesting that TMM generally promotes meristemoid activity. Our results are consistent with a model in which TMM interacts with other proteins to modulate precursor cell fate and progression in an organ and domain-specific manner. Finally, the consistent presence of a small number of dedifferentiated meristemoids in mature wild-type stems suggests that precursor cell arrest is a normal feature of Arabidopsis stem development.     

Exploration of Leads from Natural Domain Targeting HER2 in Breast Cancer: An In-Silico Approach

International Journal of Peptide Research and Therapeutics - The human epidermal growth factor (HER2/neu) receptor protein target expression plays a vital role in the development of breast cancer...     

Molecular genetics of puroindolines and related genes: allelic diversity in wheat and other grasses

The hardness or texture of cereal grains is a primary determinant of their technological and processing quality. Among members of the Triticeae, most notably wheat, much of the variation in texture is controlled by a single locus comprised of the Puroindoline a, Puroindoline b and Grain Softness Protein-1 (Gsp-1) genes. Puroindolines confer the three major texture classes of soft and hard common wheat and the very hard durum wheat. The protein products of these genes interact with lipids and are associated with the surface of isolated starch (as a protein fraction known as ‘friabilin’). During the past ten years a great diversity of alleles of both Puroindoline genes have been discovered and significant advances made in understanding the relationship between the gene presence/absence, sequence polymorphism and texture of cereal grains. Efforts have also focussed on Puroindoline and Gsp-1 genes in diploid progenitors, other Triticeae grasses and synthetic wheats in order to understand the evolution of this gene family and find potentially useful variants. The puroindoline homologues in other cereals such as rye and barley are also receiving attention. This work summarises new developments in molecular genetics of puroindolines in wheat and related Triticeae grasses, and the related genes in other cereals.