Development of Functional Nanomaterials from Wheat Bran Derived Arabinoxylan for Nucleic Acid Delivery.

Wheat bran is a major byproduct of the wheat industry and a rich source of cellulosic and hemicellulosic compounds. We developed a facile and reproducible method to generate functional nanomaterials from wheat bran derived polysaccharide, Arabinoxylan (AX). We first established that AX derived from wheat bran was chemically equivalent to commercially available AX extracted from wheat flour. Through facile chemical modification, positive and negatively charged domains were introduced along AX backbone, which in turn induced local electrostatic and hydrophobic interactions promoting the formation of nanoparticulate structures. The extracted, chemically modified AX was characterized using FTIR, 1H NMR, and elemental analysis. We observed that, while both anionic and cationic AX self-assemble into stable, spherical nanoparticles with a low polydispersity index, the unmodified AX did not exhibit such self-organizational properties. To form functionally active nanomaterials, we further complexed negatively charged CRISPR-Cas9 DNA with cationic AX. Through gel electrophoretic studies, we identified that, at a feed ratio of DNA to AX of 1:15, AX is capable of forming polyplexes with DNA in the form of nanoparticles with an average hydrodynamic diameter of ∼100 nm and surface charge of -1.40 ± 0.91 mV. We envision that chemically modified AX, originally sourced from agricultural waste materials and not from food products, can be used as functional nanomaterials for gene delivery in the agrochemical sector thus catalyzing the circular approach of sustainability.

Metformin Uptake and Translocation in Chickpeas: Determination Using Liquid Chromatography-Mass Spectrometry.

Multiple chronic conditions (MCCs) such as diabetes, hypertension, heart disease, arthritis, asthma, and common respiratory problems are prevalent in over one-fourth of Americans, and separate drugs are prescribed to manage each of the diseases. The nutritive crop seeds loaded with multiple drugs could be a cheap and sustainable alternative to drugs produced by pharmaceutical companies. Our long-term goal is to produce chickpea seeds containing comparable dosages of multiple drugs regularly prescribed for managing MCC. In this work, we conducted experiments to understand the uptake and translocation of metformin into the tissues of chickpea to demonstrate the applicability of LC-HR-ToF-MS in determining metformin concentration, and to investigate responses of increased dosage of metformin and it's accumulation into the chickpea seed. We treated the chickpea plants with 100 and 500 mg/L metformin chloride and analyzed its concentration in the leaf, stem, and seeds. We observed that metformin was successfully uptaken by chickpeas plant and translocated to stem, leaf, and seeds in both treatments. We also observed that the metformin concentration is responsive and as high as 349 times increase in seed when the dosage was increased from 100 to 500 mg/L.

High-resolution radiation hybrid map of wheat chromosome 1D.

Physical mapping methods that do not rely on meiotic recombination are necessary for complex polyploid genomes such as wheat (Triticum aestivum L.). This need is due to the uneven distribution of recombination and significant variation in genetic to physical distance ratios. One method that has proven valuable in a number of nonplant and plant systems is radiation hybrid (RH) mapping. This work presents, for the first time, a high-resolution radiation hybrid map of wheat chromosome 1D (D genome) in a tetraploid durum wheat (T. turgidum L., AB genomes) background. An RH panel of 87 lines was used to map 378 molecular markers, which detected 2312 chromosome breaks. The total map distance ranged from approximately 3,341 cR(35,000) for five major linkage groups to 11,773 cR(35,000) for a comprehensive map. The mapping resolution was estimated to be approximately 199 kb/break and provided the starting point for BAC contig alignment. To date, this is the highest resolution that has been obtained by plant RH mapping and serves as a first step for the development of RH resources in wheat.

Blood Serum Affects Polysaccharide Production and Surface Protein Expression in S. Aureus.

BACKGROUND: S. aureus biofilm serves a major role in pathogenesis. Two of the major components of bacterial biofilm are Polysaccharides intercellular adhesions (PIA) and surface proteins. It is not known how PIA and surface proteins expressions are affected in presence of blood serum. Analyses of surface proteins expressions will provide more effective biomarker discovery that might lead to development of antimicrobial therapeutics to meet the challenges of biofilm-related infections. METHOD: Secondary cultures of S. aureus Philips, a biofilm-forming bacterium, were generated by inoculating 1 ml of overnight culture into 50 ml of TSB. Bacteria were cultured at several concentrations of blood serum and found that 12.5% supplemented blood serum provide s similar growth curve as normal TSB (100%). One and 2 D SASPAGE were used to separate proteins and the differentially expressed proteins were identified by nano-LC/MS. RESULTS: Polysaccharide intercellular adhesions production was significantly increased due to the addition of blood serum in the media. We also identified two serum proteins, apolipoprotein and globulin (Fc and Fab), that remained attached with the membrane fraction of bacterial proteins. CONCLUSION: These results have strongly demonstrated that blood serum influences the exopolysaccharide expression in S. aureus.

Effect of Increased Amounts of Fe, Zn, and Cd on Uptake, Translocation, and Accumulation of Human Health Related Micronutrients in Wheat.

Agricultural Scientists has lifted the crop production many folds' overs last 100 years but nutritive quality of crop products has not been addressed accordingly as a result humans in many parts of the world are suffering from malnutrition. The efficient improvement of nutritive quality of important crop species like wheat is dependent on the understanding of the acquisition of micronutrients from soil environment and subsequent translocation and distribution into different tissues. The objectives of this work were to understand the effect of increased concentrations of Fe, Zn, and Cd 1) on overall mineral and metal concentrations, 2) on acquisition, translocation, and distribution of minerals among different tissues, and 3) on the inter-relationship of the minerals and metals as reflected in changing the relationship pattern in wheat. The application of increased concentrations of Fe and Zn resulted in three and 11 folds' increase of these micronutrients in wheat respectively and significantly increased seed Ca, P, and S contents however acquisition and translocation of 20 mineral elements varied from tissue to tissue. The improvement of major crop species for health-related micronutrient is important for combating world- wide malnutrition problem. The higher concentration of one micronutrient element may not always ensure higher concentration of that element in seed but increase concentration of Fe and Zn may ensure higher concentrations of others important minerals in wheat seed. The results from our research unveiled key aspects on interrelation among some minerals and metals due to higher concentration of Fe, Zn, and Cd application in wheat.

Pretreatment of Wheat Bran for Suitable Reinforcement in Biocomposites.

Wheat bran, abundant but underutilized, was investigated for its potential as a reinforcement in biocomposites through different pretreatment methods. Pretreatment methods included were dilute sodium hydroxide (NaOH), dilute sulfuric acid (H2SO4), liquid hot water (LHW), calcium hydroxide (CaOH), organosolv such as aqueous ethanol (EtOH), and methyl isobutyl ketone (MIBK). Changes in chemical composition and fiber characteristics of the treated bran were studied using scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR). Cellulose content increased to 35.1% and 29.6% in brans treated with H2SO4 and NaOH, respectively. The SEM micrographs showed surface cleaning of treated bran while maintaining sufficient surface roughness for the H2SO4, NaOH, and MIBK treated brans. Crystallinity index increased slightly for all treatments except H2SO4. NaOH and H2SO4 pretreated brans achieved important fiber characteristics, which could be useful for making thermoplastic biocomposites. Innovative use of bran in thermoplastic will create more opportunities for growers while enhancing biodegradability.

Characterization and Expression Analysis of Common Bean Histone Deacetylase 6 during Development and Cold Stress Response.

Histone deacetylases (HDACs) are important regulators of gene transcription thus controlling multiple cellular processes. Despite its essential role in plants, HDA6 is yet to be validated in common bean. In this study, we show that HDA6 is involved in plant development and stress response. Differential expression of HDA6 was determined in various tissues and the expression was seen to be upregulated with plant age (seedling < flowering < maturity). Higher expression was observed in flowers and pods than in stem, leaf, and root. Upregulation of HDA6 gene during cold stress implies its prominent role in abiotic stress. Furthermore, the HDA6 gene was isolated from three common bean genotypes and sequence analyses revealed homology with functionally characterized homologs in model species. The 53 kDa translated product was detected using an HDA6 specific antibody and recombinant protein overexpressed in Escherichia coli showed HDAC activity in vitro. To our knowledge, this is the first report in the agriculturally important crop common bean describing the functional characterization and biological role of HDA6.

Analyses of Methylomes Derived from Meso-American Common Bean (Phaseolus vulgaris L.) Using MeDIP-Seq and Whole Genome Sodium Bisulfite-Sequencing.

Common bean (Phaseolus vulgaris L.) is economically important for its high protein, fiber, and micronutrient contents, with a relatively small genome size of ∼587 Mb. Common bean is genetically diverse with two major gene pools, Meso-American and Andean. The phenotypic variability within common bean is partly attributed to the genetic diversity and epigenetic changes that are largely influenced by environmental factors. It is well established that an important epigenetic regulator of gene expression is DNA methylation. Here, we present results generated from two high-throughput sequencing technologies, methylated DNA immunoprecipitation-sequencing (MeDIP-seq) and whole genome bisulfite-sequencing (BS-Seq). Our analyses revealed that this Meso-American common bean displays similar methylation patterns as other previously published plant methylomes, with CG ∼50%, CHG ∼30%, and CHH ∼2.7% methylation, however, these differ from the common bean reference methylome of Andean origin. We identified higher CG methylation levels in both promoter and genic regions than CHG and CHH contexts. Moreover, we found relatively higher CG methylation levels in genes than in promoters. Conversely, the CHG and CHH methylation levels were highest in promoters than in genes. This is the first genome-wide DNA methylation profiling study in a Meso-American common bean cultivar ("Sierra") using NGS approaches. Our long-term goal is to generate genome-wide epigenomic maps in common bean focusing on chromatin accessibility, histone modifications, and DNA methylation.

Radiation hybrid mapping of the species cytoplasm-specific (scsae) gene in wheat.

Radiation hybrid (RH) mapping is based on radiation-induced chromosome breakage and analysis of chromosome segment retention or loss using molecular markers. In durum wheat (Triticum turgidum L., AABB), an alloplasmic durum line [(lo) durum] has been identified with chromosome 1D of T. aestivum L. (AABBDD) carrying the species cytoplasm-specific (scsae) gene. The chromosome 1D of this line segregates as a whole without recombination, precluding the use of conventional genome mapping. A radiation hybrid mapping population was developed from a hemizygous (lo) scsae--line using 35 krad gamma rays. The analysis of 87 individuals of this population with 39 molecular markers mapped on chromosome 1D revealed 88 radiation-induced breaks in this chromosome. This number of chromosome 1D breaks is eight times higher than the number of previously identified breaks and should result in a 10-fold increase in mapping resolution compared to what was previously possible. The analysis of molecular marker retention in our radiation hybrid mapping panel allowed the localization of scsae and 8 linked markers on the long arm of chromosome 1D. This constitutes the first report of using RH mapping to localize a gene in wheat and illustrates that this approach is feasible in a species with a large complex genome.

GENETIC VARIABILITY OF MINERAL COMPOSITION IN COMMON BEAN SEED.

Common bean genotypes were grown in three different growing sites and analyzed for 17 mineral compositions. The influence of growing sites was observed on all seed mineral contents however, ratio of genotypic variance to genotype x environment variance indicated greater influence and stability of genetic factor on Ca and Sr. It was observed that the Zn concentration is highly correlated with S and Fe and Ca with Sr in common bean seed.

Minerals (Zn, Fe, Ca and Mg) and Antinutrient (Phytic Acid) Constituents in Common Bean.

In this study, the variation of zinc (Zn), iron (Fe), calcium (Ca) and magnesium (Mg) and the interference of phytic acid (PA) on their availability was investigated in 29 US grown and CIAT breeding genotypes of common bean. Fe levels showed the highest variation (8.9-112.9 mg kg-1) followed by Ca (58.67-122.98 mg kg-1) and Zn (30.90-64.60 mg kg-1) while variability of Mg concentration (6.47-11.05 mg kg-1) is the least among the mineral components. PA showed a wide range of variability (12.52-316.42 m kg-1) and inversely correlated with Fe, Ca and Mg concentrations. The results of the minerals and PA concentration can be interpreted in terms of expected bio-availability of minerals and the correlation study indicated that the presence of high concentration of PA inhibit the availability of most minerals under study in common beans. We suggest that the genotypes, MIB466, MIB465, MIB152 and JaloEEP 558 could be considered as sources of high Zn and Vista and NUA56-1770 for high seed Fe. We also identified G122 for high Ca and JaloEEP558 genotype for high Mg. We conclude that there is scope for the enhancement of mineral contents of common bean by selecting suitable genotype and bean products require processing for dephytinization for the improvement of mineral availability.

An integrated high-density linkage map of soybean with RFLP, SSR, STS, and AFLP markers using A single F2 population.

Soybean [Glycine max (L.) Merrill] is the most important leguminous crop in the world due to its high contents of high-quality protein and oil for human and animal consumption as well as for industrial uses. An accurate and saturated genetic linkage map of soybean is an essential tool for studies on modern soybean genomics. In order to update the linkage map of a F2 population derived from a cross between Misuzudaizu and Moshidou Gong 503 and to make it more informative and useful to the soybean genome research community, a total of 318 AFLP, 121 SSR, 108 RFLP, and 126 STS markers were newly developed and integrated into the framework of the previously described linkage map. The updated genetic map is composed of 509 RFLP, 318 SSR, 318 AFLP, 97 AFLP-derived STS, 29 BAC-end or EST-derived STS, 1 RAPD, and five morphological markers, covering a map distance of 3080 cM (Kosambi function) in 20 linkage groups (LGs). To our knowledge, this is presently the densest linkage map developed from a single F2 population in soybean. The average intermarker distance was reduced to 2.41 from 5.78 cM in the earlier version of the linkage map. Most SSR and RFLP markers were relatively evenly distributed among different LGs in contrast to the moderately clustered AFLP markers. The number of gaps of more than 25 cM was reduced to 6 from 19 in the earlier version of the linkage map. The coverage of the linkage map was extended since 17 markers were mapped beyond the distal ends of the previous linkage map. In particular, 17 markers were tagged in a 5.7 cM interval between CE47M5a and Satt100 on LG C2, where several important QTLs were clustered. This newly updated soybean linkage map will enable to streamline positional cloning of agronomically important trait locus genes, and promote the development of physical maps, genome sequencing, and other genomic research activities.

Molecular cytogenetic characterization of an alloplasmic durum wheat line with a portion of chromosome 1D of Triticum aestivum carrying the scs(ae) gene.

Triticum aestivum (2n = 6x = 42, AABBDD) with Triticum longissimum (2n = 2x = 14; S1S1) cytoplasm ((lo) cytoplasm) has normal fertility and plant vigor. However, the nucleus of durum wheat (Triticum turgidum (2n = 4x = 28, AABB)) is incompatible with the T. longissimum cytoplasm, producing non-viable progeny. This incompatibility is alleviated by scs(ae), a species cytoplasm-specific (scs) gene, on the long arm of chromosome 1D (1DL) of common wheat. The hemizygous (lo) durum scs(ae) line is male sterile and is maintained by crossing to normal durum wheat. After pollination, the seeds produced are either plump and viable (with scs(ae)) or shriveled and inviable (without scsae). Thus, the chromosome with scs(ae) is inherited as a whole without recombination. The objectives of this study were to characterize the chromosome carrying scs(ae) and to determine the process through which this gene was introgressed into the (lo) durum background. Molecular marker analysis with 27 probes and primers mapped to homoeologous group 1 and genomic in situ hybridization using differentially labeled total genomic DNA of durum wheat and Aegilops tauschii suggest the presence of a 1AL segment in place of the distal region of 1DL. Owing to the absence of any detectable duplications or deletions, homoeologous recombination is the most likely mechanism by which this introgression occurred.

The organization and rate of evolution of wheat genomes are correlated with recombination rates along chromosome arms.

Genes detected by wheat expressed sequence tags (ESTs) were mapped into chromosome bins delineated by breakpoints of 159 overlapping deletions. These data were used to assess the organizational and evolutionary aspects of wheat genomes. Relative gene density and recombination rate increased with the relative distance of a bin from the centromere. Single-gene loci present once in the wheat genomes were found predominantly in the proximal, low-recombination regions, while multigene loci tended to be more frequent in distal, high-recombination regions. One-quarter of all gene motifs within wheat genomes were represented by two or more duplicated loci (paralogous sets). For 40 such sets, ancestral loci and loci derived from them by duplication were identified. Loci derived by duplication were most frequently located in distal, high-recombination chromosome regions whereas ancestral loci were most frequently located proximal to them. It is suggested that recombination has played a central role in the evolution of wheat genome structure and that gradients of recombination rates along chromosome arms promote more rapid rates of genome evolution in distal, high-recombination regions than in proximal, low-recombination regions.

Synteny perturbations between wheat homoeologous chromosomes caused by locus duplications and deletions correlate with recombination rates.

Loci detected by Southern blot hybridization of 3,977 expressed sequence tag unigenes were mapped into 159 chromosome bins delineated by breakpoints of a series of overlapping deletions. These data were used to assess synteny levels along homoeologous chromosomes of the wheat A, B, and D genomes, in relation to both bin position on the centromere-telomere axis and the gradient of recombination rates along chromosome arms. Synteny level decreased with the distance of a chromosome region from the centromere. It also decreased with an increase in recombination rates along the average chromosome arm. There were twice as many unique loci in the B genome than in the A and D genomes, and synteny levels between the B genome chromosomes and the A and D genome homoeologues were lower than those between the A and D genome homoeologues. These differences among the wheat genomes were attributed to differences in the mating systems of wheat diploid ancestors. Synteny perturbations were characterized in 31 paralogous sets of loci with perturbed synteny. Both insertions and deletions of loci were detected and both preferentially occurred in high recombination regions of chromosomes.

Comparative DNA sequence analysis of wheat and rice genomes.

The use of DNA sequence-based comparative genomics for evolutionary studies and for transferring information from model species to crop species has revolutionized molecular genetics and crop improvement strategies. This study compared 4485 expressed sequence tags (ESTs) that were physically mapped in wheat chromosome bins, to the public rice genome sequence data from 2251 ordered BAC/PAC clones using BLAST. A rice genome view of homologous wheat genome locations based on comparative sequence analysis revealed numerous chromosomal rearrangements that will significantly complicate the use of rice as a model for cross-species transfer of information in nonconserved regions.