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Background Barley seed proteins are of prime importance to the brewing industry, human and animal nutrition and in plant breeding for cultivar identification. To obtain comprehensive proteomic data from seeds, total protein from a two-rowed (Conrad) and a six-rowed (Lacey) barley cultivar were precipitated in acetone, digested in-solution , and the resulting peptides were analyzed by nano-liquid chromatography coupled with tandem mass spectrometry. Results The raw mass spectra data searched against Uniprot’s Barley database using in-house Mascot search engine identified 1168 unique proteins. Gene Ontology (GO) analysis indicated that the majority of the seed proteins were cytosolic, with catalytic activity and associated with carbohydrate metabolism. Spectral counting analysis showed that there are 20 differentially abundant seed proteins between the two-rowed Conrad and six-rowed Lacey cultivars. Conclusion This study paves the way for the use of a top-down gel-free proteomics strategy in barley for investigating more complex traits such as malting quality. Differential abundance of hordoindoline proteins impact the seed hardness trait of barley cultivars.

Barley seeds are one of the main ingredients of the malting industry for brewing beer. The barley rootlets that are separated from the kilned seeds at the end of the malting process and used as animal feed are one of the byproducts of this industry. In this study, the proteome of rootlets derived from two stages of the malting process, germination and kilning, from a popular malting barley variety were analyzed. A label-free shotgun proteomics strategy was used to identify more than 800 proteins from the barley rootlets. A high coverage and high confidence Gene Ontology annotations of the barley genome was used to facilitate the functional annotation of the proteins that were identified in the rootlets. An analysis of these proteins using Kellogg Encyclopedia of Genes and Genomes (KEGG) and Plant Reactome databases indicated the enrichment of pathways associated with phytohormones, protein biosynthesis, secondary metabolism, and antioxidants. Increased levels of jasmonic acid and auxin in the rootlets further supported the in silico analysis. As a rich source of proteins and amino acids use of these by-products of the malting industry for animal feed is validated. This study also indicates rootlets as a potential source of naturally occurring phenylpropanoids and antioxidants that can be further exploited in the development of functional foods.

Maximizing microbial functions for improving crop performance requires better understanding of the important drivers of plant-associated microbiomes. However, it remains unclear the forces that shapes microbial structure and assembly, and how plant seed-microbiome interactions impact grain quality. In this work, we characterized the seed endophytic microbial communities of malting barley from different geographical locations and investigated associations between microbial (bacterial and fungal) species diversity and malt quality traits. Host genotype, location, and interactions (genotype x location) significantly impacted the seed endophytic microbial communities. Taxonomic composition analysis identified the most abundant genera for bacterial and fungal communities to be Bacillus (belonging to phylum Firmicutes) and Blumeria (belonging to phylum Ascomycota), respectively. We observed that a greater proportion of bacterial amplicon sequence variants (bacterial ASVs) were shared across genotypes and across locations while the greater proportion of the fungal ASVs were unique to each genotype and location. Association analysis showed a significant negative correlation between bacterial alpha diversity indices (Faith PD and Shannon indices) and malt quality traits for barley protein (BP), free amino nitrogen (FAN), diastatic power (DP) and alpha amylase (AA), while fungal alpha diversity (Shannon and Simpson) showed significant negative relationship with β-D-glucan content. In addition, some bacterial and fungal genera were significantly associated with malt extract (ME) -a key trait for maltsters and brewers. We conclude that barley genotype, location, and their interactions shape the seed endophytic microbiome and is key to microbiome manipulation and management during barley production and/or malting.

Drought stress significantly reduces crop yields at all stages of plant development. Barley, known for its abiotic-stress adaptation among cereals was used to examine the genetic basis of drought tolerance. A population of 164 spring barley lines was subjected to polyethylene glycol (PEG) induced drought stress during germination and seedling development. Six traits were measured, including germination percentage and rate, seedling length and weight, and root-to-shoot ratios. Seedling area, volume, and root and shoot diameter was acquired with a flatbed scanner. This population was also subjected to short-term drought during the heading stage in the greenhouse. Root and shoot weight and grain yield data were collected from well watered and droughted plants. Significant variation within traits were observed and several of them exhibited strong correlations with each other. In this population, two genotypes had 100% germination under PEG-induced drought and drought tolerance throughout the heading stage of plant development. A genome-wide association scan (GWAS) revealed 64 significant marker-trait associations across all seven barley chromosomes. Candidate genes related to abiotic stress and germination were identified within a 0.5Mbp interval around these SNPs. In silico analysis indicated a high frequency of differential expression of the candidate genes in response to stress. This study enabled identification of barley lines useful for drought tolerance breeding and pinpointed candidate genes for enhancing drought resiliency in barley.

Drought and heat stress substantially impact plant growth and productivity. When subjected to drought or heat stress, plants exhibit reduction in growth resulting in yield losses. The occurrence of these two stresses together intensifies their negative effects. Unraveling the molecular changes in response to combined abiotic stress is essential to breed climate-resilient crops. In this study, transcriptome profiles were compared between stress-tolerant (Otis), and stress-sensitive (Golden Promise) barley genotypes subjected to drought, heat, and combined heat and drought stress for five days during heading stage. The major differences that emerged from the transcriptome analysis were the overall number of differentially expressed genes was relatively higher in Golden Promise (GP) compared to Otis. The differential expression of more than 900 transcription factors in GP and Otis may aid this transcriptional reprogramming in response to abiotic stress. Secondly, combined heat and water deficit stress results in a unique and massive transcriptomic response that cannot be predicted from individual stress responses. Enrichment analyses of gene ontology terms revealed unique and stress type-specific adjustments of gene expression. Weighted Gene Co-expression Network Analysis identified genes associated with RNA metabolism and Hsp70 chaperone components as hub genes that can be useful for engineering tolerance to multiple abiotic stresses. Comparison of the transcriptomes of unstressed Otis and GP plants identified several genes associated with biosynthesis of antioxidants and osmolytes were higher in the former that maybe providing innate tolerance capabilities to effectively combat hostile conditions. Lines with different repertoire of innate tolerance mechanisms can be effectively leveraged in breeding programs for developing climate-resilient barley varieties with superior end-use traits.

Objectives Seeds host microbes that function in plant growth and phytopathogen resistance. The aim of the work was to investigate total bacterial community in malting barley seeds and whether their bacterial seed endophytes have dual functional roles in plant growth-promotion and inhibition of Fusarium graminearum , the causative agent of Fusarium head blight (FHB) in barley. We used culture dependent and culture independent methods. Results Phylogenetic classification of seed endophytic bacteria based on sequencing data identified B. subtilis , B. licheniformis and B. pumilis as predominant subgroups. Location driven divergence in bacterial endophytic communities was evident based on a clear separation of the samples from Crookston and other location samples. The bio-primed seeds using one hundred and seventy bacterial isolates showed that 3.5% (6/170) of the bacterial isolates conferred greater than 10% increase in both root length (RL) and shoot length (SL), while 19.4% (33/170) and 26.5% (45/170) showed RL and SL specific growth effects, respectively, relative to controls. Among the six bacterial isolates that increased RL and SL, five (#29, #63, #109, #124 and #126) also significantly inhibit the growth of F. graminearum based on in vitro assays. This study identified novel seed bacterial endophytes that could be further exploited for promoting growth during seedling establishment and as biocontrol for combating the devastating scab disease.

Controlled generation of reactive oxygen species (ROS) is pivotal for normal plant development and adaptation to changes in the external milieu. One of the major enzymatic sources of ROS in plants are the plasma-membrane localized NADPH oxidases, also called as Respiratory Burst Oxidase Homologs (RBOH). In addition to the six previously reported, seven new members of RBOH gene family were identified in barley using in silico analysis. Conservation of genomic structure and key residues important for catalytic activity and co-factor binding was observed in barley RBOH genes. Phylogenetic analysis of plant RBOHs revealed distinct clades for monocot and dicot RBOH proteins. Hence, we propose to use the rice nomenclature for naming barley RBOH genes. Temporal changes in ROS profiles were observed during barley malting and was accompanied by changes in protein carbonylation, lipid peroxidation, and antioxidant capacity. Among the nine differentially expressed HvRBOHs during various malting stages, HvRBOHA and HvRBOHC showed most significant sustained changes in expression. RNAi knockdown lines with reduced expression of HvRBOHA/C gene exhibited genetic compensation via inducible expression of other gene family members during malting. However, the physiological consequence of reduced expression of HvRBOHA/C manifested as a poor malting quality profile attributable to low alpha-amylase activity and high levels of beta-glucan. We propose that the HvRBOHs play a critical role in modulating the redox milieu during the early stages of malting, which in turn can significantly impact carbohydrate metabolism.

Background With ongoing climate change, drought events are severely limiting barley production worldwide and pose a significant risk to the malting, brewing and food industry. The genetic diversity inherent in the barley germplasm offers an important resource to develop stress resiliency. The purpose of this study was to identify novel, stable, and adaptive Quantitative Trait Loci (QTL), and candidate genes associated with drought tolerance. A recombinant inbred line (RIL) population (n = 192) developed from a cross between the drought tolerant ‘Otis’ barley variety, and susceptible ‘Golden Promise’(GP) was subjected to short-term progressive drought during heading in the biotron. This population was also evaluated under irrigated and rainfed conditions in the field for yields and seed protein content. Results Barley 50k iSelect SNP Array was used to genotype the RIL population to elucidate drought-adaptive QTL. Twenty-three QTL (eleven for seed weight, eight for shoot dry weight and four for protein content) were identified across several barley chromosomes. QTL analysis identified genomic regions on chromosome 2 and 5 H that appear to be stable across both environments and accounted for nearly 60% variation in shoot weight and 17.6% variation in seed protein content. QTL at approximately 29 Mbp on chromosome 2 H and 488 Mbp on chromosome 5 H are in very close proximity to ascorbate peroxidase ( APX ) and in the coding sequence of the Dirigent ( DIR ) gene, respectively. Both APX and DIR are well-known key players in abiotic stress tolerance in several plants. In the quest to identify key recombinants with improved tolerance to drought (like Otis) and good malting profiles (like GP), five drought tolerant RILs were selected for malt quality analysis. The selected drought tolerant RILs exhibited one or more traits that were outside the realms of the suggested limits for acceptable commercial malting quality. Conclusions The candidate genes can be used for marker assisted selection and/or genetic manipulation to develop barley cultivars with improved tolerance to drought. RILs with genetic network reshuffling necessary to generate drought tolerance of Otis and favorable malting quality attributes of GP may be realized by screening a larger population.

Background Nudix hydrolases play a key role in maintaining cellular homeostasis by hydrolyzing various nuceloside diphosphate derivatives and capped mRNAs. Several independent studies have demonstrated that Arabidopsis nudix hydrolase 7 (AtNUDT7) hydrolyzes NADH and ADP-ribose. Loss of function Atnudt7-1 mutant plants (SALK_046441) exhibit stunted growth, higher levels of reactive oxygen species, enhanced resistance to pathogens. However, using the same T-DNA line, two other groups reported that mutant plants do not exhibit any visible phenotypes. In this study we analyze plausible factors that account for differences in the observed phenotypes in Atnudt7 . Secondly, we evaluate the biochemical and molecular consequences of increased NADH levels due to loss of function of AtNUDT7 in Arabidopsis. Results We identified a novel conditional phenotype of Atnudt7-1 knockout plants that was contingent upon nutrient composition of potting mix. In nutrient-rich Metro-Mix, there were no phenotypic differences between mutant and wild-type (WT) plants. In the nutrient-poor mix (12 parts vermiculite: 3 parts Redi-earth and 1 part sand), mutant plants showed the characteristic stunted phenotype. Compared with WT plants, levels of glutathione, NAD + , NADH, and in turn NADH:NAD + ratio were higher in Atnudt7-1 plants growing in 12:3:1 potting mix. Infiltrating NADH and ADP-ribose into WT leaves was sufficient to induce AtNUDT7 protein. Constitutive over-expression of AtNudt7 did not alter NADH levels or resistance to pathogens. Transcriptome analysis identified nearly 700 genes differentially expressed in the Atnudt7-1 mutant compared to WT plants grown in 12:3:1 potting mix. In the Atnudt7- 1 mutant, genes associated with defense response, proteolytic activities, and systemic acquired resistance were upregulated, while gene ontologies for transcription and phytohormone signaling were downregulated. Conclusions Based on these observations, we conclude that the differences observed in growth phenotypes of the Atnudt7-1 knockout mutants can be due to differences in the nutrient composition of potting mix. Our data suggests AtNUDT7 plays an important role in maintaining redox homeostasis, particularly for maintaining NADH:NAD + balance for normal growth and development. During stress conditions, rapid induction of AtNUDT7 is important for regulating the activation of stress/defense signaling and cell death pathways.

Switchgrass (Panicum virgatum L.) is a C4 perennial grass and widely popular as an important bioenergy crop. To accelerate the pace of developing high yielding switchgrass cultivars adapted to diverse environmental niches, the generation of genomic resources for this plant is necessary. The large genome size and polyploid nature of switchgrass makes whole genome sequencing a daunting task even with current technologies. Exploring the transcriptional landscape using next generation sequencing technologies provides a viable alternative to whole genome sequencing in switchgrass. Switchgrass cDNA libraries from germinating seedlings, emerging tillers, flowers, and dormant seeds were sequenced using Roche 454 GS-FLX Titanium technology, generating 980,000 reads with an average read length of 367 bp. De novo assembly generated 243,600 contigs with an average length of 535 bp. Using the foxtail millet genome as a reference greatly improved the assembly and annotation of switchgrass ESTs. Comparative analysis of the 454-derived switchgrass EST reads with other sequenced monocots including Brachypodium, sorghum, rice and maize indicated a 70-80% overlap. RPKM analysis demonstrated unique transcriptional signatures of the four tissues analyzed in this study. More than 24,000 ESTs were identified in the dormant seed library. In silico analysis indicated that there are more than 2000 EST-SSRs in this collection. Expression of several orphan ESTs was confirmed by RT-PCR. We estimate that about 90% of the switchgrass gene space has been covered in this analysis. This study nearly doubles the amount of EST information for switchgrass currently in the public domain. The celerity and economical nature of second-generation sequencing technologies provide an in-depth view of the gene space of complex genomes like switchgrass. Sequence analysis of closely related members of the NAD(+)-malic enzyme type C4 grasses such as the model system Setaria viridis can serve as a viable proxy for the switchgrass genome.