Genome-Wide Identification,Evolution and Expressional Analysis of OSCA Gene Family in Barley (Hordeum vulgare L.)

The hyperosmolality-gated calcium-permeable channel gene family (OSCA) is one kind of conserved osmosensors, playing a crucial role in maintaining ion and water homeostasis and protecting cellular stability from the damage of hypertonic stress. Although it has been systematically characterized in diverse plants, it is necessary to explore the role of the OSCA family in barley, especially its importance in regulating abiotic stress response. In this study, a total of 13 OSCA genes (HvOSCAs) were identified in barley through an in silico genome search method, which were clustered into 4 clades based on phylogenetic relationships with members in the same clade showing similar protein structures and conserved motif compositions. These HvOSCAs had many cis-regulatory elements related to various abiotic stress, such as MBS and ARE, indicating their potential roles in abiotic stress regulation. Furthermore, their expression patterns were systematically detected under diverse stresses using RNA-seq data and qRT-PCR methods. All of these 13 HvOSCAs were significantly induced by drought, cold, salt and ABA treatment, demonstrating their functions in osmotic regulation. Finally, the genetic variations of the HvOSCAs were investigated using the re-sequencing data, and their nucleotide diversity in wild barley and landrace populations were 0.4966 × 10⁻³ and 0.391 × 10⁻³, respectively, indicating that a genetic bottleneck has occurred in the OSCA family during the barley evolution process. This study evaluated the genomic organization, evolutionary relationship and genetic expression of the OSCA family in barley, which not only provides potential candidates for further functional genomic study, but also contributes to genetically improving stress tolerance in barley and other crops.     

Search for Barley (Hordeum vulgare L.) with higher lipid content

The search for a barley with a higher lipid content was concentrated on the USDA Barley World Conllection. Seeds of 14,000 entries were examined visually for an embryo size: total seed size ratio greater than the cultivated barley variety Prilar, and 60 entries were selected. Seeds of the 60 entries were assayed for lipid content by NMR spectroscopy, and the 7 entries with the highest oil content, along with Prilar, were prepared for further analysis. Barley lipids were solvent extracted, classified by silicic acid column chromatography, and separated by thin layer chromatography, and the fatty acid composition was determined by gas liquid chromatography. Total lipid contents of the 8 barleys ranged from 3.4% for Prilar to 4.6% for CI 12116. The seven selections had lipid contents which ranged from 9–35% higher than Prilar. Only slight qualitative differences were noted among the lipid classes of the eight barleys analyzed.     

Genome-Wide Identification and Characterization of the Brassinazole-resistant (BZR) Gene Family and Its Expression in the Various Developmental Stage and Stress Conditions in Wheat (Triticum aestivum L.)

Brassinosteroids (BRs) play crucial roles in various biological processes, including plant developmental processes and response to diverse biotic and abiotic stresses. However, no information is currently available about this gene family in wheat (Triticum aestivum L.). In the present investigation, we identified the BZR gene family in wheat to understand the evolution and their role in diverse developmental processes and under different stress conditions. In this study, we performed the genome-wide analysis of the BZR gene family in the bread wheat and identified 20 TaBZR genes through a homology search and further characterized them to understand their structure, function, and distribution across various tissues. Phylogenetic analyses lead to the classification of TaBZR genes into five different groups or subfamilies, providing evidence of evolutionary relationship with Arabidopsis thaliana, Zea mays, Glycine max, and Oryza sativa. A gene exon/intron structure analysis showed a distinct evolutionary path and predicted the possible gene duplication events. Further, the physical and biochemical properties, conserved motifs, chromosomal, subcellular localization, and cis-acting regulatory elements were also examined using various computational approaches. In addition, an analysis of public RNA-seq data also shows that TaBZR genes may be involved in diverse developmental processes and stress tolerance mechanisms. Moreover, qRT-PCR results also showed similar expression with slight variation. Collectively, these results suggest that TaBZR genes might play an important role in plant developmental processes and various stress conditions. Therefore, this work provides valuable information for further elucidate the precise role of BZR family members in wheat.     

Characterization of Volatile Organic Compounds Emitted by Barley (Hordeum vulgare L.) Roots and Their Attractiveness to Wireworms

Root volatile organic compounds (VOCs), their chemistry and ecological functions have garnered less attention than aboveground emitted plant VOCs. We report here on the identification of VOCs emitted by barley roots (Hordeum vulgare L.). Twenty nine VOCs were identified from isolated 21-d-old roots. The detection was dependent on the medium used for root cultivation. We identified 24 VOCs from 7-d-old roots when plants were cultivated on sterile Hoagland gelified medium, 33 when grown on sterile vermiculite, and 34 on non-sterile vermiculite. The major VOCs were fatty acid derived compounds, including hexanal, methyl hexanoate, (E)-hex-2-enal, 2-pentylfuran, pentan-1-ol, (Z)-2-(pentenyl)-furan, (Z)-pent-2-en-1-ol, hexan-1-ol, (Z)-hex-3-en-1-ol, (E)-hex-2-en-1-ol, oct-1-en-3-ol, 2-ethylhexan-1-ol (likely a contaminant), (E)-non-2-enal, octan-1-ol, (2E,6Z)-nona-2,6-dienal, methyl (E)-non-2-enoate, nonan-1-ol, (Z)-non-3-en-1-ol, (E)-non-2-en-1-ol, nona-3,6-dien-1-ol, and nona-2,6-dien-1-ol. In an olfactometer assay, wireworms (larvae of Agriotes sordidus Illiger, Coleoptera: Elateridae) were attracted to cues emanating from barley seedlings. We discuss the role of individual root volatiles or a blend of the root volatiles detected here and their interaction with CO₂ for wireworm attraction.     

Industrial-scale Malting Barley (Hordeum vulgare L.) Seed Disinfection by Fog of Ozonated Water Application

The aim of this study was to underscore that seed treatment by the fog of ozonated water constitutes a promising alternative tool, in terms of health and environmental gains, regarding traditional ozone treatment. In order to obtain a clear vision of this performance, the technology was implemented on an industrial scale (malting industry). Under an exposition of barley seeds to 9.8 ppm of dissolved ozone into water, our results showed a significant disinfection effect of 80% for Fusarium sp. and 70% for Aspergillus sp., but no effect was established on Alternaria sp.     

Physiological and Epigenetic Reaction of Barley (Hordeum vulgare L.) to the Foliar Application of Silicon under Soil Salinity Conditions

Soil salinity is an important environmental factor affecting physiological processes in plants. It is possible to limit the negative effects of salt through the exogenous application of microelements. Silicon (Si) is widely recognized as an element improving plant resistance to abiotic and biotic stresses. The aim of the research was to determine the impact of foliar application of Si on the photosynthetic apparatus, gas exchange and DNA methylation of barley (Hordeum vulgare L.) grown under salt stress. Plants grown under controlled pot experiment were exposed to sodium chloride (NaCl) in the soil at a concentration of 200 mM, and two foliar applications of Si were made at three concentrations (0.05%, 0.1% and 0.2%). Measurements were made of relative chlorophyll content in leaves (CCl), gas exchange parameters (C_i, E, g_s, and P_N), and selected chlorophyll fluorescence parameters (F_v/F_m, F_v/F₀, PI and RC/ABS). Additionally, DNA methylation level based on cytosine methylation within the 3′CCGG 5′ sequence was analyzed. Salinity had a negative effect on the values of the parameters examined. Exogenous application of Si by spraying leaves increased the values of the measured parameters in plants. Plants treated with NaCl in combination with the moderate (0.1%) and highest (0.2%) dose of Si indicated the lowest methylation level. Decrease of methylation implicated with activation of gene expression resulted in better physiological parameters observed in this group of barley plants.     

Identification and Characterization of Malate Dehydrogenases in Tomato (Solanum lycopersicum L.)

Malate dehydrogenase, which facilitates the reversible conversion of malate to oxaloacetate, is essential for energy balance, plant growth, and cold and salt tolerance. However, the genome-wide study of the MDH family has not yet been carried out in tomato (Solanum lycopersicum L.). In this study, 12 MDH genes were identified from the S. lycopersicum genome and renamed according to their chromosomal location. The tomato MDH genes were split into five groups based on phylogenetic analysis and the genes that clustered together showed similar lengths, and structures, and conserved motifs in the encoded proteins. From the 12 tomato MDH genes on the chromosomes, three pairs of segmental duplication events involving four genes were found. Each pair of genes had a Ka/Ks ratio < 1, indicating that the MDH gene family of tomato was purified during evolution. Gene expression analysis exhibited that tomato MDHs were differentially expressed in different tissues, at various stages of fruit development, and differentially regulated in response to abiotic stresses. Molecular docking of four highly expressed MDHs revealed their substrate and co-factor specificity in the reversible conversion process of malate to oxaloacetate. Further, co-localization of tomato MDH genes with quantitative trait loci (QTL) of salt stress-related phenotypes revealed their broader functions in salt stress tolerance. This study lays the foundation for functional analysis of MDH genes and genetic improvement in tomato.     

Identification and Dynamic Regulation of microRNAs Involved in Salt Stress Responses in Functional Soybean Nodules by High-Throughput Sequencing

Both symbiosis between legumes and rhizobia and nitrogen fixation in functional nodules are dramatically affected by salt stress. Better understanding of the molecular mechanisms that regulate the salt tolerance of functional nodules is essential for genetic improvement of nitrogen fixation efficiency. microRNAs (miRNAs) have been implicated in stress responses in many plants and in symbiotic nitrogen fixation (SNF) in soybean. However, the dynamic regulation of miRNAs in functioning nodules during salt stress response remains unknown. We performed deep sequencing of miRNAs to understand the miRNA expression profile in normal or salt stressed-soybean mature nodules. We identified 110 known miRNAs belonging to 61 miRNA families and 128 novel miRNAs belonging to 64 miRNA families. Among them, 104 miRNAs were dramatically differentially expressed (>2-fold or detected only in one library) during salt stress. qRT-PCR analysis of eight miRNAs confirmed that these miRNAs were dynamically regulated in response to salt stress in functional soybean nodules. These data significantly increase the number of miRNAs known to be expressed in soybean nodules, and revealed for the first time a dynamic regulation of miRNAs during salt stress in functional nodules. The findings suggest great potential for miRNAs in functional soybean nodules during salt stress.     

Isolation and Characterization of Barley (Hordeum vulgare) Extracellular Vesicles to Assess Their Role in RNA Spray-Based Crop Protection

The demonstration that spray-induced gene silencing (SIGS) can confer strong disease resistance, bypassing the laborious and time-consuming transgenic expression of double-stranded (ds)RNA to induce the gene silencing of pathogenic targets, was ground-breaking. However, future field applications will require fundamental mechanistic knowledge of dsRNA uptake, processing, and transfer. There is increasing evidence that extracellular vesicles (EVs) mediate the transfer of transgene-derived small interfering (si)RNAs in host-induced gene silencing (HIGS) applications. In this study, we establish a protocol for barley EV isolation and assess the possibilities for EVs regarding the translocation of sprayed dsRNA from barley (Hordeum vulgare) to its interacting fungal pathogens. We found barley EVs that were 156 nm in size, containing predominantly 21 and 19 nucleotide (nts) siRNAs, starting with a 5′-terminal Adenine. Although a direct comparison of the RNA cargo between HIGS and SIGS EV isolates is improper given their underlying mechanistic differences, we identified sequence-identical siRNAs in both systems. Overall, the number of siRNAs isolated from the EVs of dsRNA-sprayed barley plants with sequence complementarity to the sprayed dsRNA precursor was low. However, whether these few siRNAs are sufficient to induce the SIGS of pathogenic target genes requires further research. Taken together, our results raise the possibility that EVs may not be mandatory for the spray-delivered siRNA uptake and induction of SIGS.     

Lipids of six cultivated barley (Hordeum vulgare L.) varieties

The lipids of representative varieties of 2-row spring, 6-row spring, and 6-row winter-type barleys were studied. Total barley lipids were classified by silicic acid gel column chromatography and separated by thin layer chromatography, and the fatty acid composition was determined by gas liquid chromatography. Total lipid content of the 6 barley varieties ranged from 3.12%–3.56% (dry wt basis). The average values for neutral lipids, glycolipids, and phospholipids were 71, 9, and 20%, respectively. The fatty acid composition of barley was rather typical of plant tissue. The neutral lipids and glycolipids from all the varieties contained a higher percent of linoleic and linolenic (C 18∶2 and C 18∶3) acids than the phospholipid fraction. South Dakota Experiment Station Paper 1248.     

Genome-Wide Identification and Characterization of PIN-FORMED (PIN) Gene Family Reveals Role in Developmental and Various Stress Conditions in Triticum aestivum L.

PIN-FORMED (PIN) genes play a crucial role in regulating polar auxin distribution in diverse developmental processes, including tropic responses, embryogenesis, tissue differentiation, and organogenesis. However, the role of PIN-mediated auxin transport in various plant species is poorly understood. Currently, no information is available about this gene family in wheat (Triticum aestivum L.). In the present investigation, we identified the PIN gene family in wheat to understand the evolution of PIN-mediated auxin transport and its role in various developmental processes and under different biotic and abiotic stress conditions. In this study, we performed genome-wide analysis of the PIN gene family in common wheat and identified 44 TaPIN genes through a homology search, further characterizing them to understand their structure, function, and distribution across various tissues. Phylogenetic analyses led to the classification of TaPIN genes into seven different groups, providing evidence of an evolutionary relationship with Arabidopsis thaliana and Oryza sativa. A gene exon/intron structure analysis showed a distinct evolutionary path and predicted the possible gene duplication events. Further, the physical and biochemical properties, conserved motifs, chromosomal, subcellular localization, transmembrane domains, and three-dimensional (3D) structure were also examined using various computational approaches. Cis-elements analysis of TaPIN genes showed that TaPIN promoters consist of phytohormone, plant growth and development, and stress-related cis-elements. In addition, expression profile analysis also revealed that the expression patterns of the TaPIN genes were different in different tissues and developmental stages. Several members of the TaPIN family were induced during biotic and abiotic stress. Moreover, the expression patterns of TaPIN genes were verified by qRT-PCR. The qRT-PCR results also show a similar expression with slight variation. Therefore, the outcome of this study provides basic genomic information on the expression of the TaPIN gene family and will pave the way for dissecting the precise role of TaPINs in plant developmental processes and different stress conditions.     

Genome-Wide Identification and Characterization of the Cystatin Gene Family in Bread Wheat (Triticum aestivum L.)

Cystatins, as reversible inhibitors of papain-like and legumain proteases, have been identified in several plant species. Although the cystatin family plays crucial roles in plant development and defense responses to various stresses, this family in wheat (Triticum aestivum L.) is still poorly understood. In this study, 55 wheat cystatins (TaCystatins) were identified. All TaCystatins were divided into three groups and both the conserved gene structures and peptide motifs were relatively conserved within each group. Homoeolog analysis suggested that both homoeolog retention percentage and gene duplications contributed to the abundance of the TaCystatin family. Analysis of duplication events confirmed that segmental duplications played an important role in the duplication patterns. The results of codon usage pattern analysis showed that TaCystatins had evident codon usage bias, which was mainly affected by mutation pressure. TaCystatins may be regulated by cis-acting elements, especially abscisic acid and methyl jasmonate responsive elements. In addition, the expression of all selected TaCystatins was significantly changed following viral infection and cold stress, suggesting potential roles in response to biotic and abiotic challenges. Overall, our work provides new insights into TaCystatins during wheat evolution and will help further research to decipher the roles of TaCystatins under diverse stress conditions.     

Exogenous Silicon Modulates Growth,Physio-Chemicals and Antioxidants in Barley (Hordeum vulgare L.) Exposed to Different Temperature Regimes

Silicon - The exogenous application of silicon (Si) is reported to enhance tolerance of plants against various environmental stresses. Therefore, the present study was carried out to examine the...     

Barley Seeds miRNome Stability during Long-Term Storage and Aging

Seed aging is a complex biological process that has been attracting scientists’ attention for many years. High-throughput small RNA sequencing was applied to examine microRNAs contribution in barley seeds senescence. Unique samples of seeds that, despite having the same genetic makeup, differed in viability after over 45 years of storage in a dry state were investigated. In total, 61 known and 81 novel miRNA were identified in dry seeds. The highest level of expression was found in four conserved miRNA families, i.e., miR159, miR156, miR166, and miR168. However, the most astonishing result was the lack of significant differences in the level of almost all miRNAs in seed samples with significantly different viability. This result reveals that miRNAs in dry seeds are extremely stable. This is also the first identified RNA fraction that is not deteriorating along with the loss of seed viability. Moreover, the novel miRNA hvu-new41, with higher expression in seeds with the lowest viability as detected by RT-qPCR, has the potential to become an indicator of the decreasing viability of seeds during storage in a dry state.