Characteristic of the gene candidate SecARS encoding alkylresorcinol synthase in Secale.

BACKGROUND: Alkylresorcinols (ARs) are compounds belonging to the class of phenolic lipids. A rich source of ARs are cereal grains such as rye, wheat, triticale or barley. ARs found in plants are characterized by a variety of biological properties such as antimicrobial, antifungal and cytotoxic activity. Moreover, they are proven to have a positive influence on human health. Here, we aimed to find and characterize the gene with ARs synthase activity in the species Secale cereale. METHODS AND RESULTS: Using BAC library screening, two BAC clones containing the gene candidate were isolated and sequenced. Bioinformatic analyses of the resulting contigs were used to examine the structure and other features of the gene, including promoter, intron, 3'UTR and 5'UTR. Mapping using the FISH procedure located the gene on the 4R chromosome. Comparative analysis showed that the gene is highly similar to sequences coding for type III polyketide synthase. The level of gene expression in various parts of the plant was investigated, and the biochemical function of the gene was confirmed by heterologous expression in yeast. CONCLUSIONS: The conducted analyses contributed to a better understanding of the processes related to ARs synthesis. Although the research concerned the rye model, the knowledge gained may help in understanding the genetic basis of ARs biosynthesis in other species of the Poaceae family as well.

Profiling of Barley, Wheat, and Rye FPG and OGG1 Genes during Grain Germination.

This research is about the profiling of barley (Hordeum vulgare L.), wheat (Triticum aestivum L.), and rye (Secale cereale L.) FPG and OGG1 genes during grain germination. During seed germination, reactive oxygen species accumulate, which leads to DNA damage. In the base excision repair (BER) system, the enzymes formamidopyrimidine DNA glycosylase (FPG) and 8-oxoguanine DNA glycosylase (OGG1), among others, are responsible for repairing such damage. We decided to check how the expression of genes encoding these two enzymes changes in germinating grains. Spring varieties of barley, wheat, and rye from the previous growing season were used in the study. Expression level changes were checked using Real-Time PCR. After analyzing the obtained results, the maximum expression levels of FPG and OGG1 genes during germination were determined for barley, wheat, and rye. The results of the study show differences in expression levels specific to each species. The highest expression was observed at different time points for each of them. There were no differences in the highest expression for FPG and OGG1 within one species. In conclusion, the research provides information on how the level of FPG and OGG1 gene expression changes during the germination process in cereals. This is the first study looking at the expression levels of these two genes in cereals.

Characterization of the Moroccan Barley Germplasm Preserved in the Polish Genebank as a First Step towards Selecting Forms with Increased Drought Tolerance.

In marginal, arid, and semi-arid areas of Morocco, crops are often exposed to multiple abiotic and biotic stresses that have a major impact on yield. Farmer-maintained Moroccan landraces have been shaped by the impact of very strong selection pressures, gradually adapting to the local ecosystem and obsolete low-input agricultural practices without improvement towards high yield and quality. Considering the increasing threat of drought in Poland, it is necessary to introduce germplasm with tolerance to water deficit into barley breeding programs. The aim of this research was a DArTseq-based genetic characterization of a collection of germplasm of Moroccan origin, conserved in the Polish genebank. The results showed that all conserved landraces have a high level of heterogeneity and their gene pool is different from the material developed by Polish breeders. Based on the analysis of eco-geographical data, locations with extremely different intensities of drought stress were selected. A total of 129 SNPs unique to accessions from these locations were identified. In the neighborhood of the clusters of unique SNPs on chromosomes 5H and 6H, genes that may be associated with plant response to drought stress were identified. The results obtained may provide a roadmap for further research to support Polish barley breeding for increased drought tolerance.

Inhibition of the Glycogen Synthase Kinase 3 Family by the Bikinin Alleviates the Long-Term Effects of Salinity in Barley.

Crops grown under stress conditions show restricted growth and, eventually, reduced yield. Among others, brassinosteroids (BRs) mitigate the effects of stress and improve plant growth. We used two barley cultivars with differing sensitivities to BRs, as determined by the lamina joint inclination test. Barley plants with the 2nd unfolded leaf were sprayed with a diluted series of bikinin, an inhibitor of the Glycogen Synthase Kinase 3 (GSK3) family, which controls the BR signaling pathway. Barley was grown under salt stress conditions up to the start of the 5th leaf growth stage. The phenotypical, molecular, and physiological changes were determined. Our results indicate that the salt tolerance of barley depends on its sensitivity to BRs. We confirmed that barley treatment with bikinin reduced the level of the phosphorylated form of HvBZR1, the activity of which is regulated by GSK3. The use of two barley varieties with different responses to salinity led to the identification of the role of BR signaling in photosynthesis activity. These results suggest that salinity reduces the expression of the genes controlling the BR signaling pathway. Moreover, the results also suggest that the functional analysis of the GSK3 family in stress responses can be a tool for plant breeding in order to improve crops' resistance to salinity or to other stresses.

Brassinazole Resistant 1 Activity Is Organ-Specific and Genotype-Dependent in Barley Seedlings.

Brassinosteroids (BRs) control many plant developmental processes by regulating different groups of transcription factors, and consequently gene expressions. The most known is BZR1, the main member of the BES1 family. However, to date, it is poorly characterized in crop species. The main goal of the presented study was to identify HvBZR1 and determine its activity in 5-day-old barley (the stage is related to one leaf on the main shoot and a few seminal roots) using two cultivars with different sensitivities to BRs. Using the anti-OsBZR1 antibody, we identified the forms of HvBZR1 transcription factor with different molecular weights, which can be related to different phosphorylated forms of serine/threonine residues. Two phosphorylated forms in the shoots and one dephosphorylated form in the roots were determined. A minor amount of the dephosphorylated form of the HvBZR1 in the Haruna Nijo shoots was also found. The phosphorylated forms gave a higher band intensity for Golden Promise than Haruna Nijo. The bands were similar in their intensity, when two different phosphorylated forms were compared in Golden Promise, while a reduced intensity was detected for the phosphorylated form with a lower molecular weight for Haruna Nijo. Degradation of the phosphorylated forms in the shoots (complete degradation in Golden Promise and significant but not complete in Haruna Nijo) and the presence of the dephosphorylated form in the roots were proven for the etiolated barley. In the case of Haruna Nijo, a wider range of the regulators of the BR biosynthesis and signaling pathways induced the expected effects, 24-EBL (0.001 µM) and bikinin (10 and 50 µM) caused low amount of the phosphorylated forms, and at the same time, a tiny band of dephosphorylated form was detected. However, the expression of genes related to the BR biosynthesis and signaling pathways was not a determinant for the protein amount.

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.

Low RIN Value for RNA-Seq Library Construction from Long-Term Stored Seeds: A Case Study of Barley Seeds.

Seed aging is a complex biological process and its fundamentals and mechanisms have not yet been fully recognized. This is a key issue faced by research teams involved in the collection and storage of plant genetic resources in gene banks every day. Transcriptomic changes associated with seed aging in the dry state have barely been studied. The aim of the study was to develop an efficient protocol for construction of RNA-Seq libraries from long-term stored seeds with very low viability and low RNA integrity number (RIN). Here, barley seeds that have almost completely lost their viability as a result of long-term storage were used. As a control, fully viable seeds obtained in the course of field regeneration were used. The effectiveness of protocols dedicated to RNA samples with high and low RIN values was compared. The experiment concluded that library construction from low viable or long-term stored seeds with degraded RNA (RIN < 3) should be carried out with extraordinary attention due to the possibility of uneven degradation of different RNA fractions.

Annotation and profiling of barley GLYCOGEN SYNTHASE3/Shaggy-like genes indicated shift in organ-preferential expression.

GLYCOGEN SYNTHASE KINASE3/Shaggy-like kinases (GSKs) represent a highly conserved group of proteins found in all eukaryotes. In plants they are encoded by multigene families and integrate signaling of brassinosteroids, auxin and abscisic acid in wide range of physiological and developmental processes with a strong impact on plant responses to environmental and biotic factors. Based on comprehensively studied structures of 10 Arabidopsis thaliana GSK genes and encoded proteins we report identification and phylogenetic reconstruction of 7 transcriptionally active GSK genes in barley. We re-evaluated annotation of the GSK genes in the current barley genome (Hv_IBSC_PGSB_v2) and provided data that a single gene annotated in the previous barley genome ensemble should be retained in the current one. The novel structure of another GSK, predicted in Hv_IBSC_PGSB_v2 to encode both GSK and amine oxidase domains, was proposed and experimentally confirmed based on the syntenic region in Brachypodium distachyon. The genes were assigned to 4 groups based on their encoded amino acid sequences and protein kinase domains. The analysis confirmed high level of conservation of functional protein domains and motifs among plant GSKs and the identified barley orthologs. Each of the seven identified HvGSK genes was expressed indicating semi-constitutive regulation in all tested organs and developmental stages. Regulation patterns of GSKs from the indicated groups showed a shift in organ-preferential expression in A. thaliana and barley illustrating diversification of biological roles of individual HvGSKs in different plant species.

Identification and VIGS-based characterization of Bx1 ortholog in rye (Secale cereale L.).

The first step of the benzoxazinoid (BX) synthesis pathway is catalyzed by an enzyme with indole-3-glycerol phosphate lyase activity encoded by 3 genes, Bx1, TSA and Igl. A gene highly homologous to maize and wheat Bx1 has been identified in rye. The goal of the study was to analyze the gene and to experimentally verify its role in the rye BX biosynthesis pathway as a rye ortholog of the Bx1 gene. Expression of the gene showed peak values 3 days after imbibition (dai) and at 21 dai it was undetectable. Changes of the BX content in leaves were highly correlated with the expression pattern until 21 dai. In plants older than 21 dai despite the undetectable expression of the analyzed gene there was still low accumulation of BXs. Function of the gene was verified by correlating its native expression and virus-induced silencing with BX accumulation. Barley stripe mosaic virus (BSMV)-based vectors were used to induce transcriptional (TGS) and posttranscriptional (PTGS) silencing of the analyzed gene. Both strategies (PTGS and TGS) significantly reduced the transcript level of the analyzed gene, and this was highly correlated with lowered BX content. Inoculation with virus-based vectors specifically induced expression of the analyzed gene, indicating up-regulation by biotic stressors. This is the first report of using the BSMV-based system for functional analysis of rye gene. The findings prove that the analyzed gene is a rye ortholog of the Bx1 gene. Its expression is developmentally regulated and is strongly induced by biotic stress. Stable accumulation of BXs in plants older than 21 dai associated with undetectable expression of ScBx1 indicates that the function of the ScBx1 in the BX biosynthesis is redundant with another gene. We anticipate that the unknown gene is a putative ortholog of the Igl, which still remains to be identified in rye.

Structural characteristics of ScBx genes controlling the biosynthesis of hydroxamic acids in rye (Secale cereale L.).

Benzoxazinoids (BX) are major secondary metabolites of gramineous plants that play an important role in disease resistance and allelopathy. They also have many other unique properties including anti-bacterial and anti-fungal activity, and the ability to reduce alfa-amylase activity. The biosynthesis and modification of BX are controlled by the genes Bx1 ÷ Bx10, GT and glu, and the majority of these Bx genes have been mapped in maize, wheat and rye. However, the genetic basis of BX biosynthesis remains largely uncharacterized apart from some data from maize and wheat. The aim of this study was to isolate, sequence and characterize five genes (ScBx1, ScBx2, ScBx3, ScBx4 and ScBx5) encoding enzymes involved in the synthesis of DIBOA, an important defense compound of rye. Using a modified 3D procedure of BAC library screening, seven BAC clones containing all of the ScBx genes were isolated and sequenced. Bioinformatic analyses of the resulting contigs were used to examine the structure and other features of these genes, including their promoters, introns and 3'UTRs. Comparative analysis showed that the ScBx genes are similar to those of other Poaceae species, especially to the TaBx genes. The polymorphisms present both in the coding sequences and non-coding regions of ScBx in relation to other Bx genes are predicted to have an impact on the expression, structure and properties of the encoded proteins.