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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. [ABSTRACT FROM AUTHOR]

Long noncoding RNAs (lncRNAs) are a class of RNA molecules with gene regulatory functions in plant development and the stress response. Although the number of lncRNAs identified in plants is rapidly increasing, very little is known about their role in barley development. In this study, we performed global identification of barley lncRNAs based on 53 RNAseq libraries derived from nine different barley tissues and organs. In total, 17,250 lncRNAs derived from 10,883 loci were identified, including 8954 novel lncRNAs. Differential expression of lncRNAs was observed in the developing shoot apices and grains, the two organs that have a direct influence on the final yield. The regulatory interaction of differentially expressed lncRNAs with the potential target genes was evaluated. We identified 176 cis-acting lncRNAs in shoot apices and 424 in grains, while the number of trans-acting lncRNAs in these organs was 1736 and 540, respectively. The potential target protein-coding genes were identified, and their biological function was annotated using MapMan ontology. This is the first insight into the roles of lncRNAs in barley development on the genome-wide scale, and our results provide a solid background for future functional studies. [ABSTRACT FROM AUTHOR]

Since its demonstration in 2012, CRISPR/Cas9 system has been applied as a break-through technology for precise genome editing in a broad range of organisms. The main advantages of this system are among others, the technical simplicity, easy multiplex gene targeting by single construct and the ability to cleave methylated DNA. The Cas9 nuclease can also be re-engineered or inactivated and used for other genetic engineering approaches, such as gene expression regulation or for labeling of specific chromosomal regions in vivo. In this study we demonstrate the simple and efficient CRISPR/Cas9-mediated genome editing system for barley in which the target sequence is cloned into Gateway compatible vector for stable Agrobacterium-mediated transformation. To introduce site-directed mutations we chose two different barley genes, HvCKX1 and Nud, both determining important agronomical traits. HvCKX1gene is a member of the CKX gene family encoding oxidase/ dehydrogenase enzymes which metabolize cytokinins in plants and control developmental processes. Nud gene is a member of the gene family of ethylene-response transcription factor ERF. Mutations in this gene result in a phenotype with naked (hulless) caryopses. The crucial role of CKX genes in plant development and the possibility of direct detection of phenotypic changes in plants modified by site-directed mutagenesis of Nud gene were two main reasons for choosing these genes. Immature embryos of barley were transformed with vectors containing codon-optimized gene of nuclease Cas9 and single guided RNA sequence (sgRNA) targeting either HvCKX1 or Nud gene under wheat U6 RNA promoter. Induced mutations were detected by restriction analysis of amplified fragments of both genes. 71 transgenic plants were obtained after transformation with CRISPR/Cas9 construct for HvCKX1 gene and 28 transgenic plants for Nud gene. The mutation frequency was 66% and 64% of HvCKX1 and Nud genes respectively. In part of the plants the sequences carrying mutations were cloned and sequenced. Single nucleotide deletions or insertions were the most frequent types of mutations however, larger deletions (up to 10 bp) were also detected. Moreover, in most of the plants with altered Nud gene, the naked caryopses with non-adherent hulls were observed. Our results show that the CRISPR/ Cas9 technology is a simple and powerful tool for breeding improvement of crop plants. [ABSTRACT FROM AUTHOR]

Barley is among four of the most important cereal crops with respect to global production. Increasing barley yields to desired levels can be achieved by the genetic manipulation of cytokinin content. Cytokinins are plant hormones that regulate many developmental processes and have a strong influence on grain yield. Cytokinin homeostasis is regulated by members of several multigene families. CKX genes encode the cytokinin oxidase/dehydrogenase enzyme, which catalyzes the irreversible degradation of cytokinin. Several recent studies have demonstrated that the RNAi-based silencing of CKX genes leads to increased grain yields in some crop species. To assess the possibility of increasing the grain yield of barley by knocking out CKX genes, we used an RNA-guided Cas9 system to generate ckx1 and ckx3 mutant lines with knockout mutations in the HvCKX1 and HvCKX3 genes, respectively. Homozygous, transgene-free mutant lines were subsequently selected and analyzed. A significant decrease in CKX enzyme activity was observed in the spikes of the ckx1 lines, while in the ckx3 lines, the activity remained at a similar level to that in the control plants. Despite these differences, no changes in grain yield were observed in either mutant line. In turn, differences in CKX activity in the roots between the ckx1 and ckx3 mutants were reflected via root morphology. The decreased CKX activity in the ckx1 lines corresponded to greater root length, increased surface area, and greater numbers of root hairs, while the increased CKX activity in the ckx3 mutants gave the opposite results. RNA-seq analysis of the spike and root transcriptomes revealed an altered regulation of genes controlling cytokinin metabolism and signaling, as well as other genes that are important during seed development, such as those that encode nutrient transporters. The observed changes suggest that the knockout of a single CKX gene in barley may be not sufficient for disrupting cytokinin homeostasis or increasing grain yields. [ABSTRACT FROM AUTHOR]