Organ development in plants predominantly occurs postembryonically through combinatorial activity of meristems; therefore, meristem and organ fate are intimately connected. Inflorescence morphogenesis in grasses (Poaceae) is complex and relies on a specialized floral meristem, called spikelet meristem, that gives rise to all other floral organs and ultimately the grain. The fate of the spikelet determines reproductive success and contributes toward yield-related traits in cereal crops. Here, we examined the transcriptional landscapes of floral meristems in the temperate crop barley (Hordeum vulgare L.) using RNA-seq of laser capture microdissected tissues from immature, developing floral structures. Our unbiased, high-resolution approach revealed fundamental regulatory networks, previously unknown pathways, and key regulators of barley floral fate and will equally be indispensable for comparative transcriptional studies of grass meristems.

Background: The naked caryopsis character in barley is a domestication-associated trait defined by loss-of-function of the NUD gene. The functional NUD gene encodes an Apetala 2/Ethylene-Response Factor (AP2/ERF) controlling the formation of a cementing layer between pericarp and both lemma and palea. The downstream genes regulated by the NUD transcription factor and molecular mechanism of a cementing layer formation are still not sufficiently described. A naturally occurring 17-kb deletion in the nud locus is associated with the emergence of naked barley. Naked barley has been traditionally used for food and nowadays is considered as a dietary component for functional nutrition. Results: In the present study, we demonstrate that targeted knockout of the NUD gene using RNA-guided Cas9 endonuclease leads to the phenotype conversion from hulled to naked barley. Using in vivo pre-testing systems, highly effective guide RNAs targeting the first exon of the NUD gene were selected. Expression cassettes harboring the cas9 and guide RNA genes were used to transform barley cv. Golden Promise via Agrobacterium-mediated DNA transfer. The recessive naked grain phenotype was observed in 57% of primary transformants, which indicates a frequent occurrence of homozygous or biallelic mutations. T-DNA-free homozygous lines with independently generated mutations in the NUD gene were obtained in the T1 generation. At homozygous state, all obtained mutations including one- and two-amino acid losses with the translational reading frame being retained invariably caused the naked grain phenotype. Conclusions: The hulled and naked barley isogenic lines generated are a perfect experimental model for further studies on pleiotropic consequences of nud mutations on overall plant performance under particular consideration of yield-determining traits. Due to the high β-glucan content of its grains, naked barley is considered as being of particular dietary value. The possibility to convert hulled into naked barley cultivars by targeted mutagenesis allows breeders to extend the potential utilization of barley by the provision of functional food.

Domestication and breeding have created productive crops that are adapted to the climatic conditions of their growing regions. Initially, this process solely relied on the frequent occurrence of spontaneous mutations and the recombination of resultant gene variants. Later, treatments with ionizing radiation or mutagenic chemicals facilitated dramatically increased mutation rates, which remarkably extended the genetic diversity of crop plants. However, a major drawback of conventionally induced mutagenesis is that genetic alterations occur simultaneously across the whole genome and at very high numbers per individual plant. By contrast, the newly emerging Cas endonuclease technology allows for the induction of mutations at user-defined positions in the plant genome. In fundamental and breeding-oriented research, this opens up unprecedented opportunities for the elucidation of gene functions and the targeted improvement of plant performance. This review covers historical aspects of the development of customizable endonucleases, information on the mechanisms of targeted genome modification, as well as hitherto reported applications of Cas endonuclease technology in barley and wheat that are the agronomically most important members of the temperate cereals. Finally, current trends in the further development of this technology and some ensuing future opportunities for research and biotechnological application are presented.

The modifcation of crop genomes employing functional components of the microbial CRISPR/Cas immune system is a rapidly developing area of applied research. Site-directed plant genome modifcation by this technology involves the construction of Cas endonuclease- and guide-RNA-encoding vectors, delivery of the plasmid DNA into plant cells, processing of the chosen genomic target site by the corresponding gene products and regeneration of plants from modifed cells. The utilization of this technology in local breeding programs is mainly limited by the typically strong genotype dependence of gene transfer andin vitroregeneration procedures, which holds particularly true in cereals. In the present study, an evaluation ofin vitroregeneration efciency of immature embryos of ten Siberian barley cultivars revealed that only one of these is on a par with the experimental standard cultivar Golden Promise. This cultivar, namely cv. Aley, was consequently chosen for further experiments on site-directed mutagenesis in leaf mesophyll protoplasts. Two genes controlling hulledvsnaked (Nud) and two-rowedvssix-rowed barley (Vrs1) were used as targets to be modifed via polyethyleneglycol-mediated cellular uptake of guide-RNA/Cas9-encoding plasmid DNA. Deep-sequencing of amplicons obtained from protoplast genomic DNA revealed that 6 to 22 percent of the target sites were mutated. The detected modifcations comprised deletions in all three target sites and of various sizes, whereas insertions were observed in only one of the target genes (Vrs1) and were confned to the size of 1 nucleotide. This study demonstrates the possibility of site-directed genome modifcation in Siberian barley. Further steps in technology advancement will require the development of protocols with reduced genotype dependence in terms of both the gene transfer to totipotent cells and the subsequent plant regeneration originating from such cells.

Blue barley grain pigmentation results from anthocyanin accumulation in the aleurone layer. Anthocyanins are known for their beneficial effects on human health. The gene encoding the MYELOCYTOMATOSIS 2 (MYC2) transcription factor is potentially responsible for the blue coloration of the aleurone. In non-pigmented barley, a single nucleotide insertion in this gene causes a frameshift mutation with a premature stop codon. It was hypothesized that restoring the MYC2 reading frame could activate anthocyanin accumulation in the aleurone. Using a targeted mutagenesis approach in the present study, the reading frame of MYC2 was restored in the non-pigmented cultivar Golden Promise. Genetic constructs harboring cas9 and gRNA expression units were developed, pre-validated in protoplasts, and then functional MYC2 alleles were generated at the plant level via Agrobacterium-mediated transformation. Anthocyanin accumulation in the aleurone layer of grains from these mutants was confirmed through microscopy and chemical analysis. The expression of anthocyanin biosynthesis genes was analyzed, revealing that the restoration of MYC2 led to increased transcript levels of F3H and ANS genes. These results confirm the critical role of the MYC2 transcription factor in the blue aleurone trait and provide a biotechnological solution for enriching barley grain with anthocyanins.

Cas endonuclease-mediated genome editing provides a long-awaited molecular biological approach to the modification of predefined genomic target sequences in living organisms. Although cas9/guide (g)RNA constructs are straightforward to assemble and can be customized to target virtually any site in the plant genome, the implementation of this technology can be cumbersome, especially in species like triticale that are difficult to transform, for which only limited genome information is available and/or which carry comparatively large genomes. To cope with these challenges, we have pre-validated cas9/gRNA constructs (1) by frameshift restitution of a reporter gene co-introduced by ballistic DNA transfer to barley epidermis cells, and (2) via transfection in triticale protoplasts followed by either a T7E1-based cleavage assay or by deep-sequencing of target-specific PCR amplicons. For exemplification, we addressed the triticale ABA 8′-HYDROXYLASE 1 gene, one of the putative determinants of pre-harvest sprouting of grains. We further show that in-del induction frequency in triticale can be increased by TREX2 nuclease activity, which holds true for both well- and poorly performing gRNAs. The presented results constitute a sound basis for the targeted induction of heritable modifications in triticale genes.