Germination and Growth Characteristics of nud Knockout and win1 Knockout Barley Lines under Salt Stress.

Hordeum vulgare genes NUD (HvNUD) and WIN1 (HvWIN1) play a regulatory role in cuticle organization. Because the cuticle is a key evolutionary acquisition of plants for protection against environmental factors, a knockout (KO) of each gene may alter their ability to adapt to unfavorable conditions. A potential pleiotropic effect of HvNUD or HvWIN1 gene mutations can be assessed under salt stress. Initial developmental stages are the most sensitive in living organisms; therefore, we evaluated salt tolerance of nud KO and win1 KO barley lines at the seedling stage. Air-dried barley grains of the KO lines and of a wild-type (WT) line were germinated in NaCl solutions (50, 100, or 150 mM). Over 30 physiological and morphological parameters of seedlings were assessed. Potential pleiotropic effects of the HvNUD gene KO under salt stress included the stimulation of root growth (which was lower under control conditions) and root necrosis. The pleiotropic effects of the HvWIN1 gene KO under the stressful conditions manifested themselves as maintenance of longer root length as compared to the other lines; stable variation of most of morphological parameters; lack of correlation between root lengths before and after exposure to NaCl solutions, as well as between shoot lengths; and the appearance of twins. Salt tolerance of the analyzed barley lines could be ranked as follows: nud KO > win1 KO ≈ WT, where nud KO lines were the most salt-tolerant. A comparison of effects of salinity and ionizing radiation on nud KO and win1 KO barley lines indicated differences in tolerance of the lines to these stressors.

Enabling genome editing in tropical maize lines through an improved, morphogenic regulator-assisted transformation protocol.

The recalcitrance exhibited by many maize (Zea mays) genotypes to traditional genetic transformation protocols poses a significant challenge to the large-scale application of genome editing (GE) in this major crop species. Although a few maize genotypes are widely used for genetic transformation, they prove unsuitable for agronomic tests in field trials or commercial applications. This challenge is exacerbated by the predominance of transformable maize lines adapted to temperate geographies, despite a considerable proportion of maize production occurring in the tropics. Ectopic expression of morphogenic regulators (MRs) stands out as a promising approach to overcome low efficiency and genotype dependency, aiming to achieve 'universal' transformation and GE capabilities in maize. Here, we report the successful GE of agronomically relevant tropical maize lines using a MR-based, Agrobacterium-mediated transformation protocol previously optimized for the B104 temperate inbred line. To this end, we used a CRISPR/Cas9-based construct aiming at the knockout of the VIRESCENT YELLOW-LIKE (VYL) gene, which results in an easily recognizable phenotype. Mutations at VYL were verified in protoplasts prepared from B104 and three tropical lines, regardless of the presence of a single nucleotide polymorphism (SNP) at the seed region of the VYL target site in two of the tropical lines. Three out of five tropical lines were amenable to transformation, with efficiencies reaching up to 6.63%. Remarkably, 97% of the recovered events presented indels at the target site, which were inherited by the next generation. We observed off-target activity of the CRISPR/Cas9-based construct towards the VYL paralog VYL-MODIFIER, which could be partly due to the expression of the WUSCHEL (WUS) MR. Our results demonstrate efficient GE of relevant tropical maize lines, expanding the current availability of GE-amenable genotypes of this major crop.

WAX INDUCER 1 Regulates ß-Diketone Biosynthesis by Mediating Expression of the Cer-cqu Gene Cluster in Barley.

Plant surface properties are crucial determinants of resilience to abiotic and biotic stresses. The outer layer of the plant cuticle consists of chemically diverse epicuticular waxes. The WAX INDUCER1/SHINE subfamily of APETALA2/ETHYLENE RESPONSIVE FACTORS regulates cuticle properties in plants. In this study, four barley genes homologous to the Arabidopsis thaliana AtWIN1 gene were mutated using RNA-guided Cas9 endonuclease. Mutations in one of them, the HvWIN1 gene, caused a recessive glossy sheath phenotype associated with ß-diketone deficiency. A complementation test for win1 knockout (KO) and cer-x mutants showed that Cer-X and WIN1 are allelic variants of the same genomic locus. A comparison of the transcriptome from leaf sheaths of win1 KO and wild-type plants revealed a specific and strong downregulation of a large gene cluster residing at the previously known Cer-cqu locus. Our findings allowed us to postulate that the WIN1 transcription factor in barley is a master mediator of the ß-diketone biosynthesis pathway acting through developmental stage- and organ-specific transactivation of the Cer-cqu gene cluster.

Seedling Biometry of nud Knockout and win1 Knockout Barley Lines under Ionizing Radiation.

The genes NUD and WIN1 play a regulatory role in cuticle organization in barley. A knockout (KO) of each gene may alter plant mechanisms of adaptation to adverse environmental conditions. A putative pleiotropic effect of NUD or WIN1 gene mutations in barley can be assessed in a series of experiments in the presence or absence of a provoking factor. Ionizing radiation is widely used in research as a provoking factor for quantifying adaptive potential of living organisms. Our aim was to evaluate initial stages of growth and development of barley lines with a KO of NUD or WIN1 under radiation stress. Air-dried barley grains with different KOs and wild-type control (WT) were exposed to γ-radiation at 50, 100, or 200 Gy at a dose rate of 0.74 R/min. Approximately 30 physiological parameters were evaluated, combined into groups: (1) viability, (2) radiosensitivity, and (3) mutability of barley seed progeny. Seed germination, seedling survival, and shoot length were similar among all barley lines. Naked nud KO lines showed lower weights of seeds, roots, and seedlings and shorter root length as compared to win1 KO lines. The shoot-to-root length ratio of nud KO lines' seedlings exceeded that of win1 KO and WT lines. In terms of the number of seedlings with leaves, all the KO lines were more sensitive to pre-sowing γ-irradiation. Meanwhile, the radioresistance of nud KO lines (50% growth reduction dose [RD50] = 318-356 Gy) and WT plants (RD50 = 414 Gy) judging by seedling weight was higher than that of win1 KO lines (RD50 = 201-300 Gy). Resistance of nud KO lines to radiation was also demonstrated by means of root length (RD50 = 202-254 Gy) and the shoot-to-root length ratio. WT seedlings had the fewest morphological anomalies. In nud KO lines, mainly alterations of root shape were found, whereas in win1 KO lines, changes in the color and shape of leaves were noted. Thus, seedlings of nud KO lines are characterized mainly by changes in the root system (root length, root number, and root anomalies). For win1 KO lines, other parameters are sensitive (shoot length and alterations of leaf shape and color). These data may indicate a pleiotropic effect of genes NUD and WIN1 in barley.

Conversion of hulled into naked barley by Cas endonuclease-mediated knockout of the NUD gene.

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 ß-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.

The mechanism of potato resistance to Globodera rostochiensis: comparison of root transcriptomes of resistant and susceptible Solanum phureja genotypes.

BACKGROUND: Globodera rostochiensis belongs to major potato pathogens with a sophisticated mechanism of interaction with roots of the host plants. Resistance of commercial varieties is commonly based on specific R genes introgressed from natural populations of related wild species and from native potato varieties grown in the Andean highlands. Investigation of molecular resistance mechanisms and screening the natural populations for novel R genes are important for both fundamental knowledge on plant pathogen interactions and breeding for durable resistance. Here we exploited the Solanum phureja accessions collected in South America with contrasting resistance to G. rostochiensis. RESULTS: The infestation of S. phureja with G. rostochiensis juveniles resulted in wounding stress followed by activation of cell division and tissue regeneration processes. Unlike the susceptible S. phureja genotype, the resistant accession reacted by rapid induction of variety of stress response related genes. This chain of molecular events accompanies the hypersensitive response at the juveniles' invasion sites and provides high-level resistance. Transcriptomic analysis also revealed considerable differences between the analyzed S. phureja genotypes and the reference genome. CONCLUSION: The molecular processes in plant roots associated with changes in gene expression patterns in response to G. rostochiensis infestation and establishment of either resistant or susceptible phenotypes are discussed. De novo transcriptome assembling is considered as an important tool for discovery of novel resistance traits in S. phureja accessions.

Differential expression of NBS-LRR-encoding genes in the root transcriptomes of two Solanum phureja genotypes with contrasting resistance to Globodera rostochiensis.

BACKGROUND: The characterization of major resistance genes (R genes) in the potato remains an important task for molecular breeding. However, R genes are rapidly evolving and frequently occur in genomes as clusters with complex structures, and their precise mapping and identification are complicated and time consuming. RESULTS: Comparative analysis of root transcriptomes of Solanum phureja genotypes with contrasting resistance to Globodera rostochiensis revealed a number of differentially expressed genes. However, compiling a list of candidate R genes for further segregation analysis was hampered by their scarce annotation. Nevertheless, combination of transcriptomic analysis with data on predicted potato NBS-LRR-encoding genes considerably improved the quality of the results and provided a reasonable number of candidate genes that provide S. phureja with strong resistance to the potato golden cyst nematode. CONCLUSION: Combination of comparative analyses of tissue-specific transcriptomes in resistant and susceptible genotypes may be used as an approach for the rapid identification of candidate potato R genes for co-segregation analysis and may be used in parallel with more sophisticated studies based on genome resequencing.