HvGSK1.1 Controls Salt Tolerance and Yield through the Brassinosteroid Signaling Pathway in Barley.

Brassinosteroids (BRs) are a class of plant steroid hormones that are essential for plant growth and development. BRs control important agronomic traits and responses to abiotic stresses. Through the signaling pathway, BRs control the expression of thousands of genes, resulting in a variety of biological responses. The key effectors of the BR pathway are two transcription factors (TFs): BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMSSUPPRESSOR 1 (BES1). Both TFs are phosphorylated and inactivated by the Glycogen synthase kinase 3 BRASSINOSTEROID INSENSITIVE2 (BIN2), which acts as a negative regulator of the BR pathway. In our study, we describe the functional characteristics of HvGSK1.1, which is one of the GSK3/SHAGGY-like orthologs in barley. We generated mutant lines of HvGSK1.1 using CRISPR/Cas9 genome editing technology. Next Generation Sequencing (NGS) of the edited region of the HvGSK1.1 showed a wide variety of mutations. Most of the changes (frameshift, premature stop codon, and translation termination) resulted in the knock-out of the target gene. The molecular and phenotypic characteristics of the mutant lines showed that the knock-out mutation of HvGSK1.1 improved plant growth performance under salt stress conditions and increased the thousand kernel weight of the plants grown under normal conditions. The inactivation of HvGSK1.1 enhanced BR-dependent signaling, as indicated by the results of the leaf inclination assay in the edited lines. The plant traits under investigation are consistent with those known to be regulated by BRs. These results, together with studies of other GSK3 gene members in other plant species, suggest that targeted editing of these genes may be useful in creating plants with improved agricultural traits.

Silencing of HvGSK1.1-A GSK3/SHAGGY-Like Kinase-Enhances Barley (Hordeum vulgare L.) Growth in Normal and in Salt Stress Conditions.

Glycogen synthase kinase 3 (GSK3) is a highly conserved kinase present in all eukaryotes and functions as a key regulator of a wide range of physiological and developmental processes. The kinase, known in land plants as GSK3/SHAGGY-like kinase (GSK), is a key player in the brassinosteroid (BR) signaling pathway. The GSK genes, through the BRs, affect diverse developmental processes and modulate responses to environmental factors. In this work, we describe functional analysis of HvGSK1.1, which is one of the GSK3/SHAGGY-like orthologs in barley. The RNAi-mediated silencing of the target HvGSK1.1 gene was associated with modified expression of its paralogs HvGSK1.2, HvGSK2.1, HvGSK3.1, and HvGSK4.1 in plants grown in normal and in salt stress conditions. Low nucleotide similarity between the silencing fragment and barley GSK genes and the presence of BR-dependent transcription factors' binding sites in promoter regions of barley and rice GSK genes imply an innate mechanism responsible for co-regulation of the genes. The results of the leaf inclination assay indicated that silencing of HvGSK1.1 and the changes of GSK paralogs enhanced the BR-dependent signaling in the plants. The strongest phenotype of transgenic lines with downregulated HvGSK1.1 and GSK paralogs had greater biomass of the seedlings grown in normal conditions and salt stress as well as elevated kernel weight of plants grown in normal conditions. Both traits showed a strong negative correlation with the transcript level of the target gene and the paralogs. The characteristics of barley lines with silenced expression of HvGSK1.1 are compatible with the expected phenotypes of plants with enhanced BR signaling. The results show that manipulation of the GSK-encoding genes provides data to explore their biological functions and confirm it as a feasible strategy to generate plants with improved agricultural traits.

Polyamine Oxidation Is Indispensable for Wheat (Triticum aestivum L.) Oxidative Response and Necrotic Reactions during Leaf Rust (Puccinia triticina Eriks.) Infection.

Hydrogen peroxide is a signal and effector molecule in the plant response to pathogen infection. Wheat resistance to Puccinia triticina Eriks. is associated with necrosis triggered by oxidative burst. We investigated which enzyme system dominated in host oxidative reaction to P. triticina infection. The susceptible Thatcher cultivar and isogenic lines with defined resistance genes were inoculated with P. triticina spores. Using diamine oxidase (DAO) and polyamine oxidase (PAO) inhibitors, accumulation of H2O2 was analyzed in the infection sites. Both enzymes participated in the oxidative burst during compatible and incompatible interactions. Accumulation of H2O2 in guard cells, i.e., the first phase of the response, depended on DAO and the role of PAO was negligible. During the second phase, the patterns of H2O2 accumulation in the infection sites were more complex. Accumulation of H2O2 during compatible interaction (Thatcher and TcLr34 line) moderately depended on DAO and the reaction of TcLr34 was stronger than that of Thatcher. Accumulation of H2O2 during incompatible interaction of moderately resistant plants (TcLr24, TcLr25 and TcLr29) was DAO-dependent in TcLr29, while the changes in the remaining lines were not statistically significant. A strong oxidative burst in resistant plants (TcLr9, TcLr19, TcLr26) was associated with both enzymes' activities in TcLr9 and only with DAO in TcLr19 and TcLr26. The results are discussed in relation to other host oxidative systems, necrosis, and resistance level.

TaWAK6 encoding wall-associated kinase is involved in wheat resistance to leaf rust similar to adult plant resistance.

In wheat, adult plant resistance (APR) to leaf rust (Puccinia triticina), is effective in restricting pathogen growth and provides durable resistance against a wide range of virulent forms of P. triticina. Despite the importance, there is limited knowledge on the molecular basis of this type of resistance. We isolated and characterized the wall-associated kinase encoding gene in wheat, and assigned it as TaWAK6. Localization of TaWAK6 homeologs in A and B wheat subgenomes was consistent with the presence of the gene's orthologs in T. urartu (AA) and T. dicoccoides (AABB) and with the absence of its orthologs in Aegilops tauschii (DD). Overexpression of TaWAK6 did not change the wheat phenotype, nor did it affect seedling resistance. However, the adult plants overexpressing TaWAK6 showed that important parameters of APR were significantly elevated. Infection types scored on the first (flag), second and third leaves indicated elevated resistance, which significantly correlated with expression of TaWAK6. Analysis of plant-pathogen interactions showed a lower number of uredinia and higher rates of necrosis at the infection sites and this was associated with smaller size of uredinia and a longer latent period. The results indicated a role of TaWAK6 in quantitative partial resistance similar to APR in wheat. It is proposed that TaWAK6, which is a non-arginine-aspartate (non-RD) kinase, represents a novel class of quantitative immune receptors in monocots.