Barley (Hordeum vulgare L.) is an economically important crop cultivated in temperate climates all over the world. Adverse environmental factors negatively affect its survival and productivity. RNA silencing is a conserved pathway involved in the regulation of growth, development and stress responses. The key components of RNA silencing are the Dicer-like proteins (DCLs), Argonautes (AGOs) and RNA-dependent RNA polymerases (RDRs). Despite its economic importance, there is no available comprehensive report on barley RNA silencing machinery and its regulation. In this study, we in silico identified five DCL (HvDCL), eleven AGO (HvAGO) and seven RDR (HvRDR) genes in the barley genome. Genomic localization, phylogenetic analysis, domain organization and functional/catalytic motif identification were also performed. To understand the regulation of RNA silencing, we experimentally analysed the transcriptional changes in response to moderate, persistent or gradient heat stress treatments: transcriptional accumulation of siRNA- but not miRNA-based silencing factor was consistently detected. These results suggest that RNA silencing is dynamically regulated and may be involved in the coordination of development and environmental adaptation in barley. In summary, our work provides information about barley RNA silencing components and will be a ground for the selection of candidate factors and in-depth functional/mechanistic analyses.

Micro RNAs (miRNAs) are processed from precursor RNA molecules with precisely defined secondary stem-loop structures. ARGONAUTE1 (AGO1) is the main executor component of miRNA pathway and its expression is controlled via the auto-regulatory feedback loop activity of miR168 in plants. Previously we have shown that AGO1 loading of miR168 is strongly restricted leading to abundant cytoplasmic accumulation of AGO-unbound miR168. Here, we report, that intrinsic RNA secondary structure of MIR168a precursor not only defines the processing of miR168, but also precisely adjusts AGO1 loading efficiency determining the biologically active subset of miR168 pool. Our results show, that modification of miRNA duplex structure of MIR168a precursor fragment or expression from artificial precursors can alter the finely adjusted loading efficiency of miR168. In dcl1-9 mutant where, except for miR168, production of most miRNAs is severely reduced this mechanism ensures the elimination of unloaded AGO1 proteins via enhanced AGO1 loading of miR168. Based on this data, we propose a new competitive loading mechanism model for miR168 action: the miR168 surplus functions as a molecular buffer for controlled AGO1 loading continuously adjusting the amount of AGO1 protein in accordance with the changing size of the cellular miRNA pool.

Key message: Barley AGO4 proteins complement expressional changes of epigenetically regulated genes in Arabidopsis ago4-3 mutant and show a distinct affinity for the 5′ terminal nucleotide of small RNAs, demonstrating functional conservation and divergence. The function of Argonaute 4 (AGO4) in Arabidopsis thaliana has been extensively characterized; however, its role in monocots, which have large genomes abundantly supplemented with transposable elements (TEs), remains elusive. The study of barley AGO4 proteins can provide insights into the conserved aspects of RNA-directed DNA methylation (RdDM) and could also have further applications in the field of epigenetics or crop improvement. Bioinformatic analysis of RNA sequencing data identified two active AGO4 genes in barley, HvAGO4a and HvAGO4b. These genes function similar to AtAGO4 in an Arabidopsis heterologous complementation system, primarily binding to 24-nucleotide long small RNAs (sRNAs) and triggering methylation at specific target loci. Like AtAGO4, HvAGO4B exhibits a preference for binding sRNAs with 5′ adenine residue, while also accepting 5′ guanine, uracil, and cytosine residues. In contrast, HvAGO4A selectively binds only sRNAs with a 5′ adenine residue. The diverse binding capacity of barley AGO4 proteins is reflected in TE-derived sRNAs and in their varying abundance. Both barley AGO4 proteins effectively restore the levels of extrachromosomal DNA and transcript abundancy of the heat-activated ONSEN retrotransposon to those observed in wild-type Arabidopsis plants. Our study provides insight into the distinct binding specificities and involvement in TE regulation of barley AGO4 proteins in Arabidopsis by heterologous complementation.

Micropropagated Catharantus roseus plants infected with ‘Candidatus Phytoplasma asteris’ showed virescence symptoms, witches’ broom symptoms, or became asymptomatic after their planting in pots. Nine plants were grouped into three categories according to these symptoms, which were then employed for investigation. The phytoplasma concentration, as determined by qPCR, correlated well with the severity of symptoms. To reveal the changes in the small RNA profiles in these plants, small RNA high-throughput sequencing (HTS) was carried out. The bioinformatics comparison of the micro (mi) RNA and small interfering (si) RNA profiles of the symptomatic and asymptomatic plants showed changes, which could be correlated to some of the observed symptoms. These results complement previous studies on phytoplasmas and serve as a starting point for small RNA-omic studies in phytoplasma research.

The DOWNY MILDEW RESISTANCE 6 (DMR6) protein is a 2-oxoglutarate (2OG) and Fe(II)-dependent oxygenase, involved in salicylic acid (SA) metabolism, and its inactivation in tomato was found to increase SA levels and to confer disease-resistance against several pathogens. SA is also recognized as an abiotic stress-tolerance enhancer, and we tested the resistance to drought stress in Sldmr6-1 tomato mutants generated by the CRISPR/Cas9 technique. Wild-type (WT) tomato cultivar ‘San Marzano’ and its Sldmr6-1 mutants were subjected to water deprivation for 7 days. At the end of the period, while WT plants exhibited severe wilting, the T ₂ Sldmr6-1 mutant plants showed turgid leaves and maintained higher Soil Relative Water Content (SRWC). Sldmr6-1 mutants adopted a water saving behaviour reducing transpiration rate (E) by decreasing stomatal conductance (Gs). Assimilation rate (A) decreased in parallel to E under drought stress, resulting in no alteration of the CO ₂ concentration in the sub-stomatal chamber (Ci) and increasing the Water Use Efficiency (WUE, A/E). Defence mechanisms of the photosynthetic machinery triggered in Sldmr6-1 mutants, that under drought stress showed up-regulation of the genes SlAPX and SlGST (anti-oxidant related) as well as down-regulation of SlCYP707A2 gene, which is involved in ABA catabolism. Our results suggest that the disabling of SlDMR6-1 in tomato plants leads to a drought-avoidance strategy through tight control of stomatal closure controlling water loss. In addition, it was highlighted, for the first time in tomato, that Sldmr6-1 mutants showed reduced susceptibility to Phytophthora infestans , the causal agent of Late Blight.