RNA-directed DNA methylation controls seed development and heat stress memory in barley

Plant specific RNA-directed DNA methylation (RdDM) mediates the DNA methylation of specific DNA sequences directed by 24-nucleotide long (nt) small interfering (si)RNAs. In crop plants we have limited information about the biological roles of the RdDM pathway including barley (Hordeum vulgare L). Here, we show that knockout of barley NRPD/E2A gene by genome editing, bringing about the drastic inhibition of RdDM pathway, results in the early arrest of developing caryopses rendering the mutant plants sterile. The knockout of the downstream component RDR2 gene, responsible for generating double stranded precursor RNAs for the production of 24-nt siRNAs, was associated with the loss of the majority of these siRNAs and also typically induced the early arrest of caryopsis development. However, the rdr2 mutants were able to produce a limited number of seeds exhibiting smaller size, endosperm filling anomalies and inhibited germination, which phenomenon was predominantly inherited maternally. Genome-wide analyses of gene expression revealed drastic up-and down-regulations in rdr2 mutants compared to the wild type. We did not find direct correlation between the changes of gene expression and the localization of 24-nt siRNA producing clusters indicating the indirect action of RdDM in these regulatory events. We also demonstrate that rdr2 mutant shows reduced heat stress memory capacity rendering the mutant plants more vulnerable to high temperature. Altogether, our data show the RdDM pathway is a major regulatory contributor to generative development and heat stress responses in barley.