A comparative survey of small RNA and their targets in grapevine embryogenic callus cultures and young leaves

Plant embryogenic callus (EC) cultures are important both for clonal propagation and as a source of explant material for transgenesis experiments. In grapevine, much work has been done to develop protocols for establishing EC cultures from multiple cultivars. However, tissue culture provides a stress effect that can alter both the genome, through the activation of transposable elements and the epigenome. The host cell response to genomic stress is two-fold: post-transcriptional gene silencing (PTGS) targets aberrant transcripts for cleavage and sequestration and transcriptional gene silencing (TGS) targets specific DNA loci for RNA-dependent DNA methylation (RdDM) and histone modifications. Small RNA molecules direct these systems to their targets based on sequence complementarity. To better understand silencing activity in EC cultures, we undertook a survey of small RNA from these cultures using parallel short read sequencing and compared these with small RNA from young leaf tissue. Our results show that small RNA profiles differ greatly among the two tissue types. One third of the small RNAs found in leaf were known microRNA (miRNA) sequences that regulate the development of vegetative tissue. MicroRNAs were also present in EC, but comprised ‹1% of the small RNA complement. Comparison of the gene ontology (GO) networks of target genes for each sample revealed complex and distinct patterns of gene regulation in each tissue type. Trans-acting siRNAs (ta-siRNAs) from 18 loci were also present at over 100 reads million-1 (RPM) in leaf, all of which were decreased or absent in EC. In contrast, EC contained approximately twice the amount of 24 nt small interfering RNAs (siRNAs) associated with maintaining the repression of repeat-associated DNA as was found in the leaf tissue. The results indicate that not only do the targets of RNA interference (RNAi) vary in EC with respect to leaf tissue, but that the levels of activity for the various silencing systems differ greatly. Researchers studying plants recovered through somatic embryogenesis would be advised to take these observations into consideration, particularly when the introduction of foreign DNA is involved.