Functional characterization of Arabidopsis ARGONAUTE 3 in reproductive tissues

Arabidopsis encodes ten ARGONAUTE (AGO) effectors of RNA silencing, canonically loaded with either 21‐22 nucleotide (nt) long small RNAs (sRNA) to mediate post‐transcriptional‐gene‐silencing (PTGS) or 24nt sRNAs to promote RNA‐directed‐DNA‐methylation. Using full‐locus constructs, we characterized the expression, biochemical properties, and possible modes of action of AGO3. Although AGO3 arose from a recent duplication at the AGO2 locus, their expression patterns differ drastically, with AGO2 being expressed in both male and female gametes whereas AGO3 accumulates in aerial vascular terminations and specifically in chalazal seed integuments. Accordingly, AGO3 down‐regulation alters gene expression in siliques. Similar to AGO2, AGO3 binds sRNAs with a strong 5’‐adenosine bias, but unlike Arabidopsis AGO2, it binds most efficiently 24nt sRNAs. AGO3 immunoprecipitation experiments in siliques revealed that these sRNAs mostly correspond to genes and intergenic regions in a manner reflecting their respective accumulation from their loci‐of‐origin. AGO3 localizes to the cytoplasm and co‐fractionates with polysomes to possibly mediate PTGS via translation inhibition.