Further disruption of the TAS3 pathway via the addition of the AGO7 mutation to the DRB1, DRB2 or DRB4 mutations severely impairs the reproductive competence of Arabidopsis thaliana

The previous assignment of functional roles for AGO7, and the DOUBLE-STRANDED RNA BINDING (DRB) proteins, DRB1, DRB2, and DRB4, in either microRNA (miRNA) or <i>trans</i>-acting small-interfering RNA (tasiRNA) production allowed for use of the loss-of-function mutant lines, <i>drb1, drb2, drb4</i>, and <i>ago7</i>, to further functionally characterize the TAS3 pathway in <i>Arabidopsis thaliana (Arabidopsis)</i>. Towards achieving this goal, we also describe the developmental and molecular phenotypes expressed by three newly generated <i>Arabidopsis</i> lines, the <i>drb1ago7, drb2ago7</i>, and <i>drb4ago7</i> double mutants. We show that the previously reported developmental abnormalities displayed by the <i>drb1, drb2, drb4</i>, and <i>ago7</i> single mutants, are further exacerbated in the <i>drb1ago7, drb2ago7</i>, and <i>drb4ago7</i> double mutants, with rosette area, silique length, and seed set all impaired to a greater degree in the double mutants. Molecular assessment of the <i>TAS3</i> pathway in the floral tissues of the seven analyzed mutants revealed that DRB1 is the sole DRB required for miR390 sRNA production. However, DRB2 and DRB4 appear to play secondary roles at this stage of the <i>TAS</i>3 pathway to ensure that miR390 sRNA levels are tightly maintained. We further show that the expression of the <i>TAS3</i>-derived tasiARF target genes, <i>AUXIN RESPONSE FACTOR2 (ARF2), ARF3</i>, and <i>ARF4</i>, was altered in <i>drb1ago7, drb2ago7</i>, and <i>drb4ago7</i> flowers. Altered <i>ARF2, ARF3</i>, and <i>ARF4</i> expression was in turn demonstrated to lead to changes in the level of expression of <i>KAN1, KAN3</i>, and <i>KAN4</i>, three <i>KANADI</i> transcription factor genes known to be transcriptionally regulated by ARF2, ARF3, and ARF4. Taken together, the demonstrated relationship between altered <i>ARF</i> and <i>KAN</i> gene expression in <i>drb1ago7, drb2ago7</i> and <i>drb4ago7</i> flowers, could, in part, explain the more severe developmental defects displayed by the double mutants, compared to milder impact that loss of only a single piece of <i>TAS3</i> pathway protein machinery was demonstrated to have on <i>drb1, drb2, drb4</i> and <i>ago7</i> reproductive development.