Argonaute2 (AGO2) modulates water‐stress responses in <i>Nicotiana attenuata</i>

Argonaute2 (AGO2) largely participates in maintaining viral defenses. However, its function is not understood in species that are not commonly challenged by viruses in their native habitats. The ecological model species, Nicotiana attenuata, grows in arid/desert habitats. Natural virus infections are not commonly observed in this species even when the genes essential for viral defenses, like the RdRs, are silenced. The biological function of NaAGO2 has remained elusive. Silencing NaAGO2 with inverted-repeats (irAGO2) did not alter morphology, growth, or reproductive performance of unstressed plants compared to the wild-type (WT). irAGO2 was also able to defend against herbivores or pathogens and compete with con-species neighbors. However, irAGO2 had increased tolerance to water stress, exhibiting enhanced reproductive output during drought and recovery. Water-stressed irAGO2 accumulated significantly more abscisic acid (ABA) and proline, which are critical signaling and protective metabolites. Drought-responsive miRNA accumulation patterns were largely altered in irAGO2, potentially modulating ABA and proline gene expression during water stress and recovery. The function of three such Na-miRNAs (miR156, miR172, and miR398) was examined by transient overexpression in mitigating water stress and regulating ABA and proline pathways. We infer that AGO2 functions in fine-tuning ABA and proline homeostasis that optimizes N. attenuata's growth in complex stressful environments.