HHO5 orchestrates dose-dependent feedback regulation of organic versus inorganic nitrogen signaling in Arabidopsis

A major goal in agriculture is to engineer crops to maintain yield with less nitrogen (N) fertilizer. Important regulators of plant N-responses include HRS1 HOMOLOG (HHO) transcription factors (TFs); yet, their redundant repressive mode of action hinders functional analyses. Here, we highlight HHO5 as a unique HHO TF based on its phylogenetic position, phloem specific expression, and dual role in regulating responses to inorganic and organic N-dose signals. Our results support a model whereby HHO5 mediates feedback repression of inorganic nitrate uptake under organic N satiety: (i) Meta-analyses revealed HHO5 expression is repressed by inorganic N, but induced by organic N treatments. (ii) HHO5 directly binds and represses nitrate response genes, but indirectly induces organic N-response genes. (iii) HHO5 indirect target gene induction occurs via WRKY partner TFs, validated using a cell-based DoubleTARGET TF co-perturbation assay. (iv) HHO5 initiates a validated gene regulatory network path which encompasses ∼12% of the N-dose response genes in planta. (v) Phenotypically, single hho5 T-DNA mutants show reduced N-dose dependent growth, and decreased seed N content. (vi) HHO5 represses nitrate uptake but induces root growth responses to Glu in planta. Overall, our findings support a model wherein HHO5 orchestrates dose dependent feedback regulation of organic versus inorganic N-signaling in Arabidopsis.