Conservation and divergence of regulatory architecture in nitrate-responsive plant gene circuits

Plant roots dynamically respond to nitrogen availability by executing a signaling and transcriptional cascade resulting in altered plant growth that is optimized for nutrient uptake. The NIN-LIKE PROTEIN 7 (NLP7) transcription factor senses nitrogen and, along with its paralog NLP6, partially coordinates transcriptional responses. While the post-translational regulation of NLP6 and NLP7 is well established, their upstream transcriptional regulation remains understudied in Arabidopsis (Arabidopsis thaliana) and other plant species. Here, we dissected a known sub-circuit upstream of NLP6 and NLP7 in Arabidopsis, which was predicted to contain multiple multi-node feedforward loops suggestive of an optimized design principle of nitrogen transcriptional regulation. This sub-circuit comprises AUXIN RESPONSE FACTOR 18 (ARF18), ARF9, DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 26 (DREB26), Arabidopsis NAC-DOMAIN CONTAINING PROTEIN 32 (ANAC032), NLP6 and NLP7 and their regulation of NITRITE REDUCTASE 1 (NIR1). Conservation and divergence of this circuit and its influence on nitrogen-dependent root system architecture were similarly assessed in tomato (Solanum lycopersicum). The specific binding sites of these factors within their respective promoters and their putative cis-regulatory architectures were identified. The direct or indirect nature of these interactions was validated in planta. The resulting models were genetically validated in varying concentrations of available nitrate by measuring the transcriptional output of the network revealing rewiring of nitrogen regulation across distinct plant lineages.