Conservation and divergence of regulatory architecture in nitrate-responsive plant gene circuits
Nitrogen is an essential element for all life processes in plants. As such, plant roots dynamically respond to nitrogen availability below-ground by executing a signaling and transcriptional cascade resulting in altered plant growth, optimized for nutrient uptake. The NIN-LIKE PROTEIN 7 (NLP7) transcription factor senses nitrogen and along with its closely related paralog NLP6, partially coordinates these transcriptional responses. While post-translational regulation of NLP6/7 is well established, its upstream transcriptional regulation remains understudied in Arabidopsis and other plant species. Here, we dissect a previously identified sub-circuit upstream of NLP6/7 in Arabidopsis and 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 RESPONSE ELEMENT BINDING-PROTEIN 26 (DREB26), A NAC-DOMAIN CONTAINING PROTEIN 32 (ANAC032), NLP6 and NLP7 and their regulation of NITRITE REDUCTASE 1 (NIR1). Conservation and divergence of this circuit and their influence on N-dependent root system architecture are similarly assessed in Solanum lycopersicum. The specific binding sites of these factors within their respective promoters and their putative cis-regulatory architecture are identified. The direct or indirect nature of these interactions are 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. Significance StatementNitrogen is a critical nutrient for plant growth and yield. While external N has facilitated modern agriculture, over-application of N-containing fertilizers has drastic ecological and environmental consequences. Here, we experimentally validate a six gene regulatory circuit with extensive feedforward loops identified to act upstream of the critical NIN-LIKE PROTEIN 6/7 transcription factors which regulates a nitrogen metabolic enzyme. Our results indicate conservation and divergence in these circuits between Arabidopsis and tomato despite the similar role of NLP7 in N-dependent changes in root system architecture. The resulting network models complement existing knowledge of NLP7 regulation by providing a framework for targeted transcriptional engineering to increase plant nitrogen use efficiency.