ABSTRACT: Transciptome profileing analysis of wt, nlp6, nlp7 and nlp6nlp7 before nitrate treatment and after 35 minutes of 25mM nitrate induction. NLP7 (NIN-LIKE-PROTEIN 7) (NLP7) is the major transcriptional factor responsible for the primary nitrate response (PNR), but the role of its closest homologue , NLP6, in nitrogen signaling and the interplay between NLP6 and NLP7 remain to be elucidated. In this study, we show that, like NLP7, nuclear localization of NLP6 via a nuclear retention mechanism is nitrate-dependent, but nucleocytosolic shuttling of both NLP6 and NLP7 is independent of each other. Compared to single mutants, the nlp6 nlp7 double mutant displays a synergistic growth retardation phenotype in response to nitrate. The transcriptome analysis of primary nitrate response (the PNR) showed that NLP6 and NLP7 govern ~50% of nitrate-induced genes, with cluster analysis highlighting two distinct patterns. In the A1 cluster, NLP7 plays the major role, whereas in the A2 cluster, NLP6 and NLP7 are partially functionally redundant. Interestingly, comparing the growth phenotype and PNR under high and low nitrate conditions demonstrated that NLP6 and NLP7 exert a more dominant role in the response to high nitrate. Apart from nitrate signaling, NLP6 and NLP7 also participated in ammonium conditions . Growth phenotypes and transcriptome data uncovered revealed that NLP6 and NLP7 are completely functionally redundant, and may acting as repressors in response to ammonium. Other NLP family members also participated in the PNR, with NLP2 and NLP7 acting as broader regulators and NLP4, -5, -6, and -8 regulating PNR in a gene-dependent manner. Thus, our findings indicate that multiple modes of interplay exist between NLP6 and NLP7 that, which differ depending on nitrogen sources and gene clusters. To determine the genome-wide contributions of NLP7 and NLP6 to the overall nitrate response, we performed a transcriptomics analysis on nlp6, nlp7 and nlp6nlp7 for comparison to wild-type (Col-0) plants. A similar approach has been applied previously for NLP7 (Marchive et al., 2013), but not for NLP6. We collected root tissue before nitrate induction and 35 min after nitrate induction and assessed alterations in gene expression.
