Transcription Factors HBIs-Mediated ROS Homeostasis Regulates Nitrate Signal Transduction in Arabidopsis

Nitrate is both an important nutrient and a critical signaling molecule that regulates plant metabolism, growth, and development. Although several components of the nitrate signaling pathway have been identified, the molecular mechanism of nitrate signaling remains unclear. Here, we showed that the growth-related transcription factors HBI1 and its three closest homologs (HBIs) positively regulate nitrate signaling in plants. HBI1 is rapidly induced by nitrate through NLP6 and NLP7, which are master regulators of nitrate signaling pathway. Mutations in HBIs result in the reduced effects of nitrate on plant growth and approximately 22% nitrate-responsive genes no longer to be regulated by nitrate. HBIs increase the expression levels of a set of antioxidant genes to reduce the accumulation of reactive oxygen species (ROS) in plants. Nitrate treatment induces the nuclear localization of NLP7, whereas such promoting effects of nitrate are significantly impaired in the hbi-q and cat2cat3 mutants, which accumulate high levels of H2O2. These results demonstrate that HBI-mediated ROS homeostasis regulates nitrate signal transduction through modulating the nucleocytoplasmic shuttling of NLP7. Overall, our findings reveal that nitrate treatment reduces the accumulation of H2O2, and H2O2 inhibits nitrate signaling, thereby forming a feedback regulatory loop to regulate plant growth and development. Overall design: Wild-type Col-0 seedlings, nlp7-1 and hbi-q mutants growing on medium with 7 mM KNO3 for 7 days were transferred to nitrogen-free medium for 2 days then treated with 10 mM KNO3 for 3 hours. To analyse the effects of H2O2 on nitrate regulated gene expression, Col-0 seedlings growing on medium with 7 mM KNO3 for 7 days were transferred to nitrogen-free medium for 2 days then treated with 10 mM KNO3 with or without 0.5 mM H2O2 for 3 hours or treated with H2O2 for 3 hours. Total RNA was extracted with Trizaol RNA extraction kit (Transgene), and mRNA sequencing libraries construction and sequencing on the BGISEQ-500 platform were performed at Beijing Genomics Institute. The sequence reads were mapped to the Arabidopsis genome using HISAT and Bowtie2 software. The gene reads count were calculated by htseq-count with default parameters, and differential gene expression was analysed using DESeq2 tool with generalized linear model. Differentially expressed genes were defined by 2-fold expression difference with false discovery rate (FDR) <0.05.