SUMMARYNitrate acts as a potent signal to control global gene expression in Arabidopsis. Using an integrative bioinformatics approach we identified TGA1 and TGA4 as putative regulatory factors that mediate nitrate responses in Arabidopsis roots. We showed that both TGA1 and TGA4 mRNAs accumulate strongly after nitrate treatments in roots. Global gene expression analysis revealed 97% of the genes with altered expression in tga1 tga4 double mutant plants respond to nitrate treatments, indicating that these transcription factors have a specific role in nitrate responses in Arabidopsis root organs. We found TGA1 and TGA4 regulate the expression of nitrate transporter genes NRT2.1 and NRT2.2. Specific binding of TGA1 to its cognate DNA sequence on NRT2.1 and NRT2.2 promoters was confirmed by chromatin immunoprecipitation assays. The tga1 tga4 double mutant plants exhibit nitrate-dependent lateral and primary root phenotypes. Lateral root initiation is affected in both tga1 tga4 and nrt1.2 nrt2.2 double mutants, suggesting TGA1 and TGA4 regulate lateral root development at least partly via NRT2.1 and NRT2.2. Additional root phenotypes of tga1 tga4 double mutants indicate that these transcription factors play an important role in root developmental responses to nitrate. These results identify TGA1 and TGA4 as important regulatory factors of the nitrate response in Arabidopsis roots.

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Auxin is a key phytohormone regulating central processes in plants. Although the mechanism by which auxin triggers changes in gene expression is well understood, little is known about the specific role of the individual members of the TIR1/AFB auxin receptors, Aux/IAA repressors, and ARF transcription factors and/or molecular pathways acting downstream leading to plant responses to the environment. We previously reported a role for AFB3 in coordinating primary and lateral root growth to nitrate availability. In this work, we used an integrated genomics, bioinformatics, and molecular genetics approach to dissect regulatory networks acting downstream of AFB3 that are activated by nitrate in roots. We found that the NAC4 transcription factor is a key regulatory element controlling a nitrate-responsive network, and that nac4 mutants have altered lateral root growth but normal primary root growth in response to nitrate. This finding suggests that AFB3 is able to activate two independent pathways to control root system architecture. Our systems approach has unraveled key components of the AFB3 regulatory network leading to changes in lateral root growth in response to nitrate.systems biology | nitrogen | Sungear

Understanding how plants sense and respond to changes in nitrogen availability is the first step toward developing strategies for biotechnological applications, such as improvement of nitrogen use efficiency. However, components involved in nitrogen signaling pathways remain poorly characterized. Calcium is a second messenger in signal transduction pathways in plants, and it has been indirectly implicated in nitrate responses. Using aequorin reporter plants, we show that nitrate treatments transiently increase cytoplasmic Ca 2+ concentration. We found that nitrate also induces cytoplasmic concentration of inositol 1,4,5-trisphosphate. Increases in inositol 1,4,5-trisphosphate and cytoplasmic Ca 2+ levels in response to nitrate treatments were blocked by U73122, a pharmacological inhibitor of phospholipase C, but not by the nonfunctional phospholipase C inhibitor analog U73343. In addition, increase in cytoplasmic Ca 2+ levels in response to nitrate treatments was abolished in mutants of the nitrate transceptor NITRATE TRANSPORTER1.1/Arabidopsis (Arabidopsis thaliana) NITRATE TRANSPORTER1 PEPTIDE TRANSPORTER FAMILY6.3. Gene expression of nitrate-responsive genes was severely affected by pretreatments with Ca 2+ channel blockers or phospholipase C inhibitors. These results indicate that Ca 2+ acts as a second messenger in the nitrate signaling pathway of Arabidopsis. Our results suggest a model where NRT1.1/AtNPF6.3 and a phospholipase C activity mediate the increase of Ca 2+ in response to nitrate required for changes in expression of prototypical nitrate-responsive genes.