GAF domain is essential for nitrate-dependent AtNLP7 function

Abstract

Nitrate is an essential nutrient and an important signaling molecule in plants. However, the molecular mechanisms by which plants perceive nitrate deficiency signaling are still not well understood. Here we report that AtNLP7 protein transport from the nucleus to the cytoplasm in response to nitrate deficiency is dependent on the N-terminal GAF domain. With the deletion of the GAF domain, AtNLP7^ΔGAF always remains in the nucleus regardless of nitrate availability. AtNLP7 ^ΔGAF also shows reduced activation of nitrate-induced genes due to its impaired binding to the nitrate-responsive cis-element (NRE) as well as decreased growth like nlp7-1 mutant. In addition, AtNLP7^ΔGAF is unable to mediate the reduction of reactive oxygen species (ROS) accumulation upon nitrate treatment. Our investigation shows that the GAF domain of AtNLP7 plays a critical role in the sensing of nitrate deficiency signal and in the nitrate-triggered ROS signaling process.

Keywords: AtNLP7; GAF domain; Nitrate deficiency signaling; Nuclear localization; ROS.

Fig. 1

The GAF domain is required for the transport of AtNLP7 from the nucleus to the cytoplasm in response to nitrate deficiency and affects plant growth. A Domain structures schematic of the GFP fusions for the AtNLP7 derivatives. NLP7: construct used to express the full-length AtNLP7 protein driven by CaMV 35S promoter. NLP7^∆NES: construct used to express the AtNLP7 protein with deleted nuclear export sequence driven by CaMV 35S promoter. NLP7^∆GAF−1: construct used to express the AtNLP7 protein with deleted GAF domain driven by CaMV 35S promoter. NLP7^∆GAF−2: construct used to express the AtNLP7 protein with deleted region from GAF domain to RWP-RK domain driven by CaMV 35S promoter. NLP7.^∆PB1: construct used to express the AtNLP7 protein with deleted PB1 domain driven by CaMV 35S promoter. Each construct contained the GFP in the C-terminal. B Nucleocytosolic shuttling of AtNLP7 derivatives. Confocal imaging was performed on nlp7-1 seedlings expressing different fusion proteins. Seedlings growing on medium with 10 mM KNO₃ for five days were transferred to N-free medium for two days then treated with 10 mM KNO₃ or KCl for one hour. Scale bars, 20 μm. C Plants grown with a density of 20 plants per line on 1 mM (LN) or 10 mM (HN) KNO₃ modified MS medium for 10 days. Scale bars, 0.5 cm. D Image of 3-week-old AtNLP7 derivate lines, nlp7-1 and WT plants grew in soil. Scale bars, 1 cm. E–G The shoot fresh weight (E), chlorophyll a (F) and b (G) contents of the 10-day-old plants grown as in C. Values are the mean ± SD of three independent replications each containing 20 plants per genotype. P values are from the one-way ANOVA (The letters indicate significant differences. P < 0.05). H Shoot fresh weight of the plants grown as in D. Values are the mean ± SD of four independent replications each containing 6 plants per genotype (The letters indicate significant differences. P < 0.05)

Fig. 2

Effects of mutations in different domains of AtNLP7 on nitrate-inducible gene expression and nitrate assimilation ability. A-F Seedlings of WT, nlp7-1 and the complementation lines were grown on N-free medium for 7 days and then treated with 10 mM KNO₃ for 1 h. Transcript levels were normalized against AtUBQ5 expression. RT-qPCR data are mean ± SD (n = 3). P values are from the one-way ANOVA (The letters indicate significant differences. P < 0.05). NRT2.1, nitrate transporter 2.1; NIA1, nitrate reductase 1; NIR1, nitrite reductase 1; GS2, glutamine synthetase 2; LBD, lateral organ boundary domain. G ¹⁵NO₃⁻ uptake activity assay. 10-day-old seedlings were labeled with 5 mM ¹⁵NO₃⁻ for 1 h and the amount of ¹⁵NO₃⁻ taken into the plants was measured. Values are the mean ± SD of three replications each containing 30 plants per genotype (The letters indicate significant differences. P < 0.05). DW, dry weight. H, I Content of nitrate (H) and enzyme activity of nitrate reductase (I) in the plants grown under different nitrate conditions. 14-day-old seedlings grown on agar medium with 1 mM (LN) or 10 mM (HN) KNO₃ were used for metabolite analyses as described in Material and Methods. Values are the mean ± SD of three replications each containing 15 plants per genotype (The letters significant differences. P < 0.05). FW, fresh weight

Fig. 3

AtNLP7^ΔGAF loses its ability to bind NRE and fails to activate the expression of its target genes. A, B ChIP–qPCR enrichment of NRE-containing promoter fragments (relative to input) from AtNIR1 (A) and AtNIA1 (B) in different AtNLP7 domain deletion mutants. Data are mean ± SD (n = 3). The letters indicate significant differences (P < 0.05). C Schematic representation of the effector and reporter constructs used in the transactivation assay. D Transient transactivation assays. Different AtNLP7 domain deletion variants were used to activate AtNIR1 promoter-LUC fusion construct as described in the Materials and Methods. Renilla luciferase gene (REN) used as an internal control. Data are mean ± SD (n = 3). The letters indicate significant differences (P < 0.05)

Fig. 4

Nitrate inhibits the accumulation of ROS through the GAF domain of AtNLP7. A, B NBT and DAB staining showed that nitrate treatment significantly reduced the contents of ROS in the primary root of nlp7-1 and AtNLP7^ΔGAF plants. Bar = 100 μm. Plants growing on medium with 10 mM KNO₃ for five days were transferred to N-free medium for two days then treated with 10 mM KCl or KNO₃ for one hour. C, D Quantification of NBT and DAB intensities by Image J. Data are mean ± SD (n = 10). The letters indicate significant differences (P < 0.05). E ROS treatment attenuated nitrate-induced nuclear localization of AtNLP7 but not AtNLP7^ΔGAF. Plants growing on medium with 10 mM KNO₃ for five days were transferred to N-free medium for two days then treated with 10 mM KNO₃ or 10 mM KNO₃ + 0.5 mM H₂O₂ for one hour. Bar = 50 μm