GARP transcription factors repress Arabidopsis nitrogen starvation response via ROS-dependent and -independent pathways

Abstract

Plants need to cope with strong variations of nitrogen availability in the soil. Although many molecular players are being discovered concerning how plants perceive NO3- provision, it is less clear how plants recognize a lack of nitrogen. Following nitrogen removal, plants activate their nitrogen starvation response (NSR), which is characterized by the activation of very high-affinity nitrate transport systems (NRT2.4 and NRT2.5) and other sentinel genes involved in N remobilization such as GDH3. Using a combination of functional genomics via transcription factor perturbation and molecular physiology studies, we show that the transcription factors belonging to the HHO subfamily are important regulators of NSR through two potential mechanisms. First, HHOs directly repress the high-affinity nitrate transporters, NRT2.4 and NRT2.5. hho mutants display increased high-affinity nitrate transport activity, opening up promising perspectives for biotechnological applications. Second, we show that reactive oxygen species (ROS) are important to control NSR in wild-type plants and that HRS1 and HHO1 overexpressors and mutants are affected in their ROS content, defining a potential feed-forward branch of the signaling pathway. Taken together, our results define the relationships of two types of molecular players controlling the NSR, namely ROS and the HHO transcription factors. This work (i) up opens perspectives on a poorly understood nutrient-related signaling pathway and (ii) defines targets for molecular breeding of plants with enhanced NO3- uptake.

Keywords: Cell sorting; GARP transcription factors; ROS; TARGET; nitrogen starvation response; plant growth; root nitrate uptake; root protoplasts.

Fig. 1.

HRS1 and HHO1 are repressors of NSR sentinel genes. (A) Root response of NRT2.4, NRT2.5, and GDH3 to the NSR in Columbia, hrs1;hho1, and 35S:HRS1 genotypes. Plants were grown in sterile hydroponic conditions on N-containing medium for 14 d. At time 0, the medium was shifted to –N conditions for 0, 2, 4, or 6 d, or +N as a control. (B) Root response of NRT2.4, NRT2.5, and GDH3 to NSR in Columbia WT, 35S:HRS1, and 35S:HHO1 genotypes. Plants were grown in sterile hydroponic conditions on N-containing medium for 14 d. Then the medium was shifted to –N conditions for 0, 1, 2, 4, and 6 d (the media background was kept unchanged). All transcript levels were quantified by qPCR and normalized to two housekeeping genes (ACT and CLA); values are means ±SE (n=4). Asterisks indicate significant differences from WT plants (*P<0.05; **P<0.01; ***P<0.001; Student’s t-test).

Fig. 2.

The HHO subfamily is involved in repressing NSR sentinels after NO₃⁻ provision. (A) Phylogenetic tree representing the GARP HHO subfamily. The tree was built as previously described (Safi et al., 2017). (B) Root response of NRT2.4, NRT2.5, and GDH3 to the PNR following N starvation in Columbia WT, hrs1, hrs1;hho1, hrs1;hho1;hho2, and hrs1;hho1;hho2;hho3 genotypes. Plants were grown in sterile hydroponic conditions on full medium for 14 d, subjected to N starvation for 3 d, and then resupplied with 0.5 mM NH₄NO₃ for 0 (harvested before treatment), 15 min, 30 min, 1 h, 2 h, 3 h, and 4 h (the media background was kept unchanged). (C) NRT2.4 expression 2 h after N supply, showing an additive de-repression effect following sequential deletion of HHO genes. (D) pHRS1:HRS1:GFP is sufficient to complement the quadruple mutant. WT, hrs1;hho1;hho2;hho3, and hrs1;hho1;hho2;hho3;pHRS1:HRS1:GFP line 1 and line 2 (two independent transformation events) were grown on Petri dishes on 0.5 mM NH₄NO₃ for 12 d. Roots were harvested and transcripts were measured by qPCR. All transcript levels were quantified by qPCR and normalized to two housekeeping genes (ACT and CLA); values are means ±SE (n=4). Asterisks indicate significant differences from WT plants (*P<0.05; **P<0.01; ***P<0.001; Student’s t-test).

Fig. 3.

HRS1 and HHO1 negatively control NO₃⁻ HATS. (A) NO₃⁻ uptake is altered in 35S:HRS1 and in the double mutant hrs1;hho1. Plants were grown for 5 weeks on N-containing medium. The medium was then shifted to –N conditions or +N as a control for 1 or 3 weeks. Values are means ±SE (n=6). (B) Plants starved for 1 week were used to quantify NO₃⁻ HATS and LATS activities as well as high-affinity NO₃⁻ transporter transcript levels. qPCR data were normalized to two housekeeping genes (ACT and CLA); values are means ±SE (n=12). NO₃⁻ uptake measurements were performed on different ¹⁵NO₃⁻ concentrations (10, 100, 250 µM, 1 mM, and 5 mM) to evaluate HATS and LATS. Values are means ±SE (n=6). The experiment was performed exactly as detailed for (A). Asterisks indicate significant differences from WT plants (*P<0.05; **P<0.01; ***P<0.001; Student’s t-test).

Fig. 4.

The HHO subfamily represses NO₃⁻ uptake and growth in +N conditions. (A) NO₃⁻ uptake is altered in hrs1, hrs1;hho1, hrs1;hho1;hho2, and hrs1;hho1;hho2;hho3 mutants. Plants were grown for 6 weeks on N-containing non-sterile hydroponics (0.5 mM NH₄NO₃). NO₃⁻ uptake measurements were performed at 100 µM ¹⁵NO₃⁻ to evaluate the HATS. Values are means ±SE (n=6). Asterisks indicate significant differences from WT plants (*P<0.05; **P<0.01; ***P<0.001; Student’s t-test). (B) Representative pictures of the WT and the hrs1;hho1;hho2;hho3 quadruple mutant, grown in +N conditions, on the day of the uptake experiment show a growth phenotype.

Fig. 5.

HRS1 direct genome-wide targets are largely NO₃⁻ dependent and contain many redox-related genes. The TARGET procedure was performed with NO₃⁻ (data from Medici et al., 2015) and without NO₃⁻ (this work). An ANOVA followed by a Tukey test retrieved 1050 HRS1-regulated genes (ANOVA P-value cut-off 0.01, Tukey P-value cut-off 0.01, FDR <10%). (A) GeneCloud analysis (Krouk et al., 2015) of the 1050 direct targets of HRS1. (B) Clustering analysis (Pearson correlation) was performed using MeV software (number of clusters was determined by the FOM method). A selection of over-represented semantic terms is displayed in front of each cluster. Notable redox-related genes are displayed in the right column. The list of each cluster, their related gene list, as well as their respective semantic analysis are provided in Supplementary Dataset S1.

Fig. 6.

ROS are necessary for the NSR. (A) Altered response of NRT2.4, NRT2.5, and GDH3 by KI–mannitol treatment. Plants were grown in sterile hydroponic conditions on N-containing medium for 14 d. Thereafter, the medium was shifted to –N conditions containing or not 5 mM KI and 5 mM mannitol for 0, 2, 4, and 6 d, or +N as a control. (B) Altered response of NRT2.4, NRT2.5, and GDH3 by ROS scavenger treatment. Plants were grown in sterile hydroponic conditions on N-containing medium for 14 d. Then they were transferred to –N or +N conditions for 0, 2, and 4 d. In parallel, some of the N-starved plants were treated with 5 mM KI, 5 mM mannitol, a combination of both, or with 10 µM DPI. DMSO was used as a mock treatment of DPI. All transcript levels were quantified by qPCR and normalized to two housekeeping genes (ACT and CLA); values are means ±SE (n=4). Asterisks indicate significant differences from WT plants (*P<0.045; **P<0.01; ***P<0.001; Student’s t-test).

Fig. 7.

ROS are produced early after nitrogen deprivation, regulated by HRS1, and crucial for the NSR. (A) H₂O₂ production after N deprivation. Plants were grown in non-sterile hydroponics for 6 weeks on N-containing medium. Thereafter, the medium was shifted to –N conditions or +N as a control for 6 h. H₂O₂ accumulation was measured using Amplex^® Red (see the Materials and methods). Values are means ±SE (n=6). Different letters indicate significant differences (Student’s t-test, P<0.05). (B) ROS-scavenging treatment represses NSR sentinel genes in hho mutants. Plants were grown in sterile hydroponic conditions on N-containing medium for 14 d. They were then N deprived for 3 d and treated with 5 mM KI and 5 mM mannitol. Plants kept on the same renewed medium were used as control. Values are means ±SE (n=4).

Fig. 8.

The hho quadruple mutant displays ROS-related phenotypes following N treatment (A) Ascorbate peroxidase (APX), glutathione peroxidase (GPX), and glutathione reductase (GR) activities of the WT and hho quadruple mutant. Plants were grown for 4 weeks in hydroponic conditions +N (1 mM NH₄NO₃) then transferred to either +N (5 mM KNO₃) or –N (5 mM KCl mock control) for 3 d, and finally treated with 5 mM KNO₃ for 3 h. (B) NBT staining of the WT and the quadruple hho mutant. Plants were grown on Petri dishes for 2 weeks on 0.5 mM NH₄NO₃. Fresh plants were harvested and directly stained with NBT. Leaves of the mutant plants display a strong decrease in NBT coloration, reporting a defect in superoxide accumulation. (C) ROS signature genes are controlled by NO₃⁻ and by HRS1 entry into the nucleus. ROS signature genes (Vaahtera et al., 2014) were clustered on HRS1 TARGET data (Fig. 5B). ROS signature genes were color-coded according to their responsiveness to different ROS (red, hydrogen peroxide; blue, singlet oxygen; purple, superoxide; cyan, ozone) and according to statistical analysis. ANOVA followed by a Tukey test was performed. If a gene was significantly regulated (P-value <0.05) based on an ANOVA or a Tukey test [nitrate, DEX, nitrate×DEX, or a difference in the Tukey analysis (±DEX in the +N or –N context)], a black square is reported in the clustering.

Fig. 9.

Proposed model of the regulation of NSR by HHOs and ROS. Under –N conditions, ROS are produced and are needed for induction of the NSR. When nitrogen is present in the media, HRS1 and its homologs are rapidly and highly expressed to repress the NSR either directly by regulating NRT2 and GDH3 promoter activities or indirectly by reducing ROS production.