Nuclear translocation of OsMADS25 facilitated by OsNAR2.1 in reponse to nitrate signals promotes rice root growth by targeting OsMADS27 and OsARF7

Abstract

Nitrate is an important nitrogen source and signaling molecule that regulates plant growth and development. Although several components of the nitrate signaling pathway have been identified, the detailed mechanisms are still unclear. Our previous results showed that OsMADS25 can regulate root development in response to nitrate signals, but the mechanism is still unknown. Here, we try to answer two key questions: how does OsMADS25 move from the cytoplasm to the nucleus, and what are the direct target genes activated by OsMADS25 to regulate root growth after it moves to the nucleus in response to nitrate? Our results demonstrated that OsMADS25 moves from the cytoplasm to the nucleus in the presence of nitrate in an OsNAR2.1-dependent manner. Chromatin immunoprecipitation sequencing, chromatin immunoprecipitation qPCR, yeast one-hybrid, and luciferase experiments showed that OsMADS25 directly activates the expression of OsMADS27 and OsARF7, which are reported to be associated with root growth. Finally, OsMADS25-RNAi lines, the Osnar2.1 mutant, and OsMADS25-RNAi Osnar2.1 lines exhibited significantly reduced root growth compared with the wild type in response to nitrate supply, and expression of OsMADS27 and OsARF7 was significantly suppressed in these lines. Collectively, these results reveal a new mechanism by which OsMADS25 interacts with OsNAR2.1. This interaction is required for nuclear accumulation of OsMADS25, which promotes OsMADS27 and OsARF7 expression and root growth in a nitrate-dependent manner.

Keywords: OsARF7; OsMADS25; OsMADS27; OsNAR2.1; nitrate signaling; rice root.

Figure 1

Expression analyses of OsMADS25 and OsNAR2.1. (A) Relative expression level of OsMADS25 induced by nitrate signaling (5 mM KNO₃) in the WT and the Osnar2.1 mutant. One-week-old WT and Osnar2.1 seedlings grown hydroponically without nitrogen were given KNO₃, and the roots were then harvested for qRT–PCR analysis. OsActin was used as an internal control. Values are means ± SD (n = 3). (B) Relative expression level of OsNAR2.1 with (5 mM KNO₃) or without (5 mM KCl) nitrate supply in the WT. One-week-old seedlings grown hydroponically without nitrogen were given KNO₃ or KCl, and the roots were then harvested for qRT–PCR analysis. OsActin was used as an internal control. Values are means ± SD (n = 3).

Figure 2

OsMADS25 physically interacts with OsNAR2.1 in vivo. (A) A yeast two-hybrid assay showed that OsMADS25 interacts with OsNAR2.1. The empty pGADT7 vector was used as a negative control. (B) BiFC assays were performed to verify the interaction between OsMADS25 and OsNAR2.1 in tobacco leaves. OsMADS57, a homolog of OsMADS25, acted as a negative control. Scale bars, 20 μm. (C) A CoIP assay showed that 4HA-OsMADS25 was co-immunoprecipitated in the total leaf extract of N. benthamiana expressing 9Myc-OsNAR2.1 using anti-HA agarose beads. 4HA-OsMADS25 and 9Myc-OsNAR2.1 driven by the 35S promoter were co-expressed in tobacco leaves. Input: western blotting was used to detect the expression levels of 4HA-OsMADS25 and 9Myc-OsNAR2.1 in total protein extracts. HA immunoprecipitation products were tested with anti-Myc antibodies.

Figure 3

NO₃⁻ signaling promotes the transfer of OsMADS25 from the cytoplasm to the nucleus, which is dependent on OsNAR2.1. (A) Subcellular localization of OsMADS25 in WT protoplasts after NO₃⁻ induction. (B) Transient effect of NO₃⁻ signaling on subcellular location of OsMADS25. (a–l) Continuous images of subcellular localization of OsMADS25 induced by NO₃⁻ signaling in protoplasts of WT; each image interval is 15 s. (C) Subcellular localization of OsMADS25 in Osnar2.1 mutant protoplasts after NO₃⁻ induction. Scale bars, 20 μm.

Figure 4

OsMADS25 and OsNAR2.1 synergistically regulate root growth. (A and B) Root phenotypes of the WT, OsMADS25-RNAi lines, the Osnar2.1 mutant, and OsMADS25-RNAi Osnar2.1 lines. One-week-old seedlings were grown with different nitrogen sources (2 mM NH₄⁺ or 2 mM NO₃⁻). Scale bars, 1 cm. (C–F) Primary root length and lateral root number of 1-week-old WT, OsMADS25-RNAi, Osnar2.1, and OsMADS25-RNAi Osnar2.1 seedlings grown with different nitrogen sources (2 mM NH₄⁺ or 2 mM NO₃⁻). Data are presented as mean ± SD (n > 12). Student’s t-test was used to analyze significant differences (∗∗P < 0.01). (G) Nitrate content of 2-week-old WT, OsMADS25-RNAi, Osnar2.1 mutant, and OsMADS25-RNAi Osnar2.1 seedlings grown with different nitrogen sources (2 mM NH₄⁺ or 2 mM NO₃⁻). Data are presented as mean ± SD (n = 3). Student’s t-test was used to analyze significant differences (∗∗P < 0.01).

Figure 5

OsMADS25 binds directly to the promoters of OsMADS27 and OsARF7 to activate their expression. (A) Schematic diagram of binding sites on the promoter of OsMADS27. Red squares indicate the locations of the binding sites. (B) A ChIP–qPCR assay showed in vivo binding of OsMADS25 to the P1–P5 motifs of the OsMADS27 promoter. Samples of cross-linked chromatin were extracted from 35S:GFP-OsMADS25 transgenic plants and precipitated with anti-GFP antibody. The WT was used as a negative control. Values are means ± SD (n = 3). Student’s t-test was used to analyze significant differences (∗∗P < 0.01). (C) Expression level of OsMADS27 in OsMADS25-overexpressing lines. OsActin was used as an internal control. Values are means ± SD (n = 3). Student’s t-test was used to analyze significant differences (∗∗P < 0.01). (D) A dual-LUC assay confirmed that OsMADS25 could bind to the OsMADS27 promoter. (E) Fluorescence intensity of the dual-LUC assay. Values are mean ± SD (n = 3). Student’s t-test was used to analyze significant differences (∗∗P < 0.01). (F) Y1H validation of the interaction of OsMADS25 with the P5 site of the OsMADS27 promoter region. The pGADT7 empty vector was used as a control. The inhibition concentration of Aureobasidin A for autoactivation of the OsMADS27 promoter site was 200 ng/ml. (G) Localization of three putative OsMADS25 binding sites in the promoter of OsARF7. (H) A ChIP–qPCR assay showed in vivo binding of OsMADS25 to the P1 and P2 motifs of the OsARF7 promoter. Samples of cross-linked chromatin were extracted from 35S:GFP-OsMADS25 transgenic plants and precipitated with anti-GFP antibody. The WT was used as a negative control. Values are means ± SD (n = 3). Student’s t-test was used to analyze significant differences (∗∗P < 0.01). (I) Expression level of OsARF7 in OsMADS25-overexpressing lines. OsActin was used as an internal control. Values are means ± SD (n = 3). Student’s t-test was used to analyze significant differences (∗∗P < 0.01). (J) Constructs used in the dual-LUC assay. (K) The dual-LUC assay confirmed that OsMADS25 could bind to the P1 region of the OsARF7 promoter. (L) Fluorescence intensity of the dual-LUC assay. Values are mean ± SD (n = 3). Student’s t-test was used to analyze significant differences (∗∗P < 0.01). (M) Y1H validation of the interaction of OsMADS25 and the P1 site of the OsARF7 promoter region. The pGADT7 empty vector was used as a control. The inhibition concentration of Aureobasidin A for autoactivation of the OsARF7 promoter site was 1000 ng/ml.

Figure 6

OsMADS25 and OsMADS27 synergistically regulate root growth. (A and B) Root morphology of 10-day-old WT, OsMADS25-RNAi, OsMADS27-RNAi, and Osmads25 Osmads27 double mutant seedlings grown with different nitrogen sources (2 mM NH₄⁺ or 2 mM NO₃⁻). Scale bars, 1 cm. (C–F) Primary root length and lateral root number of 10-day-old WT, OsMADS25-RNAi, OsMADS27-RNAi, and Osmads25 Osmads27 double mutant seedlings grown with different nitrogen sources (2 mM NH₄⁺ or 2 mM NO₃⁻). Data are presented as mean ± SD (n > 12). Student’s t-test was used to analyze significant differences (∗∗P < 0.01).

Figure 7

Proposed working model of the mechanism by which OsMADS25 responds to nitrate signaling to modulate root growth in rice.