The ILR3-NRTs/NIA1/SWEET12 module regulates nitrogen uptake and utilization in apple

Abstract

Nitrogen (N) is essential for the physiological metabolism, growth, and development of plants. Plants have evolved a complex regulatory network for the efficient regulation of N uptake and utilization to adapt to fluctuations in environmental N levels. However, the mechanisms underlying the regulation of N absorption and utilization in apple remain unclear. Here, we identified MdILR3 (IAA-LEUCINE RESISTANT3) as an upstream regulator of MdNRT2.4 through yeast one-hybrid (Y1H) screening. MdILR3 overexpression significantly up-regulated the expression of MdNRT2.3/2.4 and MdNIA1, resulting in an increase in nitrate content and nitrate reductase activity. Y1H and EMSA assays revealed that MdILR3 directly interacted with the promoters of MdNRT2.3/2.4 and MdNIA1. Furthermore, MdILR3 can directly bind to the promoter of MdSWEET12 and activate its expression, thereby regulating sucrose transport to provide energy for N uptake in roots. In summary, we provide physiological and molecular evidence suggesting that MdILR3 may positively regulate nitrate response by activating the expression of genes related to N uptake and sugar transport. Our findings suggest that genetic improvements in apple could enhance its ability to absorb and utilize N.

Keywords: MdILR3 (IAA-LEUCINE RESISTANT3); Nitrate assimilation; Nitrate transporter; Nitrogen use efficiency; Sugar transport.

Fig. 1

Expression pattern of MdILR3 and accumulation of MdILR3 in response to nitrate. A Relative expression of MdILR3 in root, stem, leaf, flower, and fruit. B Subcellular localization of MdILR3. Bar, 50 μm. C The response of MdILR3 to different nitrate concentrations. One-month-old ‘Malus hupehensis’ seedlings were pre-cultured for 7 d with 10 mM KNO₃ solution; they were then subjected to treatments with 0, 1, 5, and 10 mM KNO₃ for 24 h. D 35S::GFP-MdILR3 Arabidopsis was geminated on 10 mM KNO₃ medium and transferred to HN or LN medium; photographs were taken after 2 days. Bar, 50 μm. E Relative fluorescence intensity was measured in D. F The stability of MdILR3 protein under different nitrate conditions. Two-week-old MdILR3::GFP calli were grown on HN medium and transferred to LN medium for the indicated time. The abundance of MdILR3 protein was identified using an anti-GFP antibody, with actin serving as an internal control. G ImageJ was used to determine the relative protein intensity. H Y2H assays were used to evaluate the transcriptional activity of MdILR3. The empty vectors pGAD424 and pGBT9 served as controls. Error bars represent the mean ± SD from three independent replicates, with significant differences marked by an asterisk (P < 0.05)

Fig. 2

MdILR3-OE transgenic apple plants promote nitrate uptake and assimilation. A Phenotypes of CK and MdILR3-OE apple seedlings under HN or LN conditions. The 30-day-old transgenic ‘Malus hupehensis’ seedlings were moved to vermiculite and subjected to HN and LN treatment for 2 weeks. CK: control group, transfected with an empty vector. OE represents apple roots overexpressing MdILR3. Bars, 1 cm. B The fluorescence intensity of transgenic apple roots was assessed. WT refers to uninfected ‘Malus hupehensis’ seedlings. C–D Fresh weight (C) and root length (D) were analyzed after HN and LN treatment. E–F The nitrate content (E) and NR activity (F) of the seedlings' roots were measured in A. G ¹⁵N influx in transgenic apple seedlings. The 30-day-old transgenic ‘Malus hupehensis’ seedlings were cultivated in a basic nutrient solution with 5 mM KNO₃ for 10 days. They were subsequently treated with 10 mM K¹⁵NO₃ and 0.2 mM K.¹⁵NO₃ for 30 min. FW: fresh weight, DW: dry weight. The mean ± SD of three independent replicates is represented by error bars, with significant differences marked by an asterisk (P < 0.05)

Fig. 3

MdILR3 stimulates the expression of MdNRT2s and MdNIA1. A-B Transgenic materials were grown on a basic nutrient medium with 10 mM KNO₃ (HN) for 7 days, followed by treatment with 0.2 mM KNO₃ (LN) for 3 days. The expression levels of genes related to nitrate uptake and assimilation in the MdILR3 transgenic lines were assessed using qRT-PCR. CK: control group, transfected with an empty vector. WT: wild type. The mean ± SD from three independent replicates is represented by error bars, with significant differences marked by an asterisk (P < 0.05)

Fig. 4

MdILR3 interacts with the promoters of MdNRT2.3, MdNRT2.4 and MdNIA1. A-C The recombinant protein GST-MdILR3 interacts with the G-box of MdNRT2.3, MdNRT2.4, and MdNIA1 in an EMSA assay. The mutated probe of pMdNRT2.3/2.4 and pMdNIA1 contains a mutated G-box, where the sequence CACGTG is replaced by AAAAAA. D-F Y1H assays were used to determine the binding of MdILR3 to the promoters of MdNRT2.3, MdNRT2.4, and MdNIA1. The yeast concentrations of 10^–1, 10^–2, and 10^–3 represent dilutions of 10, 100, and 1000 times, respectively. 3-AT stands for 3-Amino-1,2,4-triazole. The GST fusion protein serves as a negative control, with the black arrow indicating the bound probe and the free probe

Fig. 5

MdILR3 activate the expression of MdNRT2.3/2.4 and MdNIA1. A-F The expression of MdNRT2.3, MdNRT2.4 and MdNIA1 driven by MdILR3 was assessed using both dual-luciferase and GUS staining assays. The promoters of MdNRT2.3/2.4 and MdNIA1 were incorporated as reporter genes into the pGreenII 0800-LUC vector, while the effector MdILR3 was cloned into the pGreenII 62-SK vector. Different colors represent the intensity of the LUC signal. Photograph was captured by a living imaging system (Xenogen, Alameda, CA, USA). The 35S::MdILR3 construct was transiently introduced into transgenic calli containing the pMdNRT2.3/2.4/NIA1::GUS reporter. After 24 h of treatment on MS medium, a staining assay was conducted at 37 °C for 3 h. The relative transcriptional level of the GUS gene was detected by qRT-PCR. The mean ± SD from three independent replicates is represented by error bars, with significant differences marked by an asterisk (P < 0.05)

Fig. 6

MdILR3 promotes the transport of sucrose from shoot to root under low nitrate conditions. A-C Determination of sucrose content of root (A), sucrose content of shoot (B) and root:shoot ratio of sucrose content (C) in Fig. 2A. D-F Determination of sucrose content of root (D), sucrose content of shoot (E) and root:shoot ratio of sucrose content (F) in Fig. 3A. The mean ± SD from three independent replicates is represented by error bars, with significant differences marked by an asterisk (P < 0.05)

Fig. 7

MdILR3 interacts with the promoter of MdSWEET12 to stimulate its transcription. A The EMSA assay was used to assess the interaction between MdILR3 and the MdSWEET12 promoter. The mutated probe of pMdSWEET12 contains a mutated G-box, where the sequence CACGTG is replaced by AAAAAA. B Y1H assays were used to determine the binding of MdILR3 to the promoter of MdSWEET12. C MdILR3-62SK and MdSWEET12-LUC were co-transformed into tobacco leaves, with varying colors indicating the intensity of the LUC signal. The image was obtained using a live imaging system (Xenogen, Alameda, CA, USA). D Relative expression level of GUS was determined. The 35S::MdILR3 construct was transiently introduced into transgenic calli containing the pMdSWEET12::GUS reporter. The mean ± SD of three independent replicates is represented by error bars, with significant differences marked by an asterisk (P < 0.05)

Fig. 8

A proposed model illustrating the role of MdILR3 in nitrate response. Low nitrate levels induce the expression and protein accumulation of MdILR3. The activated MdILR3 directly upregulates the expression of MdNRT2.3/2.4 and MdNIA1, thereby promoting plant growth and enhancing tolerance to low nitrogen stress. Additionally, MdILR3 directly activates the transcription of MdSWEET12, facilitating sucrose allocation to provide energy for root growth and nitrogen mechanism