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Regulatory functions of cellular energy sensor SnRK1 for nitrate signalling through NLP7 repression

Nature Plants volume 8, pages 1094–1107 (2022)Cite this article

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Abstract

The coordinated metabolism of carbon and nitrogen is essential for optimal plant growth and development. Nitrate is an important molecular signal for plant adaptation to a changing environment, but how nitrate regulates plant growth under carbon deficiency conditions remains unclear. Here we show that the evolutionarily conserved energy sensor SnRK1 negatively regulates the nitrate signalling pathway. Nitrate promoted plant growth and downstream gene expression, but such effects were repressed when plants were grown under carbon deficiency conditions. Mutation of KIN10, the α-catalytic subunit of SnRK1, partially suppressed the inhibitory effects of carbon deficiency on nitrate-mediated plant growth. KIN10 phosphorylated NLP7, the master regulator of the nitrate signalling pathway, to promote its cytoplasmic localization and degradation. Furthermore, nitrate depletion induced KIN10 accumulation, whereas nitrate treatment promoted KIN10 degradation. Such KIN10-mediated NLP7 regulation allows carbon and nitrate availability to control optimal nitrate signalling and ensures the coordination of carbon and nitrogen metabolism in plants.

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Fig. 1: KIN10 negatively regulates nitrate-promoted plant growth.
Fig. 2: KIN10 negatively regulates nitrate-mediated gene expression.
Fig. 3: KIN10 phosphorylates NLP7 at ser-125 and ser-306.
Fig. 4: Functional analysis of KIN10-mediated phosphorylation on NLP7.
Fig. 5: KIN10 decreases NLP7 protein stability.
Fig. 6: KIN10 promotes cytoplasmic localization of NLP7.
Fig. 7: Nitrate treatment decreases KIN10 protein stability.
Fig. 8: KIN10-mediated phosphorylation promotes NLP7 cytoplasmic localization under nitrate depletion.

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Data availability

All data generated or analysed during this study are included in the main text and Supplementary Information. All Arabidopsis genes involved in this study can be found at TAIR (www.arabidopsis.org), with the following accession numbers: NLP7 (AT4G24020), KIN10 (AT3G01090), NIA1 (AT1G77760), NIR (AT2G15620), NRT1.1 (AT1G12110), NRT2.1 (AT1G08090), HBI1 (AT2G18300) and LBD37 (AT5G67420). Other species genes involved in this study can be found at UniProt (https://www.uniprot.org/), with the following accession numbers: A0A1B1FGW6_MARPO (https://www.uniprot.org/uniprotkb/A0A1B1FGW6/entry), A0A2K1JP51_PHYPA (https://www.uniprot.org/uniprotkb/A0A2K1JP51/entry), D8T5B1_SELML (https://www.uniprot.org/uniprotkb/D8T5B1/entry), W1PF37_AMBTC (https://www.uniprot.org/uniprotkb/W1PF37/entry), NLP3_ORYSJ (https://www.uniprot.org/uniprotkb/Q5NB82/entry), A0A3B6GRE7_WHEAT (https://www.uniprot.org/uniprotkb/A0A3B6GRE7/entry), K7VRG4_MAIZE (https://www.uniprot.org/uniprotkb/K7VRG4/entry) and A0A2Z5V8A6_LOTJA (https://www.uniprot.org/uniprotkb/A0A2Z5V8A6/entry). Extra data are available from the corresponding author upon reasonable request. All sequencing data that support the findings of this study have been deposited at the National Center for Biotechnology Information Gene Expression Omnibus (GEO) and are accessible through the GEO series accession number GSE206841. Source data are provided with this paper.

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Acknowledgements

We thank H. Yu, Y. Guo and X. Zhao from the Analysis and Testing Center of SKLMT (State Key Laboratory of Microbial Technology, Shandong University) for assistance with the laser scanning confocal microscopy. This work was supported by grants from the National Natural Science Foundation of China (32070210 to M.-Y.B., 31970306 to M.F. and 31870262 to M.-Y.B.) and the Science and Technology Department of Shandong Province (2019LZGC015 to M.-Y.B. and ZR2019ZD16 to M.-Y.B.).

Author information

Author notes

  1. These authors contributed equally: Honglei Wang, Chao Han.

Authors and Affiliations

  1. The Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education, School of Life Sciences, Shandong University, Qingdao, China

    Honglei Wang, Chao Han, Jia-Gang Wang, Xiaoqian Chu, Wen Shi, Lianmei Yao, Jie Chen, Wei Hao, Min Fan & Ming-Yi Bai

  2. Shanxi Key Laboratory of Minor Crops Germplasm Innovation and Molecular Breeding, College of Agriculture, Shanxi Agricultural University, Taigu, China

    Jia-Gang Wang & Xiaoqian Chu

  3. State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Virology and Biotechnology, Zhejiang Academy of Agricultural Sciences, Hangzhou, China

    Zhiping Deng

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Contributions

H.W., C.H. and M.-Y.B. together designed the experiments. H.W. performed statistical analysis of plant growth, transient expression and RT–qPCR. H.W. and C.H. performed western blot and subcellular location analysis. H.W., C.H. and Z.D. performed the kinase assays and mass spectrophotometric analysis. J.-G.W., X.C., W.S., L.Y., J. C., W.H. and M.F. generated p35S:NLP7S2A-YFP/nlp7-1, p35S:NLP7S2D-YFP/nlp7-1, p35S:NLP7S2E-YFP/nlp7-1, p35S:NLP7-YFP/p35S:KIN10-Myc and p35S:NLP7-YFP/kin10 transgenic plants. H.W. performed all other experiments. Z.D. and M.F. provided the critical discussion. H.W., C.H. and M.-Y.B. wrote the manuscript.

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Correspondence to Ming-Yi Bai.

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Nature Plants thanks Shuichi Yanagisawa and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 KIN10 inhibits nitrate-promoted plant growth.

a, KIN10 interacts with NLP6 in yeast. b, KIN11 interacts with NLP7 in yeast. c-f, Seedlings of Ler, p35S:KIN10-HA, Col-0, p35S:KIN10-Myc and kin10 were grown on the MGRL medium supplemented with indicated nitrate concentrations under 16 h Light/8 h Dark photoperiod for 7 days with leaf areas measurement, or for 28 days with fresh weight measurement, respectively. Scale bars represent 5 mm. Error bars indicate S.D. (n = 18). Different letters above the bars indicate statistically significant differences between the samples (One-way ANOVA analysis followed by Uncorrected Fisher’s LSD multiple comparisons test, p < 0.05). Asterisk between bars indicate statistically significant differences between the samples (Unpaired t test, *p < 0.05; ***p < 0.001; **** p < 0.0001). g,h, Seedlings of Ler, p35S:KIN10-HA and tps1-11 were grown on medium supplemented with different nitrogen sources under 16 h Light/8 h Dark photoperiod for 7 days. Scale bar represents 5 mm. Error bars indicate S.D. (n = 18). Different letters above the bars indicate statistically significant differences between the samples (One-way ANOVA analysis followed by Uncorrected Fisher’s LSD multiple comparisons test, p < 0.05).

Source data

Extended Data Fig. 2 KIN10 contributes to the inhibition of carbon deficiency on the nitrate-promoted plant growth.

a-f, Carbon deficiency caused by short photoperiod, low light intensity or DCMU treatment inhibited the nitrate-promoted plant growth. Seedlings of Col-0 and kin10 were grown on the MGRL medium supplemented with different nitrogen sources under 16 h Light/8 h Dark photoperiod or 4 h light/20 h Dark photoperiod with 80 µM/m2/s light intensity for 7 days (a-b), under 20 µM/m2/s or 80 µM/m2/s light intensity in 16 h Light/8 h Dark for 14 days (c-d), or under 16 h Light/8 h Dark photoperiod with 80 µM/m2/s light intensity in the presence or absence of 5 µM DCMU treatment for 7 days (e-f). Scale bars represent 5 mm. Error bars indicate standard deviation (S.D.). (n = 30). Different letters above the bars indicate statistically significant differences between the samples (Brown-Forsythe and Welch ANOVA tests followed by Unpaired t with Welch’s correction, p < 0.05). Asterisk between the bars indicated statistically significant differences between the ratios of inhibited plant growth by low light intensity, short photoperiod or DCMU treatment in wild-type plants and kin10 mutants (Unpaired t test, ****p < 0.0001).

Source data

Extended Data Fig. 3 KIN10 and carbon deficiency regulate nitrate-responsive gene expression.

a, qRT-PCR analysis the expression of KIN10 in Ler, p35S:KIN10-HA, Col-0 and p35S:KIN10-Myc plants. Seedlings of Col-0, p35S:KIN10-Myc, Ler and p35S:KIN10-HA transgenic plants were grown on MGRL medium containing 5 mM nitrate for 7 days. Asterisk between bars indicate statistically significant differences between samples (Unpaired t test, **p < 0.01; ***p < 0.001). b-e, Quantitative RT-PCR analysis the expression of nitrate-responsive genes in wild-type, kin10 mutant and KIN10 overexpression plants. Seedlings of Col-0, p35S:KIN10-Myc and kin10 were grown on the MGRL medium containing 5 mM nitrate for 7 days, subjected to nitrate starvation for 2 days, and then treated with 5 mM KCl or 5 mM KNO3 for 1 h. Actin gene was used as an internal control. f-i, qRT-PCR analysis the expression of nitrate responsive genes in wild-type plants grown under different light intensities. Seedling of Col-0 plant were grown on medium supplemented with or without 5 mM KNO3 under 20 µM/m2/s or 80 µM/m2/s light intensity in 16 h Light/8 h Dark for 7 days. PP2A gene was used as an internal control. j-m, qRT-PCR analysis the expression of nitrate responsive gene in response to DCMU treatment. Seedling of Col-0 plant were grown on medium supplemented with/without 5 mM KNO3 and/or 5 µM DCUM under 80 µM/m2/s light intensity in 16 h Light/8 h Dark for 7 days. PP2A gene was used as an internal control. Error bars indicate SD of three biologic repeats. Different letters above the bars indicated statistically significant differences between the samples (Student t test, p < 0.05). Asterisk between the bars indicate statistically significant differences between the ratios of inhibited gene expression by low light intensity or DCUM treatment (Unpaired t test, *p < 0.05; **p < 0.01; ***p < 0.001).

Source data

Extended Data Fig. 4 KIN10 negatively regulates NLP7-regulated gene expression.

a, Venn diagram showing the overlap between sets of genes regulated by NLP7 with or without KIN10. The Arabidopsis mesophyll protoplast of Col-0 plants were transiently expressed GFP, NLP7-GFP or Co-expressed KIN10-Myc and NLP7-GFP. b, Hierarchical cluster analysis of the expression data of 1984 genes regulated by NLP7-GFP in the presence or absence of KIN10-Myc. The numerical values for the yellow-to-blue gradient bar represent log2 of the ratio. c, Scatter plot of log2-fold change values of 1984 genes regulated by NLP7-GFP in the presence or absence of KIN10-Myc. The red line represents the trend line of the scatter plot. d, Quantitative RT-PCR analysis of the expression of genes regulated by NLP7-GFP or by KIN10-Myc and NLP7-GFP in mesophyll protoplast. PP2A gene was used as an internal control. e, Transient expression assays showed that KIN10 inhibited NLP7-induced NRT2.1 expression. The promoters of NRT2.1 and HBI1 fused to the luciferase reporter gene were co-transfected with p35S:GFP, p35S:NLP7-GFP and p35S:NLP7-GFP/p35S:KIN10-Myc into mesophyll protoplasts of wild-type plants. Error bars indicate SD of three biologic repeats. Different letters above the bars indicate statistically significant differences between the samples (Unpaired t test, p < 0.05).

Source data

Extended Data Fig. 5 KIN10-dependent phosphorylation sites on NLP7 protein.

a, Scheme of the NLP7 protein indicating KIN10 phosphorylation sites. The green dots mark in vitro KIN10 phosphorylation targets identified by mass spectrometer (this study), the purple dots refer to the previously identified CPK10/30/32 phosphorylated residue. b,c, Mass spectrometry analysis of KIN10 phosphorylation sites on NLP7.

Extended Data Fig. 6 KIN10 phosphorylates NLP7 at Ser-125 and S-306.

a, Seedlings of p35S:NLP7-YFP and p35S:NLP7-YFP/kin10 were grown on the MGRL medium containing 10 mM KNO3 under 16 h Light/ 8 h Dark photoperiod for 5 days, and then treated with 20 µM DCMU for 6 h. The immunoblots were probed with anti-GFP antibody and biotinylated Phos-tag bound with streptavidin conjugated HRP. b, The protein alignment is performed using NLPs protein sequences from green algae (Micromonas pusilla CCMP1545, A0A1B1FGW6_MARPO), moss (Physcomitrella patens, A0A2K1JP51_PHYPA), fern (Selaginella moellendorffii, D8T5B1_SELML), basal angiosperm (Amborella trichopoda, W1PF37_AMBTC), monocots (Oryza sativa, NLP3_ORYSJ; Triticum aestivum, A0A3B6GRE7_WHEAT; Zea mays, K7VRG4_MAIZE), and eudicots (Lotus japonicus, A0A2Z5V8A6_LOTJA; Arabidopsis thaliana, NLP7_ARATH). The alignment was made using the ClustalW sequence alignment program and analyzed using Vector NTI. The KIN10-mediated phosphorylation amino acid residues are indicated by red box. c, The promoter of NRT2.1 fused to the luciferase reporter gene was co-transfected with NLP7-YFP, NLP7S125A-YFP, NLP7S306A-YFP, NLP7S125AS306A-YFP (NLP7S2A-YFP) and KIN10-Myc into mesophyll protoplast of wild-type plants. The luciferase activities were normalized by Renilla luciferase as an internal control. Error bars represent S.D. (n = 3). Different letters above the bars indicate statistically significant differences between the samples (One-way ANOVA analysis followed by Uncorrected Fisher’s LSD multiple comparisons test, p < 0.05).

Source data

Extended Data Fig. 7 Phosphorylation mimic versions of NLP7 failed to rescue the growth defective phenotypes of nlp7-1.

a, The growth phenotype of wild type, nlp7-1 and transgenic plants expressing different versions of NLP7 grown in soil for 3 weeks under long-day condition. Scale bar represents 1 cm. b, RT-PCR showed the expression levels of NLP7 and mutation forms of NLP7 in wild type, nlp7-1 and different transgenic plants. PP2A represented the equal cDNA loading. c, Immunoblot analysis showed the protein levels of NLP7 and mutation forms of NLP7 in wild type, nlp7-1 and different transgenic plants. Actin was used as protein loading control. d, The promoting effects of nitrate on the expression of HBI1 were reduced in the NLP7S2D/nlp7-1 and NLP7S2E/nlp7-1 transgenic plants. Seedlings of wild-type plants and indicated mutants were grown on the MGRL medium containing 5 mM KNO3 for 5 days, transferred to nitrogen-free medium for 2 days, and then treated with 10 mM KNO3 or 10 mM KCl for 3 h. PP2A was used as an internal control. Error bars indicate SD of three biologic repeats. Different letters above the bars indicated statistically significant differences between the samples (Unpaired t test, p < 0.05). e, Transient assays showed the expression of HBI1 was induced by NLP7-YFP and NLP7S2A-YFP, but not by NLP7S2D-YFP and NLP7S2E-YFP. The promoter of HBI1 fused to the luciferase reporter gene was co-transfected with NLP7-YFP, NLP7S2A-YFP, NLP7S2D-YFP and NLP7S2E-YFP into mesophyll protoplast of wild-type plants. The luciferase activities were normalized by Renilla luciferase as an internal control. Error bars represent S.D. (n = 3). Different letters above the bars indicate statistically significant differences between the samples (Brown-Forsythe and Welch ANOVA tests followed by Unpaired t with Welch’s correction, p < 0.05).

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Extended Data Fig. 8 Overexpression KIN10 decreased the NLP7 protein stability.

a, Immunoblot analysis of the protein levels of pNLP7:NLP7-YFP in Col-0 and p35S:KIN10-Myc backgrounds. Seedlings of pNLP7:NLP7-YFP and pNLP7:NLP7-YFP/p35S:KIN10-Myc were grown on the medium containing 5 mM KNO3 for 6 days. b, qRT-PCR analysis of NLP7 expression in pNLP7:NLP7-YFP and pNLP7:NLP7-YFP/p35S:KIN10-Myc plants. Seedlings of pNLP7:NLP7-YFP and pNLP7:NLP7-YFP/p35S:KIN10-Myc were grown on the medium containing 5 mM KNO3 for 6 days. PP2A gene was used as an internal control. Error bars indicate SD of three biologic repeats. ns above bars indicates that there was no statistically significant differences between the samples (Welch’s t test, p < 0.05). c-f. Immunoblot analysis of the protein levels of NLP7-Myc in Col-0, kin10 and p35S:KIN10-Myc backgrounds with or without CHX treatment. Seedlings of p35S:NLP7-Myc, p35S:NLP7-Myc/kin10 and p35S:NLP7-Myc/p35S:KIN10-Myc were grown on ½ MS liquid medium for 7 days, then treated with 50 µM cycloheximide (CHX) for different time. The band intensities of Myc and actin were quantified by Image J software. Error bars represent S.D. (n = 3). Different letters above the dots indicate statistically significant differences between the samples (Unpaired t test, p < 0.05).

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Extended Data Fig. 9 Carbon deficiency promotes the cytoplasmic localization of NLP7.

a, b, KIN11 promotes NLP7 protein cytoplasmic localization in protoplasts. The Arabidopsis mesophyll protoplasts were transfected with NLP7-YFP and/or KIN11-Myc and Histone3−RFP. c,d, The nuclear localization of NLP7-YFP was inhibited by KIN10, but such inhibiting effects of KIN10 were reduced in NLP7S2A-YFP. The Arabidopsis mesophyll protoplasts were transfected with NLP7S2A-YFP or NLP7-YFP and/or KIN10-Myc and Histone3−RFP. e,f, DCMU treatment promotes the cytoplasmic localization of NLP7 in the tobacco leaves. The tobacco leaves that were scooped out from soil and grown on the nitrate free medium for 2 days were transformed with the Agrobacterium containing p35S:NLP7-YFP construct. After 24 h, these plants were treated with 10 mM KNO3 and/or 50 μM DCMU for 24 h. g-j, Carbon deficiency promotes the cytoplasmic localization of NLP7. Seedlings of p35S:NLP7-YFP/nlp7-1 and p35S:NLP7S2A-YFP/nlp7-1 were grown on the 10 mM KNO3 medium for 6 days under 16 h Light/8 h Dark photoperiod with 20 µM/m2/s or 80 µM/m2/s light or under 16 h Light/8 h Dark photoperiod with 80 µM/m2/s light treated with/without 50 µM DCMU for 24 h. The box plots showed the ratio of nuclear to cytoplasmic signals of NLP7-YFP or mutant form of NLP7-YFP. Scale bars represent 20 μm. Error bars represent S.D. (n = 80). Asterisk between bars indicate statistically significant differences between the samples (Welch’s t test, ****p < 0.0001). Different letters above the bars indicate statistically significant differences between the samples. (Brown-Forsythe and Welch ANOVA tests followed by Unpaired t with Welch’s correction, p < 0.05).

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Extended Data Fig. 10 Nitrate regulates SnRK1 activity and NLP7 subcellular localization.

a, Nitrate depletion induced the expression of SnRK1 target genes DIN1 and TPS8. Seedlings of Col-0 were grown on the MGRL medium containing 10 mM KNO3 for 5 days, and transferred to medium containing 10 mM KNO3 (Mock) or nitrogen-free medium (N depletion) for 24 h. b, Nitrate resupply reduced the expression of SnRK1 target genes DIN1 and TPS8. Seedlings of Col-0 were grown on the MGRL medium containing 10 mM KNO3 for 5 days, then transferred to nitrogen-free medium (Mock) for 24 h and resupplied with 10 mM KNO3 for 12 h (N resupply). Actin gene was used as an internal control. Error bars indicate SD of three biologic repeats. Asterisk above the bars indicate statistically significant differences between the samples (Unpaired t test, *p < 0.05; **p < 0.01, ***p < 0.001). c, Seedlings of p35S:KIN10-Myc were grown on the MGRL medium containing 10 mM KNO3 for 5 days, transferred to nitrogen-free medium for 2 days, then treated with 10 mM NH4+ for indicated time. Total KIN10 proteins were probed with anti-Myc and phosphorylation KIN10 were probed with anti-P-AMPK antibodies. d, The leaves of tobacco plants that were scooped out from soil and grown on the nitrate free medium were transformed with the Agrobacterium containing p35S:NLP7-YFP and p35S:NLP7S2A-YFP construct. After 36 h, these plants were treated with 10 mM KNO3 or 10 mM KCl for 3 h. The box plots showed the ratio of nuclear to cytoplasmic signals of NLP7-YFP and mutant versions of NLP7-YFP. Scale bars represent 20 μm. Error bars represent S.D. (n = 80). Different letters above the bars indicated statistically significant differences between the samples. (Brown-Forsythe and Welch ANOVA tests followed by Unpaired t with Welch’s correction, p < 0.05).

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Supplementary information

Reporting Summary

Supplementary Table

Table 1. Nitrate-regulated genes in wild-type plants; Table 2. Nitrate-regulated genes in KIN10-Ox plants; Table 3. NLP7-regulated genes in protoplasts; Table 4. NLP7andKIN10 co-regulated genes in protoplasts; Table 5. Oligo used in this study and ANOVA.

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Wang, H., Han, C., Wang, JG. et al. Regulatory functions of cellular energy sensor SnRK1 for nitrate signalling through NLP7 repression. Nat. Plants 8, 1094–1107 (2022). https://doi.org/10.1038/s41477-022-01236-5

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