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Arabidopsis bZIP19 and bZIP23 act as zinc sensors to control plant zinc status

Abstract

Zinc (Zn) is an essential micronutrient for plants and animals owing to its structural and catalytic roles in many proteins1. Zn deficiency affects around 2 billion people, mainly those who live on plant-based diets relying on crops from Zn-deficient soils2,3. Plants maintain adequate Zn levels through tightly regulated Zn homeostasis mechanisms involving Zn uptake, distribution and storage4, but evidence of how they sense Zn status is lacking. Here, we use in vitro and in planta approaches to show that the Arabidopsis thaliana F-group bZIP transcription factors bZIP19 and bZIP23, which are the central regulators of the Zn deficiency response, function as Zn sensors by binding Zn2+ ions to a Zn-sensor motif. Deletions or modifications of this Zn-sensor motif disrupt Zn binding, leading to a constitutive transcriptional Zn deficiency response, which causes a significant increase in plant and seed Zn accumulation. As the Zn-sensor motif is highly conserved in F-group bZIP proteins across land plants, the identification of this plant Zn sensor will promote new strategies to improve the Zn nutritional quality of plant-derived food and feed, and contribute to tackling the global Zn-deficiency health problem.

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Fig. 1: In vitro Zn-protein binding assay.
Fig. 2: In planta analysis of the ability of bZIP19 Cys/His-rich Zn-sensor-motif variants to activate transcription of GUS from the ZIP4 promoter in a bzip19/23-pZIP4::GUS background.
Fig. 3: Effects on gene expression and Zn concentration after expression of the bZIP19(del1 del2) variant in the bzip19/23 and bzip19/23-pZIP4::GUS backgrounds.
Fig. 4: Schematic of cellular Zn sensing in Arabidopsis.

Data availability

Accession numbers of genes mentioned in this study are as follows: bZIP19, AT4G35040; bZIP23, AT2G16770; ZIP4, AT1G10970; ZIP5, AT1G05300; NAS2, AT5G56080; ACT2, AT3G18780. All data supporting the findings of this study are available in the main text, Extended Data Figs. 16 or in the Supplementary Information. Additional data related to this study are available from the corresponding author on request. All biological materials used in this study are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by the Independent Research Fund Denmark, DFF-YDUN-program (4093–00245B); Portuguese Foundation for Science and Technology, FCT-IF program (IF/01641/2014), FCT-MCTES (PTDC/BAA-AGR/31122/2017; POCI-01-0145-FEDER- 031122); Novo Nordisk Foundation, Biotechnology-based Synthesis and Production Research program (NNF18OC0034598; to G.H.L., P.H.C., F.L. and A.G.L.A.); DFF-FTP-program (50544600-1126521001-112652; to D.P.P.) and the Netherlands Genome Initiative (40-41009-98-11084; to R.A. and M.G.M.A.). Element analysis was performed at CHEMI Center, and imaging data were collected at the CAB Center at PLEN, University of Copenhagen.

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Contributions

G.H.L., D.P.P., P.H.C., F.L., R.D.A. and A.G.L.A. designed and performed experiments. G.H.L. and A.G.L.A. analysed and interpreted data. G.H.L., M.G.M.A. and A.G.L.A. wrote the manuscript. All of the authors reviewed and approved the manuscript.

Corresponding author

Correspondence to Ana G. L. Assunção.

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The authors declare no competing interests.

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Peer review information: Nature Plants thanks Marc Hanikenne 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 SEC-ICP-MS chromatogram of MBP incubated with 67Zn stable isotope.

SEC-ICP-MS chromatogram showing a sample of maltose binding protein (MBP) incubated with 67Zn stable isotope. The MBP eluted after ~590 s and was detected both a, by UV and b, by a weak 48SO signal. The 48SO signal is weak due to the low-amount of S-containing amino acids in the MBP. c, There is no co-elution of 67Zn with the MBP at this retention time, showing that there is no Zn-binding to MBP. Free 67Zn eluted after ~680 s.

Extended Data Fig. 2 Shoot ionome analysis of the motif deletion mutant lines (bZIP19 del1 del2).

Shoot ionome analysis of the motif deletion mutant lines (bZIP19 del1 del2). Plants (aged 6 weeks) of WT (Col), bzip19/23 (-), bzip19/23-bZIP19, bzip19/23-bZIP19 del1 del2 and bzip19/23-pZIP4::GUS-bZIP19 del1 del2 lines grown in hydroponics with control nutrient solution. a, Fe b, Cu c, Mn and d, P concentration, and e, dry weight (DW), of shoot tissue. # represents independently transformed T3 homozygous lines. Bars represent element concentration or DW of shoots as mean ± s.e.m. (n = 4 or n = 5). Different letters indicate significant differences (p < 0.05), determined using one-way analysis of variance followed by Tukey post-hoc test.

Source data

Extended Data Fig. 3 Seed ionome analysis of the motif deletion mutant lines (bZIP19 del1 del2).

Seed ionome analysis of the motif deletion mutant lines (bZIP19 del1 del2). Seeds from soil-grown plants of WT (Col), bzip19/23 (-), bzip19/23-bZIP19, bzip19/23-bZIP19 del1 del2 and bzip19/23-pZIP4::GUS-bZIP19 del1 del2 lines were analyzed. a, Fe b, Cu c, Mn and d, P concentration in seeds. # represents independently transformed T3 homozygous lines. Bars represent element concentration in seeds as mean ± s.e.m. (n = 4–7). Different letters indicate significant differences (p < 0.05), determined using one-way analysis of variance followed by Tukey post-hoc test.

Source data

Extended Data Fig. 4 Histochemical GUS staining analysis under different micronutrient deficiencies.

Histochemical GUS staining analysis under different micronutrient deficiencies. a, Phenotypic analysis and b, histochemical GUS staining of seedlings (aged 12 d) of pZIP4::GUS and bzip19/23-pZIP4::GUS lines grown on ½MS medium with control or under different micronutrient deficiencies; that is, -Zn, -Fe, -Cu and -Mn. Four to six plates (a), and 3–5 seedlings (b) per treatment and genotype were analyzed.

Extended Data Fig. 5 Deletion mutant lines grown with Zn deficient (-Zn) or sufficient (control) media.

Deletion mutant lines grown with Zn-deficient (-Zn) or -sufficient (control) media. a, Seedlings (aged 14 d) of Arabidopsis WT (Col), bzip19/23 double mutant (-), bzip19/23-bZIP19 and bzip19/23-bZIP19 del1 del2 lines, grown with control or -Zn ½MS medium. b, Plants (aged 6 weeks) of WT (Col), bzip19/23 (-), bzip19/23-bZIP19, bzip19/23-bZIP19 del1 del2 and bzip19/23-pZIP4::GUS-bZIP19 del1 del2 lines grown in hydroponics with control nutrient solution. # represents independently transformed T3 homozygous lines.

Extended Data Fig. 6 Protein gel electrophoresis of purified bZIP19 and bZIP19 del1 del2 proteins.

Protein gel electrophoresis (SDS-PAGE) of purified MBP-bZIP19 and MBP-bZIP19 del1 del2 proteins stained with Coomassie Blue. Forty µg of each purified protein was loaded. The purified proteins show the expected molecular weight (arrow) ca. 68 KDa (28 KDa from bZIP19 + 40 KDa from MBP). The analysis was performed twice with two independent protein extractions. Molecular weight markers (MW) are displayed.

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Supplementary information

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Source Data Extended Data Fig. 2

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Statistical source data.

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Unprocessed gel.

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Lilay, G.H., Persson, D.P., Castro, P.H. et al. Arabidopsis bZIP19 and bZIP23 act as zinc sensors to control plant zinc status. Nat. Plants 7, 137–143 (2021). https://doi.org/10.1038/s41477-021-00856-7

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