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The SOD7/DPA4–GIF1 module coordinates organ growth and iron uptake in Arabidopsis

Nature Plants volume 9, pages 1318–1332 (2023)Cite this article

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Abstract

Organ growth is controlled by both intrinsic genetic factors and external environmental signals. However, the molecular mechanisms that coordinate plant organ growth and nutrient supply remain largely unknown. We have previously reported that the B3 domain transcriptional repressor SOD7 (NGAL2) and its closest homologue DPA4 (NGAL3) act redundantly to limit organ and seed growth in Arabidopsis. Here we report that SOD7 represses the interaction between the transcriptional coactivator GRF-INTERACTING FACTOR 1 (GIF1) and growth-regulating factors (GRFs) by competitively interacting with GIF1, thereby limiting organ and seed growth. We further reveal that GIF1 physically interacts with FER-LIKE IRON DEFICIENCY-INDUCED TRANSCRIPTION FACTOR (FIT), which acts as a central regulator of iron uptake and homeostasis. SOD7 can competitively repress the interaction of GIF1 with FIT to influence iron uptake and responses. The sod7-2 dpa4-3 mutant enhances the expression of genes involved in iron uptake and displays high iron accumulation. Genetic analyses support that GIF1 functions downstream of SOD7 to regulate organ and seed growth as well as iron uptake and responses. Thus, our findings define a previously unrecognized mechanism that the SOD7/DPA4–GIF1 module coordinates organ growth and iron uptake by targeting key regulators of growth and iron uptake.

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Fig. 1: SOD7 interacts with GIF1 in vitro and in vivo.
Fig. 2: GIF1 genetically works with SOD7 to control organ and seed size.
Fig. 3: SOD7 competes suppressing the interaction between GIF1 and GRFs, and GRFs are involved in seed size control.
Fig. 4: SOD7 and DPA4 negatively regulate Fe homeostasis.
Fig. 5: Characterization of Fe uptake activities in roots of wild-type and mutant plants.
Fig. 6: GIF1 interacts with FIT, and SOD7 competes that interaction.
Fig. 7: A proposed model for the SOD7/DPA4–GIF1 module coordinating organ growth and Fe uptake in Arabidopsis.

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Data availability

All materials in this study are available from the corresponding authors upon request. The authors declare that all data supporting the findings of this study are available within the article and its supplementary information files. Arabidopsis reference genome (TAIR10) was used in this study. The primers used in this study are provided as Supplementary Table 2. Source data are provided with this paper.

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Acknowledgements

We thank Jianmin Zhou (Institute of Genetics and Developmental Biology, CAS) for kindly providing the split luciferase complementation vectors, Mingyi Bai (School of Life Science, Shandong University) for kindly providing the MiR396a and MIM396 overexpression lines, and Feng Yu for kindly providing PE3308 and PE3449 vectors (School of Life Science, Hunan University). This work was supported by grants from National Natural Science Foundation of China (31571499, 31872663, 31961133001) and the strategic priority research programme of the Chinese Academy of Sciences (XDB27010102).

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  1. These authors contributed equally: Leiying Zheng, Huilan Wu, Anbin Wang.

Authors and Affiliations

  1. Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China

    Leiying Zheng & Xian-Jun Song

  2. State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China

    Huilan Wu, Anbin Wang, Yueying Zhang, Zupei Liu, Hong-Qing Ling & Yunhai Li

  3. College of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing, China

    Anbin Wang, Yueying Zhang, Zupei Liu, Hong-Qing Ling, Xian-Jun Song & Yunhai Li

  4. Hainan Yazhou Bay Seed Laboratory, Sanya, China

    Hong-Qing Ling

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Contributions

Y.L., H.L., L.Z. and H.W. conceived and designed the experiments. Y.L., H.L. supervised this project. L.Z., H.W. and A.W. performed most of the experiments. Y.Z. screened the SOD7 interaction protein GIF1 and did some phenotype analysis. Z.L. analysed outer integument development. L.Z., H.W., A.W., Y.L., H.L. and X.S. analysed and discussed the data. L.Z. wrote the paper. Y.L., H.L., X.S., H.W. and A.W. revised paper.

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Correspondence to Hong-Qing Ling, Xian-Jun Song or Yunhai Li.

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Extended data

Extended Data Fig. 1 SOD7 interacts with GIF2/3 in vitro and in vivo.

a, SOD7 interacts with GIF2/3 in pull-down assays. MBP–SOD7 was pulled down by GST–GIF2/3 immobilized on GST beads and analysed by immunoblotting with an anti-GST or anti-MBP antibody. b, The interaction between SOD7 and GIF2/3 was detected by split luciferase complementation assays. N. benthamiana leaves were co-infiltrated with the Agrobacterium GV3101 containing different plasmids combinations for 48 h and then images were determined by a CCD camera. The pseudocolor scale bar was used to indicate the range of luminescence intensity. All experiments were repeated independently twice with similar results.

Source data

Extended Data Fig. 2 DPA4 interacts with GIF1/2/3 in vitro and in vivo.

a, DPA4 interacts with GIF12/3 in pull-down assays. MBP–DPA4 was pulled down by GST–GIF1/2/3 immobilized on GST beads and analysed by immunoblotting with an anti-GST or anti-MBP antibody. b, The interaction between DPA4 and GIF1/2/3 was detected by split luciferase complementation assays. N. benthamiana leaves were co-infiltrated with the Agrobacterium GV3101 containing different plasmids combinations for 48 h and then images were determined by a CCD camera. The pseudocolor scale bar was used to indicate the range of luminescence intensity. All experiments were repeated independently twice with similar results.

Source data

Extended Data Fig. 3 The relative expression analysis.

a. The relative expression level of SOD7 in flowers of Col-0 and gif1. b. The relative expression level of GIF1 in flowers of Col-0 and sod7-2 dpa4-3. c. GRFs expression levels in flowers of Col-0 and sod7-2 dpa4-3. All the data were shown as mean ± SD with three biological repeats. Two-tailed unpaired t-test for a nd b, Multiple t-test followed two-tailed unpaired t-test per row for c.

Source data

Extended Data Fig. 4 The interactions between GIF1 and GRF2/3 were not affected by GFP protein.

a. The GFP did not affect the interaction between GIF1 and GRF2/GRF3 detected by split luciferase complementation assays. The leaves of N. benthamiana were co-infiltrated with the Agrobacterium GV3101 containing combinations as indicated. The pseudocolor scale bar was used to indicate the range of luminescence intensity. b. Quantification of LUC signals from a. Values represent mean ± SD (n = 5 biologically independent samples). One-way ANOVA (Dunnett’s multiple comparisons test, P = 0.05) was used for statistical analysis.

Source data

Extended Data Fig. 5 SOD7 competes suppressing the interaction between GIF1 and GRF2.

The combinations of different plasmids as indicated were overexpressed in N. benthamiana leaves. The N. benthamiana leaves were grown for another 3 days before the total proteins were extracted. The proteins were immunoprecipitated with GFP–Trap-A beads, and detected with anti-Myc and anti-GFP antibodies, respectively. The experiments were repeated independently twice with similar results.

Source data

Extended Data Fig. 6 SOD7 competes suppressing the interaction between GIF1 and GRF2/3 in sod7-2 dpa4-3 protoplast.

a. GIF1–nVenus and rGRF2/3–cCFP, plus different concentration of Myc–SOD7 as indicated were co-transformed into sod7-2 dpa4-3 protoplasts. the 10xMyc was used as a negative control. bar = 40 μm. b. Quantification of GFP signals from a. Values represent mean ± SD (n = 16 protoplasts for GRF2 and n = 20 for GRF3). Asterisk indicates significant difference, **P < 0.01 compared with GIF1–nVenus+rGRF2/3–cCFP samples, and the corresponding GIF1–nVenus+rGRF2/3–cCFP set as 100. One-way ANOVA (Dunnett’s multiple comparisons test) was used for statistical analysis. In the box plots for b, the centre lines are the median and the edges of the box are the lower and upper quartiles. Whiskers extend to the lowest and highest data points.

Source data

Extended Data Fig. 7 qRT-PCR analysis of the expression level of GRFs in 7d seedlings of Col-0, 35Spro:MIM396 and 35Spro:miR396a.

The ACTIN2 gene was used as an internal control. Data are shown as means ± SD (n = 3 biologically independent samples). * p < 0.05, ** p < 0.01 compared with Col-0 and the corresponding Col-0 data set as 1(Multiple t-test followed two-tailed unpaired t-test per row).

Source data

Extended Data Fig. 8 The relative expression level of –Fe-responsive genes in roots of Col-0 and mutants grown on MS medium with or without 100uM Fe.

Data represent mean ± SD, n = 3 for three biological replicates. Asterisk indicates significant difference, *P < 0.05 and **P < 0.01 compared with the wild type. One-way ANOVA (Dunnett’s multiple comparisons test) was used for statistical analysis.

Source data

Extended Data Fig. 9 SOD7 did not interact with FIT in BiFC assays.

The experiment was repeated independently three times with similar results.

Extended Data Fig. 10 The interaction between GIF1 and FIT was not affected by GFP protein.

a. The GFP did not affect the interaction between GIF1 and FIT detected by split luciferase complementation assays. The leaves of N. benthamiana were co-infiltrated with the Agrobacterium GV3101 containing combinations as indicated. The pseudocolor scale bar was used to indicate the range of luminescence intensity. b. Quantification of LUC signals from a. Values represent mean ± SD (n = 5 biologically independent samples). One-way ANOVA ((Dunnett’s multiple comparisons test, p = 0.05) was used for statistical analysis, cLUC-GIF1 + FIT-nLUC was used as control.

Source data

Supplementary information

Supplementary Information

Supplementary Fig. 1, Tables 1 and 2, with source data.

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Zheng, L., Wu, H., Wang, A. et al. The SOD7/DPA4–GIF1 module coordinates organ growth and iron uptake in Arabidopsis. Nat. Plants 9, 1318–1332 (2023). https://doi.org/10.1038/s41477-023-01475-0

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