Regulation of AUXIN RESPONSE FACTOR condensation and nucleo-cytoplasmic partitioning

Abstract

Auxin critically regulates plant growth and development. Auxin-driven transcriptional responses are mediated through the AUXIN RESPONSE FACTOR (ARF) family of transcription factors. ARF protein condensation attenuates ARF activity, resulting in dramatic shifts in the auxin transcriptional landscape. Here, we perform a forward genetics screen for ARF hypercondensation, identifying an F-box protein, which we named AUXIN RESPONSE FACTOR F-BOX1 (AFF1). Functional characterization of SCF^AFF1 revealed that this E3 ubiquitin ligase directly interacts with ARF19 and ARF7 to regulate their accumulation, condensation, and nucleo-cytoplasmic partitioning. Mutants defective in AFF1 display attenuated auxin responsiveness, and developmental defects, suggesting that SCF^AFF1 -mediated regulation of ARF protein drives aspects of auxin response and plant development.

Fig. 1. Identification At3g49150/AFF1 for ARF19 hypercondensation.

a EMS-mutagenized M₂ seeds of arf19-1 35 S:YFP-ARF19 were screened for individuals with increased numbers of YFP-ARF19 condensates using a fluorescence dissecting microscope. Isolate DH8 was backcrossed to the parental line (arf19-1 35 S:YFP-ARF19) and the resultant F₂ individuals displaying YFP-ARF19 hypercondensation identified and used for whole-genome sequencing of bulk segregants. b Confocal images of 3d-old Wt (Col-0) and DH8 (aff1-1) seedlings carrying 35 S:YFP-ARF19 (false-colored yellow) with cell walls counterstained with propidium iodide (false-colored magenta). Scale bar = 25 µm. c Confocal images of 3d-old wild type (Wt; Col-0) and aff1-1 seedlings carrying pARF19:ARF19-mVenus, or pARF7:ARF7-YFP (false-colored yellow) with cell walls counterstained with propidium iodide (false-colored magenta). Scale bar = 25 µm. d Time course confocal images showing fusion of condensates in root transition zone cells of 3d-old Wt (Col-0) and aff1-1 seedlings carrying UBQ10:YFP-ARF19. Scale bar = 5 µm. e Condensate circularity measurements after condensate fusion events in 3d-old Wt (Col-0) and aff1-1 seedlings carrying UBQ10:YFP-ARF19 (mean ± SD; n = 20). f Half-condensate FRAP recovery profiles after photobleaching condensates from 3d-old Wt (Col-0) and aff1-1 seedlings in root tip or upper root region carrying UBQ10:YFP-ARF19 (mean ± SD; n = 20). g Map positions of homozygous EMS-caused mutations identified in DH8 bulk segregants. h At3g49150/AFF1 schematic depicting the exons (blue), UTRs (gray), and introns (black). Locations of the aff1-1 point mutation and aff1–2 (Salk_053818), aff1–3 (Salk_083453), and aff1–4 (Sail_427_G06) insertion sites are indicated. AFF1 encodes a putative F-box protein with an N-terminal F-box domain, leucine rich repeat (LRR) region, and C-terminal F-box domain (FBD) motif. Three independent experiments were performed for (b), (c) and (d) with similar results. The source data for (e) and (f) are provided as a Source Data files.

Fig. 2. AFF1 alters ARF19 and ARF7 nucleo-cytoplasmic partitioning.

a Confocal images of root tip cells 3d-old Wt (Col-0) and aff1-1 seedlings carrying 35 S:YFP-ARF19. b Confocal images of root tip cells 3d-old Wt (Col-0) and aff1-1 seedlings carrying pARF19:ARF19-mVenus. c Confocal images of root tip cells 3d-old Wt (Col-0) and aff1-1 seedlings carrying pARF7:ARF7-YFP. For each confocal image, the ARF signal is false-colored yellow with cell walls counterstained with propidium iodide (false-colored magenta). In each image a representative nucleus is circled with an orange line. Scale bar = 10 µm. d, e, f Quantification of subcellular fluorescence of 3d-old Wt (Col-0) and aff1-1 seedlings carrying 35 S:YFP-ARF19 (d), pARF19:ARF19-mVenus (e), or pARF7:ARF7-YFP (f) n = 63, n = 52 and n = 52 independent cells were examined in d, e and f, respectively. Data are mean ± SD of three independent experiments and gray dots represent the individual values. Different letters indicate individual groups for multiple comparisons with significant differences (one-way ANOVA, Duncan, p  <  0.05). T (total), C (cytoplasmic), and N (nuclear). g Immunoblot analysis and quantification of YFP-ARF19 fractionation from 4d-old seedlings. h Immunoblot analysis and quantification of ARF7-HA fractionation from 4d-old seedlings. HSC70 and histone H3 served as loading controls (l.c.) for the cytosol (C) and the nucleus (N), respectively. Data in g and h are mean ± SD from four independent experiments and gray dots represent the individual values. Different letters indicate individual groups for multiple comparisons with significant differences (one-way ANOVA, Duncan, p  <  0.05). The source data in d, e, f, g and h are provided as a Source Data file.

Fig. 3. AFF1 regulates ARF19 and ARF7 accumulation.

a Confocal images of YFP-ARF19 fluorescence from various tissues of 3d-old wild type (Wt; Col-0) and aff1-1 seedlings carrying 35 S:YFP-ARF19 (false-colored yellow). Three independent experiments were performed with similar results. Scale bar = 50 µm. b Immunoblot analysis of 3d-, 4d-, or 5d-old wild type (Wt; Col-0) or aff1-1 seedlings carrying 35 S:YFP-ARF19. Anti- GFP antibodies were used to detect YFP-ARF19, and anti-HSC70 antibodies were used to detect HSC70 (l.c.; loading control). c Quantification of YFP-ARF19 protein levels of 3d-, 4d-, or 5d-old wild type (Wt; Col-0) or aff1-1 seedlings carrying 35 S:YFP-ARF19. Data are mean ± SD from three independent experiments and gray dots represent the individual values. Different letters indicate individual groups for multiple comparisons with significant differences (one-way ANOVA, Duncan, p  <  0.05). d Immunoblot analysis of HA₃-ARF1, ARF7-HA, and YFP-ARF19 in seedlings treated with mock (DMSO) or MG132. Anti-HA antibodies were used to detect HA₃-ARF1, ARF7-HA, anti-GFP antibodies were used to detect YFP-ARF19, and anti-HSC70 antibodies were used to detect HSC70 (l.c.; loading control). e Quantification of HA₃-ARF1, ARF7-HA, and YFP-ARF19 accumulation in seedlings treated with mock (DMSO) or MG132. Data are mean ± SD from three independent experiments and the gray dots represent the individual values. The statistical significance was determined by a two-sided Student’s t-test (Paired two sample for means). P values = 0.00043 (HA₃-ARF1), 0.0093 (ARF7-HA), 0.0019 (YFP-ARF19). **P < 0.01 when compared to the mock. The source data in (b), (c), (d) and (e) are provided as a Source Data file.

Fig. 4. AFF1 regulates ARF19 and ARF7 protein degradation.

a In vitro YFP-ARF19 degradation. Plant lysate from aff1-1 arf19-1 35 S:YFP-ARF19 was incubated with GST, GST-AFF1, or GST-ΔF-box-AFF1 recombinant proteins for the indicated times. Immunoblot analysis (top) and quantification (bottom) of YFP-ARF19, GST, GST-AFF1, or GST-ΔF-box-AFF1 using the indicated antibodies. Anti-HSC70 used for loading control (l.c.). b In vitro ARF7-HA degradation. Plant lysate from aff1-1 35 S:ARF7-HA was incubated with GST, GST-AFF1, or GST-ΔF-box-AFF1 recombinant proteins for the indicated times. Immunoblot analysis (top) and quantification (bottom) of ARF7-HA, GST, GST-AFF1, or GST-ΔF-box-AFF1 using the indicated antibodies. Anti-HSC70 used for loading control (l.c.). Data are mean ± SD of three independent experiments. Different letters indicate individual groups for multiple comparisons with significant differences (one-way ANOVA, Duncan, p  <  0.05) and gray dots represent the individual values. The source data in (a) and (b) are provided as a Source Data file.

Fig. 5. AFF1 interacts with ARF proteins and ASK1.

a GST, GST-AFF1 or GST-ΔF-box-AFF1 recombinant proteins were incubated with arf19-1 35 S:YFP-ARF19 plant lysate. Pull-down fractions and inputs were examined by immunoblot analysis using anti-GFP or anti-GST antibodies. b GST, GST-AFF1 or GST-ΔF-box-AFF1 were incubated with arf19-1 35 S:YFP-ARF19 plant lysate prior to immunoprecipitation with anti-GFP antibody. Immunoprecipitates and inputs were examined by immunoblot analysis using anti-GFP or anti-GST antibodies. c GST, GST-AFF1 or GST-ΔF-box-AFF1 were incubated with 35 S:ARF7-HA plant lysate prior to immunoprecipitation with anti-HA antibody. Immunoprecipitates and inputs were examined by immunoblot analysis using anti-HA or anti-GST antibodies. d GST, GST-AFF1 or GST-ΔF-box-AFF1 were incubated with His-ASK1 prior to pull down. Pull-down fractions and inputs were examined by immunoblot analysis using anti-His or anti-GST antibodies. e Bimolecular fluorescence complementation (BiFC; yellow) assays were used to analyze protein interactions between nEYFP-ΔF-box-AFF1 and cEYFP-ARF19, nEYFP-ΔF-box-AFF1 and cEYFP-IAA7, nEYFP-ARF19 and cEYFP-ARF19, or nEYFP-ARF19 and cEYFP-IAA7. The nuclear marker WPP-mCherry (magenta) was co-expressed to determine nuclear signal. Scale bar = 50 µm. See Supplementary Fig. 3 for extended data. f Left, confocal images of 3d-old wild type (Wt; Col-0) or aff1-1 seedlings carrying UBQ10:YFP-ARF19 or UBQ10:YFP-ARF19^K962A (false-colored yellow) with cell walls counterstained with propidium iodide (false-colored magenta). Scale bar = 25 µm. Right, quantification of subcellular fluorescence. T (total), C (cytoplasmic), and N (nuclear). See Supplementary Fig. 4 for images from additional regions of the root. Three independent experiments were performed on (a), (b), (c), (d), (e) and (f) with similar results. n = 56 (ARF19) and n = 54 (ARF19^K962A) independent cells were examined in (f). Data in (f) are mean ± SD and gray dots represent the individual values. Different letters indicate individual groups for multiple comparisons with significant differences (one-way ANOVA, Duncan, p  <  0.05). The source data in (a), (b), (c), (d) and (f) are provided as a Source Data file.

Fig. 6. aff1 exhibits developmental defects and attenuated auxin responsiveness.

a Photograph of 22d-old wild type (Wt; Col-0), aff1-1, aff1–2, aff1–3, and aff1–4 plants. Scale bar = 1 cm. b Photograph of 9d-old wild type (Wt; Col-0), aff1-1, aff1–2, aff1–3, and aff1–4 seedlings grown on media supplemented with 40 nM 2,4-D. Scale bar = 1 cm. c Mean primary root lengths of 9d-old wild type (Wt; Col-0), aff1-1, aff1-2, aff1–3, and aff1–4 seedlings vertically grown on media supplemented with mock (EtOH) or 40 nM 2,4-D. n = 80 biologically independent seedlings were examined. Data are mean ± SD from three independent experiments and gray dots represent the individual values. The statistical significance was determined by a two-sided Student’s t-test (Paired two sample for means). P values = 0.9815 (aff1-1_mock), 0.3943 (aff1-2_mock), 0.1653 (aff1–3_mock), 0.8700 (aff1–4_mock), 7.50E-17 (aff1-1_2,4-D), 1.315E-11 (aff1-2_2,4-D), 4.355E-12 (aff1-3_2,4-D), and 7.405E-13 (aff1-4_2,4-D). **P < 0.01 when compared to Wt. d Volcano plots displaying pairwise transcript accumulation differences after two hours of Mock (EtOH) or auxin (10 μM IAA) treatment in wild type (Wt; Col-0), aff1-1, and aff1-3. e Volcano plots displaying pairwise transcript accumulation differences between wild type (Wt; Col-0) and aff1-1, Wt and aff1-3, or aff1-1 and aff1–3 after 2h treatment with Mock (EtOH) or Auxin (10 μM IAA). FDR ≤ 0.01. f Venn diagrams showing the number of genes that are overlap between the datasets of differentially expressed genes (FDR < 0.01). g Relative transcript abundance (±SD, n = 3) of auxin response targets in wild type (Wt; Col-0), aff1-1 and aff1–3 with or without 10 μM IAA treatment for 2 h. Data are mean ± SD and gray dots represent the individual values. The source data in (c) and (g) are provided as a Source Data file. See Supplementary Fig. 6 for RNA Sequencing (RNA-seq) quality assessments.

Fig. 7. A proposed model for the SCF^AFF1 role in regulating ARF condensation and nucleo-cytoplasmic partitioning.

SCF^AFF1 directly interacts with ARF19 and ARF7, suggesting that these transcription factors, and perhaps additional ARFs, are substrates of this putative E3 ubiquitin ligase. Further, the distinct aff1 mutant effects on ARF protein accumulation/condensation and localization, along with the identity of AFF1 as an E3 ubiquitin ligase, raises the possibility that distinct ubiquitin moieties promote these distinct fates of the ARF transcription factors, both of which may be mediated by SCF^AFF1.