ZINC-FINGER interactions mediate transcriptional regulation of hypocotyl growth in Arabidopsis

Abstract

Integration of environmental signals and interactions among photoreceptors and transcriptional regulators is key in shaping plant development. TANDEM ZINC-FINGER PLUS3 (TZP) is an integrator of light and photoperiodic signaling that promotes flowering in Arabidopsis thaliana Here we elucidate the molecular role of TZP as a positive regulator of hypocotyl elongation. We identify an interacting partner for TZP, the transcription factor ZINC-FINGER HOMEODOMAIN 10 (ZFHD10), and characterize its function in coregulating the expression of blue-light-dependent transcriptional regulators and growth-promoting genes. By employing a genome-wide approach, we reveal that ZFHD10 and TZP coassociate with promoter targets enriched in light-regulated elements. Furthermore, using a targeted approach, we show that ZFHD10 recruits TZP to the promoters of key coregulated genes. Our findings not only unveil the mechanism of TZP action in promoting hypocotyl elongation at the transcriptional level but also assign a function to an uncharacterized member of the ZFHD transcription factor family in promoting plant growth.

Keywords: Arabidopsis; development; light; transcription; zinc-finger.

Fig. 1.

Identification of ZFHD10 as an interacting partner for TZP, using a genome-wide Arabidopsis TF library. (A) Yeast-two-hybrid analysis of pDEST32-TZP (GAL4BD-bait) and pDEST22-ZFHD10 (GAL4AD-prey) interactions by assessing growth on nonselective media (L⁻W⁻), selective media (L⁻W⁻H⁻ 100 mM 3-AT), or the x-galactosidase assay. pD22, pDEST22; pD32, pDEST32; 3-AT, 3-amino-1,2,4-triazole. (B) Schematic representation of TZP and ZFHD10 domain composition as a guide for the deletion constructs used for the interaction studies shown in Fig. 1C and SI Appendix, Fig. S1 E and F. (C) Deletion analysis of the interaction between TZP and ZFHD10. Yeast-two-hybrid analysis of pDEST32-TZP, pDEST32-TZP^Nt, pDEST32-TZP^ZFPLUS, pDEST32-TZP^PLUS (GAL4BD-bait), pDEST22-ZFHD10, pDEST22-ZFHD10^ZF, pDEST22-ZFHD10^HD (GAL4AD-prey) interaction, using the quantitative β-galactosidase assay. Data shown are representative of three independent experimental repeats.

Fig. 2.

TZP colocalizes and interacts with ZFHD10 in planta. (A) Bimolecular fluorescence complementation assay shows YFP reconstitution between TZP-spyCe and ZFHD10-spyNe when coexpressed transiently in N. benthamiana leaves. (Negative and positive controls are shown in SI Appendix, Fig. S2C.) (B) Representative images of N. benthamiana leaves coexpressing TZP-mCherry and ZFHD10-GFP. (C) Coimmunoprecipitation of TZP-GFP and ZFHD10-RFP coexpressed transiently in N. benthamiana. Single infiltration of TZP-GFP or ZFHD10-RFP were used as negative controls. Plants were grown in white light before examination using confocal microscopy and coimmunoprecipitation. Data shown are representative of three independent experimental repeats. (Scale bars, 20 μm.)

Fig. 3.

ZFHD10 and TZP promote hypocotyl elongation. (A) Hypocotyl measurements and representative images of homozygous transgenic Arabidopsis lines overexpressing ZFHD10 or TZP in Col-0. Plants were grown for 7 d in blue light (1 μmol m⁻²⋅s⁻¹). (B) Hypocotyl measurements of the indicated genotypes. Plants were grown for 7 d in blue light (1 μmol m⁻²⋅s⁻¹). Col-0 was used as the wild-type control. Representative images are shown in SI Appendix, Fig. S5A. The data presented are mean ± SE (n = 15 seedlings). (C) qRT-PCR analysis of HFR1, ATHB2, ATXTH17, and PIF7 mRNA normalized to housekeeping gene ISU1 of the indicated genotypes. Seedlings were grown in continuous blue light (1 μmol m⁻²⋅s⁻¹) for 7 d. Bars are means ± SE (n = 4 technical replicates). Graphs are representative of three independent experimental repeats. Asterisks indicate difference to Col-0 at P < 0.05. An independent biological repeat is shown in SI Appendix, Fig. S5C.

Fig. 4.

TZP and ZFHD10 associate with common genomic regions of growth-promoting genes. (A–D) Relative enrichment of TZP and ZFHD10 on HFR1, ATHB2, ATXTH17, and PIF7 loci. Col-0, a region in the 3′ untranslated region of each locus, and the IAA1 promoter were used as negative controls. Seedlings were grown for 7 d under blue light (1 μmol m⁻²⋅s⁻¹). Bars are means ± SE (n = 4 technical replicates). Graphs shown are representative of three independent experimental repeats. An independent experimental repeat is shown in SI Appendix, Fig. S5D. G, G-box promoter element; HUD, Hormone Up at Dawn promoter element (CACATG); ZfHD, Zinc finger Homeo-Domain binding site TAAATTG.

Fig. 5.

ZFHD10 is important for the recruitment of TZP on light-regulated promoters. (A) Western blot analysis of TZP protein levels in OXTZP/Col-0 and OXTZP/zfhd10-1 transgenic lines. Col-0 was used as a negative control for the anti-GFP antibody, and UGPase was used a loading control. Relative enrichment of TZP on HFR1 (B), ATHB2 (C), PIF7 (D), and ATXTH17 (E) loci when expressed in Col-0 or zfhd10 mutant background. Col-0 and the 3′ untranslated region were used as negative controls. Seedlings were grown for 7 d under blue light (1 μmol m⁻²⋅s⁻¹). Bars are means ± SE (n = 4 technical replicates). Graphs shown are representative of two independent experimental repeats. An independent experimental repeat is shown in SI Appendix, Fig. S5E. G, G-box.

Fig. 6.

TZP and ZFHD10 bind to common genomic regions. (A) Relative binding-peak distribution of TZP and ZFHD10 across genomic regions. Values were normalized to those of Col-0. (B) Venn diagram depicting the overlap between TZP and ZFHD10 targets (promoters and TSS) determined by whole-genome ChIP sequencing analysis. (C) Enriched motifs identified within 1 kb of the surrounding peak summits for overlapping TZP and ZFHD10 ChIP-seq peaks.

Fig. 7.

ChIP sequencing identifies overlapping targets for TZP and ZFHD10. (A, D, and G) Visualization of ChIP-seq data in the genomic regions encompassing three representative genes, ARF6, SUVR1, and EFL4. The ChIP tracks show the pile-up distribution of the raw reads from three pooled biological replicates of ChIP-seq data. Gray bars underneath the peaks indicate differences identified by SICER between TZP vs. Col-0 and ZFHD10 vs. Col-0 (TZP peaks ≥ 1.5 and ZFHD10 ≥ twofold change to Col-0). Gene regions are represented as arrow-shaped boxes, which shows transcriptional orientation. (B, E, and H) Relative enrichment of TZP and ZFHD10 on ARF6, SUVR1, and EFL4 promoter regions identified by SICER. Col-0 was used as a negative control. Bars are means ± SE (n = 4 technical replicates). (C, F, and I) qRT-PCR analysis of absolute ARF6, SUVR1, and EFL4 mRNA levels normalized to housekeeping gene ISU1 of the above-indicated genotypes. For all the experiments, seedlings were grown in continuous blue light (1 μmol m⁻²⋅s⁻¹) for 7 d. Bars are means ± SE (n = 4 technical replicates). Graphs are representative of two independent experimental replicates.

Fig. 8.

The role of TZP and ZFHD10 in regulating hypocotyl elongation. TZP acts as a positive regulator of growth downstream of the clock and the photoreceptors. TZP and ZFHD10 directly interact through their ZFPLUS3 and ZF domains, respectively. ZFHD10 recruits TZP to light-regulated promoter elements (G-box, SORLIP), and they act in concert to control the expression of growth-promoting transcriptional regulators in response to blue light (LBL). Whether TZP and ZFHD10 act cooperatively or independent of the PIF TFs remains to be investigated.