Transcriptional repression of BODENLOS by HD-ZIP transcription factor HB5 in Arabidopsis thaliana

Abstract

In Arabidopsis thaliana, the phytohormone auxin is an important patterning agent during embryogenesis and post-embryonic development, exerting effects through transcriptional regulation. The main determinants of the transcriptional auxin response machinery are AUXIN RESPONSE FACTOR (ARF) transcription factors and AUXIN/INDOLE-3-ACETIC ACID (AUX/IAA) inhibitors. Although members of these two protein families are major developmental regulators, the transcriptional regulation of the genes encoding them has not been well explored. For example, apart from auxin-linked regulatory inputs, factors regulating the expression of the AUX/IAA BODENLOS (BDL)/IAA12 are not known. Here, it was shown that the HOMEODOMAIN-LEUCINE ZIPPER (HD-ZIP) transcription factor HOMEOBOX PROTEIN 5 (HB5) negatively regulates BDL expression, which may contribute to the spatial control of BDL expression. As such, HB5 and probably other class I HD-ZIP proteins, appear to modulate BDL-dependent auxin response.

Keywords: Arabidopsis; BODENLOS (BDL); HOMEOBOX PROTEIN 5 (HB5); auxin; embryo; transcriptional regulation..

Fig. 1.

Identification of a conserved regulatory element in the 5′ region of BDL/IAA12. (A) mVISTA analysis of 1kb upstream regulatory regions of the indicated genes with reference to the AtIAA12 (BDL) sequence. Blue arrowhead/pink block, region of conservation. A.l., Arabidopsis lyrata; A.t., Arabidopsis thaliana; B.r., Brassica rapa; P.t., Populus trichocarpa. (B) Sequence alignment of a 50bp stretch of the respective 5′ regulatory regions indicated by the blue arrowhead in (A) (corresponding to 245–196bp upstream of the start codon in BDL/AtIAA12). PF _36bp indicates the fragment used for EMSA (see Fig. 3). (C) Sequence logo of the conserved 50bp stretch.

Fig. 2.

pBDL deletion study. (A) GUS-stained pBDL ₂₄₅ ::bdl:GUS seedling root tip. (B) Hypophysis division defect (vertical instead of horizontal division) in pBDL ₂₄₅ ::bdl:GUS embryos. (C) GUS-stained pBDL ₁₉₅ ::bdl:GUS seedling root tip. (D) Normal hypophysis division in pBDL ₁₉₅ ::bdl:GUS embryos. (E, F) p3×PF:m35S::NLS:3×GFP expression in a seedling root counterstained with propidium iodide (E) or in a torpedo-stage embryo (basal part shown) (F). Bars, 25 μm.

Fig. 3.

Direct binding of HB5 to a BDL promoter fragment (see Fig. 1). Results of an EMSA with PF _36bp and mPF _36bp in the absence or presence of HB5. Mutations are indicated by asterisks and the shifted band by an arrowhead.

Fig. 4.

HB5 expression. (A–C) pHB5::HB5:3×GFP expression (green) in a globular-stage embryo (A), heart-stage embryo (B), and seedling root counterstained with propidium iodide (red) (C). Bars, 50 μm.

Fig. 5.

Transactivation of bdl by pLTP1 results in cotyledon defects. (A–C) Seedlings with cotyledon defects caused by protoderm-specific transactivation of bdl expression (B, C) compared with the wild-type (A). Bar, 1mm. (D–F) Cotyledon development is already impaired in embryogenesis in pLTP1>>bdl (compare panels D and F with panel E). Bars, 10 μm.

Fig. 6.

Rescue of the bdl rootless phenotype by HB5 overexpression. (A) Segregation analysis of independent pRPS5A::HB5 transgenic lines in the pBDL::bdl:GUS background (T₂ seedlings were counted). Bars for the pBDL::bdl:GUS controls are shown in red and the bar for the homozygous pBDL::bdl:GUS line with two pRPS5A::HB5 transgenes in blue. (B) Five-week-old plant homozygous for pBDL::bdl:GUS ‘rescued’ by pRPS5A::HB5. Bar, 1cm. (C) One-week-old rootless pBDL::bdl:GUS plant. Bar, 1mm.

Fig. 7.

Effect of HB5 on the expression of different pBDL reporter constructs. (A–C) Transient activity assays using p3×PF::LUC (A), p4×PF _36bp ::LUC (B), or pBDL::LUC (C) as reporter constructs in the presence or absence of HB5, MP, and 1-naphthaleneacetic acid (NAA), as indicated in the respective panels. Values represent mean ±standard error. Statistically significant differences (P <0.05) are indicated by asterisk (Student’s t-test; for statistical details see Table S1).