Adaptor proteins GIR1 and GIR2. II. Interaction with the co-repressor TOPLESS and promotion of histone deacetylation of target chromatin

Abstract

Understanding how root hair development is controlled is important for understanding of many fundamental aspects of plant biology. Previously, we identified two plant-specific adaptor proteins GIR1 and GIR2 that interact with the major regulator of root hair development GL2 and suppress formation of root hair. Here, we show that GIR1 and GIR2 also interact with the co-repressor TOPLESS (TPL). This interaction required the GIR1 protein EAR motif, and was essential for the transcriptional repressor activity of GIR1. Both GIR1 and GIR2 promoted histone hypoacetylation of their target chromatin. Potentially, GIR1 and GIR2 might may link TPL to and participate in epigenetic regulation of root hair development.

Keywords: Arabidopsis; Epigenetic regulation; GL2-Interacting proteins; Root hair development; TPL.

Fig. 1.

GIR1 interacts with TPL. (A) The amino acid sequence of GIR1. The conserved EAR motif is indicated and highlighted in red. (B) GIR1-TPL interaction in the yeast two-hybrid system and its dependence on the GIR1 EAR motif. The indicated dilutions of yeast cells were grown in the absence or presence of histidine as shown. Protein interaction was detected as cell growth on histidine-deficient medium and as β-galactosidase (β-gal) activity on a nitrocellulose filter. (C) GIR1-TPL interaction in planta, and accumulation of the interacting proteins in the cell nucleus. Protein interaction was detected by BiFC. YFP signal is in yellow. Fluorescence images are single confocal sections. Scale bars = 20 μm.

Fig. 2.

GIR2 interacts with TPL. (A) GIR2-TPL interaction in the yeast two-hybrid system. The indicated dilutions of yeast cells were grown in the absence or presence of histidine as shown. Protein interaction was detected as cell growth on histidine-deficient medium and as β-galactosidase (β-gal) activity on a nitrocellulose filter. (B) GIR2-TPL interaction in planta, and accumulation of the interacting proteins in the cell nucleus. Protein interaction was detected by BiFC. YFP signal is in yellow. Fluorescence images are single confocal sections. Scale bars = 20 μm.

Fig. 3.

GIR1 promotes gene repression. (A) Schematic structure of the transcriptional repression reporter construct. The transcriptional activity of GIR1 was tested using the p35Smin-(UAS)₃-GUS reporter construct composed of the minimal CaMV 35S promoter (CaMV 35S minimal prom.), three copies of the GAL4 binding site [(UAS)₃], TATA box, β-glucuronidase coding sequence (GUS), and terminator of the nopaline synthase gene (nos term.). (B) GIR1-mediated transcriptional repression of GUS. In the presence of the DBD-VP16 transcriptional activator, p35Smin-(UAS)₃-GUS expressed the GUS enzymatic activity, which was detected as blue histochemical staining of leaf disks from the indicated plant lines transiently transformed with the reporter construct. Inhibition of GUS expression by tested GIR1 sequences fused to DBD-VP16 indicated transcriptional repression activity of these sequences.

Fig. 4.

GIR1, GIR2, and GL2 repress PLDζ1 expression via histone hypoacetylation. (A) RT-qPCR analysis of PLDζ1 gene expression in the indicated plant lines. (B) RT-qPCR analysis of MYB23 gene expression in the indicated plant lines. The expression level in the wild-type (WT) plants is set to 1.0, and error bars represent SEM of N = 3 independent biological replicates. (C) Schematic representation of locations of regions #1–5 within the PLDζ1 promoter sequence used for qChIP analyses. Region #1, −1045 to −813; region #2, −859 to −594; region #3, −604 to −385; region #4, −404 to −208; region #5, −130 to +8, relative to the translation initiation codon (ATG). The conserved L1-box within region #3 and the ATG codon of PLDζ1 are indicated. (D) qChIP analysis of relative acetylation levels of histone H3 (H3ac) in the promoter regions #1–5 of PLDζ1 in the indicated plant lines. Error bars represent SEM of N = 3 independent biological replicates.