Non-cell-autonomous regulation of root hair patterning genes by WRKY75 in Arabidopsis

Abstract

In Arabidopsis (Arabidopsis thaliana), root hairs are formed in cell files over the cleft of underlying cortex cells. This pattern is established by a well-known gene regulatory network of transcription factors. In this study, we show that WRKY75 suppresses root hair development in nonroot hair files and that it represses the expression of TRIPTYCHON and CAPRICE. The WRKY75 protein binds to the CAPRICE promoter in a yeast one-hybrid assay. Binding to the promoter fragment requires an intact WRKY protein-binding motif, the W box. A comparison of the spatial expression of WRKY75 and the localization of the WRKY75 protein revealed that WRKY75 is expressed in the pericycle and vascular tissue and that the WRKY75 RNA or protein moves into the epidermis.

Figure 1.

Root hair phenotypes in the wild type and mutants. A, Wild-type root. B, wrky75-25 root with ectopic root hairs in nonhair positions indicated by arrows. C, WRKY75 RNAi root displaying ectopic root hairs. D, p35S:WRKY75 root lacking root hairs in hair positions indicated by dashed arrows. Bars = 100 µm.

Figure 2.

Transcriptional regulation of root hair patterning genes by WRKY75. The relative expression of patterning genes was compared by real-time PCR (three biological samples, each with three technical repeats). The wild-type (wt) level was set to 1, and the expression changes in the mutants and overexpression line were plotted. Changes significantly different from the wild type are marked with asterisks (Student’s t test, P < 0.05). Error bars represent sd.

Figure 3.

Histochemical GUS expression analysis of CPC, GL2, and WRKY75 in roots. The expression of CPC and GL2 is monitored in pCPC:GUS (A–D) and pGL2:GUS (E–H) transgenic plants. The genetic background is indicated below each image. The expression of WRKY75 is monitored in the pWRKY75:GUS:3′-WRKY75 (I and J) and pWRKY75:YFP-GUS:3′-WRKY75 (K and L) wild-type (wt) Col-0 background. Bars = 50 μm.

Figure 4.

Yeast one-hybrid studies showing WRKY75 binding to a CPC promoter fragment. A, The 80-bp promoter sequence of CPC used for the yeast one-hybrid assay. The W box is highlighted in gray, and the WER-binding site is underlined. B, Yeast one-hybrid assay. Yeast strain YM4271 carrying a prey construct with three tandem repeats of 80 bp (−459 to −378) of the CPC promoter was cotransformed with WER (positive control) and WRKY75 fusions to the GAL4 activation domain or with an empty vector (EV; negative control). Binding to the wild-type sequence of the promoter fragment (WBOX) was compared with a fragment in which the W box was mutated (mWBOX). –LU, Medium lacking Leu and uracil; –HLU, medium lacking His, Leu, and uracil.

Figure 5.

Genetic analysis of WRKY75. Light micrographs of roots in the wild type (A), cpc-2 wrky75-25 (B), and cpc-2 p35S:WRKY75 (C) are shown. Bars = 50 μm.

Figure 6.

Intercellular motility of WRKY75. A, Root of a wrky75-25 plant carrying pWRKY75:YFP-WRKY75:3′-WRKY75. B, Root of a Col-0 plant carrying pWRKY75:YFP-GUS:3′-WRKY75. Squares mark the region shown at higher magnification. Black stars mark lateral root cap cells, white stars label epidermal cells, and red stars mark cortical cells. Note that high laser intensity and high-brightness/contrast modifications were applied on the images to allow the visualization of the signal, resulting in overexposure to the outer layers (lateral root cap cells). Bars = 40 µm.

Figure 7.

Movement of WRKY75 when expressed under the control of the SCR promoter. A, Root of a wrky75-25 plant carrying pSCR:YFP-WRKY75. B, Root of a Col-0 plant carrying pSCR:GFPer. Squares mark the region shown at a higher magnification. Black stars mark lateral root cap cells, white stars label epidermal cells, and red stars mark cortical cells. Bars = 40 µm.