Arabidopsis ZINC FINGER PROTEIN1 Acts Downstream of GL2 to Repress Root Hair Initiation and Elongation by Directly Suppressing bHLH Genes

Abstract

Cys2His2-like fold group (C2H2)-type zinc finger proteins promote root hair growth and development by regulating their target genes. However, little is known about their potential negative roles in root hair initiation and elongation. Here, we show that the C2H2-type zinc finger protein named ZINC FINGER PROTEIN1 (AtZP1), which contains an ERF-associated amphiphilic repression (EAR) motif, negatively regulates Arabidopsis (Arabidopsis thaliana) root hair initiation and elongation. Our results demonstrate that AtZP1 is highly expressed in root hairs and that AtZP1 inhibits transcriptional activity during root hair development. Plants overexpressing AtZP1 lacked root hairs, while loss-of-function mutants had longer and more numerous root hairs than the wild type. Transcriptome analysis indicated that AtZP1 downregulates genes encoding basic helix-loop-helix (bHLH) transcription factors associated with root hair cell differentiation and elongation. Mutation or deletion of the EAR motif substantially reduced the inhibitory activity of AtZP1. Chromatin immunoprecipitation assays, AtZP1:glucocorticoid receptor (GR) induction experiments, electrophoretic mobility shift assays, and yeast one-hybrid assays showed that AtZP1 directly targets the promoters of bHLH transcription factor genes, including the key root hair initiation gene ROOT HAIR DEFECTIVE6 (RHD6) and root hair elongation genes ROOT HAIR DEFECTIVE 6-LIKE 2 (RSL2) and RSL4, and suppresses root hair development. Our findings suggest that AtZP1 functions downstream of GL2 and negatively regulates root hair initiation and elongation, by suppressing RHD6, RSL4, and RSL2 transcription via the GL2/ZP1/RSL pathway.

Figure 1.

Bioinformatics Analysis of AtZP1. (A) Conserved domain and sequence analysis of AtZP1. Different numbers (1 to 204) and lines represent all the contiguous amino acids of AtZP1 from N terminus to C terminus. Boxes represent conserved domains of the AtZP1 protein. The C2H2 zinc finger domain is the amino acids from 46 to 68, the QALGGH sequence is from 59 to 64, and the EAR motif is from 194 to 199. (B) Phylogenetic relationships among different C2H2 zinc finger protein family members in Arabidopsis. Phylogenetic analysis of 22 C2H2 zinc finger proteins using the neighbor-joining method. The proteins were divided into four groups (I to IV) corresponding to different branches in the neighbor-joining tree. Bar = 0.1.

Figure 2.

Expression Analysis of AtZP1. (A) Relative expression levels of AtZP1 in different Arabidopsis tissues. Data represent the means of three biological replicates ± sd. Statistical significance was determined by one-way ANOVA, Duncan’s multiple range test. Significant differences at P < 0.05 are represented by different letters (a to e) above the bars. (B) to (F) GUS analysis of AtZP1 promoter activity in young seedlings (B), mature roots (C), epidermal cells of young leaves (D), flowers (E), and siliques (F). Bar in (B), (D), (E), and (F) = 0.5 cm; bar in (C) = 200 μm.

Figure 3.

Root Hair Phenotypes of Wild Type, Overexpression Lines (L2 and L7), Loss-of-Function atzp1 Mutants (zp1-1 and zp1-2), and AtZP1 Complementation Lines in the atzp1 Mutant Background (ProZP1:ZP1:zp1-1 and ProZP1:ZP1:zp1-2). (A) Root hair phenotypes of the wild type (WT), overexpression lines (L2 and L7), atzp1 mutants (zp1-1 and zp1-2), and AtZP1 complementation lines in the atzp1 mutant background (ProZP1:ZP1:zp1-1 and ProZP1:ZP1:zp1-2). Bar = 400 μm. (B) Average root hair length in the wild type (WT), overexpression lines (L2 and L7), atzp1 mutants (zp1-1 and zp1-2), and complementation lines in the atzp1 mutant background (ProZP1:ZP1:zp1-1 and ProZP1:ZP1:zp1-2). Error bars indicate sd (n = 20). Statistical significance was determined by one-way ANOVA, Duncan’s multiple range test. Significant differences at P < 0.01 are represented by different letters (a to c) in different columns.

Figure 4.

AtZP1 Is a Transcriptional Repressor of Root Hair Growth and Development. (A) Schematic representation of the reporter and various effector constructs used in the luciferase activity assay. Red font represents mutated amino acids. (B) Bioluminescence (showing relative luciferase activity) in Arabidopsis protoplasts. Error bars indicate sd (n = 3). Statistical significance was determined by one-way ANOVA, Duncan’s multiple range test. Significant differences at P < 0.01 are represented by different letters (a to c) above the bars. Cont, control group. (C) Root hair phenotypes of the controls (the wild type; Cont), 35S:ZP1 lines, 35S:ZP1m lines, and 35S:ZP1d lines. Bar = 200 μm. (D) Relative expression levels of AtZP1 in different types of transgenic Arabidopsis lines determined by RT-qPCR; UBQ10 was used for the internal reference. Cont indicates the control group (the wild-type Arabidopsis). Data are means of three biological replicates ± sd. Statistical significance was determined by one-way ANOVA, Duncan’s multiple range test. Significant differences at P = 0.05 are represented by different letters (a and b) above the bars. (E) Average root hair length for the controls (the wild type; Cont), 35S:ZP1 lines, 35S:ZP1m lines, and 35S:ZP1d lines. Statistical significance was determined by one-way ANOVA, Duncan’s multiple range test. For each column, significant differences at P < 0.01 are represented by different letters (a to c) above the bars.

Figure 5.

ChIP Analysis Showing That AtZP1 Binds to the A[AG/CT]CNAC Regions of bHLH Gene Promoters. (A) Conserved sequence A[AG/CT]CNAC (gray bars) and various promoter positions in the candidate bHLH genes examined by ChIP-PCR (lines 1 to 6). The positions in the promoters are relative to the start codons of the bHLH genes. Numbers (1 to 6) and lines represent the possible binding sequences of AtZP1 in the different promoter region. Different bars represent the binding sequence of AtZP1 at the corresponding position of the promoters; numbers and bases show the position and sequence information of AtZP1 binding to the promoter. (B) to (F) ChIP-PCR enrichment (fold) of the RHD6 gene promoter regions (B), ChIP-PCR enrichment (fold) of the RSL2 gene promoter regions (C), ChIP-PCR enrichment (fold) of the RSL4 gene promoter regions (D), ChIP-PCR enrichment (fold) of the LRL1 gene promoter regions (E), ChIP-PCR enrichment (fold) of the LRL3 gene promoter regions (F). The ChIP experiment was performed with wild-type (Cont) and ProAtZP1:AtZP1:GFP transgenic Arabidopsis. Error bars indicate ± sd from three biological repeats. Values are relative to each Cont value. *P < 0.05 and **P < 0.01 (Student’s t test) represent significant differences from the control value.

Figure 6.

AtZP1 Represses the Expression of RSL2, RSL4, and RHD6. (A) Root hair phenotypes of the wild-type and AtZP1:GR plants after transfer to DEX-containing medium for 2 d. The control shows the wild-type Arabidopsis on standard medium. Bar = 300 μm. (B) Root hair length of the wild-type and AtZP1:GR plants after transfer to DEX-containing medium for 2 d. Six biological replicates were performed. Error bars indicate sd (n = 6). Statistical significance was determined by one-way ANOVA, Duncan’s multiple range test. Significant differences at P < 0.01 are represented by different letters (a and b) above the bars. (C) to (E) RT-qPCR analysis of RSL2 (C), RSL4 (D), and RHD6 (E) expression after AtZP1 activation in the presence of DEX or CHX+DEX. Three biological replicates were performed. Error bars indicate sd (n = 3). Statistical significance was determined by one-way ANOVA, Duncan’s multiple range test. Significant differences at P < 0.05 are represented by different letters (a and b) above the bars. Cont, control group.

Figure 7.

EMSAs and Yeast One-Hybrid Analysis. (A) EMSAs showing the binding of AtZP1 to the A[AG/CT]CNAC regions of bHLH gene promoters. glutathione S-transferase–tagged AtZP1 and biotinylated A[AG/CT]CNAC probe were used. The competitor was nonbiotinylated A[AG/CT]CNAC at 10-, 100-, and 200-fold the amount of biotinylated probe. WT, wild type. (B) Interaction of AtZP1 with the bHLH gene promoters verified by yeast one-hybrid analysis. Detection of LacZ activity in cotransformed yeast with different expression vectors pB42AD-ZP1 and pLacZi-A[AG/CT]CNAC. Three cotransformation combinations, pB42AD and pLacZi, pB42AD and pLacZi-A[AG/CT]CNAC, and pB42AD-ZP1 and pLacZi, were used as the controls.

Figure 8.

AtZP1 Functions in the Same Genetic Pathway as RHD6, RSL4, and RSL2. (A) to (C) Relative expression levels of RHD6 (A), RSL4 (B), and RSL2 (C) in different lines. Three biological replicates were performed. Error bars indicate sd (n = 3). Statistical significance was determined by one-way ANOVA, Duncan’s multiple range test. Significant differences at P < 0.05 are represented by different letters (a to d) above the bars. (D) Root hair phenotype of the 5-d-old wild-type (Cont) plants; AtZP1 overexpression lines (L2 and L7); and RHD6, RSL4, and RSL2 overexpression lines in the L2 and L7 background. Bar = 500 μm.

Figure 9.

AtZP1 Is Regulated by GL2. (A) Expression analysis of AtZP1 in various mutants and the wild type (WT). Three biological replicates were performed. Error bars indicate sd (n = 3). Statistical significance was determined by Student’s t tests. *P < 0.05 represents a significant difference. (B) Root hair phenotypes of the wild-type (WT), 35S:AtZP1, atzp1-1, gl2, atzp1-1 gl2, and 35S:AtZP1×gl2 plants. Bar = 500 μm. (C) Mean root hair lengths of the wild-type (WT), 35S:AtZP1, atzp1-1, gl2, atzp1-1 gl2, and 35S:AtZP1×gl2 genotypes. Six biological replicates were performed. Error bars indicate sd (n = 6). Statistical significance was determined by one-way ANOVA, Duncan’s multiple range test. Significant differences at P < 0.01 are represented by different letters (a to e) above the bars. (D) Schematic diagram of the AtZP1 promoter region (3 kb from the start codon, L1 box sequence TAAATGT [gray bars] and its location [lines marked 1 and 2]) and ChIP-PCR enrichment (fold) of the regions (the ChIP experiment was performed with the wild type [Cont] and ProGL2:GL2:GFP transgenic Arabidopsis). The error bars indicate the ± sds from three biological repeats. The values are relative to each Cont value. *P < 0.05 (Student’s t test) represents significant differences from the control value. Numbers (1 and 2) and lines represent the possible binding sequences of GL2 in the different promoter region of AtZP1. Different bars represent the binding sequence of GL2 at the corresponding position of the AtZP1 promoter, numbers (−2227 and −1873) show the position information of AtZP1 promoter.

Figure 10.

A Model Showing How AtZP1 Regulates the Expression of bHLH Transcription Factor Genes to Promote Root Hair Development. GL2 positively regulates AtZP1, and AtZP1 directly negatively regulates RHD6, RSL4, and RSL2. RHD6 directly regulates RSL4. The thickness of the arrow represents the relative level of gene expression.