Regulation of Root Hair Initiation and Expansin Gene Expression in Arabidopsis

Root Hair Cell–Specific Expression Pattern of AtEXP7 in the Arabidopsis Root.

(A), (B), and (H) to (V) show AtEXP7 promoter::GUS expression; (C) and (D) show AtEXP7 promoter::GFP expression; and (E) to (G) show AtEXP7 promoter::genomic AtEXP7-GFP expression.

(A) to (D) In the wild-type root, reporter gene expression occurs in the root hair cell files. The weaker blue staining between the strong stains are from the hair cell files of the opposite side. (C) shows an optical longitudinal section demonstrating GFP expression at the root hair cell files. The red area from propidium iodide indicates the cell boundary. (D) shows an optical cross-section of the root demonstrating gene expression at the eight root hair cells. The arrowheads in (B) and (D) indicate emerging root hair bulges.

(E) to (G) Expression of the AtEXP7-GFP fusion protein shows the same pattern as expression of GUS or GFP alone. (G) shows an optical cross-section. Arrowheads indicate emerging root hair bulges.

(H) and (I) In the ttg-1 (H) and gl2-1 (I) backgrounds, reporter gene expression is observed in cells from both the H and N positions.

(J) axr2-1 background. Arrowheads indicate some root hair bulges.

(K) to (N) rhd6 background with no treatment (K) or with 5 μM ACC (L), 30 nM IAA (M), or separation of the root from the medium (N). The bases of the arrows in (L) and (M) indicate the approximate starting points of hormone treatments. The vertical bar in (N) indicates where the root was separated from agar.

(O) to (Q) Wild-type roots treated with 5 μM ACC (O), 5 μM AVG (P), or 50 μM silver ion (Q). Stars in (O) indicate ectopic expression of GUS in the N positions.

(R) ctr1-1 background. Stars indicate ectopic expression of GUS in the N positions.

(S) to (U) Dominant ethylene receptor mutants etr1-1 (S), ein4 (T), and ers2-1 (U).

(V) ein2-1 background.

Bars = 100 μm in (K) to (N), 50 μm in (A), (P), (Q), and (S) to (V), and 20 μm in (B) to (J), (O), and (R).

RNA Gel Blot Analyses of AtEXP7 and AtEXP18 Transcripts in Different Mutant Backgrounds and under Treatment with Ethylene Precursor and Inhibitor.

Total RNA was prepared from roots of 4-day-old wild-type and mutant seedlings. For ACC (5 μM) and AVG (5 μM) treatments, the seedlings were transferred to chemical-containing plates on day 3. Ten micrograms of total RNA, except for Wassilewskija and rhd6 (30 μg), was analyzed. The transcript level of Arabidopsis actin2 (AtACT2) served as a loading control. Col, Columbia wild type; WS, Wassilewskija wild type.

Effect of 1-MCP on ACC-Induced Root Hair Formation and Expansin Gene Expression in the rhd6 Root.

(A) and (B) Bright-field microscopy images of roots grown in 5 μM ACC without (A) or with (B) 1 μL/L 1-MCP.

(C) and (D) Relative expression levels of AtEXP7 (C) and AtEXP18 (D) in the root when induced by 5 μM ACC without (−MCP) or with (+MCP) 1-MCP. Relative expression levels were evaluated from GFP expression (fluorescence) driven by the AtEXP7 promoter or the AtEXP18 promoter. Bars indicate standard errors (n = 11 to 18).

(E) to (H) Confocal microscopy images of the roots harboring AtEXP7 promoter::GFP ([E] and [F]) and AtEXP18 promoter::GFP ([G] and [H]). Seedlings were incubated in 5 μM ACC without ([E] and [G]) or with ([F]) and [H]) 1-MCP.

Effect of 1-MCP on IAA-Induced Root Hair Formation and Expansin Gene Expression in the rhd6 Root.

(A) and (B) Bright-field microscopy images of the roots grown in 30 nM IAA without (A) or with (B) 1 μL/L 1-MCP.

(C) and (D) Relative expression levels of AtEXP7 (C) and AtEXP18 (D) in the root when induced by IAA without (−MCP) or with (+MCP) 1-MCP. Relative expression levels were evaluated from GFP expression (fluorescence) driven by the AtEXP7 promoter or the AtEXP18 promoter. Bars indicate standard errors (n = 7 to 12).

(E) to (H) Confocal microscopy images of the roots harboring AtEXP7 promoter::GFP ([E] and [F]) and AtEXP18 promoter::GFP ([G] and [H]). Seedlings were incubated in IAA without ([E] and [G]) or with ([F] and [H]) 1-MCP.

Effect of 1-MCP on Root Separation-Induced Root Hair Formation and Expansin Gene Expression in the rhd6 Root.

(A) and (B) Bright-field microscopy images of the roots separated from the agar medium without (A) or with (B) 1 μL/L 1-MCP.

(C) Effect of 1-MCP on root hair number in separation-treated roots. Total root hairs were counted from the separated region of the root. Bars indicate standard errors (n = 13 to 19).

(D) and (E) Relative expression levels of AtEXP7 (D) and AtEXP18 (E) in the root when induced by separation of the root without (−MCP) or with (+MCP) 1-MCP. Relative expression levels were evaluated from GFP expression (fluorescence) driven by the AtEXP7 promoter or the AtEXP18 promoter. Bars indicate standard errors (n = 11 to 15).

(F) to (I) Confocal microscopy images of roots harboring AtEXP7 promoter::GFP ([F] and [G]) and AtEXP18 promoter::GFP ([H] and [I]). Seedlings whose roots were separated from the medium were incubated without ([F] and [H]) or with ([G] and [I]) 1-MCP.

Effect of 1-MCP on Expansin Gene Expression and Root Hair Formation in the Wild Type.

(A) Relative expression levels of AtEXP7 without (−MCP) or with (+MCP) 1 μL/L 1-MCP. Relative expression levels were evaluated from GFP expression (fluorescence) driven by the AtEXP7 promoter. Bars indicate standard errors (n = 7 to 10). WT, wild type.

(B) and (C) Confocal microscopy images of roots harboring AtEXP7 promoter::GFP without (B) or with (C) 1 μL/L 1-MCP.

Deletion Analysis of the AtEXP7 Promoter.

(A) Deletions of the AtEXP7 promoter (ΔpE7) that are fused to the coding region of GFP. Numbers indicate nucleotide positions relative to the transcription initiation site (+1).

(B) Relative activities (GFP expression) of the truncated AtEXP7 promoters in the wild-type (WT) root. Bars indicate standard errors.

(C) to (E) Relative activities of the truncated AtEXP7 promoters in the rhd6 root. For gene induction, the transformed mutant seedlings were treated with 5 μM ACC (C) or 30 nM IAA (D) or roots were separated from agar to expose them to air (E) for 1 day before observation. Bars indicate standard errors.

In (B) to (E), n = 27 to 62.

Substitution and Gain-of-Function Analyses of the AtEXP7 Promoter.

(A) The proximal promoter region of AtEXP7 between −134 and −46 bp. For substitution mutations (E7M1∼E7M7), the underlined regions were replaced by the nucleotides shown in lowercase letters. These substitution mutations were generated from the region between −386 and +48 bp. E7G1∼E7G3 are the gain-of-function promoter fragments. The substituted promoters were fused to the coding region of GFP, and the gain-of-function promoter fragments were connected to the minimal 35S promoter of Cauliflower mosaic virus (mp35S) before the GFP gene. The putative DOF (AAAG) and MYBSt1 (GGATA) core motifs are indicated.

(B) and (C) Relative activities (GFP expression) of the substituted AtEXP7 promoters in the wild-type (WT) root (B) and in the rhd6 root with 5 μM ACC treatment (C). Bars indicate standard errors (n = 15 to 32).

(D) Relative activities of the gain-of-function AtEXP7 promoters in the wild-type root. Bars indicate standard errors (n = 9 to 14). E7G4 contains the −134/−46 region (wild-type promoter), and E7G4M6 and E7G4M7 are the same as the E7G4 construct but with E7M6 and E7M7 substitution mutations, respectively.

(E) to (G) Confocal microscopy images of roots harboring the gain-of-function AtEXP7 promoters mp35S (E), E7G4 (F), and E7G4M6 (G) (a similar expression pattern was observed with E7G4M7).

Deletion Analysis of the AtEXP18 Promoter.

(A) Deletions of the AtEXP18 promoter (ΔpE18) that are fused to the coding region of GFP. Numbers indicate nucleotide positions relative to the transcription initiation site (+1).

(B) Relative activities (GFP expression) of the truncated AtEXP18 promoters in the wild-type (WT) root. Bars indicate standard errors.

(C) to (E) Relative activities of the truncated AtEXP18 promoters in the rhd6 root. For gene induction, the transformed mutant seedlings were treated with 5 μM ACC (C) or 30 nM IAA (D) or roots were separated from the agar medium (E) for 1 day before observation. Bars indicate standard errors.

In (B) to (E), n = 25 to 40.

Summary of Promoter Analyses of AtEXP7 and AtEXP18.

The hatched boxes represent elements for hair cell specificity, and the open boxes represent elements that are likely to be relevant to promoter strength. The environmental (root separation) and hormonal signals converge on the elements for hair cell specificity. Numbers indicate nucleotide positions relative to the transcription initiation site (+1). TATA indicates the TATA box.