SOMBRERO, BEARSKIN1, and BEARSKIN2 regulate root cap maturation in Arabidopsis

Abstract

The root cap has a central role in root growth, determining the growth trajectory and facilitating penetration into the soil. Root cap cells have specialized functions and morphologies, and border cells are released into the rhizosphere by specific cell wall modifications. Here, we demonstrate that the cellular maturation of root cap is redundantly regulated by three genes, SOMBRERO (SMB), BEARSKIN1 (BRN1), and BRN2, which are members of the Class IIB NAC transcription factor family, together with the VASCULAR NAC DOMAIN (VND) and NAC SECONDARY WALL THICKENING PROMOTING FACTOR (NST) genes that regulate secondary cell wall synthesis in specialized cell types. Lateral cap cells in smb-3 mutants continue to divide and fail to detach from the root, phenotypes that are independent of FEZ upregulation in smb-3. In brn1-1 brn2-1 double mutants, columella cells fail to detach, while in triple mutants, cells fail to mature in all parts of the cap. This complex genetic redundancy involves differences in expression, protein activity, and target specificity. All three genes have very similar overexpression phenotypes to the VND/NST genes, indicating that members of this family are largely functionally equivalent. Our results suggest that Class IIB NAC proteins regulate cell maturation in cells that undergo terminal differentiation with strong cell wall modifications.

Figure 1.

Structure of the Arabidopsis Root Meristem. A schematic diagram with relevant tissues indicated. Asterisks indicate QC cells. Stem cells are shown in lighter shading. [See online article for color version of this figure.]

Figure 2.

Relationship of SMB with Class IIB NAC Proteins. (A) Genomic structure of BRN1 (At1g33280), BRN2 (At4g10350), and SMB (At1g79580); black boxes indicate coding sequence. The positions of the insertion sites are shown. (B) Multiple sequence alignment between BRN1, BRN2, and SMB. Fully conserved amino acids are indicated with black shading. The C-terminal motifs (LP and WQ box) are boxed, and below the alignment, the consensus sequences for these motifs (based on VND1-VND7/NST1-NST3) are shown. (C) The NAC domain and the two motifs (LP and WQ box) were used to infer a maximum likelihood tree. The numbers above the nodes are the result of a RAxML bootstrap analysis (100 replicates). Only bootstrap values >50% are shown, and branch lengths are proportional to the number of substitutions per site (see scale bars). We used the sequences from the moss Physcomitrella patens as an outgroup to root the tree. [See online article for color version of this figure.]

Figure 3.

Mutants in SMB Have a Defect in Root Cap Maturation . (A) to (H) LRC structure in Col-0 ([A] and [E]), smb-3 ([B] and [F]), fez-2 ([C] and [G]), and fez-2 smb-3 ([D] and [H]). (I), (L), and (M) Electron micrographs of the root meristem of Col-0 (I) and smb-3 ([L] and [M]). (J), (K), and (N) Cross sections stained with toluidine blue, through the root meristem ([J] and [K]) or DZ (N) of Col-0 (J) and smb-3 ([K] and [N]) roots. (O) LRC cells still attached to the root in the DZ of fez-2 smb-3. Bars = 100 μm in (A) to (D), (I), (L), and (O) and 10 μm in (E) to (H). White arrows indicate the epidermal layer; black, magenta, and cyan arrows indicate LRC layers in the mature, maturing, and developing positions, respectively. Green stars indicate the position of the Epi/LRC stem cells. Red arrowheads indicate the end of the LRC layer. Green arrowheads indicate root hairs emerging through LRC. Red arrow indicates partially detached root cap cells.

Figure 4.

SMB-Like Genes Are Expressed in the Root Cap. (A) to (D) Staining for GUS activity in SMB_pro:GUS in root tips ([A] to [C]) and cotyledons (D). (E) to (H) Staining for GUS activity in BRN1_pro:GUS in root tips ([E] to [G]) and cotyledons (H). (I) to (L) Staining for GUS activity in BRN2_pro:GUS in root tips ([I] to [K]) and cotyledons (L). The length of staining is indicated in each panel. Bars = 200 μm.

Figure 5.

SMB, BRN1, and BRN2 Act Redundantly. (A) to (D) Root meristem structure in brn1-1 (A), brn2-1 (B), smb-3 brn1-1 (C), and smb-3 brn2-1 (D). (E) Expression of BRN1 and BRN2 in brn1-1 and brn2-1 mutants. (F) to (H) Root meristem structure in the brn1-1 brn2-1 double mutant, in the COL (F), stem cell area (G), and LRC (H). (I) to (L) Root meristem structure of the smb-3 brn1-1 brn2-1 triple mutant, in the COL ([I] and [J]), old root (K), and LRC (L). Asterisk indicates position of the QC, black arrows indicate position of mature LRC layers, white arrows indicate position of epidermal layers, red arrows indicate undetached LRC cells, and black arrowheads indicate cells failing to cease expansion.

Figure 6.

BRN1 and BRN2 Can Rescue smb-3 When Overexpressed. Root meristem structure, visualized by confocal laser scanning microscopy after cell wall staining with propidium iodide, in smb-3 ([A] and [B]), SMB:SMB-GR ([C] and [D]), SMB:BRN1-GR ([E] and [F]), SMB:BRN2-GR ([G] and [H]), 35S:BRN1-GR ([I] and [J]), and 35S:BRN2-GR ([K] and [L]) after treatment with or without DEX (as indicated in each panel). Asterisks indicate positions of the QC; white arrowheads indicate positions of COL stem cell–like layers.

Figure 7.

Class IIB NAC Proteins Can Generically Activate SCW Synthesis. (A) to (F) Phenotypes in 35S:SMB-GR seedlings treated with DEX, in whole seedlings (A), the root meristem (B), root DZ (C), hypocotyl (D), shoot meristem (E), and cotyledon (F) in tissue stained with 1% phloroglucinol ([C] to [F]), which indicates the presence of lignin prior to visualization. (G) and (H) Phenotypes in 35S:SMB-GR seedlings without DEX treatment in the DZ (G) and hypocotyl (H). (I) and (J) Phenotypes in Col-0 seedlings treated with DEX in the DZ (I) and hypocotyl (J). (K) to (T) Phenotypes in the DZ ([K], [M], [O], [Q], and [S]) and hypocotyls ([L], [N], [P], [R], and [T]) of 35S:NST1-GR ([K] and [L]), 35S:VND6-GR ([M] and [N]), 35S:VND7-GR ([O] and [P]), 35S:BRN1-GR ([Q] and [R]), and 35S:BRN2-GR ([S] and [T]) hypocotyls stained with 1% phologlucinol prior to visualization.

Figure 8.

SMB, BRN1, and BRN2 Can Regulate Diverse Target Genes. (A) Expression of VND/NST target genes in whole seedlings (5 d.p.g.) of 35S:SMB-GR, 35S:BRN1-GR, and 35S:BRN2-GR overexpressing lines after 24 h treatment with or without DEX and assessed by RT-PCR. All transcripts were amplified for 30 cycles. (B) Expression of potential SMB/BRN1/BRN2 targets in the root meristem (5 d.p.g.) Col-0 or smb-3 brn1-1 brn2-1 seedlings. All transcripts were amplified for 35 cycles. (C) to (F) Staining for GUS activity in CEL5_pro:GUS in root tips of Col-0 (C), smb-3 (D), and smb-3 brn1-1 brn2-1 (overstained to indicate maximum response) ([E] and [F]).