Fig 1.
PHB regulates root meristem activity downstream of SHR-SCR.
(A) Seedlings of shr phb (7 DAG) mutants display significant recovery in root length. (B) Growth restoration in shr phb roots compared with other shr HD-ZIP III mutants and shr phb phv-5 mutants. (C) Meristem size over time in the shr, shr phb, and wild-type roots. (D) Root length measured over a time course. The shr phb roots grow in a more determinate manner than do the wild-type roots. (E) Expression of pCycB1.2:GUS in the shr, shr phb, and wild-type roots (5 DAG). (F) phb-mediated recovery in root meristem/growth is specific to the SHR-SCR pathway. The error bars represent the standard error (n = 20–40 plants). Scale bars: A, 1 cm; E, 20 μm.
Fig 2.
Recovery of root meristem/growth activity in the absence of a functional QC.
(A–C) Expression analysis of QC markers in the wild-type, shr, and shr phb roots (5 DAG). (A) pWOX5:erGFP. (B) pPLT2:CFP. (C) pQC25:GUS. (D) Analysis of QC status in a shr phb root expressing pWOX5:SHR:nlsGFP, by confocal microscopy (upper panel) and Lugol staining (lower panel). Black arrowhead indicates the QC where SHR-nlsGFP is observed in the upper panel. Red arrowhead indicates the recovered columella stem cell layer where starch granules are absent. (E) The comparison of root length between shr-2 phb-6 and pWOX5:SHR:GFP;shr-2 phb-6. Root lengths were measured from seedlings 3–14 DAG. Scale bars: A–C, 25 μm; D, 20 μm. The error bars represent the standard error (n = 5–10 plants).
Fig 3.
PHB in the stele regulates root meristem and growth activity in a QC-independent manner.
(A) A comparison of root lengths in wild-type, pWOL:PHB-m:GFPNLS, pWOL:PHB-em:GFPNLS and shr-2 plants (7 DAG). The error bars represent the standard error (n = 9–14 plants). (B, C) Quantitative comparison of PHB-GFP levels in the root stele cells expressing pWOL:PHB-m:GFPNLS and pWOL:PHB-em:GFPNLS. Fluorescence intensity was measured for GFP in the boxed area of the panel (B). Error bars represent standard error (n = 11) (D) pCycB1.2:GUS expression shows drastic reduction in cell division potential in the proximal meristem. Expression of pQC25:GUS (E) and pWOX5:YFP (F) in the PHB-em roots. (G) Starch granule accumulation as visualized by Lugol’s staining in the pWOL:PHB-em:GFPNLS roots. The black arrowheads and red arrows indicate the QC and columella stem cells, respectively. Scale bars: B, 20 μm; D-G, 25 μm.
Fig 4.
Downstream genes of SHR-PHB in the root stele.
(A) Hierarchical clustering of expression in the Arabidopsis root along the longitudinal axis for genes that are repressed (blue; genes in cluster 2 and 3 in S6C Fig.) or activated (red; genes in cluster 1 in S6C Fig.) by a high level of PHB in shr mutants. Expression values are normalized by row. (B) Over-represented biological functions of genes that are repressed (P-value marked in blue) or (C) activated (P-value marked in red) by a high level of PHB in shr mutants.
Fig 5.
The SHR-PHB pathway controls root growth in a cytokinin-dependent manner (A) Relative mRNA levels of IPT3 and IPT7 in shr, shr phb, and pWOL:PHB-em:GFPNLS (PHB-em) roots.
Data are normalized to Expression levels in wild-type roots. (B) Root lengths (10 DAG) and (C) meristem images (5 DAG) in shr, shr ipt3-2 ipt7-1 mutants. The error bars represent the standard error (n = 20 plants). Scale bar: 50 μm. The white arrowhead marks the end of the meristem.
Fig 6.
The SHR-PHB pathway and cytokinin signaling.
(A) Cytokinin (CK) expression in wild-type, shr, and shr phb roots. tZ, Transzeatin; tZR, tZ Riboside; iP9G, N6-(Δ2-isopentenyl) adenine-9-glucoside; tZ9G, tZ-9-glucoside. (B) Expression of pTCS:GFP in wild-type, shr, and shr phb roots. (C) Real-time PCR and ChIP showing PHB binding to the ARR7 promoter. Line diagram represents the ARR7 promoter region. Arrows and blue bars indicate primers and B-ARR binding elements (GGATT/AATCT), respectively. (D) Recovery in pAHP6:erGFP expression in the shr phb roots in comparison with shr roots. The error bars represent the standard deviation (A) and standard error (C) (n = 3 biological replicates). Scale bars: 25 μm. CK, cytokinin.
Fig 7.
PHB suppresses B-ARR activities in the presence of high cytokinin.
A protoplast assay that measures the ARR10 activities in the presence of PHB. (A) A high dosage of PHB (p35S:PHB-em and p35S:PHB) suppressed ARR10 activities under the high cytokinin. For the reporter assay, pUBQ:rLUC was co-transfected as an internal transfection control. Relative TCS activities with effectors (fold induction) were inferred by measuring the ratios between luminescence from fire fly luciferase (pTCS:fLUC) and luminescence from renilla luciferase (pUBQ:rLUC) and then dividing those values with the ratio obtained from control without an effector and BAP treatment. The error bar represents standard deviation. (B) PHB expression from 100 nM BAP-treated protoplasts is analyzed by real-time RT-PCR. Relative PHB expression was obtained by measuring fold changes against PHB expression in the control protoplasts. The error bar represents standard deviation (n = 3). (C) Increasing ARR10 levels by expressing it under WOL promoter restores the root growth in the phb-7D. BAP, 6-benzylaminopurine.
Fig 8.
A model of SHR-PHB-cytokinin signaling in post-embryonic root growth.
SHR/SCR posttranscriptionally regulates PHB expression in the stele via the miRNA 165/6. PHB in the stele promotes cytokinin biosynthesis, thereby enhancing B-ARR activities in one hand. On the other hand, PHB interferes with B-ARR activities depending on its concentration. Such regulation generates a negative feedback loop, which likely contributes to the homeostasis of cytokinin signaling.