Article Figures & Data
Figures
- Figure 1.
Comparison of WOX4pro:YFP and DR5revpro:GFP Activities in the Arabidopsis Inflorescence Stem.
(A) and (B) WOX4pro:YFP activity (arrows) in the hypocotyl of 30-cm-tall plants (A) and in cotyledons of 21-d-old seedlings (B).
(C) and (D) Schematic representations of tissue patterns in primary ([C]; before onset of secondary growth) and secondary ([D]; after onset of secondary growth) stems. The IC is indicated by arrows.
(E) to (J) Analysis of reporter gene activity 10 mm above the uppermost rosette leaf ([E] to [G]) and at the stem base ([H] to [J]) of 30-cm-tall plants. Tissues are marked in (E) and (H) according to the color coding used in (C) and (D). (F), (G), (I), and (J) show overlays of the YFP- and GFP-specific channels with the respective bright-field image. Details shown in (G) and (J) are marked in (F) and (I), respectively. The yellow bracket in (H) indicates the extension of the IC-derived tissue. WOX4pro:YFP signal in red, DR5revpro:GFP signal in green (green arrows), and overlapping signal in yellow (white arrows).
Bars = 50 μm in (A), 500 μm in (B), 100 μm in (C) and (D), and 25 μm (E) to (J). The position of primary vascular bundles is indicated by asterisks. Note that autofluorescence of secondary cell walls generates background signals (cf. Supplemental Figure 3 online). The combination of the YFP- and GFP-specific channels shown in (G) and (J) is also depicted in magenta and green, respectively, in Supplemental Figure 2 online.
- Figure 2.
Analysis of WOX4pro:YFP and DR5revpro:GFP Activities 5 mm above the Uppermost Rosette Leaf at Different Growth Stages.
(A) to (C) A 5-cm-tall plant.
(D) to (F) A 15-cm-tall plant.
(G) to (I) A 30-cm-tall plant.
(G), (F), and (I) show details marked in (B), (H), and (E), respectively. In the gray-channel images in (A), (D), and (G), tissues are marked according to the color coding used in Figures 1C and 1D. WOX4pro:YFP signal in red and DR5revpro:GFP signal in green (green arrows). Bars = 25 μm. The combination of the YFP- and GFP-specific channels shown in (C), (F), and (I) is also depicted in magenta and green, respectively, in Supplemental Figure 4 online.
- Figure 3.
WOX4 Is an Essential Factor for Cambium Activity in the Inflorescence Stem.
(A) to (C) Histological analysis of wild-type (A), wox4-1 (B), and WOX4pro:WOX4/wox4-1 plants (C) at the stem base. Brackets indicate the lateral extensions of the IC-derived (red) or the FC-derived (yellow) tissue. The red arrow in (B) indicates the expected position of the IC.
(D) Quantitative analysis of cambium activity in wild-type, wox4-1, and WOX4pro:WOX4/wox4-1 plants. The extensions of the FC- and the IC-derived tissue were measured. Significance levels are calculated for the differences between the wild type and wox4-1 and between the wild type and WOX4pro:WOX4/wox4-1 plants. n.s., not significant; double asterisks indicate significance levels of P < 0.01.
(E) to (L) Results of RNA in situ hybridization experiments using histone H4 ([E] and [F]), WOX4 ([G] and [H]), At5g57130 ([I] and [J]), and PXY ([K] and [L]) specific antisense probes in the wild type (WT) ([E], [G], [I], and [K]) and wox4-1 ([F], [H], [J], and [L]). Experiments were performed in 5-cm (H4 and WOX4 probes) and 15-cm (At5g57130 and PXY probes) tall plants. Arrows indicate sites of mRNA accumulation, and asterisks label the position of primary vascular bundles. Bars = 100 μm.
- Figure 4.
Comparison of the Effects of NPA and NAA Applied Locally to a Narrow Region of the Bottommost Elongated Internode of Wild-Type Plants.
(A) to (C) Toluidine-stained sections collected from the site of treatment showing the induction of periclinal cell divisions in interfascicular regions by NPA (B) and NAA (C) treatments.
(D) to (F) Activity of the DR5revpro:GFP reporter at the site of treatment.
(G) to (I) WOX4pro:GFP reporter activity at the treatment site. The position of primary vascular bundles is indicated by asterisks. Arrows indicate sites of reporter gene activity. Extensions of the newly produced tissue are indicated by brackets. Bars = 50 μm.
- Figure 5.
Short-Term Effect of Local NPA Treatments on DR5revpro:GFP and WOX4pro:GFP Activities.
(A) and (B) DR5revpro:GFP activity in mock- (A) and NPA-treated (B) samples 1 d after treatment.
(C) and (D) WOX4pro:GFP activity in mock- (C) and NPA-treated (D) samples. The position of primary vascular bundles is indicated by asterisks. Arrows indicate sites of reporter gene activity. Bars = 50 μm.
(E) qRT-PCR demonstrating that WOX4 mRNA abundance is enhanced in stems after 1 d of NPA treatment, similar to PIN1. Two biological replicates with three technical replicates each were included.
- Figure 6.
WOX4 Is Essential for Auxin-Dependent Cambium Stimulation.
(A) to (D) In contrast with wild-type plants ([A] and [B], bracket), wox4-1 mutants ([C] and [D]) treated with NPA do not show enhanced FC activity (D) and no interfascicular cell divisions are induced (arrow in [D]).
(E) Quantification of the lateral extension of the FC-derived tissues did not reveal an effect of NPA treatment on FC activity in wox4-1. The asterisk indicates a significance level of P < 0.05. n.s., not significant; WT, wild type.
(F) and (G) In contrast with mock-treated stems (F), DR5revpro:GFP/wox4-1 stems treated for 1 d with NPA (G) display DR5revpro:GFP activity in cortex cells similarly to lines with a functional WOX4 (arrows in [G]; see Figure 5B for comparison).
(H) and (I) Similarly to plants with a functional WOX4 gene (arrows in [H]), DR5revpro:GFP activity is observed at the stem base in the interfascicular regions of wox4-1 mutants (arrows in [I]). Asterisks mark the position of primary vascular bundles. Bars = 50 μm.
- Figure 7.
WOX4 Promoter Activity and WOX4-Dependent Cambium Activation Depend on the Tissue Context.
(A) and (B) In comparison to mock treatment (A), NAA treatment induces DR5pro:GUS reporter gene activity (B) 1 d after treatment.
(C) and (D) No ectopic WOX4pro:GUS reporter gene activity is observed upon NAA treatment.
(E) and (F) NPA treatment of 35Spro:WOX4 stems (F) leads to wild-type-like IC initiation (cf. with Figures 4B and 6B). The IC-derived tissue is indicated by the bracket in (F). Bars = 50 μm.
(G) RT-PCR comparing WOX4 transcript abundance in the wild type (WT), wox4-1, and 35Spro:WOX4, at the stem base (b), in rosette leaves (l), and in flowers (f). Genomic DNA (g) and water (c) were used as control samples. Two technical replicates gave identical results.
- Figure 8.
PXY Is Necessary for Auxin-Dependent Cambium Activation.
(A) to (D) In contrast with wild-type (WT) plants ([A] and [B]), pxy-4 mutants ([C] and [D]) did not respond to NPA treatment by an increase of cambium activity.
(E) WOX4 mRNA abundance was not elevated in pxy mutant backgrounds after 7 d of NPA treatment, although WOX4 activation took place 1 d after treatment. Bars = 50 μm.
[See online article for color version of this figure.]
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