Article Figures & Data
Figures
- Figure 1.
CLV3 Protein Is Localized to the Apoplast.
(A) to (C) Phase-contrast optics used to detect GUS.
(A) Cell transiently expressing the GFP/GUS protein alone, which is confined to the nucleus (arrow).
(B) Cells transiently expressing the CLV3Δsp-GFP/GUS fusion protein, which is present throughout the cytoplasm.
(C) Cells transiently expressing the CLV3-GFP/GUS fusion protein, which is detected only between cells (arrows).
(D) Inflorescence apex of a clv3-2 mutant plant showing massive meristem enlargement (arrow) and production of supernumerary flowers and floral organs.
(E) Inflorescence apex of a clv3-2 mutant plant stably expressing the 35S::CLV3-G fusion protein. This transgenic T2 plant resembles the wild type, indicating that CLV3 function has been restored by the transgene.
(F) Seedling with a prematurely terminated meristem from a 35S::CLV3-G clv3-2 line. The shoot apical meristem has formed a terminal leaf.
(G) GFP fluorescence in a root from a 35S::CLV3-G wild-type T2 transgenic plant.
(H) GFP fluorescence in a root from a 35S::CLV3Δsp-G wild-type T2 transgenic plant.
(I) Autofluorescence in the cell wall of an untransformed wild-type hypocotyl.
(J) GFP fluorescence in the hypocotyl of a 35S::CLV3-G wild-type T2 transgenic plant.
Bars = 20 μm in (G) and (H) and 50 μm in (I) and (J).
- Figure 2.
Targeting to the Vacuole Blocks the Activity of CLV3.
(A) Scheme of the fusion constructs. The C-terminal vacuolar sorting signal from barley lectin (ctVSSbl) was fused at the C terminus of CLV3 (Vac1) or CLV3 fused to a T7 peptide tag (Vac2). The ctVSS from tobacco chitinase A (ctVSSchi) was fused at the C terminus of CLV3 (Vac3). The ctVSSbl with two additional Gly residues (GG), which no longer functions as a vacuole-sorting signal, was attached either at the C terminus of CLV3 (Sec1) or CLV3-T7 (Sec2). Sec3 is obtained from Vac2 by deleting a single nucleotide that inserts a stop codon after the final amino acid of CLV3. All constructs are driven by the 35S promoter of Cauliflower mosaic virus.
(B) clv3-2 plants transformed with the constructs described in (A). Primary transformants were grouped into six phenotypic classes according to the severity of meristem termination. Class 1 plants showed no transgene activity and resembled clv3-2 mutants. Class 2 plants showed weak transgene activity and displayed the phenotype of weak clv3 alleles. (The inset shows the mass of cells accumulating in the SAM of these plants 10 weeks after transplanting.) Class 3 plants showed intermediate transgene activity that complemented the clv3 phenotype. These plants looked essentially like untransformed wild-type plants, although the SAMs terminated prematurely in some cases (inset). Most class 4 plants showed SAM termination before the production of floral organs, although some flowers formed and set seed. Class 5 and class 6 plants showed strong transgene activity, and all of the SAMs were terminated before producing flower organs (class 5) or inflorescence meristems (class 6). The graph at bottom shows for each construct the percentage of transformed plants that belong to each phenotypic class.
(C) Wild-type Columbia plants transformed with the constructs described in (A). Primary transformants were grouped into classes 3, 4, 5, and 6 as described in (B). The graph at bottom shows for each construct the percentage of transformed plants that belong to each phenotypic class.
- Figure 3.
The Extent of Meristem Termination Is Related Directly to Transgene Expression Level.
(A) Progeny from two independent primary transformants are shown. Eight weeks after kanamycin-resistant T2 plants were transplanted to soil, segregation of class 3 and class 5 phenotypes was observed. The T2 plants were genotyped by scoring the segregation of kanamycin resistance in their progeny (class 5 plants eventually set some seed). Plants with class 3 phenotypes were heterozygous for the transgene, whereas plants with class 5 phenotypes were homozygous.
(B) RNA gel blot analysis of transgene expression. Five micrograms of total RNA from rosette leaves of plants transformed with the CLV3 fusion constructs was hybridized with probes corresponding to CLV3 and eIF4A. Two different exposures of the hybridization with the CLV3 probe are shown (20 min and 2 hr). The mRNA abundance of the eIF4A transcript was used as a loading control. The top panel shows primary transformants in a clv3-2 background; the bottom panel shows primary transformants in the wild-type Columbia background. The phenotypic class of the plant is indicated at top, and the genotype is indicated at bottom. All constructs except for Vac3 contain the 3′ untranslated region of barley lectin and two transcription termination sites, which explains the larger transcript sizes and doublet bands in these lanes compared with the Vac3 lanes.
- Figure 4.
CLV3 Is Localized in the Extracellular Space.
(A) and (B) Localization of a Sec2 fusion protein was analyzed in roots of a transgenic line that showed complementation of the clv3-2 mutation.
(C) Section from an untransformed clv3-2 mutant plant showing no labeling.
(D) Section from the same plant as in (A) and (B) incubated with no primary antibody showing no labeling.
(E) Vac2 transformant with no transgene activity showing no immunolabeling in the cell wall.
(F) Vac2 transformants with complementation of the clv3-2 mutation showing immunolabeling in the cell wall.
(A) to (D) show streptavidin conjugated to 15-nm gold particles; (E) and (F) show streptavidin conjugated to 10-nm gold particles. Bars = 200 nm.
- Figure 5.
Model of CLV3 Localization and Action.
The Arabidopsis SAM consists of three clonally distinct cell layers. The L1 epidermal layer and the L2 subepidermal layer are each single cell layers (outlined in green), whereas the interior L3 cells make up the bulk of the meristem (cells outlined in red). CLV3 is expressed at the apex of the SAM, predominantly in the superficial L1 and L2 cell layers and in a few cells of the L3. CLV1, which encodes a receptor for CLV3, is expressed in the underlying L3 cell layers. CLV3 is localized to the extracellular space and is predicted to travel through the apoplast from the L1 and L2 to the CLV1-expressing cells in the L3, which are isolated symplastically from the tunica (L1 and L2). Signaling by CLV3 through the CLV1 receptor complex causes the downregulation of WUS, restricting the WUS expression domain to the interior cells of the meristem.
