Article Figures & Data
Figures
- Figure 1.
Apex Differentiation in clv cna-1 Meristems.
Scanning electron micrographs of clv3-2 cna-1 ([A] to [H]) and clv1-1 cna-1 ([I] and [J]) shoot apices. Bars = 50 μm in (A), 100 μm in (B) to (D), and 250 μm in (E) to (J). (I) and (J) are shown at the same magnification.
(A) Apex from an 8-d-old clv3-2 cna-1 plant. Arrow indicates central defect in the meristem.
(B) Apex from an 11-d-old clv3-2 cna-1 plant. Arrow indicates central defect in the meristem.
(C) Apex from a 13-d-old clv3-2 cna-1 plant. Note the lack of organ primordia around the periphery.
(D) Apex from a 13-d-old clv3-2 cna-1 plant. Note the tissue buckling across the center.
(E) Apex from a 15-d-old clv3-2 cna-1 plant. Note the lack of younger primordia around most of the periphery.
(F) Apex from a 15-d-old clv3-2 cna-1 plant. Note the large carpeloid organs formed in the center of the meristem.
(G) A portion of the apex from a 21-d-old clv3-2 cna-1 plant. Note the extensive initiation of filamentous organs with stigmatic tissues at their tips.
(H) A portion of the apex from a 27-d-old clv3-2 cna-1 plant. Note the absence of any meristem-like region.
(I) Apex from a 15-d-old clv1-1 cna-1 plant. Note the ectopic organs in the center of the meristem (arrow).
(J) Apex from a 15-d-old clv1-1 cna-1 plant. Note the ectopic flowers in the center of the meristem and the lack of primordia around the periphery.
- Figure 2.
Effects of cna Mutations on Organogenesis and Vascular Development.
(A) to (E) clv3-2 cna-1 apices shortly after the transition to flower. Note the variable expressivity in terms of the numbers of leaf and flower organs initiated. cot, cotyledon.
(F) clv3-2 apex shortly after the transition to flower, with the full complement of leaves and flower primordia apparent. Wild-type Ler apex shortly after the transition to flowering is shown in the inset.
(G) Phloroglucinol-stained cross section of clv3-2 inflorescence stem. Note the large number of peripherally located bundles. VB, vascular bundles.
(H) Phloroglucinol-stained cross section of clv3-2 cna-1 inflorescence stem. Note of the presence of ectopic bundles in the center of the stem. VB, vascular bundle.
(I) High-magnification view of a toluidine-blue stained section of an ectopic internal bundle from a clv3-2 cna-1 stem. Note the apparent inside-out orientation of the ectopic bundles. x, xylem; p, phloem.
(J) Apex of a clv3-2 mutant.
(K) Apex of a clv3-2 cna-2 double mutant.
(L) clv3-4 single mutant harboring the REV:cna-1 transgene. Note the lack of organ primordia other than a single flower (f). SM, shoot meristem.
- Figure 3.
wus and stm Are Epistatic to clv3-2 cna-1.
(A) wus-1 single mutant.
(B) wus-1 clv3-2 cna-1 triple mutant.
(C) stm-1 single mutant.
(D) stm-1 clv3-2 cna-1 triple mutant.
- Figure 4.
Map-Based Isolation of the CNA Gene.
(A) The thick line represents a portion of chromosome I, and the sequenced BAC clones in the region are represented as pentagons. Positions of molecular markers used in fine mapping during the identification of the CNA locus (star) are indicated by vertical lines below the chromosome (see Supplemental Table 1 online). The double-headed arrows show the distances between markers in the Landsberg ecotype, and the fractions report the numbers of recombinant chromosomes out of the total number screened in the mapping population. The three triangles indicate the locations of large blocks of sequences present in the Col ecotype but absent in Ler that appear to be associated with transposon insertions.
(B) Schematic diagram of the CNA gene with the positions of the cna-1 and cna-2 mutations indicated. Protein-coding exons are indicated by boxes, and introns are indicated by lines. The regions corresponding to the homeodomain, Leu zipper, and START domain are denoted by the solid, vertically hatched, and diagonally hatched boxes, respectively.
(C) Alignment of the sequences around the mutations sites in rev-3 and cna-1. The positions of mutated residues are indicated in bold.
(D) A comparison of the putative null allele rev-8 and the missense mutant rev-3, which exhibits a more severe flower meristem defect.
- Figure 5.
WUS Expression in Vegetative Meristems of clv3-2 and clv3-2 cna-1 Plants.
(A) and (B) WUS mRNA was detected in clv3-2 (A) and clv3-2 cna-1 (B) torpedo-stage embryos using dioxygenin-labeled riboprobes. WUS signal is indicated by indigo staining.
(C) and (D) WUS mRNA was detected in 3-d-old clv3-2 (C) and clv3-2 cna-1 (D) seedlings using 35S-labeled riboprobes. WUS mRNA signal is shown in yellow. Note the gap in WUS signal in the center of the clv3-2 cna-1 apex.
(E) to (I) Detection of PWUS:GUS expression in clv3-2 ([E] and [G]) and clv3-2 cna-1 ([F], [H], and [I]) seedlings on day 3 (F), day 6 ([E] and [H]), and day 9 ([G] and [I]). The blue color indicates tissues with GUS activity. The insets in (F) and (G) show higher magnification views of the meristems. Note the loss of PWUS:GUS signal from the day 9 clv3-2 sample and the loss of central PWUS:GUS signal from the clv3-2 cna-1 plants as early as day 3.
- Figure 6.
WUS Expression in Inflorescences of clv3-2 and clv3-2 cna-1 Plants.
Detection of PWUS:GUS expression ([A], [C], [E], [G], [H], and [L]). Photographs were taken from above the apices, showing GUS activity in blue. Detection of WUS mRNA using 35S-labeled probes in longitudinal sections ([B], [D], [F], [I], [J], and [K]). WUS mRNA signal is shown in yellow. SM, shoot meristem; FM, floral meristem.
(A) The apex of an 11-d-old clv3-2 plant. The meristem is outlined in white. Note the lack of GUS signal in most of the shoot apical meristem.
(B) The apex of a 14-d-old clv3-2 plant. Note the robust expression within flower meristems, whereas the shoot apical meristem lacks robust signal.
(C) The apex of a 15-d-old clv3-2 plant. The meristem is outlined in white.
(D) The apex of a 17-d-old clv3-2 plant.
(E) The apex of an 11-d-old clv3-2 cna-1 plant. Note the robust GUS signal around the periphery of the meristem and within the flower meristems both outside and inside the ring. The signal at the bottom of the ring is obscured by a leaf.
(F) The apex of a 14-d-old clv3-2 cna-1 plant. Note the robust expression within flower meristems, whereas the shoot apical meristem lacks robust signal.
(G) The apex of a 14-d-old clv3-2 cna-1 plant. Note the ring of GUS signal around the periphery.
(H) The apex of a 20-d-old clv3-2 cna-1 plant. Note the increase in GUS signal across the center of the apex associated with differentiation.
(I) The apex of a 22-d-old clv3-2 cna-1 plant. Note the pockets of WUS signal.
(J) Developing anther of a 22-d-old clv3-2 plant.
(K) Older anther of a 22-d-old clv3-2 plant.
(L) The apex of a wild-type Ler plant shortly after the transition to flowering.
- Figure 7.
CLV3 Expression in clv3-2 and clv3-2 cna-1 Apices.
Detection of PCLV3:GUS expression ([A] to [I]). Photographs were taken from above the apices, showing GUS activity in blue. Detection of CLV3 mRNA using 35S-labeled riboprobes in longitudinal sections ([J] to [L]). CLV3 signal is shown in yellow. Insets in (A) and (B) show higher-magnification views of the shoot apical meristem. SM, shoot apical meristem. FM, flower meristem.
(A) The apex of a 9-d-old clv3-2 plant.
(B) The apex of a 13-d-old clv3-2 plant. Note that only weak peripheral GUS signal remains.
(C) The apex of a 16-d-old clv3-2 plant.
(D) The apex of a 22-d-old clv3-2 plant. Note that the signal is restricted to flower meristems.
(E) The apex of a 6-d-old clv3-2 cna-1 plant.
(F) The apex of a 13-d-old clv3-2 cna-1 plant.
(G) The apex of a 16-d-old clv3-2 cna-1 plant. Note the relative paucity of flower meristems around the periphery.
(H) The apex of a 22-d-old clv3-2 cna-1 plant. Note the sustained GUS signal in the apex despite the onset of differentiation.
(I) The apex of a wild-type Ler plant shortly after the transition to flowering.
(J) The apex of an 18-d-old clv1-4 plant. Note the patch of the shoot apical meristem lacking signal (arrow).
(K) The apex of a 22-d-old clv1-4 plant.
(L) The apex of a 21-d-old clv1-4 cna-1 plant. Note the robust CLV3 signal in the filamentous structures (arrows).
- Figure 8.
Phenotypes of cna-1 phb phv Plants.
(A) A 13-d-old phb phv plant was identical in phenotype to wild-type plants.
(B) A 13-d-old cna-1 phb plant that underwent only a short developmental arrest.
(C) A 13-d-old cna-1/+ phb plant with a strong defect in organogenesis.
(D) A 13-d-old cna-1 phb phv triple mutant that arrested after the production of two leaves, but then recovered.
(E) A 19-d-old cna-1 phb phv seedling with a mass of undifferentiated cells and radially symmetric organs coming from the midst of the meristem.
(F) A cna-1 phb phv seedling at day 34. Note the entire apex has differentiated into flowers, but the inflorescence stem did not elongate.
(G) The plant in (E) at day 54, with axillary meristems that emerged and elongated relatively normally.
(H) A secondary inflorescence from a 65-d-old cna-1 phb phv plant exhibiting a Cna− phenotype.
(I) A 44-d-old cna-2 phb phv plant. Note the much weaker phenotypes compared with the cna-1 triple mutant.
(J) Phloroglucinol-stained hand sections of cna-1 phb phv and cna-1 phb stems. The unelongated primary inflorescence stems of 54-d-old triple mutants had an unorganized pattern of vascular and lignified tissue (stained pink). The cna-1 phb double mutants had relatively normal patterns of lignified cells around the stem periphery, but ectopic vascular bundles appeared in the center of the stem.
Tables
Genotype Day 12 Day 24 Ler 55.6 ± 0.99 (n = 5) 57.3 ± 1.48 (n = 10) cna-1 72.7 ± 1.72 (n = 10) 61.1 ± 1.67 (n = 15) The size of the shoot apical meristem (in micrometers) of cna-1 and Ler plants at 12 and 24 d old was determined. The mean ± standard error is presented. The difference between means is statistically significant on day 12 (t test, P < 0.0001) but is less statistically significant on day 24 (t test, P = 0.1).
Genotype Shoot Meristem Size (μm)a Age (d)b nc clv2-1 58 ± 2.5d 13 12 clv2-1 cna-1/cna-1 109 ± 16.6 13 12 clv3-2 163 ± 11.1e 13 18 clv3-2 cna-1/+ 196 ± 9.1 13 18 clv3-2 cna-1/cna-1 276 ± 9.3 13 32 clv3-2 cna-2/cna-2 223 ± 12.1 13 16 clv1-7 71 ± 2.8f 15 8 clv1-7 cna-1/cna-1 95 ± 2.1 15 8 ↵a Values represent the mean shoot apical meristem diameter ± standard error (see Methods).
↵b Age indicates the days after germination plants were collected.
↵c n indicates the number of measurements used to determine size.
↵d The mean meristem diameter for clv2-1 is significantly different from that of clv2-1 cna-1 plants (t test, P < 0.01).
↵e The mean meristem diameter for clv3-2 is significantly different from that of clv3-2 cna-1/CNA plants (P = 0.03), clv3-2 cna-1 plants (t test, P < 0.01), and clv3-2 cna-2 plants (P < 0.01).
↵f The mean meristem diameter for clv1-7 is significantly different from that of clv1-7 cna-1 plants (t test, P < 0.01).
Genotype Shoot Meristem Size (μm)a Ring Meristemb Loss of Organogenesisc nd clv3-2 209 ± 21 0% 0% 20 clv3-2 cna-2/+ 228 ± 17 0% 0% 22 clv3-2 cna-1/+ 266 ± 23 22% 0% 18 clv3-2 cna-2/cna-2 260 ± 17 17% 0% 24 clv3-2 cna-1/cna-2 441 ± 20 100% 0% 18 clv3-2 cna-1/cna-1 478 ± 19 91% 36% 22 ↵a Values represent the mean shoot apical meristem diameter ± standard error of plants collected simultaneously at the onset of bolting (see Methods).
↵b The percentage of plants showing a distinct ring shape to the meristem.
↵c The percentage of plants showing a severe loss of organogenesis.
↵d n indicates the number of measurements used to determine size.
Genotype Carpels per Flowera Pb clv3-2 5.66 ± 0.11 (n = 59) – clv3-2 cna-1 5.34 ± 0.12 (n = 67) 0.04 clv3-2 cna-2 5.79 ± 0.13 (n = 62) 0.44 ↵a Values represent the mean number of carpels per flower ± standard error. The total number of flowers scored for each genotype is indicated in parentheses (n). Only the first 10 flowers of any given plant were included in the analysis.
↵b The probability associated with differences in the mean carpel numbers for clv3-2 and clv3-2 cna lines was assessed using the t test.
Supplemental Data
Files in this Data Supplement:
- Supplemental Data 1
- Supplemental Table 1
- Supplemental Table 2
- Supplemental Table 3
- Supplemental Figure 1 - Supplemental Figure 1. Meristems of 12 Day-Old cna-1 Mutants Are Larger than those of Wild-Type Plants. Inflorescence shoot apical meristems were collected from multiple wild-type Ler and cna-1 plants. Scanning electron micrographs of the smallest and largest meristem of each genotype are shown. See Table 1 for means and sample sizes. Bar = 50 μm. All images shown at same magnification. (A) Smallest wild-type Ler meristem (B) Largest wild-type Ler meristem (C) Smallestcna-1 meristem (D) Largest cna-1 meristem
- Supplemental Figure 2 - Supplemental Figure 2. The cna-1 Mutation Enhances Weak, Intermediate, and Strong Clv? Mutant Phenotypes. Scanning electron micrographs of 15 day-old inflorescence apices of clvsingle and clv cna-1 double mutant apices. (A) and (B), (C) and (D), and (E) and (F) are shown at the same magnification, respectively. Bars = 100μm in (A) and (D), and 200 μm in (F). (A) clv1-7 meristem (B) clv1-7 cna-1 meristem. (C) clv2-1 meristem (D) clv2-1 cna-1 meristem (E) clv1-4meristem (F) clv1-4 cna-1 meristem
- Supplemental Figure 3 - Supplemental Figure 3. Transcript Accumulation in Mutant Lines. For each allele used in this study, RT-PCR was used to detect the presence of transcripts. The locations of the mutations and primers used for RT-PCR are indicated for each gene. Arrows in T-DNA insertion rectangles indicate the likely locations of P35S promoter sequences. With the exception of clv3-2, clv3-2 cna-1, and clv3-2 cna-2, RNA was extracted from the aerial portions of two-week-old seedlings and reverse transcribed using a poly-dT primer. Inflorescence apices were used for RNA extraction from clv3-2, clv3-2 cna-1, and clv3-2 cna-2plants. After amplification, samples were electrophoresed on agarose gels and photographed. Primers for the β-ATPase gene were assayed for each RNA sample to control for quality and quantity of cDNA (Prigge and Wagner, 2001).
- Supplemental Figure 4 - Supplemental Figure 4. CNA is Expressed in Developing Vascular Elements, Flowers, and Ovules Sections of wild-type Ler ([A] to [E]), clv3-2 cna-1 (F) and clv3-2 (G) tissues were hybridized with anti-sense ([A] to [F]) and sense (G) dioxygenin-labeled CNA riboprobe. (A) Stage 4 to stage 7 flowers showing CNA signal diffusely throughout flower meristem and young organs. (B) Stage 9 and older flowers showing CNA signal strongest within developing stamens and carpels. (C) Ovules at early stages of initiation showing CNA signal. (D) Older ovules continue to express CAN. (E) CNA is most strongly expressed in developing vascular tissue. (F) cna-1 continues to be expressed in apices of clv3-2 cna-1 plants undergoing differentiation. (G) Sense controls show no signal.
