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First published online December 1, 2003
doi: 10.1242/10.1242/dev.00909

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VARICOSE, a WD-domain protein, is required for leaf blade development

¹ Department of Biology, University of Utah, Salt Lake City, Utah, 84112, USA
² Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada T6G 2E9

View larger version (106K): [in a new window]   Fig. 1. vcs mutants show temperature-dependent defects in leaf development. (A-C) Plants grown at 22°C; 15-day wild type (A) has broad leaves, whereas the 15-day vcs mutant (B) produces narrow curled leaves. Following prolonged growth (C; 25-day shown), vcs develops flowers, but no seeds are produced. (D-F) Plants grown at 16°C; 16-day wild type (D) produces broad leaves, and the 16-day vcs produces broad, but pointed leaves (E). At this temperature, vcs can be grown on soil, produce flowers and set seed (F), but the mutant plant has short inflorescence stems and reduced apical dominance (wild type on the left and vcs on the right). (G-I) Plants grown at 29°C; 11-day wild type produces broad leaves (G), whereas the 11-day vcs mutant appears chlorotic and produces small epinastic leaves (H). Prolonged growth at 29°C in vcs results in a few additional leaf-like organs, and an enlarged callus-like apex (I). (J-P) Roots of wild type and vcs mutants: (J,K) 16°C-grown 7-day wild type and vcs, respectively; (L,M) 22°C-grown 4-day wild type and vcs, respectively; (N-P) 29°C-grown 4-day wild type (N) and vcs (O,P). A high magnification image of the vcs root (P) shows the swollen root hairs. Scale bars: (A-O) 1 mm; (P) 100 µm.

View larger version (145K): [in a new window]   Fig. 2. vcs mutants have reduced SAM size. (A-F) SEM images of the SAM (all shown at the same magnification) of Ler (A-C) and vcs (D-F) from seedlings grown at 16°C for 15 days (A,D), 22°C for 12 days (B,E) and 29°C for 9 days (C,F). (G,H) Toluidine Blue-stained paraffin section of 14-day 29°C-grown Ler (G) and vcs-1 (H). (I,J) CSLM images of meristems from 7-day 29°C-grown Ler (I) and vcs (J). m indicates meristem. Scale bars: (A-F, I,J) 50 µm; (G,H) 100 µm.

View larger version (102K): [in a new window]   Fig. 3. Leaf defects in internal tissues of vcs mutants. (A-F) Cross sections of the first leaf from 12-day Ler (A-C) and vcs (D-F), grown at (A,D) 29°C; (B,E) 22°C; (C,F) 16°C. (G-L) Dark-field images of cleared first leaf showing the patterns of veins in Ler (G,I,K) and vcs (H,J,L) grown for (G,H) 12 days at 22°C; (I,J) 11 days at 29°C; (K,L) 15 days at 16°C. (M,N) DIC images of the primary vein from leaves shown in G and H. (O,P) Cotyledon vein pattern of 12-day 22°C-grown Ler (O) and vcs (P) show similar vein patterns, although cotyledon shape differs slightly. (Q,R) DIC images of the primary vein of cotyledons of Ler (Q) and vcs (R), showing ectopic tracheary elements surrounding the vcs primary vein. Scale bars: (A-F) 50 µm; (G-L,O,P) 1 mm; (M,N,Q,R) 100 µm.

View larger version (71K): [in a new window]   Fig. 4. SEM analysis of vcs leaf defects. (A) Ler 9-day 29°C-grown; (B-D) vcs 9-day 29°C-grown, showing adaxial (B), side (C) and abaxial (D) views. (E-G) Trichome from 9-day 29°C-grown Ler (E) and vcs (F-G) leaves. (H-M) Leaf adaxial cells; the images were taken at a position half way down the leaf and midway between the margin and the center of the leaf. Typical arrangements of stomatal guard cells and pavement cells are present in Ler grown at 16°C for 15 days (H), 22°C for 12 days (I) and 29°C for 9 days (J), and in vcs grown at 16°C for 15 days (K) and 22°C for 12 days (L), however in vcs grown for 9 days at 29°C (M) no typical pavement cells could be detected. The abaxial leaf surface is typically uneven, and composed of stomatal guard cells and small irregularly shaped cells, such as shown for Ler grown at 16°C for 15 days (N), 22°C for 12 days (O) and 29°C for 9 days (P). The abaxial leaf surface of vcs grown at 16°C for 15 days (Q) and 22°C for 12 days (R) appeared similar to the wild type. The abaxial surface of the 9-day 29°C-grown leaf (S) contained stomatal complexes, but no small irregularly shaped cells were present. Scale bars: (A) 1 mm; (B,C,D) 500 µm; (E) 200 µm, (F, G) 100 µm; (H-M) 40 µm, (N-S) 50 µm.

View larger version (100K): [in a new window]   Fig. 5. Temperature shift experiments reveal a requirement for VCS throughout leaf development. (A) Seedling morphology of 14-day temperature-shifted vcs mutants. The top row shows plants germinated at 29°C and transferred to 16°C on the day indicated under each plant. The lower row shows plants germinated at 16°C, and transferred to 29°C. (B) Representative vein patterns from selected temperature shift time points. The red bars indicate the duration of time at 29°C, and whether this exposure was at the beginning or the end of the 14-day growth period. Scale bars: (A) 5 mm; (B) 200 µm.

View larger version (21K): [in a new window]   Fig. 6. Vascular development in leaves of temperature shifted and control plants. (A) Counts of completed leaf areoles (a region fully delimited by veins) in the first leaves of 14-day temperature-shifted plants. Bars indicate the standard error of the mean, 18-26 leaves were examined for each time-point. (B) Depiction of the developmental progression of primary and secondary veins in leaves of Ler plants from a developmental time course carried out at 16°C or 29°C. For each time-point, the height of the white, stippled or black area represent the fraction of leaves with 1° or 2° veins totally absent (white), present only as procambium (stippled), or at least some of that vein class differentiated (black).

View larger version (80K): [in a new window]   Fig. 7. Cotyledon and leaf vein patterns in axr1-3, vcs, and vcs axr1-3 double mutants. All tissue is from 15-day 16°C-grown plants. Cotyledon (A,C,E,G) and leaf (B,D,F,H) vein patterns from Ler (A,B), axr1-3 (C,D), vcs-1 (E,F), and axr1-3 vcs-1 double mutants (G,H). Scale bars: 1 mm.

View larger version (137K): [in a new window]   Fig. 8. DR5 expression and auxin inducibility in vcs leaves. DR5 expression in developing leaves of wild type (A-C) grown at 22°C (wild-type DR5 expression was similar regardless of growth temperature). (D-F) vcs mutants show DR5 expression at the distal end of the developing leaf (D, 29°C-grown) and in procambium (E,F, 22°C-grown). DR5 auxin induction was compared for 8-hour incubation in water (G,I) or 5 µM 2,4-D (H,J) in the wild type (G,H) and vcs (I-J). Data shown is for 29°C-grown tissue; similar results were observed for plants grown at 22°C and 16°C. Scale bars: (A-F) 100 µm; (G-J) 1 mm.

View larger version (68K): [in a new window]   Fig. 9. vcs mutants show heightened sensitivity to polar auxin transport inhibitor NPA regardless of growth temperature. (A-H) 29°C-grown 9-day plants, (I-P) 22°C-grown 12-day plants, and (Q-X) 16°C-grown 15-day plants. (A,I,Q) Ler controls and (B,J,R) Ler grown in medium containing 1 µM NPA. (C,K,S) vcs controls and (D,L,T) vcs grown in 1 µm NPA. Dark-field images of cleared first leaves of Ler and vcs grown for 9 days at 29°C (E-H), 12 days at 22°C (M-P) and 15 days at 16°C (U-X). Ler controls (E,M,U), Ler 1 µM NPA (F,N,V), vcs controls (G,O,W) and 1 µM NPA (H,P,X). Scale bars: 1 mm.

View larger version (14K): [in a new window]   Fig. 10. Molecular identification of VARICOSE. (A) Mapping strategy within a 1.6 MB region of chromosome 3. The approximate positions of the polymorphisms used in this study for high resolution mapping of recombination breakpoints are indicated at the top. Using 750 vcs F2 mapping cross plants, we identified six recombinants at the polymorphism indicated as VCSA, and 48 at the VCSI polymorphism. The DNA from the plants containing recombinant chromosomes were further analyzed at polymorphisms VCSB, MDC11, MRP15, and MAG2. The number of chromosomes that were still heterozygous at each position is indicated. (B) Depiction of the VCS gene; black boxes represent exons, the arrow on the bar indicates transcription direction for orientation of the 5' end of this gene. The position and nature of mutations in each vcs allele is indicated.

View larger version (80K): [in a new window]   Fig. 11. Alignment of the Arabidopsis VCS and VCR amino acid sequences. Grey shading indicate amino acid identity. The N-terminal proline-rich domain is indicated by an asterisk above each proline of the VCS sequence. The two robust WD domains are indicated by boxes with solid lines, and the third less well conserved WD domain is indicated by a box with a dashed line.

View larger version (93K): [in a new window]   Fig. 12. VCS expression patterns. (A) RT-PCR analysis of VCS and -tubulin (control) expression in RNA isolated from roots, hypocotyls, cotyledon, apices (leaves plus meristem), mixed age siliques, and entire seedlings (Whole). Genomic controls show the amplified fragment size that includes intron sequences. (B-H) GUS staining from a pVCS::GUS transgene. (B) 6-day cotyledon; (C) first leaf from a 4-day plant; (D) young leaves from a 9-day plant; (E) first leaf from a 9-day plant. (F) leaf trichome; (G) leaf vein; (H) stomata in the leaf. Scale bars: (B-H) 100 µm.