First published online 30 August 2006
doi: 10.1242/dev.02554
Development 133, 3755-3766 (2006)
Published by The Company of Biologists 2006
The HVE/CAND1 gene is required for the early patterning of leaf venation in Arabidopsis
María Magdalena Alonso-Peral1,*,
Héctor Candela1,*,
,
Juan Carlos del Pozo2,
Antonio Martínez-Laborda3,
María Rosa Ponce1 and
José Luis Micol1,
1 División de Genética and Instituto de Bioingeniería,
Universidad Miguel Hernández, Campus de Elche, 03202 Elche, Alicante,
Spain.
2 Instituto Nacional de Investigación y Tecnología Agraria y
Alimentaria, Departamento de Biotecnología, Carretera de la
Coruña Km. 7, 28040 Madrid, Spain.
3 División de Genética, Universidad Miguel Hernández,
Campus de San Juan, 03550 Alicante, Spain.
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Fig. 1. Map-based cloning and characterization of hve mutations.
(A) Strategy followed to identify the HVE gene. After
analyzing 3090 chromosomes, 41 recombinants (shown in parentheses) placed the
HVE gene in the interval flanked by the nga1145 and T17M13B markers.
Sequencing of segments of the candidate region in the informative recombinants
rendered five novel SNPs, which allowed us to map the HVE gene within
a 61 kb interval encompassed by the T8K22 BAC. (B-D) Structure of the
HVE gene of Arabidopsis with the position and nature of
hve mutations indicated. (B) Schematic representation of the
HVE gene. Exons and introns are depicted as black boxes and lines,
respectively. The predicted translation start (ATG) and stop (TAA) codons are
indicated. The region harboring the 16 nt deletion found in the hve-1
allele is aligned with those of the wild-type HVE alleles of Col-0
and Ws-2, the splicing donor signals of which are boxed. (C) PCR amplification
products encompassing the 14th exon and 14th intron of HVE, using as
templates genomic DNA (gDNA) and cDNA from Col-0, Ws-2 and
hve-1/hve-1 plants. The three different splicing products identified
in hve-1 cDNA are numbered. M, molecular weight marker; NC, negative
control. (D) Partial alignment of the predicted HVE proteins from Col-0 and
two of the splicing products of hve-1. Numbers correspond to amino
acid positions. Asterisks represent premature stop codons.
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Fig. 2. Some phenotypic traits of hve mutants. (A-D)
Rosettes of (A) Col-0, (B) hve-1/hve-1, (C) hve-2/hve-2 and
(D) hve-3/hve-3 plants. (E-G) Bushy inflorescences of (E)
hve-1/hve-1, (F) hve-2/hve-2 and (G) hve-3/hve-3
plants. (H-J) Effects of hve alleles on root waving. (H) Ws-2,
(I) hve-1/hve-1 and (J) hve-3/hve-3 seedlings grown on
vertically oriented 1.5% agar Petri dishes. Pictures were taken 8 (H-J), 21
(A-D) and 55 (E-G) days after sowing. Scale bars: 1 cm (A-D,H-J) and 2 cm
(E-G).
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Fig. 3. Venation pattern of assorted lateral organs in hve mutants.
Cleared cotyledons, rosette leaves, cauline leaves, sepals and petals are
shown from (A-G) Col-0, (H-N) hve-1/hve-1, (O-U)
hve-2/hve-2 and (V-AB) hve-3/hve-3 plants. Plant
material was collected 15 (cotyledons), 21 (rosette leaves), 28 (remaining
lateral organs of Col-0 plants), 37 (remaining lateral organs of
hve-2 and hve-3 homozygotes) and 64 (remaining lateral
organs of hve-1 homozygotes) days after sowing. Scale bars: 1 mm
(cotyledons and leaves); 0.5 mm (sepals and petals).
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Fig. 4. Expression pattern of the HVE gene as uncovered by the
PETA2-GUS transgene. (A) GUS staining
of Col-0 seedlings carrying (right) or not (left) the
PETA2-GUS transgene. All the remaining pictures
(B-R) correspond to transgenic plants. (B) Roots; (C) a rosette,
magnified in D; (E) cotyledon; (F) first, (G)
third and (H) seventh vegetative leaves; (I) cauline leaf;
(J) sepal; (K) petal and stamen; (L) inflorescence;
(M) several immature flowers; and (N) a single mature flower.
(O-Q) Details of the venation of a first leaf at several magnification
levels; (R) a vein before xylem differentiation (red arrow) in a
seventh leaf. Plant material was collected 10 (A,B), 21 (C-H,O-R) and 35 (I-N)
days after sowing. Scale bars: 1 mm in A-I,N; 2 mm in L; 500 µm in J,K,O;
250 µm in M; 5 µm in P; 2.5 µm in R; 1.25 µm in Q.
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Fig. 5. Expression of ATHB-8-GUS. hve-3/hve-3 (A-H)
and Col-0 (I-P) transgenic leaves. Pictures of vegetative leaves from
the first (A,I), third (B,J), seventh (C,K) and tenth (D,L) nodes were taken
21 days after sowing. Diagrams of hve-3/hve-3 (E-H) and Col-0 (M-P)
obtained from the pictures are also shown. Red lines are used to indicate
poorly stained vein segments. Leaves in I and J had to be incised to allow
flattening. Scale bars: 1 mm.
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Fig. 6. Expression of the HVE and ATHB-8 genes during first
leaf expansion. Plants carrying the (A-K)
PETA2-GUS and (L-U) ATHB-8-GUS
transgenes. Numbers indicate days after germination. Pictures were taken with
differential interference contrast microscopy. Scale bars: 5 µm in A,L,G;
1.25 µm in H; 50 µm in B-E,M-Q; 100 µm in F,I,R-T; 250 µm in
J,K,U.
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Fig. 7. Venation pattern of single and double mutants with altered vascular
development. First leaf venation pattern diagrams are shown for the
phenotypically wild-type (wt; left column), and single (second and third
columns) and double (right column) mutant siblings of several F2 populations.
Diagrams were obtained from leaves collected 21 days after sowing. Scale bars:
1 mm.
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© The Company of Biologists Ltd 2006
