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First published online 30 June 2004
doi: 10.1242/dev.01221

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GRAMINIFOLIA promotes growth and polarity of Antirrhinum leaves

John F. Golz^1,*, Mario Roccaro^1,, Robert Kuzoff^2, and Andrew Hudson^1,

¹ Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, UK
² Section of Plant Biology, University of California Davis, 1 Shields Avenue, Davis, CA 95616, USA

View larger version (81K): [in a new window]   Fig. 1. gram mutations affect leaf growth and adabaxial asymmetry. (A) Comparisons of the length and width of fully expanded leaves of wild-type (filled bars) and gram-1 (open bars) at nodes 1 to 6. Values represent means of eight replicates and error bars, one standard deviation. (B) The adaxial wild-type leaf (top left) appears darker than the abaxial (top right). In gram mutant leaves, strips of darker tissue extend abaxially (arrowheads). (C) The wild-type adaxial leaf surface (left) consists of large, irregular pavement cells and rectangular cells towards the leaf edge (left in this image). Abaxial pavement cells (centre) are smaller and interspersed with frequent stomata (s). The edge of the leaf (right) consists of elongated edge cells (e). (D) In gram mutants, adaxial pavement cells are unaltered whereas cells at the margins are more irregular (left), edge cells are found abaxially (centre) and larger abaxial cells are seen, and stomata and hairs (h) are found at the leaf edge. (E) A transverse section of a wild-type leaf shows that, the junction between adaxial palisade mesophyll (pm) and abaxial spongy mesophyll (sm) cells runs to the edge of the leaf (arrowhead). In the gram mutant leaf (F), palisade mesophyll cells (pm) are found abaxially at the margins and adaxial cells away from the margin resemble spongy mesophyll. (G,H) gram mutants occasionally produce needle-like leaves (arrowhead in G), that appear radial in transverse section (H). (I) The arrangement of xylem elements internal to phloem in central vascular bundles of these leaves suggests a loss of adaxial leaf identity.

View larger version (55K): [in a new window]   Fig. 2. gram mutations affect growth and asymmetry of petals. (A-D) A wild-type flower. (A) A lateral (left) and dorsal (right) view. (B) Two rows of dense yellow hairs (*), are present on the adaxial surface at the junction between the ventral and lateral petals within the corolla tube. (C) In a section, taken at the position of the bar in B, the junction between petals is flanked by veins in which xylem elements (x) are adaxial to phloem (p; D). (E-H) gram mutant flower. (E) Mutant flowers are smaller than wild-type ones and the dorsal petals are free laterally and more symmetrical in shape. (F) The junction between ventral and lateral petals (arrowhead in F) is flanked by ridges containing enlarged abaxialised veins (G, and enlarged in H) that have central xylem elements (x) surrounded by phloem (p). Epidermal tissue between the ridges (arrowhead in G) has abaxial identity, as evidenced by a higher level of anthocyanin pigmentation characteristic of abaxial petal epidermis and absence of yellow hairs.

View larger version (84K): [in a new window]   Fig. 3. GRAM promotes marginal leaf growth. (A) The leaves of periclinal chimeras in which L2-derived cells carry an oli mutation that reduces chlorophyll content. L2-derived cells contribute a variable proportion of the internal cells of the leaf - seen as a yellow marginal region in the surface view (above) or as cells showing no red chlorophyll auto-fluorescence under UV light in section (below). (B) The loss of L2-derived tissues at the leaf margins of gram mutants is partially compensated for by an increased growth of the L3 layer. (C,D) Transverse sections of wild-type (C) and gram (D) vegetative apices probed with the CYCLIND3a probe. Note the shift in CYCLIN expression to more internal regions of gram mutant leaves.

View larger version (15K): [in a new window]   Fig. 4. Structure and evolution of GRAM and PROL. (A) A neighbour-joining tree showing the relative similarity of the full-length Antirrhinum and Arabidopsis YABBY proteins, suggesting their evolutionary relationships. Bootstrap values (1000 replicates) are given. (B) The structure of the GRAM and PROL loci and mutant alleles. Boxes represent exons (black are translated, white are untranslated). The regions encoding the N-terminal zinc finger domain and the C-terminal YAB domain are stippled. Transposon insertions are shown as triangles (not to scale); numbers denote Tam2 or Tam3.

View larger version (127K): [in a new window]   Fig. 5. Expression of GRAM, PROL and AMPHB RNA. In situ hybridisation to detect GRAM (A-C), PROL (D-F) and AmPHB (G-I) RNA. (A,D,G) Longitudinal sections and (B,E,H) transverse sections of wild-type apices; (C,F,I) transverse sections of gram mutant apices. The youngest leaf primordia are towards the centre of each apex. (A) GRAM transcript (detected as a dark precipitate) is abaxially restricted and becomes most abundant in the abaxial margins (B). PROL expression in wild type (D,E) is abaxial in newly initiated primordia (inset in E) and later confined to the developing midveins of leaves. (G,H) Expression of AmPHB RNA is detected within the wild-type SAM and throughout leaf initials within it, becomes restricted to an adaxial region of leaf primordia after initiation then to adaxial leaf margins and to the vasculature of leaves and stem. In gram mutant leaves, GRAM (C) and PROL (F) expression is lost from the marginal, abaxial domain of developing leaves, whereas AmPHB now extends into this region (I).

View larger version (91K): [in a new window]   Fig. 6. Interactions between gram, phan and prol mutations. phan single mutants (A,C) and phan gram double mutants (B,D) grown at either 20°C or 25°C. gram enhances the abaxialised phenotype of phan leaves and removes their sensitivity to temperature. Wild type, gram and phan single mutants produce a functional embryonic SAM, seen to have a layered structure in an optical section of a newly germinated seedling (G), that gives rise to a shoot between the cotyledons (E). phan gram double mutants have abaxialised cotyledons (F) and fail to form an organised or functional SAM during embryogenesis (H). The apices of phan mutants grown at 20°C, seen in transverse section (I) and longitudinal section (J) express GRAM RNA ectopically in adaxial regions of developing leaves. The needle-like leaves of phan gram double mutants are radially symmetrical in transverse section (K), have an abaxialised arrangement of xylem internal to phloem (L) and express gram RNA ubiquitously (M). (N) prol gram double mutants also fail to form an embryonic SAM and produce radially symmetrical leaves (Q) that have the long glandular hairs characteristic of the wild-type abaxial (P) rather than adaxial (O) midrib; show abaxialisation of internal cell types (R); ubiquitous expression of gram RNA (S) and reduced expression of AmPHB RNA (T), although AmPHB expression remains in the SAM (arrowhead).

View larger version (98K): [in a new window]   Fig. 7. GRAM RNA expression in a periclinal chimera. In situ hybridisation with either a full-length GRAM cDNA probe (A,C,E) or a transcribed region of GRAM downstream from the transposon insertion in gram-3 (3' probe; B,D,F). Both probes detect wild-type GRAM transcripts (A,B). Transcripts from gram-3 are detected, at a reduced level, by the full-length probe (C) but not the 3' probe (D). In the phenotypically wild-type shoot of a periclinal chimera, the full-length probe detects GRAM transcripts in all cell layers of developing leaves (E) whereas the 3' probe detects a high level of transcripts only in the epidermal cell layer (F), indicating that the shoot carries a GRAM+ revertant allele in the epidermal, L1 cell layer. Each row shows adjacent sections from the same shoot apex and the sections in each column were probed together on the same microscope slide.

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