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Manipulation of leaf shape by modulation of cell division

Joanna Wyrzykowska^1,2, Stéphane Pien^1,, Wen Hui Shen³ and Andrew J. Fleming^1,*

¹ Institute of Plant Sciences, Swiss Federal Institute of Technology (ETH), Universitätstrasse 2, 8092 Zurich, Switzerland
² Department of Genetics and Cytology, University of Gdansk 80-822, Poland
³ IBMP-CNRS, 12 rue du Général Zimmer, 67084 Strasbourg Cedex, France
Present address: Institute of Plant Biology, University of Zurich, Switzerland

View larger version (22K): [in a new window]   Fig. 1. Analysis of Ahtet-inducible Nicta;CycA3;2 transcript accumulation. (A) Northern blot analysis of Nicta;CycA3;2 gene expression in Tet::Nicta;CycA3;2 (lines S7b and S7c) and Tet::GUS (Con) seedlings treated either with (+) or without (–) Ahtet. Blots were hybridised with a probe for Nicta;CycA3;2 (upper panel) or histone H4 (lower panel) (10 µg RNA/lane). (B) RT-PCR analysis of Nicta;CycA3;2 transcript accumulation in leaf discs from Tet::Nicta;CycA3;2 plants (line S7b) induced for 24 hours with various concentrations of Ahtet. (C) As in (B) except that leaf discs were incubated with (+) or without (–) Ahtet (0.02 mg/ml), for the times given, before analysis.

View larger version (121K): [in a new window]   Fig. 2. Local induction of Nicta;CycA3;2 and Sp;cdc25 gene expression leads to cell proliferation on the leaf margin. (A) Cross section through a tobacco shoot apex to show site of Ahtet application (red spot) on the abaxial flank of a P2 primordium. (B) Cross section of a P2 primordium from a Tet::Sp:cdc25 plant 24 hours after local induction on one flank (red line). (C) Cross section of a P2 primordium from a Tet::Nicta;CycA3;2 plant 24 hours after local induction on one flank (red line). (D) Tangential section through a primordium from a Tet::Nicta.CycA3;2 plant 72 hours after Ahtet induction on one flank. An accumulation of small cytoplasmically dense cells is apparent on the induced flank (i) compared with the non-induced flank (ni). (E,F) Magnifications of the induced flank (E) and non-induced flank (F) shown in D. Cells in E are small, dense and meristematic. Cells in F have undergone vacuolation. (G) In situ hybridisation of a section of a P2 primordium from a Tet::Nicta;CycA3;2 plant 72 hours after Ahtet induction on one flank. The section has been hybridised with a probe for Nicta;CycA3;2. A larger area of signal (blue/purple) is present in the induced flank (i) compared with the non-induced flank (ni). (H) As in G except that the primordium was mock-induced (i) with buffer on one flank. (I) Cross section of primordium of Tet::Sp:cdc25 plant 72 hours after Ahtet induction on one flank. The induced flank (i) is slightly thicker than the non-induced (ni) and the lateral vein (v) differentiation in the non-induced flank is not apparent in the induced flank. (J,K) Examples of Ahtet-induced flanks of Tet::Sp:cdc25 primordia. Note the disorganised cellular patterns leading in some areas to apparent cell death and compression and poorly developed vascular tissue (v). (L) Cross section of leaf blade from a non-induced flank showing highly ordered cellular structure and appropriate vascular differentiation (v). Bars: 40 µm in E and F; 60 µm in G and H; 75 µm in B and C; 100 µm in D, J, K, and L; 120 µm in I.

View larger version (52K): [in a new window]   Fig. 3. Altered lamina growth and leaf shape resulting from local induction of Nicta;CycA3;2 and Sp;cdc25 gene expression. (A) Microinductions were performed at various positions along the flanks of a number of Tet::Nicta;CycA3;2 primordia, as shown in the schematic diagrams (red = area of treatment, t = Ahtet induction, b = buffer application). The resultant leaf shape is shown below each schematic. (B) As in A except that the microinductions were performed on primordia of Tet::Sp;cdc25 plants. Bars: 5 mm.

View larger version (74K): [in a new window]   Fig. 4. Histology of asymmetric leaves. (A) Cross section of an asymmetric leaf resulting from local induction of Nicta;CycA3;2 in a primordium at stage P2. The lamina resulting from the induced flank (i, inset) shows altered cellular patterning compared with the non-induced flank (ni, inset) and a lack of epidermal hairs. (B) Cross section of a symmetrical leaf formed after local mock induction (i) of a Tet::Nicta;CycA3;2 primordium with buffer only. Bars: 80 µm in A (inset); 250 µm in B.

View larger version (79K): [in a new window]   Fig. 5. Induction of ectopic lamina by local application of roscovitine. (A) Leaf shape resulting from application of roscovitine-loaded lanolin on one flank of a P2 primordium. Ectopic lamina has formed on one side (arrowhead). (B) Leaf shape resulting from mock-induced control primordium. (C) Cross section through ectopic lamina resulting from roscovitine treatment. (D) Cross section through lamina of a normally formed leaf. (E) Leaf shape resulting from local induction of expansin gene expression on one flank of a primordium at the P2 stage. Ectopic lamina has formed on one side (arrowhead). (F) Leaf shape resulting from local induction of Nicta;CycA3;2 gene expression on one flank of a primordium at the P2 stage. Retarded lamina growth has occurred at the site of induction (arrowhead). Bars: 2 mm in A and B; 250 µm in C and D; 5 mm in E and F.

View larger version (141K): [in a new window]   Fig. 6. Altered cell division pattern does not affect meristem function. (A) Cross section of tobacco apex to show position of Ahtet induction (red spot) on the meristem (m) between primordia P2 and P1. (B) Scanning electron micrograph of shoot apex 72 hours after micro-induction of Nicta;CycA3;2 gene expression. A primordium has arisen at the expected position (I1) with no overt change in morphology at the I2 position. (C) Section through the apical meristem 24 hours after local induction of Nicta;CycA3;2 gene expression. Proliferation has occurred on one flank (arrowhead) to generate smaller cells than in the surrounding meristem. (D) As C, with cellular boundaries drawn in. (E) In situ hybridisation of an Ahtet-induced meristem from a Tet::Nicta;CycA3;2 plant hybridised with an antisense probe for histone H 4. Signal (blue/black) is localised to individual cells in the apex. (F) As in E except the meristem was mock-induced with buffer. Bars: 30 µm in C; 50 µm in E and F; 100 µm in B.