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First published online 24 November 2004
doi: 10.1242/dev.01544

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Microsurgical and laser ablation analysis of leaf positioning and dorsoventral patterning in tomato

Didier Reinhardt^1,*, Martin Frenz², Therese Mandel¹ and Cris Kuhlemeier^1,

¹ Institute of Plant Science, University of Berne, Altenbergrain 21, 3013 Bern, Switzerland
² Institute of Applied Physics, University of Berne, Sidlerstrasse 5, 3012 Bern, Switzerland

View larger version (141K): [in a new window]   Fig. 1. Isolation of the site of incipient leaf formation (I1) from the meristem affects phyllotaxis. (A,E-I) Scanning electron micrographs (SEM); (B-D) light stereomicrographs. (A) Tomato meristem in top view. The site of incipient leaf formation (I1), which can be predicted to be on the upper left part of the meristem (white arrowhead), was separated from the remainder of the meristem by an incision (black arrowhead) just before imaging. (B) Tomato apex just after operation as in A. (C) The same apex as is shown in B 1 day after operation. (D) The same apex as is shown in B, 3 days after operation. P1 was removed to expose the meristem. I2 has initiated at the expected position. Note an apparent post-meristematic increase in the divergence angle between P1 and P2 (blue lines), compare with (B). (E) The same apex as is shown in B, 6 days after operation. Note that I2 (bottom) and I3 (top) diverge by approximately 180°. (F) Control apex with normal divergence angle between I2 and I3. (G) Tomato apex 6 days after operation as in A. Note the extended width of the I3 primordium. (H) Apex 6 days after operation as in A. The vertical distance between I1 and I2, is strongly increased compared with a control (I). P3 and P2 indicate the bases of pre-existing leaf primordia that were removed at the beginning of the experiment, and P1 represents the youngest primordium; I1, I2 and I3 indicate primordia formed after the operation. Black arrowheads indicate the incisions. Blue lines in B and D represent the divergence angle between P1 and P2. Scale bar: 100 µm.

View larger version (11K): [in a new window]   Fig. 2. Effect of I1 isolation from the meristem on the divergence angles. Each angle was determined at the actual time point of primordium initiation, not at the end of the experiment. The angle between I2 and I3 deviates by approximately 30° from the mean phyllotactic divergence angle (137°).

View larger version (98K): [in a new window]   Fig. 3. Laser ablation of the site of incipient leaf formation (I1) affects phyllotaxis. (A-H) Two individual tomato apices imaged from the top, 1 day (A,E), 2 days (B,F), 3 days (C,G) and 4 days (D,H) after laser ablation of I1. (A-C,E-G) light stereomicrographs; (D,H) SEM images. (A) The site of incipient leaf formation (black arrowhead) was ablated with 10 successive laser pulses as described previously (Reinhardt et al., 2003b). (B) I1* initiates adjacent to the ablation. The divergence angle between P1 and I1* is increased to approximately 175°, whereas the angle between I1* and I2 (C) is smaller than normal (107°). The following angle between I2 and I3 (D) is normal again (138°). (E) Ablation as in A. I1* was formed just above P2 (F), resulting in the reversal of the phyllotactic spiral from an anti-clockwise to a clockwise direction (G,H). P4, P3 and P2, indicate the bases of pre-existing leaf primordia that were removed at the beginning of the experiment, and P1 represents the youngest primordium; I1, I2 and I3 indicate primordia formed after the ablation. Scale bar: 100 µm.

View larger version (110K): [in a new window]   Fig. 4. Isolation of the meristem from all primordia but P1 leads to wider leaves. (A) Control tomato apex in top view. The approximate delimitation of the meristem is represented by a circle. The arc encompassed by the primordia is represented by thickened portions of the circles. (B) Top view of a meristem 2 days after its isolation. Note the increased lateral width of P1 and I1 (compare with A). (C) Meristem as shown in B, with I2 becoming visible as a small bulge (white arrowhead). (D) An oversized P1 primordium with two tips, one week after isolation of the meristem. (E) Control apex shown in A showing the direct contact neighbours of P1 (P3 and P4) and of I1 (P2 and P3). P4, P3 and P2, indicate the bases of pre-existing leaf primordia that were removed at the beginning of the experiment, and P1 represents the youngest primordium; I1, I2 and I3 indicate primordia formed after the ablation. Scale bar: 100 µm.

View larger version (132K): [in a new window]   Fig. 5. Isolation of the site of incipient leaf formation (I1) from the meristem leads to defects in dorsoventral patterning of the isolated primordium. (A) Leaf primordium of an untreated control apex. Note the difference in trichome morphology on the adaxial side compared with on the abaxial side. Abaxial trichomes are mostly long and linear, with interspersed short linear and globular trichomes. Adaxial trichomes are exclusively globular, and are arranged in a row along the central axis of the primordium. The lateral leaflets (white arrowheads) emerge from the edge between the adaxial and abaxial domain, and point to the meristem (removed for better visibility). (B) Leaf primordium with weak dorsoventral defects. The upper half of the primordium was removed for better visual access. Note the more central position of the second leaflet pair (white arrowheads), and the absence of globular trichomes on the basal portion of the adaxial side (asterisk). (C) Leaf primordium with intermediate dorsoventral defects. The upper half of the primordium was removed for clarity. Note the fused single leaflet in the centre (white arrowhead), and the absence of globular trichomes below the leaflet (asterisk). (D) Leaf primordium with strong dorsoventral defects from the same apex as is shown in Fig. 1B-E. I1 is completely radialized, except for a small distal portion (between arrows). (E,F) Leaf primordia that lack any sign of dorsoventral patterning. The primordium in E is retarded, whereas the primordium in F grew out to a normal length. Note that the trichomes around the entire circumference of the primordia correspond to abaxial trichomes (compare with A and B). (GJ) Development of an apex after incision through the meristem centre. The meristem continues to grow and to form leaf primordia (H, 1 day; I, 3 days), and finally splits (J). I1 and the following primordia exhibit normal dorsoventral curvature. P3, P2 and P1 indicate the bases of pre-existing leaf primordia that were removed at the beginning of the experiment; I1, I2 and I3 indicate primordia formed after the operation. Black arrowheads indicate the incision. Scale bar: 100 µm.

View larger version (92K): [in a new window]   Fig. 6. Formation of radially symmetric primordia after ablations of the L1 layer. (A) Control tomato apex after 7 days in tissue culture. Besides P1, which was the youngest primordium at the beginning of the experiment, four new primordia were formed in clockwise phyllotaxis. The youngest (I4) is just becoming evident at the flank of the meristem. (B-F) Tomato apices 7 days after the removal of 75-100% of the L1 layer. The apices had one preformed primordium (P1) at the time of the operation. (B) I1 has developed into a radially symmetric organ with a small adaxial domain at the distal end (between arrows). (C) P1 has developed into a radially symmetric primordium. The trichomes on its surface are typical for the abaxial side of normal leaf primordia, indicating that the radially symmetric primordium has only abaxial identity. The white arrow indicates an axillary meristem growing from the leaf base of P3. (D) P1 developed almost normally in the distal part; however, at the base, the adaxial domain is lost after the formation of one pair of leaflets (white arrowheads; compare with P1 in Fig. 5A). One additional primordium was initiated (I1), which developed to be completely radially symmetric with only abaxial identity, based on the morphology and the distribution of the trichomes. (E) P1 has developed similarly to the primordium shown in D. After the formation of one pair of leaflets (arrowheads), the adaxial domain terminates with a single central leaflet (cl). (F) P1 has developed normally (compare with P1 in A), and exhibits two pairs of leaflets (white arrowheads). Axilllary meristems of older primordia are induced to grow out (arrow). M, meristem; P1, youngest preformed primordium; I1, I2, I3 and I4, first, second, third and fourth primordium, respectively, formed after the ablation. Scale bar: 200 µm.

View larger version (154K): [in a new window]   Fig. 7. Partial isolation of the site of incipient leaf formation (I1), or of the youngest primordium (P1), from the meristem by a superficial incision leads to defects in dorsoventral patterning of the isolated primordium. (A) Tomato apex immediately after isolation of I1 by an ablation of the superficial L1 layer (black arrowheads). (B) Semi-thin section of a tomato apex immediately after isolation of P1 by an ablation as in A (black arrowhead). (C) Tomato apex 8 days after isolation of P1 as described in A. P1 lacks any sign of leaflets or of a developing leaf blade. The trichomes exhibit only abaxial features (compare with Fig. 5A). (D-I) Tomato apices 8 days after operation as in A. (D) P1 lacks leaflets but has developed a leaf blade (white arrowheads). (E) Close up view of D. Note the lack of leaflets in P1, compared with I1 (white arrowheads). (F) P1 exhibits only one pair of leaflets, compared with I1, which has already formed two pairs of leaflets (white arrowheads). (G) I1 is retarded and lacks any sign of dorsoventral patterning. (H) Completely radialized I1 of approximately normal size. (I) Initiation of an accessory meristem (white arrowhead) above the operated I1 position. P4, P3 and P2 indicate the bases of pre-existing leaf primordia that were removed at the beginning of the experiment, and P1 represents the youngest primordium; I1 indicates the first primordium formed after the ablation. Scale bar: 200 µm.