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First published online July 21, 2003
doi: 10.1242/10.1242/dev.00596

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Microsurgical and laser ablation analysis of interactions between the zones and layers of the tomato shoot apical meristem

¹ Institute of Plant Sciences, University of Berne, Switzerland
² Institute of Applied Physics, University of Berne, Switzerland

View larger version (63K): [in a new window]   Fig. 1. Organisation of the shoot apical meristem, and schematic representation of the ablations performed in this study. (A) SEM micrograph of a vegetative shoot apical meristem of tomato. P3, P2 and P1 indicate young primordia. P1 is just being initiated at the flank. (B-F) Schematic representation of a meristem as in A. (B) The meristem consists of the central zone (CZ, light blue) that harbours the stem cells (dark blue) and the peripheral zone (PZ, yellow) in which organs are formed. An organising centre that expresses the marker gene LeWUS (red) induces stem cell identity in the above cells. Superimposed on the zonation (CZ and PZ), the meristem is organised in layers, namely the external L1 layer, the subepidermal L2 layer and the remaining cells, called L3. All three layers cooperate in organ formation (arrowhead). (C) Ablation of the entire CZ. (D) Ablation of the stem cells. (E) Ablation of the L1 layer in the CZ. (F) Ablation of the entire L1 layer. Scale bar: 100 µm.

View larger version (89K): [in a new window]   Fig. 2. Organ formation and meristem maintenance after ablations of the CZ. Yellow arrowheads indicate the position of the lesion. (A) Longitudinal section through a meristem immediately after ablation. The ablation eliminated the entire CZ and projected more than 100 µm into the meristem. (B) Longitudinal section through a control meristem hybridised with a dig-labelled antisense probe against LeWUS. The signal in the meristem centre appears brown. (D,F,H) Transverse sections; (C,E,G,I,J) scanning electron micrographs. (C) Scanning electron micrograph of a meristem with ablated CZ, in top view. (D) Transverse section of a meristem after ablation of the CZ. (E-J) Development of the meristem after ablation of the CZ. After 2 days, a new primordium was formed at the expected site (E), and the hole closed (F). After 4 days, four new primordia had been formed (G), the lesion was displaced from the centre towards the flank, and a new meristem centre (star) was established (G,H). After 6 days, the lesion was removed from the meristem and displaced onto the stem (I), or two new meristem centres had been established on either side of the lesion (J). P3, P2, and P1 indicate leaf primordia that were present at the beginning of the experiment; I1, I2, I3, and I4 indicate primordia formed after the ablation. In some cases, primordia were removed to allow manipulation or visualisation. Scale bars: 100 µm.

View larger version (135K): [in a new window]   Fig. 3. Expression of the meristem marker genes LeWUS and LeT6 in the meristem after ablation of the CZ. Gene expression was visualised by in situ hybridisation with 35S-labelled riboprobes. Signal appears as yellow grains. All images are transverse sections, except for I and J which are longitudinal sections. Position of the lesion is indicated by yellow arrowheads. (A-G) Expression of LeWUS. (H-L) Expression of LeT6. (A) Expression of LeWUS in a control meristem. (B) Expression of LeWUS immediately after ablation of the CZ (yellow arrowhead). No LeWUS expression can be detected. (C) Expression of LeWUS 1 day after ablation of the CZ. A ring-shaped area around the lesion expresses LeWUS at low levels (arrows). (D) Expression of LeWUS 2 days after ablation of the CZ. The lesion (arrowhead) is closed, and the LeWUS signal becomes confined to one side of the lesion. (E) Expression of LeWUS 4 days after ablation of the CZ. The LeWUS-expressing zone at the flank has resolved into a new WUS centre with normal dimensions (compare with A). (F) Expression of LeWUS 6 days after ablation of the CZ. A new functional meristem centre with a normal WUS centre has been re-established. (G) Expression of LeWUS 4 days after ablation of the CZ. Two new WUS centres are evident at opposite sides of the lesion. (H) Expression of LeT6 in a control meristem. Note down-regulation of LeT6 in the youngest primordium (P1) and at the site of incipient leaf formation (I1). (I) Expression of LeT6 in a control meristem. Note down-regulation of LeT6 in the youngest (P1) and second youngest primordium (P2). (J) Expression of LeT6 6 hours after ablation of the CZ. LeT6 remains active at the periphery of the meristem and is only decreased in the vicinity of the lesion. (K) Expression of LeT6 5 days after ablation of the CZ. The lesion is displaced, and LeT6 is expressed in the new meristem centre and excluded from leaf primordia. (L) Expression of LeT6 4 days after ablation of the CZ. Two new LeT6-expressing meristems are induced on opposite sides of the lesion. P3, P2, and P1 indicate leaf primordia that were present at the beginning of the experiment; I1, I2, I3, and I4 indicate primordia formed after the ablation. Scale bars: 100 µm.

View larger version (120K): [in a new window]   Fig. 4. Control treatments do not influence the central zone. (A-H) Laser ablations (yellow arrowheads) as in Fig. 2C, but at the periphery instead of the centre. (A) Laser ablation at the periphery approximately at the site of incipient leaf formation. (B,C) Consecutive video images of a single meristem with an ablation as in (A). Cut primordia are dark green, and the youngest primordia are highlighted in yellow for clarity. (B) Immediately after ablation (t0); (C) 2 days after ablation. Leaf formation at the site of the ablation is suppressed (arrowheads), while two new primordia (I1 and I2) were induced at the next two expected positions. (D,E) Consecutive video images of a single meristem with an ablation as in (A). (D) t0; (E) 2 days after ablation. I1 is initiated closer to P1 than normal, resulting in a divergence angle of approximately 90° instead of 137°. Arrowheads point to the lesion. (F) Ablation on P1. 2 days after the ablation (arrowhead), the primordium has recovered and split in two halves. (G) In situ hybridisation with a LeWUS probe 6 hours after an ablation at the periphery. The LeWUS expression domain remained normal. (H) 2 days after ablation, the lesion was displaced (arrowhead), and LeWUS continued to be expressed in the normal area. (I-L) Effects 4 days after treatments of the CZ with stress metabolites and oxidants. (I) Control. (J) Treatment with 1 mM salicylic acid. (K) Treatment with 1 mM hydrogen peroxide. (L) Treatment with 0.1 mM Paraquat. Treatments did not affect the formation rate or the positioning of leaf primordia. Lanolin paste (red) remained in the centre, indicating that the growth centre persisted. P5, P4, 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 ablation. Scale bars: 100 µm.

View larger version (48K): [in a new window]   Fig. 5. Expression of LeWUS after ablation of the stem cells. (A) Longitudinal section through a meristem after ablation of the upper four to five cell layers. (B-D) Transverse sections showing LeWUS expression at various times after ablation as in A. (B) Immediately after ablation. (C) One day after ablation. (D) Three days after ablation. P1 indicates the leaf primordium that was present at the beginning of the experiment; I1 denotes a primordium that was formed after the ablation. Scale bars: 100 µm.

View larger version (93K): [in a new window]   Fig. 6. Laser ablations of the L1 layer in the centre have local effects on cell division orientation, but not on meristem maintenance and phyllotaxis. (A) Scanning electron micrograph of a meristem immediately after superficial ablation in the centre (yellow arrowhead). (B) Longitudinal section of a meristem immediately after treatment as in A. (C-E) Consecutive video images of a single meristem after ablation as in A: (C) 1 day, (D) 2 days and (E) 3 days after ablation. Note normal phyllotaxis. (F) Longitudinal section through a meristem 5 days after ablation as in A. Note periclinal divisions in cells just below the ablation. P2 and P1 indicate leaf primordia that were present at the beginning of the experiment; I1 and I2 indicate primordia formed after the ablation. Scale bars: 100 µm.

View larger version (134K): [in a new window]   Fig. 7. Surgical ablation of the L1 layer leads to aberrant cell division and differentiation in L2 and L3. (A-C) Consecutive video images of a single meristem from which the left half of the L1 layer was removed. Cut primordia are coloured in dark green, and the youngest primordia are highlighted in yellow: (A) t0, (B) 2 days and (C) 4 days after ablation. Organ formation continues from the unperturbed half, and the meristem centre is shifted to the right. (D-F) Consecutive video images of a meristem from which most of the L1 layer has been removed: (D) t0, (E) 2 days, (F) 4 days. Meristem activity ceases. Note that a final primordium is formed at the normal position (I1). (G-I) Longitudinal sections through meristems after a surgical ablation as in D. (G) Note the continuity of the L2 layer at the site of the ablation (between arrows). (H) Two days after ablation as in (D). A last primordium was formed (I1), while the cells at the ablated site (arrowhead) became vacuolated and started to divide periclinally. (I) Five days after ablation as in (D). Note stacks of cells resulting from repeated periclinal division, and increasing vacuolisation (arrowhead). (J) Close up of (I). Note different cell division patterns and cell shape in the area where a primordium had been removed at the beginning of the experiment (arrow), compared to the site at which the L1 was ablated (arrowhead). (K-M) In situ hybridisations with a 35S-labelled antisense probe against LeT6. Tomato meristems were treated as in (D), and fixed for analysis either immediately (K), or after 2 days (L), and 5 days (M). LeT6 signal can be observed at the ablated site until 2 days after the ablation (L). After 5 days, the vacuolated cells exhibited low levels of LeT6 mRNA, whereas high levels of LeT6 remained in the lower L3 cells that exhibit less vacuolisation. P4, P3, P2 and P1 indicate the bases of preexisting leaf primordia that were removed at the beginning of the experiment; I1 and I2 indicate primordia formed after the ablation. Scale bars: 100 µm (A-I, K-M); 50 µm (J).

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