Using fs-laser pulses to selectively kill specific cells inside tomato meristems Investigation of leaf patterning

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1 Using fs-laser pulses to selectively kill specific cells inside tomato meristems Investigation of leaf patterning Dominik Marti, Yamini Deb, Cris Kuhlemeier, Martin Frenz Institute of Applied Physics Institute of Plant Sciences University of Bern 9. August / 11

2 Some plants grow leaves in Fibonacci spirals / 11

3 The meristem, source of Phyllotaxis The meristem is a specialized tissue that continuosly produces lateral organs such as leaves. The Primordium is the leaf in its earliest recognizable stage. The image shows a tomato meristem and P1 to P6 (with P5 hidden behind and P4 and P6 cut away). P2 P4 P3 P1 P6 3 / 11

4 Auxin, the hormone inducing leaf growth Auxin, a class of hormones, induce growing of leaves and flowers in plants. The flow of Auxin between cells is regulated by influx and efflux carrier proteins. Auxin efflux proteins are upregulated towards high Auxin concentration regions, resulting in a positive feedback loop. Sketch of 5 adjacent plant cells Blue: Auxin, Green: Efflux protein 4 / 11

5 Auxin, the hormone inducing leaf growth Auxin, a class of hormones, induce growing of leaves and flowers in plants. The flow of Auxin between cells is regulated by influx and efflux carrier proteins. Auxin efflux proteins are upregulated towards high Auxin concentration regions, resulting in a positive feedback loop. Sketch of 5 adjacent plant cells Blue: Auxin, Green: Efflux protein 4 / 11

6 Auxin, the hormone inducing leaf growth Auxin, a class of hormones, induce growing of leaves and flowers in plants. The flow of Auxin between cells is regulated by influx and efflux carrier proteins. Auxin efflux proteins are upregulated towards high Auxin concentration regions, resulting in a positive feedback loop. Sketch of 5 adjacent plant cells Blue: Auxin, Green: Efflux protein 4 / 11

7 The Primordia pump Auxin Primordia have an Auxin concentration peak at their tip, therefore, Auxin is pumped towards it. The pumping of Auxin results in a depletion of Auxin around every Primordium. This creates a new center of higher Auxin concentration at I1, at a divergence angle of 137, which then amplifies by upregulation of efflux carriers pointing towards it. P2 P1 5 / 11

8 The Primordia pump Auxin Primordia have an Auxin concentration peak at their tip, therefore, Auxin is pumped towards it. The pumping of Auxin results in a depletion of Auxin around every Primordium. This creates a new center of higher Auxin concentration at I1, at a divergence angle of 137, which then amplifies by upregulation of efflux carriers pointing towards it. P2 P1 5 / 11

9 Is phyllotaxis a superficial or a deeper story? If the phyllotaxis behavior can be influenced by blocking specific Auxin paths, we may be able to uncover the different parts influencing leaf patterning. P2 P1 6 / 11

10 Is phyllotaxis a superficial or a deeper story? If the phyllotaxis behavior can be influenced by blocking specific Auxin paths, we may be able to uncover the different parts influencing leaf patterning. P2 P1 6 / 11

11 Femtosecond laser affects only the focal spot 7 / 11

12 Setup coherent chameleon nm 130 fs coherent RegA nm, 150 fs Nikon A1R MP confocal microscope 8 / 11

13 MP Microscopy arrests growth P2 P1 Transgenic tomatoes Green: GFP, efflux protein Red: PI, life-dead staining 9 / 11

14 MP Microscopy arrests growth Transgenic tomatoes Green: GFP, efflux protein Red: PI, life-dead staining 9 / 11

15 Ablation of the forming midvein Transgenic tomatoes Green: GFP, efflux protein Red: PI, life-dead staining Laser parameters: Wavelength 800 nm Pulsewidth ~150 fs Intensity ~10 TW/cm 2 9 / 11

16 Ablation of the forming midvein Laser parameters: Wavelength 800 nm Pulsewidth ~150 fs Intensity ~10 TW/cm 2 9 / 11

17 Ablation of the forming midvein Laser parameters: Wavelength 800 nm Pulsewidth ~150 fs Intensity ~10 TW/cm 2 (too low to induce optical breakdown in water) We need 2 shots with a time separation of 4 ms to kill a cell, a single shot is not sufficient What kills the cells? 9 / 11

18 Ablation of the forming midvein Laser parameters: Wavelength 800 nm Pulsewidth ~150 fs Intensity ~10 TW/cm 2 (too low to induce optical breakdown in water) We need 2 shots with a time separation of 4 ms to kill a cell, a single shot is not sufficient What kills the cells? 9 / 11

19 Outcomes Not shot, normal growth Shot, flattened tip 10 / 11

20 Outcomes Loss of leaf polarity, i.e., the leaf is either peg like or flattened Change in divergence angle, i.e., the next leaf doesn t grow at / 11

21 Outcomes Loss of leaf polarity, i.e., the leaf is either peg like or flattened Change in divergence angle, i.e., the next leaf doesn t grow at / 11

22 Open questions Why does scanning using multiphoton microscopy arrest the growth of the plant, although we don t see any effect immediately after scanning? Why do we need to successive shots tho kill a single cell? What kills the cells? 11 / 11

23 Open questions Why does scanning using multiphoton microscopy arrest the growth of the plant, although we don t see any effect immediately after scanning? Why do we need to successive shots tho kill a single cell? What kills the cells? 11 / 11