Supplemental Information. Spatio-temporal mapping of variation potentials in leaves of Helianthus annuus L. seedlings in

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1 Supplemental Information Spatio-temporal mapping of variation potentials in leaves of Helianthus annuus L. seedlings in situ using multi-electrode array Dong-Jie Zhao 1, Zhong-Yi Wang 1, Lan Huang 1*, Yong-Peng Jia 2, and John Q. Leng 3 1 College of Information and Electrical Engineering, China Agricultural University, Beijing , China 2 Bio-gene Technology Ltd., Beijing , China 3 Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, CT 06520, USA Correspondence and requests for materials should be addressed to L.H. (hlan@cau.edu.cn, biomed_hl@263.net ) Inventory of Supplemental Information Supplemental Methods Supplemental Method 1 Aniline blue staining, microscopic analysis of callose. Supplemental Method 2 Cooling stimulation. Figure S1 shows that the noise levels of the recordings vary at different K + concentrations. Figure S2 shows the spatio-temporal propagation of VPs in a leaf using the standard buffer solution. Figure S3 shows the spatio-temporal propagation of VPs in a leaf that was treated with inhibitors. Figure S4 shows the recording results after the leaf petiole was killed with 10% paraformaldehyde. Figure S5 shows the course of the average aniline blue fluorescence as an indication of callose deposition and degradation in the phloem of the H. annuus leaf vein after burning the adjacent leaf tip.

2 Figure S6 shows the recorded electrical activity that is induced by the cooling stimulus that is applied to the upper side of the recorded leaf. Supplemental References Provides literature for the Supplemental Information. Supplemental Methods Aniline blue staining, microscopic analysis of callose The phloem exposure and aniline blue (0.005% aniline blue in medium containing 2 mm KCl, 1 mm CaCl2, 1 mm MgCl2, 50 mm mannitol, and 2.5 mm MES/NaOH, ph 5.7) staining techniques have been previously described by Furch et al 1. The aniline blue was excited at UV wavelengths, and emission measurements were recorded for spectra greater than 400 nm using fluorescence microscopy (XSP-63XD, Sunguang, Beijing, China) with a cooled CCD camera (TCH-1.4CICE, Tucsen, Fuzhou, Fujian, China). Callose quantification was performed according to a previously described method in the literature 2. Cooling stimulation The cooling stimulus was applied by placing 1 cm 3 of ice water sealed with plastic film on the recorded leaf near the petiole or on a neighbouring leaf. After placing the ice water, the temperature in the cooling region lowered from 26 C to approximately 10 C in 2 min.

3 Figure S1. (a) (d) The recordings were performed using a MED64 probe chamber that was filled with 1 mm, 2 mm, 5 mm, or 10 mm KCl. The noise levels of the recordings varied at different concentrations ( 50 µv at 1 mm KCl and ± 17 µv at 10 mm KCl). (e) The recording results using a standard buffer solution 1 (2 mm KCl, 1 mm CaCl 2, MgCl 2, 50 mm mannitol, and 2.5 mm MES/NaOH buffer, ph 5.7) demonstrated a noise level of approximately ± 18 µv.

4 Figure S2. Spatio-temporal propagation of VPs in a leaf using a standard buffer solution. (a) After thermal stimulation, nearly all 64 of the electrodes recorded VPs. The white arrow points to an artifact that was observed during the thermal stimulation. (b) Overlapped signals from (a). VP onset was delayed approximately 50 s after stimulation, and VP duration was approximately 30 s. (c) VP propagation was visualised through the colour coding of VP onset at each electrode. The VPs reached the blue regions first and finally travelled to the red regions. (e) Average VP propagation velocities in different directions. The mean velocity ranged from 0.7 mm/s in the main route to 2.5 mm/s in the branch route. (f) Maps of the voltage distributions for the propagating waves at individual snapshots in time.

5 Figure S3. Spatio-temporal propagation of VPs in a leaf that was treated with inhibitors. The relative positions of the planar microelectrodes and locations of stimulation are the same as those shown in Fig. 2a. (a c) Each leaf was treated with one of the following: 1 mm sodium orthovanadate, 1 mm La 3+, or 10 mm TEA +. Following thermal stimulation (white arrow points to artifacts that were observed at the time of thermal stimulation), the 64 electrodes recorded VPs after ten seconds. The distributions are

6 shown in (a i), (b i), and (c i) for leaves that were treated with 1 mm sodium orthovanadate, 1 mm La 3+, or 10 mm TEA +, respectively. The overlapping VPs in (a ii), (b ii), and (c ii) indicate that the amplitude of the VPs is reduced by the inhibitors. Furthermore, the delay times of the VP recordings over the electrodes are prolonged. (d) VP propagation as visualised by the colour-coding of VP onset at each electrode. The leaf was treated with sodium orthovanadate, and the profile is shown in (a). The VPs reached the electrodes that are depicted in blue first, then propagated to the yellow regions, and finally moved to the red regions. (e) Average VP propagation velocity in different directions is calculated from (a). The velocity equals the distance between the two numbered planar electrodes on the arrow divided by the time delay between signal detection at the two electrodes. The mean velocity ranged from 0.6 mm/s in the main route to 2 mm/s in the branch route. (f) Maps of the voltage distribution for the propagating waves in (a) at individual time points. (g) Visualisation of VP propagation using the same method as in (d), in which the leaf was treated with La 3+. The profile is shown in (b). (h) Average VP propagation velocities in the different directions of (g), in which the mean velocity ranged from 0.5 mm/s in the main route to 1.8 mm/s in the branch route. (i) Maps of the voltage distributions for the propagating waves in (b) at individual time points.

7 Figure S4. The recording results after the leaf petiole was killed with 10% paraformaldehyde. MED64 only recorded the artifacts; however, no electrical signal was recorded. To kill the recording leaf petiole, it was immersed in a 10% paraformaldehyde solution for half an hour.

8 Figure S5. Time course of the average aniline blue fluorescence as an indication of callose deposition and degradation at the phloem of the H. annuus leaf vein after the burning of the adjacent leaf tip (at a distance of 6 cm from the observation window). (a) (d) After burning, an increase in aniline blue fluorescence was observed at 26 min, indicating the deposition of callose. (e) (g) The aniline blue fluorescence decreased between 26 min and 73 min, indicating the degradation of callose. (i) Quantification of callose deposition and degradation after burning.

9 Figure S6. The recorded electrical activities that were induced by the cooling stimulus that was applied to the upper side of the recording leaf. Approximately 40 s after the stimulus was applied, the electrical activities were recorded by MED64 planar electrodes. Compared with the thermal stimulus, the cooling stimulus did not induce any electrical activity that was able to be transmitted between leaves in H. annuus. Notably, the electrical responses and temperature sensing in the plants depend upon both the cooling rate and the final temperature at which cooling occurs 3-7 Supplemental References 1 Furch, A. C. U., Zimmermann, M. R., Will, T., Hafke, J. B. & Van Bel, A. J. E. Remote-controlled stop of phloem mass flow by biphasic occlusion in Cucurbita maxima. J. Exper. Bot. 61, (2010). 2 Yi, S. Y., Shirasu, K., Moon, J. S., Lee, S.-G. & Kwon, S.-Y. The Activated SA and JA Signaling Pathways Have an Influence on flg22-triggered Oxidative Burst and Callose Deposition. PloS one 9, e88951 (2014). 3 Plieth, C., Hansen, U. P., Knight, H. & Knight, M. R. Temperature sensing by plants: the primary characteristics of signal perception and calcium response. Plant J. 18,

10 (1999). 4 Opritov, V. A., Lobov, S. A., Pyatygin, S. S. & Mysyagin, S. A. Analysis of possible involvement of local bioelectric responses in chilling perception by higher plants exemplified by Cucurbita pepo. Russ. J. Plant Physiol.52, (2005). 5 Carpaneto, A. et al. Cold transiently activates calcium-permeable channels in Arabidopsis mesophyll cells. Plant Physiol. 143, (2007). 6 Knight, M. R. Signal transduction leading to low temperature tolerance in Arabidopsis thaliana. Philos. T. Roysoc. B 357, (2002). 7 Hafke, J. B. et al. Involvement of the sieve element cytoskeleton in electrical responses to cold shocks. Plant Physiol. 162, (2013).