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Analysis of plasmodesmata permeability using cultured tobacco BY-2 cells entrapped in microfluidic chips


Plasmodesmata are unique channel structures in plants that link the fluid cytoplasm between adjacent cells. Plants have evolved these microchannels to allow trafficking of nutritious substances as well as regulatory factors for intercellular communication. However, tracking the behavior of plasmodesmata in real time is difficult because they are located inside tissues. Hence, a system was constructed to monitor the movement of substances by plasmodesmata using tobacco BY-2 cells, which are linearly organized cells, and a microfluidic device that traps them in place and facilitates observation. After targeting one cell for photobleaching, recovery of the lost H2B-GFP protein was detected within 200 min. No recovery was detected in that time frame by photobleaching the entire cell filaments. This suggested that the recovery of H2B-GFP protein was not due to de novo protein synthesis, but rather to translocation from neighboring cells. The transport of H2B-GFP protein was not observed when sodium chloride, a compound known to cause plasmodesmata closure, was present in the microfluid channel. Thus, using the microfluidic device and BY-2 cells, it was confirmed that the behavior of plasmodesmata could be observed in real time under controllable conditions.

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We thank Kazuki Yakamoto, Haruo Kassai, Ikue Yoshikawa, and Mayumi Taniguchi for technical assistance.


This work was supported by grants from the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research (18K05373 and 20H05501 to RT, 18KT0040, 18H03950, 21H00368, and 21H05657 to MN and 20H03273 to KK and MN), Grant-in-Aid for Scientific Research on Innovative Areas (20H05358 to DK), the Japan Science and Technology Agency PRESTO program (JPMJPR18K4 to DK), and the Canon Foundation (R17-0070 to MN).

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KS, RT, HH, and MN: conceived this study. YK and MK: designed and conducted the main experiments with advice from KK, KS, RT, HH, and MN. DK: constructed transgenic BY-2 cell lines. MK, and RT: maintained and prepared the BY-2 cells for experiments. KK, YK, KS, and MN wrote the paper.

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Correspondence to Kazunori Shimizu or Michitaka Notaguchi.

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Kurotani, Ki., Kawakatsu, Y., Kikkawa, M. et al. Analysis of plasmodesmata permeability using cultured tobacco BY-2 cells entrapped in microfluidic chips. J Plant Res 135, 693–701 (2022).

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  • BY-2 cell
  • FRAP imaging
  • Microfluidic chip
  • Plasmodesmata