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.

Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions.

OBJECTIVES: To develop a simple pectin-degrading microorganism screening method. RESULTS: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. CONCLUSIONS: The developed method is useful for identifying pectin-degrading microorganisms.

Development of microfluidic chip for entrapping tobacco BY-2 cells.

The tobacco BY-2 cell line has been used widely as a model system in plant cell biology. BY-2 cells are nearly transparent, which facilitates cell imaging using fluorescent markers. As cultured cells are drifted in the medium, therefore, it was difficult to observe them for a long period. Hence, we developed a microfluidic device that traps BY-2 cells and fixes their positions to allow monitoring the physiological activity of cells. The device contains 112 trap zones, with parallel slots connected in series at three levels in the flow channel. BY-2 cells were cultured for 7 days and filtered using a sieve and a cell strainer before use to isolate short cell filaments consisting of only a few cells. The isolated cells were introduced into the flow channel, resulting in entrapment of cell filaments at 25 out of 112 trap zones (22.3%). The cell numbers increased through cell division from 1 to 4 days after trapping with a peak of mitotic index on day 2. Recovery experiments of fluorescent proteins after photobleaching confirmed cell survival and permeability of plasmodesmata. Thus, this microfluidic device and one-dimensional plant cell samples allowed us to observe cell activity in real time under controllable conditions.