59022bs and Emission filter, 59022m, and Dichroic mirror,
ET460lp and Emission filter, ET645/75m (Chroma, VT,
USA) for cpsfGFP and LSSmOrange, respectively (see Note 4).
2. NIS-Elements AR 5.02.00 64-bit imaging software (Nikon,
Tokyo, Japan).
3. Fiji (https://fiji.sc/#) or ImageJ (https://imagej.nih.gov/ij/)
software for image analysis (see Note 5).
2.3 Plant Material,
Growth Medium, and
Perfusion Solutions
1. We used Arabidopsis thaliana Col-0 plants expressing
pUBQ10:: MatryoshCaMP6s
60 (Fig. 2).
2. Growth medium: 0.5Â Murashige and Skoog (MS) medium
with 1% (w/v) agar, 1% (w/v) sucrose, 5 mM MES-KOH
(pH 5.7) (see Note 6).
3. Control solution: 0.5Â MS liquid medium with 5 mM
MES-KOH (pH 5.7).
4. Treatment solution: 0.5Â MS liquid medium with 5 mM
MES-KOH (pH 5.7) and 0.1 or 1 mM L-glutamate (L-Glu)
as a stimulant.
3 Methods
3.1 Preparation of
Plastic Cones
1. Fill a 9-cm petri dish with warm growth medium containing
agar. Use a multichannel micropipette for 10-μL pipette tips,
and fill each tip with 10 μL of medium from the petri dish
before the medium solidifies. Put the pipette tips back in the tip
box and wait for at least 5 min for solidification.
2. After solidification of the medium, cut about 1 cm of the tip off
by sterilized scissors or razors and retain. Throw away the large
end of the tip.
3. Put the small ends of these plastic cones into the plant growth
medium, which has been already solidified in the petri dish (see
Note 7).
3.2 Sowing and
Germination
1. Sterilize the surface of Arabidopsis seeds with 70% ethanol for
5 min (see Note 8) in a 1.5-mL centrifuge tube and wash four
times with sterile water.
2. Incubate the sterilized seeds in sterile water for 3 days at 4
C to
synchronize seed germination.
3. Place single seeds on each plastic cone (the cut ends of the
pipette tips) in the media-filled dish.
4. Seal the dish with Parafilm (Bemis, IL, USA).
5. Transfer the petri dish into a plant growth chamber (22
C,
16-h light and 8-h dark cycle).
6. Grow plants until the root tip reaches near bottom outlets of
the plastic cone.
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Akira Yoshinari et al.
ET460lp and Emission filter, ET645/75m (Chroma, VT,
USA) for cpsfGFP and LSSmOrange, respectively (see Note 4).
2. NIS-Elements AR 5.02.00 64-bit imaging software (Nikon,
Tokyo, Japan).
3. Fiji (https://fiji.sc/#) or ImageJ (https://imagej.nih.gov/ij/)
software for image analysis (see Note 5).
2.3 Plant Material,
Growth Medium, and
Perfusion Solutions
1. We used Arabidopsis thaliana Col-0 plants expressing
pUBQ10:: MatryoshCaMP6s
60 (Fig. 2).
2. Growth medium: 0.5Â Murashige and Skoog (MS) medium
with 1% (w/v) agar, 1% (w/v) sucrose, 5 mM MES-KOH
(pH 5.7) (see Note 6).
3. Control solution: 0.5Â MS liquid medium with 5 mM
MES-KOH (pH 5.7).
4. Treatment solution: 0.5Â MS liquid medium with 5 mM
MES-KOH (pH 5.7) and 0.1 or 1 mM L-glutamate (L-Glu)
as a stimulant.
3 Methods
3.1 Preparation of
Plastic Cones
1. Fill a 9-cm petri dish with warm growth medium containing
agar. Use a multichannel micropipette for 10-μL pipette tips,
and fill each tip with 10 μL of medium from the petri dish
before the medium solidifies. Put the pipette tips back in the tip
box and wait for at least 5 min for solidification.
2. After solidification of the medium, cut about 1 cm of the tip off
by sterilized scissors or razors and retain. Throw away the large
end of the tip.
3. Put the small ends of these plastic cones into the plant growth
medium, which has been already solidified in the petri dish (see
Note 7).
3.2 Sowing and
Germination
1. Sterilize the surface of Arabidopsis seeds with 70% ethanol for
5 min (see Note 8) in a 1.5-mL centrifuge tube and wash four
times with sterile water.
2. Incubate the sterilized seeds in sterile water for 3 days at 4
C to
synchronize seed germination.
3. Place single seeds on each plastic cone (the cut ends of the
pipette tips) in the media-filled dish.
4. Seal the dish with Parafilm (Bemis, IL, USA).
5. Transfer the petri dish into a plant growth chamber (22
C,
16-h light and 8-h dark cycle).
6. Grow plants until the root tip reaches near bottom outlets of
the plastic cone.
314
Akira Yoshinari et al.