1. GFP AtNCED-GFP AAO3-GFP 35S nos-T nos-T nos-T sGFP
Supplementary Fig. S1. Schematic presentation of 35S promoter::GFP-fusion constructs.
For generation of the control construct the 35S::sGFP-nos terminator cassette was inserted into pUC18 vector (upper scheme), thereby generating a construct of 4.1 kb. Likewise, the 35S::AtNCED3-sGFP cassette with the nos terminator (nos-T) was cloned into pUC18, yielding a construct of 5.9 kb (middle scheme). In contrast, the 35S::AAO3-sGFP cassette with the nos terminator was cloned into pBluescript KS (pBS KS) whereby a construct of 8.7 kb was generated.
GFP
pUC18 (total 4.1 kb)
35S
sGFP
AAO3-GFP
pBS KS (total 8.7 kb)
AAO3
4.3 kb
1.0 kb
0.4 kb
AtNCED-GFP
pUC18 (total 5.9 kb)
nos-T
AtNCED3
1.8 kb
nos-T
nos-T

2. Transformed guard cells/leaf 3cm
Supplementary Fig. S2. Efficiency of transformation in the guard cells of broad bean leaves.
35S::GFP construct was introduced into the leaves of broad bean placed upside down on agar plates using a particle gun. To check the efficiency of the transformation rate in the guard cells, we conducted the experiment under the conditions of 650 to 1100 PSI and 3 to 12 cm distance between the gun and sample. After 24 h of bombardment, the number of guard cells that had GFP fluorescence was analysed by fluorescence microscopy. The combination of 650 PSI and 9 cm yielded the best result and was therefore chosen for all experiments.
500
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1500
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2500
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3500
4000
4500
650
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Helium pressure (psi)
Transformed guard cells/leaf
3cm
6cm
9cm
12cm

3. . a b c An (Aperture of non-transformed guard cell) = a/c
Supplementary Fig. S3. Measurement of half aperture sizes for non-transformed and transformed sides of a guard cell of broad bean.
Half aperture width of the non-transformed cell side was represented by a (dotted line). Half aperture width of the transformed cell side was represented by b (broken line). Length of stomatal pore was represented by c (solid line). Stomatal aperture of the non-transformed guard cell (An) was calculated by a/c. Stomatal aperture of the transformed guard cell (At) was calculated by b/c.
An (Aperture of non-transformed guard cell) = a/c
At (Aperture of transformed guard cell) = b/c
a( ) : half aperture of non-transgenic cell side
b( ) : half aperture of transformed cell side
c( ) : length of stomatal pore a b c .