1. Volume 9, Issue 3, Pages 417-427 (March 2016)
Rice Stomatal Closure Requires Guard Cell Plasma Membrane ATP-Binding Cassette Transporter RCN1/OsABCG5 
Shuichi Matsuda, Sho Takano, Moeko Sato, Kaoru Furukawa, Hidetaka Nagasawa, Shoko Yoshikawa, Jun Kasuga, Yoshihiko Tokuji, Kazufumi Yazaki, Mikio Nakazono, Itsuro Takamure, Kiyoaki Kato 
Molecular Plant 
Volume 9, Issue 3, Pages (March 2016)
DOI: /j.molp
Copyright © 2016 The Author Terms and Conditions

2. Figure 1
RCN1/OsABCG5 Is Localized at the Plasma Membrane in the Guard Cells and Mestome Sheath Cells of Vascular Tissue in Leaves.
(A–C) The tissue-specific localization of RCN1/OsABCG5. 10-DAG RCN1/OsABCG5Pro::RCN1/OsABCG5ORF::GFP::GUS seedlings were used for the GUS assay. A strong GUS signal was found. (A) Magnified image of the leaf blades. St, stomata; Vas, vasculature. (B) Magnified image of the basal shoot that removed second and third leaves, showing RCN1/OsABCG5-GFP-GUS localized in the vascular tissue, epidermis of a leaf and second and third tiller buds. (T2, second tiller bud; T3, third tiller bud). (C) Cross-sectional image of the basal shoot (B), showing RCN1/OsABCG5-GFP-GUS localized in the vascular tissue and epidermis of a leaf. Epi, epidermis. Scale bar represents 200 μm in (A), (B) and (C).
(D–I) Fourteen-DAG RCN1/OsABCG5Pro::GFP::RCN1/OsABCG5ORF seedlings were used for an anti-GFP immunohistochemical staining assay. GFP-RCN1/OsABCG5: image of Alexa Fluor 555 fluorescence (green pseudo color). Cell walls were stained with fluorescent brightener 28 (blue pseudo color). The fluorescence signal was observed using confocal microscopy. (D) GFP-RCN1/OsABCG5 localized at the stomata in the abaxial side of the third leaf blade. St, stomata. (E) Immunohistochemical staining assay without anti-GFP antibody, as negative control. Scale bar represents 100 μm in (D) and (E). (F) is magnified in (E). GFP-RCN1/OsABCG5 localized at the plasma membrane in the guard cell. GC, guard cell; SC, subsidiary cell. Scale bar represents 5 μm. (G) GFP-RCN1/OsABCG5 localized at the plasma membrane in the mestome sheath cell in the vasculature of the basal shoot. (H) Immunohistochemical staining assay without anti-GFP antibody, as negative control. Scale bar represents 200 μm in (G) and (H). (I) is magnified in (H). GFP-RCN1/OsABCG5 localized at the plasma membrane of mestome sheath cells. BS, bundle sheath; MS, mestome sheath; Xy, xylem; Ph, phloem. Scale bar represents 25 μm.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

3. Figure 2
GFP-RCN1/OsABCG5 Localized at the Plasma Membrane of Guard Cells.
Fourteen-DAG RCN1/OsABCG5Pro::GFP::RCN1/OsABCG5ORF seedlings were used for GFP-RCN1/OsABCG5 and were stained using a mouse anti-GFP monoclonal antibody. Fluorescence from the secondary antibody (Alexa Fluor 555 goat anti-mouse IgG; green pseudo color). P-type H+ ATPase as a plasma membrane marker was stained using rabbit anti-ABA polyclonal antibody. Fluorescence from the secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG; red pseudo color). Cell walls were stained with fluorescent brightener 28 (blue pseudo color). The fluorescence signal was observed using confocal microscopy. Scale bars represent 10 μm. Images were from in-focus images of two selected depths of one guard cell (upper and lower panels).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

4. Figure 3 GFP-RCN1/OsABCG5 and ABA Localization in Guard Cells.
(A) 14-DAG RCN1/OsABCG5Pro::GFP::RCN1/OsABCG5ORF seedlings were used for immunohistochemical staining assays. The fluorescence signal in the abaxial side of the leaf blade was observed using confocal microscopy before ABA treatment (upper panel) and after treatment with 10 μM ABA (lower panel). GFP-RCN1/OsABCG5 was stained using mouse anti-GFP monoclonal antibody. Fluorescence from the secondary antibody (Alexa Fluor 555 anti-mouse IgG; green pseudo color). Scale bars represent 100 μm.
(B) Number of guard cell pairs containing GFP-RCN1/OsABCG5 in the abaxial sides of the third leaf blades of RCN1/OsABCG5Pro::GFP::RCN1/OsABCG5ORF plants after 4 h treatment with 10 μM ABA (n = 6 leaf areas on three plants). Data are presented as mean ± SD, and different letters indicate significant differences (P < 0.05) between time points.
(C) 14-DAG RCN1/OsABCG5Pro::GFP::RCN1/OsABCG5ORF seedlings were used for immunohistochemical staining assays. The fluorescence signal in the abaxial sides of leaf blades was observed before ABA treatment (upper panel) and after treatment with 10 μM ABA (lower panel). ABA was stained using rabbit anti-ABA polyclonal antibody. Fluorescence from the secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG; red pseudo color). Scale bars represent 100 μm.
(D) Number of ABA-localized guard cell pairs in the abaxial sides of the third leaf blades from RCN1/OsABCG5Pro::GFP::RCN1/OsABCG5ORF plants after 4 h treatment with 10 μM ABA (n = 6 leaf areas on three plants). Data are presented as means ± SD, and different letters indicate significant differences (P < 0.05) between time points.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

5. Figure 4
GFP-RCN1/OsABCG5 and ABA Were Co-localized in Guard Cell Pairs.
14-DAG seedlings of RCN1/OsABCG5Pro::GFP::RCN1/OsABCG5ORF were used for immunohistochemical staining assays. The fluorescence signal in the abaxial side of third leaf blades was observed using confocal microscopy. ABA, image of Alexa Fluor 488 fluorescence (red pseudo color). GFP-RCN1/OsABCG5, Alexa Fluor 555 fluorescence (green pseudo color). Cell walls were stained with fluorescent brightener 28 (blue pseudo color).
(A) Localization of ABA and GFP-RCN1/OsABCG5 with or without ABA application. Values indicate the percentage of all observed guard cell pairs.
(B) Localization of ABA and GFP-RCN1/OsABCG5 in guard cell pairs. Merge, image made by combining images of ABA and GFP-RCN1/OsABCG5 (right panel). Scale bars represent 100 μm in the upper panel and 5 μm in the lower panel.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

6. Figure 5
RCN1/OsABCG5 Is Involved in ABA Localization in Guard Cell Pairs.
(A) 14-DAG WT (Shiokari) and rcn1-2 seedlings were used for immunohistochemical staining assays. The fluorescence signal in the abaxial side of the leaf blades was observed using confocal microscopy after ABA application. Anti-ABA, image of Alexa Fluor 488 fluorescence (red pseudo color).
(B) Number of ABA-localized guard cell pairs in the abaxial side of WT and rcn1-2 third leaf blades after treatment with 10 μM ABA (n = 10 leaf areas on three plants).
(C) Expression analysis of RCN1/OsABCG5 in RCN1-RNAi lines. The qRT–PCR products amplified using the primers described in Supplemental Table 1 from the mRNA samples taken from RCN1-RNAi (lane 1) and WT (lane 2) individuals after separation by 2% agarose gel electrophoresis.
(D) 12-DAG WT (Shiokari) and RCN1-RNAi seedlings were used for immunohistochemical staining assays. The fluorescence signal in the abaxial side of the leaf blades was observed using confocal microscopy after application of 10 μM ABA.
(E) Number of ABA-localized guard cell pairs in the abaxial side of WT and RCN1-RNAi third leaf blades after treatment with ABA (n = 10 leaf areas on three plants).
(F) 12-DAG WT and rcn1-2 seedlings were used for immunohistochemical staining assays. The fluorescence signal in the abaxial side of the leaf blades was observed using confocal microscopy after PEG application.
(G) Number of ABA-localized guard cell pairs in the abaxial side of WT and rcn1-2 third leaf blades after treatment with 10 μM ABA (n = 10 leaf areas on three plants).
Data are presented as means ± SD and different letters indicate significant differences (P < 0.05) between time points in (B), (E), and (G). Scale bars represent 100 μm in (A), (D), and (F).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

7. Figure 6 RCN1/OsABCG5 Is Involved in ABA-Induced Stomatal Closure.
(A) Scanning electron microscopy images of three levels of stomata opening. Scale bar represents 5 μm.
(B) The percentage of stomata at each of three levels of opening in WT (Shiokari) and rcn1-2 plants (50 stomata for triplicate replications). Data are presented as means ± SD.
(C and D) Water loss increased in rcn1-2, relative to WT. The ratio of water content (mg) to dry weight (mg) in 10-DAG WT and rcn1-2 mutant plants (C). Water loss from 10-DAG WT and rcn1-2 seedlings at the indicated time points (n = 4, including 10 plants) (D). Data are presented as means ± SD, and different letters indicate significant differences (P < 0.05).
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions

8. Figure 7
RCN1/OsABCG5 Forms a Homodimer and Can Heterodimerize with OsABCG2 and 3.
Yellow fluorescence indicates protein–protein interaction due to assembly of split YFP, as seen using confocal microscopy. Co-transformation of nYFP-OsABCGs and cYFP-OsABCGs into onion epidermis with particle bombardment (A). An empty vector was included as a negative control (B). Transient expression in onion epidermis cells, and yellow fluorescence was observed using confocal microscopy. Scale bar represents 100 μm.
Molecular Plant 2016 9, DOI: ( /j.molp )
Copyright © 2016 The Author Terms and Conditions