Fm4 64

Confocal analyses were performed on either an Olympus FV3000 (Tokyo, Japan) or a Zeiss LSM 880 microscope (Oberkochen, Germany). For callose staining, roots were incubated in 150 mM K₂HPO₄ and 0.01% (w/v) aniline blue in the dark for 2 h. For ROS staining, roots were incubated with 10 μM H₂DCFDA (Beyotime Biotech, Shanghai, China) in the dark for 30 min and then washed twice with deionized water. For PI or FM4-64 staining, roots were directly imaged in 10 μM PI or 4 μM FM4-64 (Thermo Fisher Scientific, Waltham, MA, USA). The excitation (ex)/emission (em) parameters for confocal analyses are as follows: GFP/YFP/H₂CDFA ex: 488 nm, em: 500–550 nm; PI/FM4-64 ex: 561, em: 570–670 nm; aniline blue ex: 405 nm, em: 475–525 nm; esculin ex: 405 nm, em: 420–480 nm; and roGFP2-Orp1 ex: 405 and 488 nm in sequence, em: 505–535 nm and 425–475 nm (auto fluorescence recorded at ex: 405 nm for subtraction in calculating the ratio 405/488 nm)²⁹ .

The coding sequences (CDSs) of three CNGC genes were amplified from mature anther cDNA and cloned into pGA3574 vectors fused with C-terminal GFP. All the cloning primers used in the experiments are listed in Supplementary Table S1. For the transfection of these constructs, rice protoplasts from leaf and stem tissue were isolated, as described previously [40 (link)]. The fluorescent tags used to determine the intracellular distribution of proteins were observed using a confocal laser scanning fluorescence microscopy (K1-Fluo; Nanoscope System, Daejeon, Republic of Korea) with 488/510 nm excitation/emission filter sets. To identify the subcellular localization in the cell membrane, the plasma and vacuolar membrane marker FM4-64 (Thermo Fisher Scientific, Waltham, MA, USA) was used; rice protoplasts were immersed in 0.1% FM4-64 solution for 10 min in the dark conditions and observed using the red fluorescence protein channel with 543/560 nm excitation/emission filter sets.

For the evaluation of intracellular localization of the transporters, hmt-1-deleted S. pombe was transformed with pREP1-HMT-1-GFP or ABCB6-GFP. Cells were grown to mid-log phase (A_600nm of 0.5–0.8) and stained with FM 4–64 as described in [58 (link)] with the following modifications. FM 4–64 (T3166 ThermoFischer Scientific Waltham, MA, USA) was dissolved in dimethyl-sulphoxide at a concentration of 1.64 mM. Cells were harvested and incubated with 1-μL FM 4–64 in 50-μL EMM medium at 30 °C for 20 min. An aliquot of 1-mL EMM was added and cells were centrifuged at 5000×g for 5 min at RT. The cell pellet was resuspended in 5-mL EMM, and the suspension was shaken at 30 °C for 90 min. The total volume was transferred to a centrifuge tube and spun for 5 min at 5000×g at RT. The cell pellet was resuspended in 1-mL sterile water, and centrifuged at 5000×g for 5 min at RT. Cells were resuspended in 25-μL EMM. An aliquot of 7 μL was spotted on ConA/polyK-coated (1:1 mixture of 2 mg/mL concanavalin A and 0.1% poly-l-lysine) glass slides covered with an 18 × 18 mm² cover slip. Confocal images were obtained using a LSM 710 confocal laser scanning microscope (Carl Zeiss AG, Oberkochen, Germany) equipped with a Plan-Apochromat 63 ×/1.4 Oil DIC M27 objective. Noise reduction and deconvolution of the images were performed with Huygens Essential (Scientific Volume Imaging B.V.).