Fig 2. Subcellular localization of choline transporter-like 1 (CTL1).

(A) CTL1- enhanced green fluorescent protein (EGFP) signals in root cortex cells of CTL1-EGFP transgenic plants expressing CTL1-EGFP in the ctl1 mutant background. Bar = 5 μm. (B–D) Comparison of CTL1-EGFP (green) signal with subcellular markers tagged with monomer red fluorescent protein (mRFP) or mCherry (red). Bar = 2 μm. (B) CTL1-EGFP is largely colocalized with the trans-Golgi network/early endosome (TGN/EE) marker, vacuolar proton ATPase subunit VHA-a isoform 1 (VHA-a1)-mRFP. Arrowheads and arrows indicate overlapping and nonoverlapping signals, respectively. (C) CTL1-EGFP is localized to distinct compartments from Syntaxin of plants 32 (SYP32)-mCherry. (D) Partial colocalization of CTL1-EGFP with Arabidopsis Rab homolog F2A (Rha1)-mCherry. Arrowheads indicate overlapping signals. (E) Root cortex cells labeled with FM4-64 (red) and CTL1-EGFP (green). The image was captured within 5 minutes of incubation with 4 μM FM4-64. Bars = 5 μm. (F) Colocalization of CTL1-EGFP and VHA-a1-mRFP in root cortex cells treated with brefeldin A (BFA). (G) CTL1-EGFP and SYP32-mCherry are not colocalized in BFA-treated cells. The images in (F) and (G) were captured after the roots were treated with 100 μM BFA for 60 minutes. Bars = 2 μm. (H) CTL1-EGFP signals in root cells after they were treated with 33 μM wortmannin (Wm) for 60 minutes. White arrow shows the ring-like structure. Bars = 2 μm. (I–K) Colocalization analysis of CTL1-EGFP with VHA-a1-mRFP (I), SYP32-mCherry (J), or Rha1-mCherry (K) using the ImageJ software with the colocalization finder plugin. The linear Pearson correlation coefficient (r_p) indicates the percentage of signal overlap, and an r_p value of 1.0 represents 100% colocalization. (L) The colocalization percentage of CTL1-EGFP with VHA-a1-mRFP, SYP32-mCherry, or Rha1-mCherry. Data are mean ± SD. Six images acquired from 3 roots were used. The raw data for panel L can be found in S1 Data.