Stable expression of bacterial transporter ArsB attached to SNARE molecule enhances arsenic accumulation in Arabidopsis

Figures & data

Figure 1. ArsB localizes to the endosome, and N- and C-terminal ends of ArsB are exoplasmic

(a) GFP-ArsB was transiently expressed in five-day-old Arabidopsis seedlings of the marker lines, ST-mRFP, mRFP-VHAa1, SNX1-mRFP, and mRFP-AtVAM3; the root elongation zone was observed by confocal microscopy. (b) For the iVenus assay, ciV-ArsB or ArsB-CiV were transiently expressed with RHM1-niV or AALP-niV in the leaves of Nicotiana benthamiana, and then observed by confocal microscopy. Bars = 10 μm. The topology of endosomal ArsB was determined by estimation.

Figure 2. VAMP711-ArsB-GFP (VBG) localizes to the vacuole membrane

(a) VAMP711 protein consists of three conservative domains. (b) VBG was constructed with VAMP711, ArsB, and GFP under 35 s promoter. Magenta: longin domain of VAMP711, green: R-SNARE domain, deep green: transmembrane region, gray: ArsB, grass green: VAMP711, pale green: GFP. (c) VBG was co-expressed with the central vacuole membrane marker (mRFP-AtVAM3) or late endosome marker (SNX1-mRFP) in the Arabidopsis root, and then observed by confocal microscopy. Bars = 10 μm. (d) Size of VBG-localized organelle was measured as the diameter.

Figure 3. N-terminus of VBG is located on the cytosolic side and C-terminus is on the vacuole lumen side

(a) VBG was transiently expressed in Nicotiana benthamiana leaf, which was then treated with the plasma membrane marker FM4-64(b) In the iVenus assay, ciV-VAMP711-ArsB or VAMP711-ArsB-ciV were transiently expressed with RHM1-niV or AALP-niV in the leaves of N. benthamiana, and then observed by confocal microscopy. Bars = 10 μm. The topology of VAMP711-ArsB was determined by estimation. Pale green: Venus, magenta: longin domain of VAMP711, gray: ArsB, grass green: VAMP711, pink: RHM1, cyan: AALP.

Figure 4. Growth assay of VBG plant at various As(III) concentrations

(a) VBG plants were grown on medium containing 0, 5, or 10 mM As(III), and the length of their primary root was measured. Data are shown as the mean ± SD *P < .001 (Student’s t-test; n > 20). Col-0 and VGB plants were grown in control medium for 4 days and then transferred to medium containing 10 μM As(III). After 10 days, the plants were harvested and As concentration was quantified in the shoot (b) and root (c) by ICP-OES. Data are shown as the mean ± SD *P < .001 (Student’s t-test; n > 4).

Figure 5. Various microelement concentrations in lines expressing VBG under As(III) stress

Col-0 and VBG plants were grown in control medium for 4 days and then transferred to medium containing 10 μM As(III). After 10 days, the plants were harvested and concentrations of elements (Ca, Cu, Fe, Mg, Mn, Zn, S, and P) were quantified in the shoot by ICP-OES. Data are shown as the mean ± SD *P < .001 (Student’s t-test; n > 4).