-gfp (a 568) mergea b anti-pma2 anti-gfp - plant cell · clc-gfp localization in naa pre-treated...

Supplemental Figure-1: Subcellular localization of GPI-anchored proteins. (A) Surface immunostaining of BY2 protoplasts expressing GFP-GPI with mouse anti-GFP and Alexa-568 labelled goat anti-mouse. (B) Protein blot probed with anti-PMA2 and anti-GFP antibodies. Total membrane from GFP-GPI expressing BY2 cells was solubilized in Triton X-114 and phase-partitioned into an aqueous fraction that contains peripheral proteins and a detergent phase that contains integral and lipid -anchored proteins. The detergent phase was treated with PI-PLC (+PIPLC) or with buffer (−PIPLC). After digestion, the reaction mixture was phase re-partitioned into detergent and aqueous phases. Total protein from each fraction was precipitated, run on SDS-PAGE and probed with respective antibodies after blotting. (C) GFP-GPI expressing BY2 cells pulsed with 5 µM FM4-64 for 5 min on ice and then chased for 30 min at room temperature. (D) GFP-GPI, BY2 cells pulsed with FM4-64 for 5 min and then treated with 50 μM BFA for one hour. White arrow mark a BFA body. (E) Arabidopsis root epidermal cells expressing ARA7-GFP and mCherry-GPI. Magnified version of boxed area shown in the right. (F) BFA treatment (50 µM, 1h) cause agglomeration of ARA7-GFP and mCherry-GPI (inset shows a BFA body in higher magnification). (G) Arabidopsis root cells expressing SYP32-RFP and GFP-GPI. GFP-GPI vesicles are marked by arrows in the magnified image on the right side. (H) SYP32-RFP, GFP-GPI Arabidopsis roots treated with BFA (50 µM, 1h). Magnified version of the boxed BFA body is shown in the inset on right side. (I-J) BY2 cells double transfected with mCherry-GPI and ARA6-GFP. Control cell (I) and cell treated with 50 μM BFA for one hour (J). White arrow mark a BFA body. (K) GFP-GPI expressing BY2 protoplast transfected with SM1-mCherry. A GFP-GPI vesicle is marked by white arrow. (n, nucleus, Scale bar: 10 µm).

GFP-GPI Anti-GFP (A-568) Merge

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ARA6-GFP mCherry-GPI Merge

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F E ARA7-GFP mCherry-GPI Merge ARA7-GFP mCherry-GPI Merge

G GFP-GPI GFP-GPI Merge Merge SYP32-RFP SYP32-RFP H

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ARA6-GFP mCherry-GPI Merge

Supplemental Data. Baral et al. (2015). Plant Cell 10.1105/tpc.115.00154

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Merge SKU5-GFP FM4-64

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Supplemental Figure 2. Trafficking properties of endogenous GPI-anchored protein SKU5-GFP. (A) Plants expressing functional SKU5-GFP expressed from the endogenous promoter, pulsed with 5 µM FM4-64 for 5 min on ice, washed and then chased for 10 min at room temperature. Arrows indicate co-localization of SKU5-GFP and endocytosed FM4-64. (B) BFA treatment causes aggregation of endocytosed SKU5-GFP together with FM4-64 in epidermal cells (arrows). (C) BFA treatment results in clumping of endocytosed SKU5-GFP in all layers of the root (left). In response to NAA pre-treatment, SKU5-GFP accumulates in BFA bodies only in the epidermal cells (right, arrows) but not in the internal layers (boxed area). (Scale bar: 10 µm).


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Supplemental Figure 3. VAEM imaging of CLC-GFP and mCherry GPI foci on plasma membrane. (A-D) Arabidopsis root epidermal cells expressing CLC-GFP and mCherry-GPI were imaged by VAEM. Snapshots of first frames of VAEM movies for control plants (A) and plants treated with 10 µM NAA (B), 2 mM MβCD (C) or 100 mM NaCl (D). The duration of each treatment was 30 min. (E) Quantification of foci containing only CLC-GFP (green bar), only mCherry-GPI (red bar) and both CLC-GFP and mCherry-GPI (yellow bar). The data is collected form10 images for individual treatments across 3 independent experiments. Error bars represent SE of the mean. Asterisks represent p-value < 0.0005 (Student’s t-test between groups indicated by brackets) (Scale bar: 5 µm)

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Supplemental Figure 4. Effect of NAA on clathrin-dependent endocytosis. (A) CLC-GFP at the PM in the absence (left, arrows) or presence (middle) of 10 µM NAA for 30 min. CLC-GFP localization in NAA pre-treated plants which are subsequently co-treated with 100 mM NaCl for 30 min (right). (B-C) VAEM imaging of CLC-GFP foci on PM of control plant (B) or plant treated with 10 µM NAA for 30 min (C). Image taken at 0 sec (green) and 20 sec (red) is superposed to reveal dynamics of the foci over 20 sec. Encircled area in (B) marks appearance of new foci. No such foci is formed in NAA-treated plant (C). (D) Quantification of CLC-GFP foci abundance on plasma membrane by VAEM imaging. The data are cumulative of two independent experiments with a total of 10 images quantified for each group. Error bars represent SE of the mean. Asterisks represent p-value < 0.0001 (Student’s t-test). (E) BFA induced aggregation of endocytosed PIN2-GFP, PIN1-GFP and LTI6b-GFP (left) is blocked by NAA pre-treatment (right). (Scale bar: 10 µm).

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Supplemental Figure 5. Effect of NAA on LTI6b-GFP and FM4-64 uptake. Uptake and clumping of LTI6b-GFP and FM4-64 in response to BFA treatment (A) or NAA + BFA treatment following NAA pre-treatment (B). In presence of NAA pre-treatment, FM4-64 containing BFA bodies are found only in the epidermal layer (White arrow). (Scale bar: 10 µm)

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Supplemental Figure 6. NAA blocks endocytosis in stele cells. (A) ARA7-GFP plants pulsed with 5 µM FM4-64 for 30 min and chased for 1h. (B) Uptake and clumping of ARA7-GFP and FM4-64 in response to BFA treatment (50 µM, 1h). Both ARA7-GFP and FM4-64 are present in BFA bodies. In plants pre-treated with NAA (10 µM, 30 min), ARA7-GFP containing BFA bodies still formed but they did not contain any internalized FM4-64 (C). (D) WT plants pulsed with 5 µM FM4-64 for 30 min. Internalization of FM in punctate structures can be seen in epidermis (green arrows) and stele (red arrows- dashed box). (E) In NAA pre-treated plants uptake is still seen in epidermis (green arrows) but no uptake is seen in stele. (Scale bar: 10 µm)

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Supplemental Figure 7. Endocytosis in the presence of Tyr-A23. (A) PIN2-GFP seedlings treated with 50 µM BFA alone (left) or along with 30 µM Tyr-A23 pre-treatment (right). (B) Dark treatment (6 h) of PIN2-GFP seedlings in absence (left) and in presence of Tyr-A23 (right ). Arrows indicate vacuolar accumulation of PIN2-GFP. (C) Quantification of vacuolar (left) and PM associated PIN2-GFP fluorescence in dark-incubated control plants or plants treated with Tyr-A23. (D) Endocytosis and BFA induced clumping of GFP-GPI in presence of Tyr-A23 in epidermal cells (left) and stele (right). (E) Endocytosis and dark induced accumulation of GFP-GPI in vacuoles of epidermal cells in absence (left ) or in presence of Tyr-A23 (right ). Arrows indicate accumulation of GFP-GPI in vacuoles. (F) Quantification of vacuolar (left) and PM associated GFP-GPI fluorescence in dark-incubated control plants or plants treated with Tyr-A23. (G) Uptake and clumping of FM4-64 in response to BFA treatment (left) or withTyr-A23 pre-treatment (right). Insets are magnified version of the outlined area in stele. (H-K) Uptake and clumping of LTI6b-GFP and FM4-64 in response to BFA treatment (H and I) or with Tyr-A23 pre-treatment (J and K). (I) and (K) are magnified images of stele region of images (H) and (J). With Tyr-A23 pre-treatment, FM4-64 containing BFA bodies are found across all the layers while uptake and aggregation of LTI6b-GFP is blocked. Formation of LTI6b-GFP and FM4-64 BFA bodies in stele is marked in BFA treated plants (I, green and red arrows). In Tyr-A23 pre-treated plants only FM containing BFA bodies are seen (K, red arrows) (Scale bar 10µm). The data presented in (C) and (F) are from two independent experiments with at least 150 cells quantified per group per experiment. Error bars represent SE of the mean. Asterisks denote p-value <0.0001 (Student’s t-test)

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Supplemental Figure 8. Salt induced bulk flow pathway in Arabidopsis roots. (A) Exposure to 100 mM NaCl induces FM4-64 uptake in internal layers of NAA pre-treated WT roots (right, inset). (B) NaCl treatment induces uptake of PIN2-GFP in epidermal cells of Tyr-A23 pre-treated roots (right, inset). Insets represent magnified versions of boxed areas. (Scale bar: 10 µm).


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Supplemental Figure 9. Salt induced increase of plasma membrane sterol content of internal cells. (A) Filipin staining of PIN1-GFP Arabidopsis root. Control (top) and salt (100 mM, 1 h) treated plant (bottom) stained with 100 µg/ml filipin for 1 h. Stele is marked by the dashed box. (B) Control and salt treated plants stained with 5 µM FM4-64 for 1 h. Stele is marked by the dashed box. (C) Quantification of FM labelling of PM of the stele cells of control and salt-treated plants. The result is cumulative of two independent experiments with mean grey value of a total of 245 ROIs for control and 257 ROIs drawn on PM of salt-treated plants quantified. Error bars represent SE of the mean (p-value is 0.41; Student’s t-test). (Scale bar: 10 µm)

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Supplemental Figure 10. Salt sensitivity associated with sterol biosynthetic defects. (A) Four days old WT and smt2smt3 plants were transferred to plates containing ½ MS medium (top) or ½ MS medium supplemented with 100 mM NaCl (bottom). After 10 d, salt-treated smt2smt3 plants turn yellow, while the WT plants remain relatively unaffected. (B) Relative reduction of fresh weight of WT and smt2smt3 plants (normalized to respective control plants) in response to salt stress. (C-D) FM4-64 uptake (5 µM, 30 min pulse) in epidermal cells of WT and smt2smt3 plants under control and salt-stressed condition. The data are cumulative of two independent experiments with 80 cells analyzed per group per experiment. Error bars represent SE of the mean. Asterisks represent p-value <0.0001 (Student’s t-test). (E) Salt sensitivity (100mM NaCl) of WT plants in absence (left) or in presence of 50µg/ml fenpropimorph (Fen). In presence of fenpropimorph, salt stressed plants die in 4-5 d. (F) Fenproimorph-treated (20 µg/ml) plants grown in absence (top) or in presence (bottom) of 100 mM NaCl for 10 d. (Scale bar: 1 cm in A, E, F and 10 µm in C).

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Supplemental Figure 11. Association of VPS9a-GFP with plasma membrane under salt-stress. (A-B) VPS9a-GFP localization in root epidermal cells in FM4-64 stained control plants (A) or salt-stressed (100 mM, 45 min) plants (B). Inset in (B) is magnified version of the GFP channel image. Arrows indicate PM association of VPS9a-GFP. (C-D) Root epidermal cells of plants expressing VPS9a-GFP and mCherry-GPI. Control plants (C) or salt-stressed (100 mM, 30min) plants (D). Arrows in (D) indicate PM association of VPS9a-GFP. (E) Immunoblotting of total membrane fraction of VPS9a-GFP plants in control or salt-stressed conditions. The blot is probed with Anti-GFP as proxy for VPS9a-GFP and Anti-PM H+ ATPase as loading control. The Anti-GFP recognizes multiple bands in the blot, of which the 80kDa band is of the calculated molecular weight of full length VPS9a-GFP (Arrow). (F) VPS9a-GFP band intensities normalized with PM H+ ATPase band intensity in control and salt-stressed (100 mM, 30min) plants. The data represented are from two independent repeats. (Error bar represents standard deviation). (Scale bar: 10 µm).

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Supplemental Figure 12. Expansion of vacuolar structures in stele in response to salt-stress. Three days old Arabidopsis seedlings expressing SYP22-GFP were incubated either on ½ MS (control) (A) or ½ MS +100 mM NaCl for 24 h (B). In stele of salt treated root, expansion of SYP22-GFP labelled vacuolar structures can be seen. Cell outline is marked by short pulse (15 min) of FM4-64. (C) Quantification of cellular area occupied by SYP22-GFP labelled vacuoles under control conditions or after 24 h salt stress. Bars represent weighted mean of percentage of cellular area occupied by vacuoles ± SE of the mean. Asterisks indicate p-value <0.0001(Student’s t-test). Plotted data is a sum of two independent experiments with at least 150 cells quantified per treatment. (Scale Bar: 10 µm)

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-gfp (a 568) mergea b anti-pma2 anti-gfp - plant cell · clc-gfp localization in naa pre-treated...

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