Figure S1. A secondary leaf vein crossing the mesophyll tissue. Optical tangential section (bright field image) through Arabidopsis leaf (‘clarified’, i.e., made transparent by soaking in 95% ethanol) showing the vein and the adjacent mesophyll cells. Note the bundle sheath cells (BSCs) tightly enwrapping the dead xylem trachea (XT) elements. Scale bar = 20 μm.

Figure S2. BSCs from main and secondary veins have similar Pf. (a) Time course (1 min) of protoplast swelling upon exposure to hypotonic challenge. Symbols: relative volumes (mean ± SE); arrow: onset of bath flush. Bundle Sheath Cells (BSCs) were isolated from main veins (n = 38) and from secondary veins (n = 19; Materials and Methods). (b) Time course of the osmotic concentration change in the bath (Cout) during the hypotonic challenge. Note the delay between the start of cell swelling and the onset of solution exchange (on average, it was 12.0 ± 1.4 sec (± SE) and 12.6 ± 2.0 sec, in main- and secondary-veins protoplasts, respectively. (c) Mean Pf (±SE) in both cell types. These values of Pf and delay are best-fit parameters obtained from analysis of the time-courses in a and b (see Supplementary Materials and methods). Within the BSCs themselves, those surrounding the leaf midrib vessels (main BSCs) and those surrounding the secondary, tertiary and quaternary) vessels (‘secondary’ BSCs; Materials and methods) did not differ in size, in the number of chloroplasts, or in their Pf and delay values. Therefore, the use of protoplasts mainly from secondary BSCs should not have affected our results and conclusions.

Figure S3. The effect of drought and ABA on the frequency distribution of Pf in protoplasts isolated from Bundle Sheath Cells (BSCs) and Mesophyll Cells (MCs). Plants were subjected to 8–10 days of drought, or protoplasts from well-watered plants were subjected directly to ABA (1 μM for 1-4 h). (a, c), BSCs, (b, d), MCs, (a,b), protoplasts from non-irrigated (‘droughted’) plants, (c, d), Protoplasts from irrigated plants, treated directly by ABA. Note the long tail of the Pf distribution in MCs protoplasts in contrast to the relatively ‘compact’ distribution in BSCs. Note also the leftward shift of the histograms of the treated BSCs. See Table 1 for summaries and statistics of comparisons.

Figure S4. HgCl2 treatment inhibits reversibly the swelling of Bundle Sheath cells (BSCs) protoplasts. (a) Time course of swelling upon exposure to hypotonic challenge (mean volumes ± SE) of control BSC protoplasts (n = 21) and protoplast pretreated (for ≤10 min) with 50 μM HgCl2 (n = 22). Arrows: onset of hypotonic challenge. Note the inhibition of swelling of the HgCl2-pretreated protoplasts. After the first minute of swelling, the cells were flushed with isotonic solution containing 2 mM DTT (No HgCl2) for 10 min followed by (b) a second hypotonic challenge and swelling containing no HgCl2 or DTT (control, n = 6; HgCl2-washed, n = 8) the Pf calculation of these cell present in Figure 4. Note the removal by DTT of the HgCl2 inhibition of swelling.

Figure S5. The effect of HgCl2 on the frequency distribution of Pf in protoplasts isolated from bundle sheath cells (BSCs). (a) Pf distribution of protoplasts from well-watered plants. (b) Pf distribution of protoplasts that were subjected directly to 50 μM HgCl2 (for up to 10 min). (c) Their merged histogram. Note the leftward shift in the histograms of the treated BSCs.

Appendix S1. Can a bundle-sheath cell (BSC) osmotic water permeability (Pf) of ~5 μm sec−1 support water transport though the whole plant?

Appendix S2. Equivalent osmotic permeability of the leaf.

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