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Fig 1
Computationally predicted SLAC1 protein structure and its potential bicarbonate binding sites. (A) Predicted structure of SLAC1 transmembrane domains. The structure was predicted as described using modeling algorithms and coordinates of the TehA template structure (35) and is pseudocolored based on sequence conservation among Arabidopsis SLAC/SLAH homologs and TehA. Blue and red respectively correspond to 17% and 100% conservation with AtSLAC1/SLAH homologous and TehA. (B) The simulation system is shown. The SLAC1 structure was embedded in a POPC lipid membrane using the CHARMM-GUI Membrane Builder. Bicarbonate was added to the model afterward (see Materials and Methods). The final simulation system of SLAC1 had dimensions of 83 × 83 × 105 Å3, with a total of ∼65,000 atoms. (C) Key residues (red) and probability of bicarbonate interaction map (orange). F450 is the proposed gate residue of SLAC1; K255, R256, and R321 are residues predicted to interact with bicarbonate during GaMD simulations; and R432 is the conserved control residue for which bicarbonate is predicted not to bind in simulations. (D) The SLAC1 protein is predicted to form 10 transmembrane α-helices with the N and C termini located in the cytoplasm. Filled red circles indicate the location of amino acids shown in A and C; the open red circle indicates the proposed channel gate residue.
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Fig 2
Impact of R256 mutation on bicarbonate enhancement of SLAC1-mediated currents. (A) Representative whole-cell Cl− current recordings in oocytes coexpressing SLAC1yc or SLAC1-R256Ayc with OST1yn. Currents were recorded in response to 3-s voltage pulses ranging from +40 mV to −160 mV in −20 mV steps, with a holding potential at 0 mV followed by −120 mV after voltage pulses. (B) Mean current–voltage curves of oocytes coexpressing the indicated proteins with or without NaHCO3 injection. (C) Average currents of the indicated SLAC1 isoforms with and without injection of 11.5 mM bicarbonate (14, 22, 29) at −160 mV. The bath solution contained 10 mM MES/Tris (pH = 7.4), 1 mM MgCl2, 1 mM CaCl2, 2 mM HCl, 24 mM NaCl, and 70 mM sodium gluconate. Three independent batches of oocytes showed similar results (n = 9 to 14 oocytes from each oocyte batch per condition). Error bars denote mean ± SEM. Means with letters (a, b, and c) are grouped based on one-way ANOVA and Tukey’s test, P < 0.05.
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Fig 3
Impact of R321 mutation on bicarbonate enhancement of SLAC1. (A) Representative whole-cell Cl− current recordings in oocytes coexpressing SLAC1yc or SLAC1-R321Ayc with OST1yn. Currents were recorded in response to 3-s voltage pulses ranging from +40 mV to −160 mV in −20 mV steps, with a holding potential of 0 mV and return to −120 mV after voltage pulses. The bath solution contained 10 mM MES/Tris (pH = 7.4), 1 mM MgCl2, 1 mM CaCl2, 2 mM HCl, 24 mM NaCl, and 70 mM sodium gluconate. (B) Mean current–voltage curves of oocytes coexpressing the indicated proteins and the indicated HCO3− injection. (C) Average currents of the indicated SLAC1 isoforms with and without injection of bicarbonate at −160 mV. Error bars denote mean ± SEM. Means with different letters are grouped based on one-way ANOVA and Tukey’s test, P < 0.05. Three independent batches of oocytes showed similar results (n = 5 to 11 oocytes from each oocyte batch per condition).
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Fig 4
Expression of SLAC1-R256A in slac1 mutant alleles impairs CO2-regulated stomatal conductance. (A) slac1-1 guard cells are shown expressing the indicated SLAC1-mVENUS fusion isoforms. All images were taken under the same confocal microscopy intensity parameters. (Scale bar: 10 μm.) (B and D) Stomatal conductance of WT and slac1 plants transformed with SLAC1pro::SLAC1 and SLAC1pro::SLAC1-R256A in the slac1-1 background (B) and the slac1-3 background (D) in response to ambient CO2 changes. For B: WT, n = 5; SLAC1pro::SLAC1::mVENUS, n = 5; SLAC1pro::R256A#5::mVENUS, n = 8; SLAC1pro::R256A#7::mVENUS, n = 9; and slac1-1, n = 3. For D: WT, n = 3; SLAC1pro::SLAC1::mVENUS, n = 5; SLAC1pro::R256A#10::mVENUS, n = 3; and SLAC1pro::R256A#11::mVENUS, n = 3). Note that in B, SLAC1pro::R256A#7::mVENUS data points are not clearly visible, as they overlap with data from SLAC1pro::R256A#5::mVENUS. (C and E) Normalized data shown in B and D, respectively. Data were normalized relative to average stomatal conductance during 30 min of 360-ppm CO2 exposure before CO2 elevation. Error bars denote mean ± SEM.
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Fig 5
Effect of point mutations R321, K255, and R432 in SLAC1 on CO2 response in slac1-transformed lines. Stomatal conductance of WT and slac1 transgenic plants expressing the indicated SLAC1 isoforms in the slac1-1 background in response to ambient CO2 changes. (A) SLAC1-R321A (WT, n = 3; SLAC1pro::SLAC1::mVENUS, n = 3; SLAC1pro::R321A#2::mVENUS, n = 4; and SLAC1pro::R321A#8::mVENUS, n = 3). (B) SLAC1-K255A (WT, n = 3; SLAC1pro::SLAC1::mVENUS, n = 3; and SLAC1pro::K255A::mVENUS, n = 3). (C) SLAC1-R432A (SLAC1pro::SLAC1::mVENUS, n = 3; and SLAC1pro::R432A::mVENUS, n = 3). Normalized data are shown Right. Data were normalized relative to the average stomatal conductance during 30 min of 360-ppm CO2 exposure before CO2 elevation. Error bars denote mean ± SEM.
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Fig 6
Point mutation SLAC1-R256A has no effect on ABA-induced stomatal closing in slac1 transgenic lines. Stomatal conductance of detached leaves from 6-wk-old SLAC1pro::SLAC1::mVENUS (n = 3), SLAC1pro::R256A#5::mVENUS (n = 4), SLAC1pro::R256A#7::mVENUS (n = 4), and slac1-1(n = 4) plants in response to 2 μM ABA added to the transpiration stream. Error bars denote mean ± SEM.
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Fig 7
Effect of point mutation SLAC1-R256A on HCO3− activation of S-type anion channel currents in guard cells of slac1-transformed lines. (A) Representative current recordings in whole-cell patch clamp configuration with guard cell protoplasts from 4- to 6-wk-old WT and transformed slac1 lines. (B) Current-voltage relationships of WT and the indicated transformed slac1 lines in the absence (control) or presence of added HCO3− in the cytosol. WT control guard cells and SLAC1-R256A#7 control cells without addition of HCO3− showed only small background currents that overlap with the illustrated controls and are not included as data points in B for clarity. Error bars show SEM. Note that Figs. 7 and 8 share the same control current–voltage curves, as they were completed within the same datasets.
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fIG 8
Mutation of R256A in SLAC1 has no effect on ABA activation of S-type anion channel currents in guard cells of slac1-transformed lines. (A) Representative current recordings in whole-cell configuration with guard cell protoplasts from 4 to 6 wk-old WT and transformed slac1 lines. (B) Current–voltage relationships of WT and the indicated slac1 lines in the absence (control) or presence of 50 µM ABA (+ABA). Note that WT control guard cells and SLAC1-R256A#7 control cells without addition of ABA showed only small background currents that overlap with the illustrated controls and are not included as data points in B for clarity. Error bars show SEM. Note that Figs. 7 and 8 share the same control current–voltage curves, as they were completed within the same datasets.
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