Supplementary Data
Central functions of bicarbonate in S-type anion channel activation and OST1
protein kinase in CO2 signal transduction in guard cells
Shaowu Xue1,3,4, Honghong Hu1,4, Amber Ries1, Ebe Merilo2, Hannes Kollist2, Julian I.
Schroeder1,*
1
Division of Biological Sciences, Cell and Developmental Biology Section, University of
California, San Diego, La Jolla, CA 92093-0116, USA
2
Institute of Technology, University of Tartu, Nooruse 1, 50411 Tartu, Estonia
3
Institute of Molecular Science, Shanxi University, Taiyuan, 030006, China
*Corresponding author: Division of Biological Sciences, Cell and Developmental Biology
Section, University of California, San Diego, La Jolla, CA 92093-0116, USA. Tel: + 1 858 534
7759; Fax: + 1 858 534 7108; E-mail: jischroeder@ucsd.edu
4
These authors contributed equally to this work.
1
Supplementary Table Ι.
Cytosolic free [Ca2+]i and free [HCO3-]i activation of anion currents at a voltage of -146 mV.
[Ca2+]i
(µM)
[HCO3]i
(mM)
I (pA)
at -146 mV
0.15
0
-16.4 ± 2.0a
2
0
-15.6 ± 4.0b
0.76 (b vs. a)
6
0.15
11.5
-22.3 ± 2.3c
0.071 (c vs. a)
7
2
5.75
-21.8 ± 3.2d
0.054 (d vs. b)
7
2
11.5
-58.7 ± 5.9e
< 0.001* (e vs. b)
10
P value
Cell number
5
0.056 (f vs. a)
7
0.35 (f vs. c)
a-f
Current values from Figure 4G for comparison. Data are mean ± s.e. *Stands for significant
difference using Student’s t-test.
0.6
11.5
-27.3 ± 4.5f
2
Supplementary Figures
Figure S1. No large S-type anion currents were activated by extracellular application of
with bicarbonate. (A) Whole-cell currents recording in Col-0 wild type guard cells (n = 6). The
bath solution contained 30 mM CsCl, 2 mM MgCl2, 1 mM CaCl2 and 10 mM Mes/Tris, pH 5.6.
The pipette solution contained 150 mM CsCl, 2 mM MgCl2, 6.7 mM EGTA, 6.03 mM CaCl2 (2
µM [Ca2+]i), 5 mM Mg-ATP, 5 mM Tris-GTP, 1 mM HEPES/Tris, pH 7.1. Liquid junction
potential was -1 mV. (B) Whole-cell recording of guard cells perfused with total 13.5 mM
bicarbonate-containing solution (11.5 mM free HCO3- and 2 mM CO2) at pH 7.1. The other
components of the bath were 30 mM CsCl, 2 mM MgCl2, 1 mM CaCl2 and 10 mM HEPES/Tris,
pH 7.1. Bath volume was 200 µl and perfused for 2 min at 1 ml/min. n = 6. Liquid junction
potential was -2 mV. (C) Steady-state current-voltage relationships of whole-cell currents as
shown in (A) and (B). At a voltage of -144 mV, the control (background) current was -13 ± 5 pA
(n = 6), and the current was -17 ± 5 pA in a bicarbonate-containing solution (n = 6), P > 0.05.
3
Figure S2. Reversal potential of S-type anion currents activated by 50 mM total
bicarbonate added to the pipette solution. (A) Typical recording of S-type anion currents
activated by intracellular 50 mM total bicarbonate. 50 mM total bicarbonate at pH 7.1 equivalent
to 43.4 mM free [HCO3-]i and 6.6 mM [CO2] was calculated using the Henderson-Hasselbalch
equation as described in the Methods. (B) Steady-state current-voltage relationship showed
reversal potential of S-type anion currents at + 26.0 ± 0.9 mV (n = 4). Data are mean ± s.e.
Liquid junction potential was + 3 mV.
4
Figure S3. Extracellular pH shifts cause measurable intracellular pH changes in guard cells.
Fluorescence ratio time series of guard cells from another transformed line expressing pH
sensitive reporter Pt-GFP during extracellular perfusion with buffers of different pH as indicated
by the top bar (See also Figure 2D). GC denotes ratiometric fluorescence in guard cells and the
ratio of non-guard cell background fluorescence (bg) is shown for the same experiments.
5
Figure S4. CO2-induced stomatal closure in ost1 and pyr1;pyl1;pyl2;pyl4 quadruple mutant
mutants. (A) Stomatal conductance responses to [CO2] in ost1-3 mutant and Col-0 wild type
intact leaves (n = 4 for each genotype). (B) Stomatal conductance in responses to [CO2] changes
in intact ost1-3 and Col-0 wild type plants (n = 8 for ost1-3, n = 6 for WT). (C) Stomatal
conductance in responses to [CO2] changes in intact ost1-1, ost1-2 and Ler wild type plants (n =
4 for each genotype). Data shown in Figure 7B, C and D were normalized in (A), (B) and (C),
respectively. (D) Stomatal conductance in responses to [CO2] changes in pyr1;pyl1;pyl2;pyl4
mutant and Col-0 wild type intact leaves (n = 4 for each genotype). Data shown in Figure 8A
were normalized in (D). Imposed CO2 concentrations are shown at the bottom. Data are mean ±
s.e.
6
Supplementary Methods
Solutions for patch clamp experiments
For analyses of S-type anion currents, the pipette solution contained 150 mM CsCl, 2 mM MgCl2,
6.7 mM EGTA, 2.61 mM CaCl2 (150 nM [Ca2+]i), 4.84 mM CaCl2 (0.6 µM [Ca2+]i), or 6.03 mM
CaCl2 (2 µM [Ca2+]i), 5 mM Mg-ATP, 5 mM Tris-GTP, 1 mM HEPES/Tris, pH 7.1. For
experiments analyzing effects of protons on S-type anion currents, the pipette solution contained
150 mM CsCl, 2 mM MgCl2, 6.7 mM EGTA, 0.6 mM CaCl2 (2 µM [Ca2+]i), 5 mM Mg-ATP, 5
mM Tris-GTP, 1 mM MES/Tris, pH 6.1. For experiments with pipette solution at pH 7.8, the
pipette medium contained 150 mM CsCl, 2 mM MgCl2, 2 µM free [Ca2+]i, 5 mM Mg-ATP, 5
mM Tris-GTP, 1 mM HEPES/Tris. Calcium affinities of EGTA and free Ca2+ concentrations
were
calculated
using
the
WEBMAXC
tool
(http://www.stanford.edu/~cpatton/webmaxc/webmaxcE.htm), which considers pH, [ATP] and
ionic conditions. The bath solution contained 30 mM CsCl, 2 mM MgCl2, 5 mM CaCl2 and 10
mM Mes/Tris, pH 5.6. Osmolalities of all solutions were adjusted to 485 mmol·kg-1 for bath
solutions and 500 mmol·kg-1 for pipette solutions by addition of D-sorbitol.
Stomatal conductance measurements
Stomatal conductance measurements of 5-week-old plants in response to the imposed [CO2] at a
light (PAR) fluence rate of 150 µmol m-2 s-1 were conducted with a Li-6400 gas exchange
analyzer with a fluorometer chamber (Li-Cor Inc.) as described previously (Hu et al, 2010).
Relative stomatal conductance values of intact leaves were calculated by normalization relative
to 365 or 400 ppm just before transition to 800 ppm [CO2]. Data shown are mean  s.e. of at
least 3 leaves per genotype in the same experimental set.
For whole-plant gas-exchange experiments, 24 to 26-day-old plants were used. Plants
were grown in pots as described previously (Kollist et al, 2007). For monitoring CO2-induced
changes in whole-plant stomatal conductance, a custom made device for Arabidopsis wholeplant gas-exchange measurements was used (Kollist et al, 2007). Before application of different
CO2 treatments, plants were acclimated in the measuring cuvettes for at least 1 h (Vahisalu et al,
2008). Experiments were performed at photosynthetic photon flux density of 150 ± 3 µmol m -2 s1
, relative humidity of 60-70% (vapour pressure deficit = 0.9-1.2 kPa) and air temperature of 247
25 ºC. Photographs of plants were taken before the experiment and rosette leaf area was
calculated using ImageJ 1.37v (National Institutes of Health, USA). Stomatal conductance for
water vapour was calculated as described previously (Kollist et al, 2007; Vahisalu et al, 2008).
Data were normalized relative to the stomatal conductance at 400 ppm [CO2] just before the
transition to 100 ppm [CO2].
Stomatal aperture measurements
Three to 4-week-old plants grown in a plant growth chamber were used for analyses of stomatal
movements in response to ambient and elevated [CO2]. Intact leaf epidermal layers with no
mesophyll cells in the vicinity and ambient or high [CO2] (800 ppm) incubation buffers were
prepared as described (Hu et al., 2010; Young et al., 2006). Leaf epidermal layers were preincubated for 1.5 h in a buffer containing 10 mM MES, 10 mM KCl, 50 µM CaCl2 at pH 6.15
and then perfused with incubation buffers continually bubbled with ambient air or 800 ppm CO2
for 30 min. For stomatal movement analyses in pyr1;pyl1;pyl2;pyl4 quadruple mutant plants,
individual stomata were imaged and individually tracked as previously reported (Siegel et al,
2009). Stomatal apertures were measured using ImageJ software and analyzed. Data shown are
from genotype blind analyses (n = 3 experiments, 40 stomata per experiment and condition).
Supplementary Reference
Hu H, Boisson-Dernier A, Israelsson-Nordstrom M, Bohmer M, Xue S, Ries A, Godoski J, Kuhn
JM, Schroeder JI (2010) Carbonic anhydrases are upstream regulators of CO 2-controlled
stomatal movements in guard cells. Nat Cell Biol 12: 87-93
Kollist T, Moldau H, Rasulov B, Oja V, Ramma H, Huve K, Jaspers P, Kangasjarvi J, Kollist H
(2007) A novel device detects a rapid ozone-induced transient stomatal closure in intact
Arabidopsis and its absence in abi2 mutant. Physiol Plantarum 129: 796-803
Siegel RS, Xue S, Murata Y, Yang Y, Nishimura N, Wang A, Schroeder JI (2009) Calcium
elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of
stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type
anion and inward-rectifying K+ channels in Arabidopsis guard cells. Plant J 59: 207-220
Vahisalu T, Kollist H, Wang YF, Nishimura N, Chan WY, Valerio G, Lamminmaki A, Brosche
M, Moldau H, Desikan R, Schroeder JI, Kangasjarvi J (2008) SLAC1 is required for plant
guard cell S-type anion channel function in stomatal signalling. Nature 452: 487-491
8
Young JJ, Mehta S, Israelsson M, Godoski J, Grill E, Schroeder JI (2006) CO2 signaling in guard
cells: calcium sensitivity response modulation, a Ca2+-independent phase, and CO2
insensitivity of the gca2 mutant. Proc Natl Acad Sci USA 103: 7506-7511
9