Supplementary Methods for "Sphingolipid signalling in Arabidopsis guard cells
involves heterotrimeric G proteins"
Sylvie Coursol, Liu-Min Fan, Hervé Le Stunff, Sarah Spiegel, Simon Gilroy & Sarah
M. Assmann
Preparation of mesophyll cell protoplasts (MCPs). Solutions used for isolation of
MCPs were as follows. Enzyme solution: 0.5% (w/v) macerozyme R-10 (Yakult
Honsha), 1% (w/v) Onozuka RS cellulase (Yakult Honsha), 0.01% (w/v) pectolyase Y23 (Seishin Pharmaceutical), 0.2% (w/v) BSA (Sigma), and 0.1% (w/v) PVP-40
(Sigma) in basic solution prepared as described1,2, except that sorbitol was added to the
solution at 0.6 M. Gradient solution: 10 mM MES-KOH (pH 5.5), and 0.6 M sucrose.
The abaxial epidermis of each leaf was removed and the leaves were floated on the
enzyme solution. Digestion was performed in a shaking water bath (30 excursions/min)
at 24 ºC for 2.5 h. The suspension was then centrifuged at 150g for 5 min, and the pellet
obtained was resuspended in 5 ml of gradient solution. Five ml of basic solution were
then layered on top. After 5 min of centrifugation at 150g, the interface containing
MCPs was transferred to a new tube, and resuspended in 5 ml of basic solution. The
suspension was then centrifuged, and the final pellet was resuspended in 5 ml of basic
solution and kept on ice in the dark until use.
Stomatal bioassay and electrophysiology. For stomatal opening, changes in stomatal
aperture reflect final minus initial (2.8 ± 0.06 μm) aperture in the dark. For stomatal
closure, changes in stomatal aperture reflect final minus initial (6.5 ± 0.07 μm) aperture.
Values are means ± s.e.m. from three independent experiments; n = 40 per experiment.
For K+ current recordings, the pH of the bath solution was 5.6 and 10 mM Mg ATP (10
mM from a 0.5 mM Mg ATP stock solution in 0.5 M Tris) was added to the pipette
solution. Whole-cell currents were recorded from a 8 s holding potential of –79 mV
with 3.9 s voltage steps from –219 mV to +61 mV in +20 mV increments, 10 min after
achieving whole-cell configuration. Time-activated currents were obtained by
subtracting the instantaneous current at 20 ms from the average steady-state current
between 3.55 and 3.87 s. Slow anion currents were measured under weak cytosolic pH
buffering, as described1, using 0.1 mM Hepes-Tris (pH 7.1) in the pipette solution. The
osmolality of bath and pipette solutions were 485 mmol kg-1 and 500 mmol kg-1,
respectively. Whole-cell currents were recorded from a 12 s holding potential of +30
mV with 50 s voltage steps from –145 mV to +35 mV in +30 mV increments, 12 min
after achieving whole-cell configuration. Steady-state currents were acquired by
subtracting the basal current at a holding potential of +30 mV from the average current
between 42.5 and 50.0 s. Data were simultaneously compared using Scheffe’s method,
and only results with a P-value  0.05 were considered statistically significant.
Preparation of sphingolipid solutions. Sphingosine, D-erythro-dihydrosphingosine,
DL-threo-dihydrosphingosine,
N,N-dimethylsphingosine, sphingosine-1-phosphate, and
D-erythro-dihydrosphingosine-1-phosphate were from
Biomol. For sphingosine kinase
(SphK) assays, sphingoid bases were prepared as complexes with fatty acid free BSA3
(final concentration 0.2 mg ml-1).
Phosphorylation assays. SphK assays4 were performed with leaf lysates. The labelled
lipids in the organic phase were dried under a stream of N2 and resuspended in 4 ml of a
basic aqueous phase [methanol/1 M NaCl/3 N NaOH (1:1:0.1, v/v/v)]. Hydrolysis
buffer [900 l, 200 mM Tris-HCl (pH 7.5), 1.2 M glycine (pH 9.0), 75 mM MgCl2] and
100 units of alkaline phosphatase were added. After 30 min at 37 °C, 80 l of
concentrated HCl were added, and sphingoid bases were extracted with 3 ml of
chloroform. The organic phase was then re-extracted with 3 ml of chloroform. The
aqueous phase fractions were combined, dried under a stream of N2, and then
resuspended in kinase buffer with 0.2 mg ml-1 BSA. Reactions were started by the
addition of [-32P]ATP (10 Ci, 1 mM) containing 10 mM MgCl2, and 10 g of
recombinant human SphK1. After 30 min at 37 °C, 40 l of 2 N HCl and 500 l of
chloroform/methanol/concentrated HCl (100:200:1, v/v/v) were added. The samples
were mixed thoroughly, and 250 µl chloroform and 250 µl 2 M KCl were added. The
labelled lipids in the organic phase were separated by TLC with
chloroform/acetone/methanol/acetic acid/water (10:4:3:2:1, v/v/v/v) and visualized with
a PhosphorImager (Molecular Dynamics Storm).
References
1. Wang, X.-Q., Ullah, H., Jones, A. M. & Assmann, S. M. G protein regulation of ion
channels and abscisic acid signaling in Arabidopsis guard cells. Science 292, 2070-2072
(2001).
2. Pandey, S., Wang, X.-Q., Coursol, S. & Assmann, S. M. Preparation and applications
of Arabidopsis thaliana guard cell protoplasts. New Phytol. 153, 517-526 (2002).
3. Liu, H. et al. Molecular cloning and functional characterization of a novel
mammalian sphingosine kinase type 2 isoform. J. Biol. Chem. 275, 19513-19520
(2000).
4. Olivera, A., Kohama, T., Tu, Z., Milstien, S. & Spiegel, S. Purification and
characterization of rat kidney sphingosine kinase. J. Biol. Chem. 273, 12576-12583
(1998).