Nature 2003-01109B:
A cell surface receptor mediates extracellular Ca2+ sensing in guard cells.
Shengcheng Han*, Ruhang Tang*, Lisa K. Anderson*, Todd E. Woerner† & Zhen-Ming Pei*
Supplementary Information
Ca2+o may trigger the IP3 signalling pathway in Arabidopsis guard cells
In epidermal peels bathed in 50 M CaCl2, [Ca2+]i increases in guard cells were observed after
addition of 2 mM CaCl2 (Fig. S-1a; control), consistent with previous studies1,2. Note that, a high
concentration of Ca2+o, which may be at the high end of the physiological Ca2+o concentration
range3-5, was used here to maximize the Ca2+o signal. Additionally, as the initial peak of CICI
might well represent the response to a Ca2+o signal and [Ca2+]i oscillations are complex and
longer-lived, we only focused on those initial peaks (here and in Figure 4d) which occur within 5
min after addition of CaCl2. We tested whether blocking phospholipase C (PLC) in the IP3
pathway6,7 could affect CICI in guard cells. Neomycin is widely utilized inhibitor of PLC in
plants8,9. Remarkably, neomycin inhibited Ca2+o-induced [Ca2+]i increases (CICI) in guard cells
(Fig. S-1a, b; P < 0.002), and completely abolished Ca2+o-induced stomatal closure (Fig. S-1c; P
> 0.5). Ca2+ channel blockers verapamil and nifedipine did not inhibit CICI and Ca2+o-induced
stomatal closure (not shown), suggesting that Ca2+o may trigger Ca2+ release from IP3-sensitive
stores in guard cells. In other words, the CICI may represent a Ca2+o-sensing process, and the
corresponding cell surface receptor is probably a Ca2+o-sensing receptor (CAS). Note that, based
on this preliminary pharmacological study we could not exclude the possibilities that the CICI is
due to Ca2+ influx or release from IP3-insensitive stores.
References
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Figure Legends
Figure S-1 Ca2+o induces a [Ca2+]i increase in guard cells and stomatal closure possibly by
activating the IP3 signalling pathway.
a,
The PLC blocker neomycin inhibited Ca2+o-induced [Ca2+]i increases (CICI) in guard
cells. Epidermal peels carrying the Ca2+-indicator cameleon were incubated in a solution
containing 50 M CaCl2 for 2 hr, and then treated with 2 mM CaCl2 at indicated times
(arrows). When used, neomycin (100 M) was added to the solutions. Relative [Ca2+]i is
shown as changes in emission fluorescence ratios (F535/F480)2.
b,
Inhibitory effects of neomycin on CICI in guard cells. Experiments are described as in a
(n = 60 cells). Changes in [Ca2+]i are normalized to that without inhibitor treatment
(control). r.u., relative unit.
c,
Ca2+o-induced stomatal closure was eliminated by neomycin. Intact leaves were floated in
a solution containing 50 M CaCl2 ( Ca2+) with or without neomycin (100 M) under
light for 2 hr. Then 2 mM CaCl2 (+ Ca2+) was added to the solutions to assay stomatal
closing. Stomatal apertures (W/L, width/length) were measured as described10. Data from
six separate experiments and total of 120 stomata are shown.
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