Planta (2007) 225:1421–14291427
Fig.8
Suppression by wortmannin or LY294002 of bicarbonateinduced H2O2 in guard cells of Arabidopsis. The pattern of H2O2production was monitored either in the absence (a–c) or presence(d–f) of 2mM bicarbonate. The inclusion of wortmannin (e) or
LY294002 (f) suppressed the rise in bicarbonate-induced H2O2?levels, but had no discernible eVect in the absence of bicarbonate(c, d). Bar represents 25 m
clear. There is very limited information on short-termresponses to CO2 of stomata. We report for the Wrsttime that bicarbonate causes a marked increase inH2O2 levels in guard cell, like in case of ABA treat-ment. The involvement of H2O2 during bicarbonate-induced stomatal closure is demonstrated by severallines of evidence: production of H2O2 in guard cells, onexposure to bicarbonate (Fig.5); partial prevention ofbicarbonate-induced stomatal closure and H2O2 pro-duction by catalase or DPI (Figs.3, 7); and suppressionof bicarbonate eVects in mutants of Arabidopsis, deW-cient in NAD(P)H oxidase and subsequently H2O2production (Figs.1, 7). The production of H2O2, cyto-solic alkalization and the involvement of calcium haveall been shown to be common signaling elements dur-ing stomatal closure caused by ABA or MJ (Pei etal.2000; Zhang etal. 2001; Suhita etal. 2004). This reportadds that marked H2O2 production occurs in guardcells after exposure to bicarbonate.
An increase in H2O2 in guard cells can lead to anincrease in the cytosolic free Ca2+ concentration(Rental and Knight 2004) through the modulation ofCa2+ channels (Webb etal. 1996; Pei etal. 2000). Ourprevious work with calcium channel blockers (Lantha-num) demonstrated the role of plasma membrane Ca2+channels during bicarbonate-induced stomatal closure(Kolla etal. 2004). Such H2O2 production in guard cellsat high CO2 may also result in an inhibition of theinward K+ channels, thus limiting stomatal opening(Zhang etal. 2001).
There have been a few attempts to examine super-oxide production in plants during long-term exposuresto high CO2. However, these are ambiguous with some
reports indicating an increase in the superoxide pro-duction (Liochev and Fridovich 2004; Wang etal.2003), while others reported a reduction (Havir andMcHale 1989; Polle etal. 1993; Schwanz etal. 1996).We could not Wnd information on short-term (withinminutes) responses of plant tissues to elevated CO2.This is the Wrst report on marked increase in H2O2 ofguard cells on exposure to high bicarbonate.Similarities and diVerences between ABA or bicarbonate eVects
There are some similarities and diVerences betweenABA and bicarbonate induced stomatal closure. Thestomatal closure induced by either bicarbonate orABA were signiWcantly reversed by DPI (NAD(P)Hoxidase inhibitor). However, the presence of externallyadded H2O2 decreased the stomatal opening, even inthe presence of either ABA or bicarbonate. Similarly,calcium channels play an important role during theaction of bicarbonate (Kolla etal. 2004) and ABA onstomata (Pei etal. 2000). In contrast to these similari-ties, the extent of stomatal closure induced by ABAwas much stronger than that with bicarbonate. Also,bicarbonate had a biphasic eVect, while promotingslightly the stomatal opening at low concentrations andinhibiting at high concentrations (Fig.1).
The amount of H2O2 production by ABA was muchmore than that with bicarbonate (Fig.5). This could beone of the reasons for the strong eVect of ABA on sto-mata. Further, there was a diVerence in the location ofH2O2 production in response to ABA or bicarbonate.Cytosolic alkalization of the guard cell cytoplasm is a
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