1422occasionally as a source of CO2 during studies on chlo-rophyll Xuorescence in guard cells and stomatal closure(Melis and Zeiger 1982; Mrinalini etal. 1982).
Webb and Hetherington (1997) reported that CO2+2-induced increase in cytosolic Ca is in a way similar toABA evoked response. Elevated CO2 levels depolar-ized the membrane potential of guard cells, possibly aresult of an inhibition of the H+-ATPase (Edwards andBowling 1985). We have earlier shown the involvementof plasma membrane calcium channels and protein kin-ases during bicarbonate-induced stomatal closure(Kolla etal. 2004). This paper reports our observationson an increase in H2O2 production in relation to bicar-bonate-induced stomatal closure.
H2O2 production occurs in plant tissues under a vari-ety of abiotic and biotic stimuli. These includeextremes of temperature, UV irradiation, excess exci-tation energy, ozone exposure, phytohormones such asABA or MJ, dehydration, wounding, elicitors andpathogen challenge (Langebartels etal. 2000; Pie etal.2000; MacKerness etal. 2001; Apel and Hirt 2004).Exposure of Vicia faba guard cells to exogenous H2O2elevated the cytosolic calcium and induced stomatalclosure (McAinsh etal. 1996). ABA induced a rapidburst of H2O2 that resulted in stomatal closure in Ara-bidopsis and V. faba (Pei etal. 2000; Zhang etal. 2001).Increase in H2O2 production in guard cells, appears tobe a common event during stomatal closure inresponse to ABA or MJ (Suhita etal. 2004). This is theWrst report on bicarbonate induced H2O2 production inguard cells.
Production of H2O2 can occur via several routes inplant cells, for e.g. NAD(P)H oxidase, lipid peroxida-tion or even photosynthetic electron transport, (Crossand Jones 1986; Neill etal. 2002; Montillet etal. 2004).Zhang etal. (2001) proposed two diVerent sources ofH2O2 in V. faba guard cells in response to ABA, onechloroplastic and another via a plasma membrane-located enzyme (potentially NAD(P)H oxidase). Theelevation of H2O2 in the presence of ABA or MJ hasbeen shown to be mediated through NAD(P)H oxi-dase based on experiments with NAD(P)H oxidaseknock-out mutants as well as the eVects of DPI, aninhibitor of NAD(P)H oxidase (Murata etal. 2001;Kwak etal. 2003; Suhita etal. 2004). The role ofNAD(P)H oxidase was therefore evaluated by usingthe Arabidopsis double mutant, deWcient in NAD(P)Hoxidase (Kwak etal. 2003).
The NADPH oxidase can be activated by phosphati-dylinositol 3-phosphate (PI3P) (Ellson etal. 2001). Theoccurrence of a type III PI3-kinase activity and PI3P inV. faba guard cells and the suppression of calciumwaves by wortmannin and LY 294002 (inhibitors of
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PI3P) suggest an important role of PI3P during ABA-induced stomatal closure (Pei etal. 2000; Jung etal.2002). Moreover, it has been previously reported inanimal cells that PI3P activates H2O2 generation.We, therefore, studied the role of NAD(P)H oxidaseas well as PI3P during bicarbonate induced H2O2production.
Materials and methodsPlant material
The wild-type Arabidopsis thaliana (ecotype Colum-bia) or atrbohD/F mutant plants (Columbia, Kwaketal. 2003) were sown in a 1:1:1 mixture of vermiculite,perlite and soilrite in plastic disposable cups and keptat 4°C in dark for 3days before transferring to growthroom maintained at 20–22°C. The plants were grown at8h light (225–250 molm¡2s¡1) and 16h dark regime.Nutrient solution was supplied daily up to three weeksand then once every week.Bioassays using epidermal strips
The abaxial (lower) epidermis was peeled oV from theleaves of Arabidopsis and cut into strips of ca. 0.4cm2.About 30 epidermal strips were transferred to 3cmdiameter petri dishes containing 3ml of 25mM MES-KOH, pH 7.0, 10mM KCl and other test compounds.The epidermal strips were kept in light Wrst for 2hunder conditions promoting stomatal opening. Furtherthe epidermal strips were incubated under a bank oftungsten lamps, whose light was Wltered through awater jacket, providing 300 molm¡2s¡1 of white light.The epidermal strips incubated along with pharmaco-logical compounds were irradiated with white light for3h. Photon Xux was measured with a Li-Cor quantumsensor (Li-Cor Instruments Ltd., Lincoln, NE, USA).All the experiments were performed at room tempera-ture (25§2°C).
The width of the stomatal aperture was measuredunder a research microscope (Nikon, Eclipse TE 200,Tokyo) with the help of a precalibrated ocular micro-meter. Twenty apertures at random were monitored ineach of Wve epidermal strips from each treatment.Thus, each value of stomatal aperture is an average of100 measurements.
Monitoring H2O2 production in guard cells
Hydrogen peroxide production in guard cells wasmonitored using 2 ,7 -dichloroXuorescein diacetate