荧光分析描述方法的好文章--Hydrogen_peroxide_production_is_an(5)

Fig.5

The pattern of H2O2 production in guard cells of Arabid-opsis as indicated by the Xuorescence of H2DCF-DA, in the ab-sence (a, d) or the presence of 2mM HCO3 (b, e) or 10 M ABA

(c, f), respectively. The panels a–c are of Columbia wild type andd–f are of Atrboh D/F mutant. Bar

represents 25 m

H2O2 production by ABA was much more than thatwith bicarbonate.

A time-course analysis of H2DCF-DA Xuorescencein guard cells after exposure to bicarbonate or ABArevealed that H2O2 production reached a maximumwithin 3min in ABA treated guard cells (Fig.6). Incase of bicarbonate, H2O2 levels continued to increaseup to about 5min. The presence of DPI prevented par-tially the bicarbonate-induced increase in H2O2 inguard cells (Fig.7). Such H2O2 production in guardcells in response to bicarbonate was strongly decreasedby wortmannin or LY 294002 (Fig.8).

Stomatal responses to bicarbonate in NAD(P)H oxi-dase deWcient mutants of Arabidopsis

The atrbohD/F double mutant of Arabidopsis is deW-cient in H2O2 production as well as ABA–induced

Fig.6Time courses of HCO3 or ABA induced H2O2 pro-duction, in guard cells of

Columbia wild and Atrboh D/F type mutant Arabidopsis, as indicated by H2DCF-DA Xuo-rescence. H2O2 level in the ab-sence of bicarbonate or ABA at the beginning of experi-ment was taken as 100%. Fur-ther details are in Materials and methods. The data are the averages of three independent experiments (§SE)

stomatal closure (Kwak etal. 2003). The stomata ofNAD(P)H oxidase mutant did not close in response tobicarbonate or ABA (Fig.1). Similarly, there was noappreciable H2O2 production on exposure to eitherbicarbonate or ABA in the mutant (Figs.5, 6). Not sur-prisingly, DPI prevented the bicarbonate-induced sto-matal closure in wild but had no eVect in atrbohD/Fmutant (Fig.3). In contrast, H2O2 promoted stomatalclosure in the wild as well as atrbohD/F mutants(Fig.7).

Discussion

It is well known that atmospheric levels of CO2 causestomatal closure (Morison 1987; MansWeld 1990). Thestimulation of stomatal opening at low bicarbonate andmarked inhibition at higher bicarbonate, as observed

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