荧光分析描述方法的好文章--Hydrogen_peroxide_production_is_an(3)

(H2DCF-DA) as previously described (Murata etal.2001; Suhita etal. 2004). Epidermal strips were stuckon glass cover slips with the help of Telesis V adhe-sive (Premiere Products Inc., Pacaima, CA, USA)and were left in incubation medium containing30mM KCl and 10mM Mes-KOH, pH 6.15 at 22°Cfor 3h under light of 300 molm¡2s¡1. The dye,H2DCF-DA (50 M) was added to the medium at thelast 30min of the 3h illumination period. After20min, the excess of dye was removed by threewashes with distilled water.Other test compounds

When used, wortmannin (10 M) or LY294002(100 M) was added to the incubation medium onwhich epidermal strips Xoated during the last 1h of the3h illumination. CAT (100unitsmL¡1) or DPI(10 M) was added 30min before the addition of thedye to the epidermal strips.Fluorescence microscopy

Guard cells were observed under Xuorescence micros-copy (Eclipse TE 200, Nikon, Japan). The Xuorescenceand bright Weld images of epidermal guard cells wereacquired with a monochrome high-resolution digitalcamera (Coolsnap cf, Roper ScientiWc). Fluorescencelevels in the guard cells were quantiWed with the helpof scientiWc image processing software (IP Labs v 3.51,Scanalytics Inc., USA). In some of the experiments, theXuorescence of guard cells was observed under a LaserScanning Confocal Microscopy (Leica, TCS SP2 withAOBS, Heidelberg GmbH) with the excitation wave-length at 488nm and emission at 525nm.

Solvent eVects and replication

ABA was dissolved in ethanol, whereas H2DCF-DA orDPI was dissolved in DMSO. Necessary controls wereincluded to test the eVect of the solvents mentionedabove. Experiments were repeated 3–5 times on diVer-ent days. The average values §SE are reported.Chemicals

Sodium bicarbonate and KCl are from Merck (India)ltd. MES, ABA, DPI, catalase, H2DCF-DA, wortman-nin and LY294002 were from Sigma Chemicals Com-pany, St Louis, USA.

Results

Stomatal closure by bicarbonate or ABA

The addition of sodium bicarbonate to the incubationmedium elicited interesting responses in stomatalopening (Fig.1). Bicarbonate promoted stomatalopening at low concentrations (<0.5mM), while pro-moting stomatal closure at higher concentration(>2mM). In contrast, ABA promoted stomatal closurein a concentration-dependent manner.

EVects of H2O2 modulators on bicarbonate-induced stomatal closure

Exogenous application of H2O2 also promoted stoma-tal closure (Fig.2). Although the concentrations of10¡4 and 10¡3M H2O2 were eYcient, the eVect was notfully reversible and stomata did not recover completely

Fig.1

EVect of increasing concentrations of bicarbonate andABA on stomatal aperture in abaxial epidermis of Columbia wild(a) or atrbohD/F mutant (b) of Arabidopsis thaliana. Stomatalapertures were measured at the end of 3h incubation. Values are

means of 100 measurements§SE. Errors, if not seen, are withinthe symbols. Averages of at least three experiments conducted ondiVerent days

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