ALA-Induced Flavonols Accumulation in Guard Cells Is Involved in Scavenging H2O2 and Inhibiting Stomatal Closure in Arabidopsis Cotyledons

Abstract

5-aminolevulinic acid (ALA), a new plant growth regulator, can inhibit stomatal closure by reducing H₂O₂ accumulation in guard cells. Flavonols are a main kind of flavonoids and have been proposed as H₂O₂ scavengers in guard cells. 5-aminolevulinic acid can significantly improve flavonoids accumulation in plants. However, whether ALA increases flavonols content in guard cells and the role of flavonols in ALA-regulated stomatal movement remains unclear. In this study, we first demonstrated that ALA pretreatment inhibited ABA-induced stomatal closure by reducing H₂O₂ accumulation in guard cells of Arabidopsis seedlings. This result confirms the inhibitory effect of ALA on stomatal closure and the important role of decreased H₂O₂ accumulation in this process. We also found that ALA significantly improved flavonols accumulation in guard cells using a flavonol-specific dye. Furthermore, using exogenous quercetin and kaempferol, two major components of flavonols in Arabidopsis leaves, we showed that flavonols accumulation inhibited ABA-induced stomatal movement by suppressing H₂O₂ in guard cells. Finally, we showed that the inhibitory effect of ALA on ABA-induced stomatal closure was largely impaired in flavonoid-deficient transparent testa4 (tt4) mutant. In addition, exogenous flavonols recovered stomatal responses of tt4 to the wild-type levels. Taken together, we conclude that ALA-induced flavonol accumulation in guard cells is partially involved in the inhibitory effect of ALA on ABA-induced H₂O₂ accumulation and stomatal closure. Our data provide direct evidence that ALA can regulate stomatal movement by improving flavonols accumulation, revealing new insights into guard cell signaling.

Keywords: 5-aminolevulinic acid (ALA); abscisic acid (ABA); flavonol; hydrogen peroxide; stomatal opening.

FIGURE 1

ALA pretreatment inhibits ABA-induced stomatal closure by reducing H₂O₂ accumulation in guard cells. (A,B) ALA pretreatment inhibits ABA-induced stomatal aperture. Two-week-old wild-type Arabidopsis pretreated with exogenous 0.5 mg L^-1 ALA under light (240 μmol m^-2 s^-1) for 4 h and YHem1-transgenic Arabidopsis incubated under the same light condition for 4 h were immersed in opening buffer (50 mM KCl, 10 mM MES, and 0.1 mM CaCl₂, pH 6.2) alone or containing 10 μM ABA for 2 h under light (240 μmol m^-2 s^-1). The cotyledons were picked with tweezers at 30-min intervals for 2 h for the record of guard cell images and determination of stomatal apertures. (A) Images of guard cell after 1 h of different treatments. Scale bar: 10 μm. (B) Time courses of stomatal responses in WT and YHem1 to different treatments. Values are the means of 90 measurements ± SE from three independent experiments. Different letters on the same time point indicate significant differences at P = 0.05 level. (C,D) ALA pretreatment reduces ABA-induced H₂O₂ accumulation in guard cells. The cotyledons of the above treated seedlings were picked with tweezers at 1 h of treatment and loaded with 50 μM H₂DCF-DA for 30 min in darkness at 25°C. Fluorescence of the cotyledons (C) was observed using a confocal microscope. For each treatment, DCF fluorescence is shown in green and chlorophyll autofluorescence is shown in blue in separate channels. Scale bar: 15 μm. (D) DCF intensity in guard cells of each treatment. Values are the means of 15 measurements ± SE from three independent experiments. Different letters indicate significant differences at P = 0.05 level.

FIGURE 2

ALA pretreatment increases flavonols accumulation in guard cells. Two-week-old wild-type (WT) Arabidopsis pretreated with exogenous 0.5 mg L^-1 ALA under light (240 μmol m^-2 s^-1) for 4 h and YHem1-transgenic Arabidopsis incubated under the same light condition for 4 h were loaded with 2.52 mg mL^-1 diphenylboric acid 2-aminoethyl ester (DPBA) for 30 min in darkness at 25°C. The cotyledons were then picked with tweezers and their fluorescence (A) was observed using a confocal microscope. For each treatment, DPBA bound to flavonols is shown in yellow and chlorophyll autofluorescence is shown in blue in separate channels. Scale bar: 15 μm. (B) DPBA intensity values in guard cells of each treatment. Values are the means of 15 measurements ± SE from three independent experiments. ^∗∗ indicates significant differences between treatments at P = 0.01 level.

FIGURE 3

Flavonols inhibit ABA-induced stomatal closure. (A,B) Exogenous flavonols increase endogenous flavonols accumulation in guard cells. Two-week-old wild-type Arabidopsis were immersed in opening buffer (50 mM KCl, 10 mM MES, and 0.1 mM CaCl₂, pH 6.2) alone or containing 1–100 μM quercetin or kaempferol for 1 h under light (240 μmol m^-2 s^-1) and then loaded with 2.52 mg mL^-1 DPBA for 30 min in darkness at 25°C. The cotyledons were then picked with tweezers and their fluorescence (A) was observed using a confocal microscope. For each treatment, DPBA bound to flavonols is shown in yellow and chlorophyll autofluorescence is shown in blue in separate channels. Scale bar: 15 μm. (B) DPBA intensity in guard cells of each treatment. Values are the means of 15 measurements ± SE from three independent experiments. The same letters indicate no significant differences at P = 0.05 level. (C,D) Flavonols inhibit ABA-induced stomatal closure. Two-week-old wild-type Arabidopsis pretreated with exogenous 10 μM quercetin or kaempferol for 1 h under light (240 μmol m^-2 s^-1) were immersed in opening buffer (50 mM KCl, 10 mM MES, and 0.1 mM CaCl₂, pH 6.2) alone or containing 10 μM ABA for 2 h under light (240 μmol m^-2 s^-1). The cotyledons were picked with tweezers at 30-min intervals for 2 h for the record of guard cell images and determination of stomatal apertures. (C) Images of guard cell after 1 h of different treatments. Scale bar: 10 μm. (D) Time courses of stomatal responses to different treatments. Values are the means of 90 measurements ± SE from three independent experiments. Different letters on the same time point indicate significant differences at P = 0.05 level.

FIGURE 4

Flavonols reduce H₂O₂ accumulation in guard cells. (A,B) Flavonols reduce ABA-induced H₂O₂ accumulation in guard cells. Two-week-old wild-type Arabidopsis pretreated with exogenous 10 μM quercetin (Q) or kaempferol (K) for 1 h under light (240 μmol m^-2 s^-1) were immersed in opening buffer (50 mM KCl, 10 mM MES, and 0.1 mM CaCl₂, pH 6.2) alone or containing 10 μM ABA for 1 h under light (240 μmol m^-2 s^-1), and then loaded with 50 μM H₂DCF-DA for 30 min in darkness at 25°C. The cotyledons were picked with tweezers and their fluorescence (A) was observed using a confocal microscope. For each treatment, DCF fluorescence is shown in green and chlorophyll autofluorescence is shown in blue in separate channels. Scale bar: 15 μm. (B) DCF intensity in guard cells of each treatment. Values are the means of 15 measurements ± SE from three independent experiments. Different letters indicate significant differences at P = 0.05 level. (C,D) Flavonols inhibit H₂O₂-induced stomatal closure. Two-week-old Arabidopsis pretreated with exogenous 10 μM quercetin or kaempferol for 1 h under light (240 μmol m^-2 s^-1) were immersed in opening buffer (50 mM KCl, 10 mM MES, and 0.1 mM CaCl₂, pH 6.2) alone or containing 200 μM H₂O₂ for 2 h under light (240 μmol m^-2 s^-1). The cotyledons were picked with tweezers at 30-min intervals for 2 h for the record of guard cell images and determination of stomatal apertures. (C) Images of guard cell after 1 h of different treatments. Scale bar: 10 μm. (D) Time courses of stomatal responses to different treatments. Values are the means of 90 measurements ± SE from three independent experiments. Different letters on the same time point indicate significant differences at P = 0.05 level.

FIGURE 5

The inhibitory effect of ALA on ABA-induced stomatal closure is impaired in tt4 mutant. (A,B) Flavonols accumulation is absent in tt4 mutant pretreated with or without ALA. Two-week-old wild-type and tt4 Arabidopsis pretreated with exogenous 0.5 mg L^-1 ALA under light (240 μmol m^-2 s^-1) for 2 h or 4 h were loaded with 2.52 mg mL^-1 DPBA for 30 min in darkness at 25°C. The cotyledons were then picked with tweezers and their fluorescence (A) was observed using a confocal microscope. For each treatment, DPBA bound to flavonols is shown in yellow and chlorophyll autofluorescence is shown in blue in separate channels. Scale bar: 15 μm. (B) DPBA intensity in guard cells of each treatment. Values are the means of 15 measurements ± SE from three independent experiments. Different letters indicate significant differences at P = 0.01 level. (C,D) The inhibition of ABA-induced stomatal closure by ALA is impaired in tt4 mutant. Two-week-old wild-type and tt4 Arabidopsis pretreated with exogenous 0.5 mg L^-1 ALA under light (240 μmol m^-2 s^-1) for 4 h were immersed in opening buffer (50 mM KCl, 10 mM MES, and 0.1 mM CaCl₂, pH 6.2) alone or containing 10 μM ABA for 2 h under light (240 μmol m^-2 s^-1). The cotyledons were picked with tweezers at 30-min intervals for 2 h for the record of guard cell images and determination of stomatal apertures. (C) Images of guard cell after 1 h of different treatments. Scale bar: 10 μm. (D) Time courses of stomatal responses in WT and tt4 to different treatments. Values are the means of 90 measurements ± SE from three independent experiments. Different letters on the same time point indicate significant differences at P = 0.05 level. (E,F) The inhibition of ABA-induced H₂O₂ accumulation in guard cells by ALA is impaired in tt4 mutant. The cotyledons of the seedlings treated as shown in Figures C and D were picked at 1 h of treatment and loaded with 50 μM H₂DCF-DA for 30 min in darkness at 25°C. Fluorescence of the cotyledons (E) was observed using a confocal microscope. For each treatment, DCF fluorescence is shown in green and chlorophyll autofluorescence is shown in blue in separate channels. Scale bar: 15 μm. (F) DCF intensity in guard cells of each treatment. Values are the means of 15 measurements ± SE from three independent experiments. Different letters indicate significant differences at P = 0.05 level.

FIGURE 6

Stomatal response of tt4 to ABA is recovered by exogenous flavonols. (A,B) Flavonols accumulation in tt4 is recovered to the WT level by exogenous flavonols. Two-week-old wild-type and tt4 Arabidopsis pretreated with or without exogenous 10 μM quercetin (Q) or kaempferol (K) under light (240 μmol m^-2 s^-1) for 1 h were loaded with 2.52 mg mL^-1 DPBA for 30 min in darkness at 25°C. The cotyledons were then picked with tweezers and their fluorescence (A) was observed using a confocal microscope. For each treatment, DPBA bound to flavonols is shown in yellow and chlorophyll autofluorescence is shown in blue in separate channels. Scale bar: 15 μm. (B) DPBA intensity in guard cells of each treatment. Values are the means of 15 measurements ± SE from three independent experiments. Different letters indicate significant differences at P = 0.01 level. (C) Stomatal response of tt4 to ABA is recovered by exogenous flavonols. Two-week-old tt4 Arabidopsis pretreated with exogenous 10 μM quercetin (Q) or kaempferol (K) under light (240 μmol m^-2 s^-1) for 1 h were immersed in opening buffer (50 mM KCl, 10 mM MES, and 0.1 mM CaCl₂, pH 6.2) alone or containing 10 μM ABA for 2 h under light (240 μmol m^-2 s^-1). The cotyledons were picked with tweezers at 30-min intervals for 2 h for the record of guard cell images and determination of stomatal apertures. Values are the means of 90 measurements ± SE from three independent experiments. Different letters on the same time point indicate significant differences at P = 0.05 level.

FIGURE 7

H₂O₂ accumulation in tt4 under ABA treatment is recovered by exogenous flavonols. Two-week-old tt4 mutants pretreated with or without exogenous 10 μM quercetin (Q) or kaempferol (K) under light (240 μmol m^-2 s^-1) for 1 h were loaded with 50 μM H₂DCF-DA for 30 min in darkness at 25°C. The cotyledons were then picked with tweezers and their fluorescence (A) was observed using a confocal microscope. For each treatment, DCF fluorescence is shown in green and chlorophyll autofluorescence is shown in blue in separate channels. Scale bar: 15 μm. (B) DCF intensity in guard cells of each treatment. Values are the means of 15 measurements ± SE from three independent experiments. Different letters indicate significant differences at P = 0.01 level.