Ethylene-induced flavonol accumulation in guard cells suppresses reactive oxygen species and moderates stomatal aperture

Abstract

Guard cell swelling controls the aperture of stomata, pores that facilitate gas exchange and water loss from leaves. The hormone abscisic acid (ABA) has a central role in regulation of stomatal closure through synthesis of second messengers, which include reactive oxygen species (ROS). ROS accumulation must be minimized by antioxidants to keep concentrations from reaching damaging levels within the cell. Flavonols are plant metabolites that have been implicated as antioxidants; however, their antioxidant activity in planta has been debated. Flavonols accumulate in guard cells of Arabidopsis thaliana, but not surrounding pavement cells, as visualized with a flavonol-specific dye. The expression of a reporter driven by the promoter of CHALCONE SYNTHASE, a gene encoding a flavonol biosynthetic enzyme, in guard cells, but not pavement cells, suggests guard cell-specific flavonoid synthesis. Increased levels of ROS were detected using a fluorescent ROS sensor in guard cells of transparent testa4-2, which has a null mutation in CHALCONE SYNTHASE and therefore synthesizes no flavonol antioxidants. Guard cells of transparent testa4-2 show more rapid ABA-induced closure than the wild type, suggesting that flavonols may dampen the ABA-dependent ROS burst that drives stomatal closing. The levels of flavonols are positively regulated in guard cells by ethylene treatment in the wild type, but not in the ethylene-insensitive2-5 mutant. In addition, in both ethylene-overproducing1 and ethylene-treated wild-type plants, elevated flavonols lead to decreasing ROS and slower ABA-mediated stomatal closure. These results are consistent with flavonols suppressing ROS accumulation and decreasing the rate of ABA-dependent stomatal closure, with ethylene-induced increases in guard cell flavonols modulating these responses.

Figure 1.

Flavonols accumulate in guard cells. A, Confocal micrograph showed yellow DPBA fluorescence in guard cells, but not pavement cells. B, DIC image overlaid on a confocal micrograph of wild-type leaves showing the location of yellow DPBA fluorescence. C, Bright-field image shows CHS:GUS expression in guard cells, but not pavement cells of wild-type plants. Bar = 15 μm.

Figure 2.

Flavonol accumulation is absent in tt4 guard cells and enhanced in eto1 guard cells. DPBA bound to flavonols is shown in yellow and chlorophyll autofluorescence is shown in blue. Bar = 15 μm.

Figure 3.

A, Subcellular flavonol accumulation in wild-type guard cells compared with eto1 and tt4. Flavonol accumulation was measured using guard cell DPBA fluorescence intensity values and the average ± se is reported relative to the levels in the cytosol of Col-0 for n = 90 stomata. Asterisks indicate significant differences (P < 0.005) between the mutant and the wild type within cellular location. Number signs indicate significant differences (P < 0.02) between cytosol and nucleus within genotype. B, LC-MS analysis of flavonol levels in whole leaves showed enhanced accumulation of quercetin and kaempferol in eto1 leaves compared with the control (P < 0.003 and P < 0.06, respectively). No flavonols were detected in tt4. Data represent results from three separate experiments each with n = 6. ND, Not detected because compound is either absent or below the level of threshold detection.

Figure 4.

Stomatal aperture of Col-0 and tt4 in response to ABA. ABA sensitivity is indirectly proportional to relative flavonol concentration. Guard cells were incubated under white light in a 20-μM ABA solution for 0, 45, 90, and 180 min. The average ± se of 90 stomata from three biological replicates are reported. Asterisks indicate significant differences (P < 0.05) between the mutant and Col-0 at each time point as determined by the Student’s t test.

Figure 5.

Subcellular flavonol accumulation in Col-0 and ein2-5 guard cells with and without ethylene treatment. Intact soil grown plants were incubated in ethylene gas at 5 μL/L concentration for 24 h. DPBA fluorescence intensity values were quantified and are reported relative to the untreated Col-0 cytosol fluorescence intensity. Asterisks indicate significant differences (P < 0.02) between the mutant and the wild type within treatment. Number signs indicate significant differences between treated and untreated controls within a genotype as determined by the Student’s t test (P < 0.02). The average ± se of 90 stomata from three biological replicates are reported.

Figure 6.

A, Confocal micrographs of H2DCF-DA-stained guard cells of 4-week-old Col-0, tt4, and eto1 plants. DCF fluorescence is shown in green, and chlorophyll autofluorescence is shown in blue in separate channels and merged images captured under identical confocal settings. B, Quantification of subcellular DCF fluorescence in guard cells. Leaf peels of 3- to 4-week-old plants were stained with 2.5 μM H2DCF-DA for 30 min and imaged using confocal microscopy. DCF intensity values in the cytosol and nucleus of guard cells were determined and are reported relative to the levels in the cytosol of Col-0. The average ± se of 90 stomata from three biological replicates are reported. C, Quantification of subcellular DCF fluorescence in guard cells. Data from two experiments are combined and the average ± se of 60 stomata are reported. Asterisks indicate significant differences (P < 0.005) between the mutant and the wild type within treatment. Number signs indicate significant differences (P < 0.05) between treated and untreated controls within a genotype as determined by the Student’s t test. Bar = 15 µm.

Figure 7.

Stomatal aperture widths in response to ABA. ABA sensitivity is indirectly proportional to relative flavonol concentration. Guard cells were incubated under white light in a 20-μM ABA solution for 0, 45, and 90 min. The average ± se of 90 stomata from three biological replicates are reported. Asterisks indicate significant differences between samples and untreated Col-0 using a Student’s t test (P < 0.05).

Figure 8.

A proposed model of the effects of ethylene and flavonols on stomatal closure. ABA is released into the cytosol where it triggers respiratory burst oxidases, which induce a burst of ROS, which act as secondary messengers to signal stomatal closure. Ethylene induces flavonols accumulation in guard cells through EIN2. Flavonols act as antioxidants to scavenge ROS and thereby inhibits stomatal closure. In the absence of flavonols, ethylene induced ROS accumulation after 24 h, but not after 3 h of treatment. Similarly, 24 h of ethylene treatment affected stomatal aperture in tt4, whereas 3 h of ethylene treatment did not. The effects of ethylene observed at 24 h are indicated with dashed lines. Arrows represent positive effects and bars represent negative effects.