Figure 1.
Chlorella-induced stomatal closure in V. faba.
(A) The dosage effect of Chlorella-induced stomatal closure. Epidermal peels of broad beans preincubated for 2 h in opening buffer under light were treated with various concentrations of Chlorella suspension, and stomatal apertures were measured after 2 h. Each bar represents the mean ± SE of three biological replicates (n = 150). (B) Time response course of stomatal closure triggered by Chlorella (1.0×109 ind mL−1) and stomatal apertures were quantified every 20 min. Data are the mean of 60 measurements ± SE from three biological repeats. Solid square: Control, solid circle: Chlorella. Means with different letters are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
Figure 2.
Effects of 20 µM DPI, 2 mM SHAM and 100 UmL−1 CAT on Chlorella-induced stomatal closure.
Epidermal peels of broad beans preincubated for 2 µM DPI, 2 mM SHAM and 100 UmL−1 CAT for 30 min, then, floated on 1.0×109 ind mL−1 Chlorella suspension and stomatal apertures were measured after 2 h. These data are the mean ± SE of the representative results from three biological repeats (n = 150 per bar). Different letters above the bars indicate mean values that are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
Figure 3.
Chlorella induces ROS production in V. faba.
Epidermal peels of broad beans without (control) (A) or with 120 min of treatment with Chlorella alone (B), with Chlorella and DPI (C), with Chlorella and SHAM (D) or with Chlorella and CAT (E) were loaded with 50 µM of H2DCF-DA for 15–20 min in the dark. After a brief wash with opening buffer, photographs were taken for representative pairs of guard cells from each treatment using fluorescence microscopy (A–E). The scale bar in E is 100 µm, and applies to all photographs. (F)The average fluorescence intensity of guard cells in images (A–E). The vertical scale represents percentage response relative to fluorescence value taken as 100% in control treatments. These data are the mean ± SE of the representative results from three biological repeats (100 total guard cells per bar). Different letters above the bars indicate mean values that are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
Figure 4.
The effects of Chlorella on stomatal movement in leave level.
After spraying uniformly different concentrations of Chlorella suspension (1.0×106, 1.0×107, 1.0×108, 1.0×109 and 1.0×1010 ind mL−1) and water onto the leaves of broad bean for 48 h, the epidermal strips were peeled off and immediately observed under a microscope. Photographs were taken for representative pairs of guard cells from each treatment using microscopy (A–F). The scale bar in F is 40 µm, and applies to all photographs. (G) The average stomatal aperture shown in images (A–F). These data are the mean ± SE of the representative results (n = 120 per bar). Different letters above the bars indicate mean values that are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
Figure 5.
The effects of Chlorella on gas exchange in leaves of broad bean.
Alterations in (A) net photosynthetic rate (Pn), (B) stomatal conductance (gs), (C) intercellular CO2 concentration (Ci), (D) transpiration rate (E), and (E) instantaneous intrinsic water use efficiency (WUEi), of broad bean leaves at 48 h after treatment with different concentrations of Chlorella suspension (1.0×106, 1.0×107, 1.0×108, 1.0×109 and 1.0×1010 ind mL−1). These data are the mean ± SE (n = 14 per bar). Different letters above the bars indicate mean values that are significantly different from one another as determined by ANOVA (LSD test, P<0.05).
Table 1.
Photosynthetic activity and chlorophyll content of broad bean leaves after treatment with different concentrations of Chlorella suspension raging from 0 to 1.0×1010 ind mL−1 for 48 h.