Effect of exogenous H2O2,Ca2+, and their inhibitors on stomatal aperture
To verify that H2O2, K+, and Ca2+ in subsidiary cells were involved in stomatal movement, the lower epidermis of maize leaves was treated with exogenous H2O2, Ca2+, and their inhibitors prior to scanning electron microscopy observation. Results showed that the treatments promoted or inhibited stomatal opening to varying degrees (Fig. 1). Both H2O2 and Ca2+ inhibited stomatal opening. In particular, exogenous H2O2 resulted in stomatal aperture reaching only 8% of that observed under light conditions, whereas exogenous Ca2+ reduced stomatal opening by 42% compared with the light control (Fig. 2). DPI and LaCl3 inhibited stomatal closure by 72% and 26%, respectively, when switching form light to dark (Fig. 2). These results showed that H2O2, Ca2+, and their inhibitors could regulate stomatal opening and closing.
Effect of exogenous H2O2, Ca2+, and their inhibitors on H2O2 distribution in guard and subsidiary cells during stomatal movement
Under light conditions (i.e., when stomata were open), H2O2 was low and distributed at both ends of guard cells, but it was negligible in subsidiary cells (Fig. 3a). Under dark conditions (i.e., when stomata were closed), H2O2 content augmented in both guard and subsidiary cells, with the latter exhibiting a significantly higher increase (Fig. 3b). A low amount of exogenously added H2O2 under light conditions significantly increased H2O2 in guard and subsidiary cells (Fig. 3c), mimicking the control situation in the dark (Fig. 3b) and promoting stomatal closure(Fig. 1c). DPI was added after 3 h of light and samples were transferred to the dark for 3 h, which significantly reduced H2O2 content in guard and subsidiary cells (Fig. 3d). Guard cells treated with exogenous H2O2 under light conditions exhibited 78.5% higher level of H2O2 than light control (Fig. 4a). In samples exposed to 3 h of light and then treated with DPI for 3 h in the dark, H2O2 amounted to only 18.8% of that in dark control, and 39% of that in guard cells of light control (Fig. 4a). Subsidiary cells treated with exogenous H2O2 under light conditions displayed a 86.7% level of H2O2 in dark control, but only 10.4% of the latter when treated with DPI (Fig. 4b). The difference between treatment and control was significant, indicating that DPI effectively inhibited the production of H2O2.
H2O2 content in guard cells was more than1.7 times as high following CaCl2 treatment than in light control (Fig. 3e, Fig. 4a). Seemingly, H2O2 content in subsidiary cells increased with the addition of CaCl2 (Fig. 4b). This finding indicated a positive correlation between Ca2+ and H2O2 in both subsidiary and guard cells. To further test this relationship in stomatal cells, LaCl3 was added for 3 h under dark conditions after 3 h in the light. Results showed a significant reduction in H2O2 content in guard cells (50.9%) and subsidiary cells (72.8%) compared with dark control (Fig. 3f, Fig. 4). This finding confirmed that H2O2 in subsidiary cells correlated positively with changes in Ca2+.
Effect of exogenous H2O2,Ca2+ and their inhibitors on K+ distribution in guard and subsidiary cells during stomatal movement
The amount of K+ in guard cells regulates cell turgor, which is very important for stomatal opening and closing. As a K+ reservoir, subsidiary cells play an important role in stomatal movement. Under light conditions, K+ could be found mainly in guard cells (Fig. 5a), which absorbed water and expanded to open stomata (Fig. 2). Under dark conditions, K+ flowed out from guard cells and into subsidiary cells (Fig. 5b), which caused the former to lose water and stomata to close (Fig. 2). Under light conditions, K+ content in subsidiary cells was only 26.8% of that in guard cells, whereas in guard cells it was only 19.5% of that in subsidiary cells under dark conditions (Fig. 6). When the epidermis of maize leaves epidermis was treated with H2O2 under light conditions, K+ was distributed mainly in subsidiary cells and less in guard cells (Fig. 5c); with the former accounting for 1.7 times as much K+ as the latter. However, upon DPI treatment for 3 h in the dark following 3 h in the light, K+ was found mainly in guard cells and less in subsidiary cells (Fig. 5d); with the latter accounting for only 36.2% as much K+ as the former (Fig. 6). Even in the presence of light, K+ content in guard cells decreased with addition of exogenous H2O2, while it increased in subsidiary cells, reflecting the situation under dark conditions (Fig. 5b, c). H2O2 inhibition following DPI addition was similar under light settings (Fig. 5a, d). Therefore, H2O2 content correlated negatively with K+ in guard cells, but positively in subsidiary cells.
K+ content in guard cells treated with CaCl2 under light conditions was 71% lower compared to light control, whereas in subsidiary cells it increased by 4.3 times over the control (Fig. 5e, Fig. 6). In addition, when samples were treated with LaCl3 for 3 h in the dark after 3 h in the light, K+ was detected mainly in guard cells, and less in subsidiary cells (Fig. 5f), where it was only half as much as in the former (Fig. 6). These results indicated that Ca2+ content correlated positively with K+ content in subsidiary cells, but negatively in guard cells.
Effect of exogenous H2O2,Ca2+ and their inhibitors on Ca2+ distribution in guard and subsidiary cells during stomatal movement
Ca2+ content in guard cells was 52.4% higher in the dark than under light conditions (Fig. 7a, b, Fig. 8). The change in Ca2+ content was similar to that observed for H2O2 in subsidiary cells, where it was almost undetectable in the light (Fig. 7a), but increased significantly in the dark (Fig. 7b). When exogenous Ca2+ was applied under light conditions, Ca2+ content increased significantly in both cell types (Fig. 7c), becoming about twice as high as in the dark control (Fig. 8). Incubation with LaCl3 for 3 h in the dark after 3 h in the light resulted in only a small amount of Ca2+ was being left in guard cells and almost none in subsidiary cells, which was similar to the light control (Fig. 7d). The difference in fluorescence intensity was only 4% following LaCl3 treatment (Fig. 8), which indicated that exogenous Ca2+ and LaCl3 augmented and reduced, respectively, Ca2+ content in stomata.
H2O2 treatment under light conditions promoted a significant increase in Ca2+ content in both cell types compared with light controls (Fig. 7e, Fig. 8). The amount of Ca2+ in guard cells treated with DPI for 3 h in the dark after 3 h in the light was essentially analogous to that under light control conditions (Fig. 7a, f) with only a 1.9% difference in fluorescence intensity between them (Fig. 8a). In contrast, in subsidiary cells there was no difference at all compared to the light control (Fig. 7a, f, Fig. 8b). These findings indicated that DPI had an inhibitory effect on the increase of Ca2+ in both guard and subsidiary cells. Taken together with the changes in H2O2, the results were point to a positive correlation between Ca2+ and H2O2.