Stomatal Movement Of Vicia Faba Epidermis In The Light / Dark Conditions A Comparative Study

Many researchs had showed that H₂O₂,NO,Ca²⁺ and PK/PP were concerned with guard cell signaling pathway, But it was not clear wether the role of H₂O₂,NO,Ca²⁺ and PK/PP in adaxial and abaxial stomatal movement were the same, and The relationship between Ca²⁺,PK/PP and H₂O₂/NO in adaxial and abaxial guard cells are same or not need to be detected, too. In the present investigation, we used epidermal strip bioassay and laser-scanning confocal microscopy to study and compare the differences of adaxial and abaxial stomatal movement regulated by light/dark in vicia faba. The main results were as followed:1. The apertures of adaxial stomata and abaxial stomata were bigger in light than in dark observably. It showed that dark induced both adaxial and abaxial stomatal closure. the adaxial stomatal aperture are smaller in light but bigger in dark than abaxial stomatal aperture, it implied that the abaixal stomata are more sensitive than the adaxial stomata.2. Both of light/dark-induced adaxial and abaxial stomatal movement were concerned with H₂O₂, the level of H₂O₂ was higher in dark than in light; in dark, the level of H₂O₂ was higher in the abaxial guard cell than the adaxial guard cell.3. Both of light/dark-induced adaxial and abaxial stomatal movement were concerned with NO, the level of NO was higher in dark than in light; in dark, the level of NO was higher in the abaxial guard cell than the adaxial guard cell.4. Both of light/dark-induced adaxial and abaxial stomatal movement were concerned with Ca²⁺, the level of endogenous Ca²⁺ was higher in abaxial guard cells than in adaxial guard cells in dark.5. Both of light/dark-induced adaxial and abaxial stomatal movement were concerned with PK/PP(incIuding MAPK, CDPK, PTP), the activity of MAPK was higher in adaxial guard cells than in abaxial guard cells in dark, but the activity of CDPK and PTP were higher in abaxial guard cells than in adaxial guard cells in dark.6. EGTA,LaCl₃,RR could restrain exogenous H₂O₂-induced stomatal closure obviously, both in adaxial epidermis and in abaxial epidermis. It implied that Ca²⁺ functioned in the downstream of H₂O₂ in light/dark-induced adaxial and abaxial stomatal movement. Exogenous Ca²⁺ reduced stomatal clousure significantly in light, but CAT(H₂O₂ scavenger),Vc(substrate of H₂O₂ removal) and DPI (an inhibitor of superoxide generating NADPH oxidases)couldn't restrain the effect of Exogenous Ca²⁺; the results of endogenous H₂O₂ detection showed that: EGTA(Ca²⁺ chelator), LaCl₃(plasma membrane Ca²⁺ channels inhibitor),RR(endocellular Ca²⁺ channels inhibitor) could restrain dark-induced stomatal closure obviously , but didn't reduce the level of H₂O₂ in guard cells both in adaxial epidermis and in abaxial epidermis. Exogenous Ca²⁺ reduced stomatal clousure significantly in light but didn't improve the level of H₂O₂. Those results proved that Ca²⁺ functioned in the downstream of H₂O₂ in light/dark-induced adaxial and abaxial stomatal movement.7. EGTA,LaCl₃,RR could restrain SNP(exogenous NO donor)-induced stomatal closure obviously, both in adaxial epidermis and in abaxial epidermis. It implied that Ca²⁺ functioned in the downstream of NO in light/dark-induced adaxial and abaxial stomatal movement. Exogenous Ca²⁺ reduced stomatal clousure significantly in light, but c-PTIO(NO scavenger), L-NAME (NO synthase inhibitor)couldn't restrain the effect of exogenous Ca²⁺; the results of endogenous NO detection showed that: EGTA,LaCl₃,RR could restrain dark-induced stomatal closure obviously, but didn't reduce the level of NO in guard cells both in adaxial epidermis and in abaxial epidermis. Exogenous Ca²⁺ reduced stomatal clousure significantly in light but didn't improve the level of NO. Those results proved that Ca²⁺ functioned in the downstream of NO in light/dark-induced adaxial and abaxial stomatal movement.8. PD98059(inhibitor of MAPK),TFP(inhibitor of CDPK),PAO(inhibitor of PTP) could restrain exogenous H₂O₂-induced stomatal closure obviously, both in adaxial epidermis and in abaxial epidermis. the results of endogenous H₂O₂ detection showed that PD98059,TFP,PAO could restrain dark-induced stomatal closure and reduce the level of H₂O₂ in guard cells , also could restrain exogenous H₂O₂-induced stomatal closure and reduce the level of H₂O₂ in guard cells. These results proved that PK/PP could function in the downstream of H₂O₂ in light/dark-induced adaxial and abaxial stomatal movement by adjusting the level of H₂O₂, but it couldn't be excluded that PK/PP could function in the upstream of H₂O₂ by adjusting the synthesizing of H₂O₂.9. PD98059,TFP,PAO could restrain SNP-induced stomatal closure obviously, both in adaxial epidermis and in abaxial epidermis. the results of endogenous NO detection showed that PD98059,TFP,PAO could restrain dark-induced stomatal closure and reduce the level of NO in guard cells, also could restrain exogenous SNP-induced stomatal closure and reduce the level of NO in guard cells. These results proved that PK/PP could function in the downstream of NO in light/dark-induced adaxial and abaxial stomatal movement by adjusting the level of NO, but it couldn't be excluded that PK/PP could function in the upstream of NO by adjusting the synthesizing of NO.