The Effect And The Mechanism Of CTK, Auxin In V-B-induced Stomatal Closure

The Enhancement of UV-B radiation is one of environmental problem,which draws great attentions recently. Cytokinin(CTK) and Auxin, are important inregulating plant growth and development. Both NO and H₂O₂ are significant signalingmolecules in plants. Researches have shown that NO and H₂O₂ are involved in bothUV-B-induced stomatal closure and CTK, Auxin-induced stomatal opening. In nature,the regulation of stomatal movement by solar radiation inevitably involves theinteraction between UV-B and CTK, Auxin. But this interaction and its relation to NOand H₂O₂ yet have not been reported. In order to integrate the intricate network ofsignaling pathways in guard cells, enrich and develop data on mechanisms of stomatalmovements, the effects and the mechanism of CTK, Auxin on UV-B-induced stomatalclosure in Vacia faba L. were studied here by epidermal strip bioassay andlaser-scanning confocal microscopy.The main results are as followed:1. Former research had proved that UV-B could enhance NO and H₂O₂ levels inguard cells, thereby induce the stomatal closure. Our results showed cytokinin(6-BA,KT, ZT) and Auxin(IAA, NAA, 2, 4-D) on reversing UV-B-induced stomatal closurecould be imitated by c-PTIO(NO scavenger), HB(NO scavenger), L-NAME(NOsynthase inhibitor) and ASA (H₂O₂ scavenger), CAT(catalase), SHAM(cell wall PODinhibitor) but not DPI (NADPH oxidase inhibitor). This probably suggested that 6-BA,KT, ZT and IAA, NAA, 2, 4-D reversed UV-B-induced stomatal closure by reducingNO or H₂O₂ in guard cells. Furthermore, the generation of endogenous H₂O₂ is likelythrough cell wall POD pathway but not NADPH oxidase pathway.2. Utilizing specific fluorescent indicator for NO or H₂O₂ and LSCM technique, ithad been found that the increase of NO in guard cells promoted by UV-B could beprevented by 6-BA, KT, ZT and IAA, NAA, 2, 4-D, which was similar to c-PTIO orL-NAME. And as the effect of ASA or SHAM, 6-BA, KT, ZT and IAA, NAA, 2, 4-Dcould prevent the increase of H₂O₂ in guard cells promoted by UV-B. These resultswere in accordance with the epidermal strip bioassay. All these results indicated that the prohibitive effects of 6-BA, KT, ZT and IAA, NAA, 2, 4-D on UV-B-inducedstomatal closure were definitely related to the decreasing of NO or H₂O₂ in guard cells.3.6-BA, KT and ZT, similar to c-PTIO or ASA, could promote the closed stomatainduced by UV-B to open again. Yet, IAA, NAA and 2, 4-D, just like DPI or L-NAME,could not promote the closed stomata induced by UV-B to open again. In other words,6-BA, KT and ZT has similar effects of NO and H₂O₂ scavengers while IAA, NAA and2, 4-D has similar effects of their synthesis inhibitors. The accordance perhapsindicated that 6-BA, KT and ZT promoted the closed stomata to open by scavengingthe high level of NO or H₂O₂ induced by UV-B directly while IAA, NAA and 2, 4-Dcould not promote the closed stomata to open for they have no scavenging ability onNO or, H₂O₂ induced by UV-B.4. Utilizing specific fluorescent indicator for NO or H₂O₂ and LSCM technique, ithad been found that, although the fluorescent level of NO and H₂O₂ in guard cells werehigh after dealt with UV-B for four hours, the level of NO would decrease clearly in900s after 6-BA, KT, ZT and c-PTIO were added and the fluorescent level wassignificantly weaker than the blank contrast two hours later. Similarly, the level ofH₂O₂ also decreased clearly in 900s after 6-BA, KT, ZT and ASA were added and thefluorescent level was significantly weaker than the blank contrast two hours later.These results suggested 6-BA and KT, as c-PTIO or ASA, could scavenge thegenerated NO or H₂O₂ in guard cells induced by UV-B. And these results were agreewith the effects of 6-BA, KT and ZT promoting the UV-B-induced closed stomata toopen again.5. Utilizing specific fluorescent indicator for NO or H₂O₂ and LSCM technique, ithad been found that, added IAA, NAA, 2, 4-D and L-NAME could not change the highlevel of NO induced by four hours' UV-B radiation in 900s, neither in 2 hours.Similarly, IAA, NAA, 2, 4-D and SHAM also could not change the high level of H₂O₂induced by four hours' UV-B radiation in 900s, neither in 2 hours. Thus, it could beconcluded that IAA, NAA and 2, 4-D, just like L-NAME or SHAM, couldn'tsignificantly reduce UV-B-induced high level of NO or H₂O₂ in guard cells. Theseresult were just in accord with the epidermal strip bioassay.To sum up, 6-BA, KT, ZT and IAA, NAA, 2, 4-D could prevent UV-B-inducedincrease of NO or H₂O₂ and reversed UV-B-induced stomatal closure. 6-BA, KT andZT decreased the high level of NO and H₂O₂ induced by four hours' UV-B radiation and promoted the closed stomata to open again. While IAA, NAA 2, 4-D couldn'tdecrease the high level of H₂O₂ or NO induced by four hours' UV-B radiation andcouldn't promote the closed stomata to open. Synthetically, it could be concluded that6-BA, KT and ZT reversed UV-B-induced stomatal closure through directlyscavenging the NO or H₂O₂ induced by UV-B, while IAA, NAA and 2, 4-D reversedUV-B-induced stomatal closure by inhibiting the synthesis of the generation of NO orH₂O₂ induced by UV-B.