Study On Mechanism Of Microtubule Involvement In Stomatal Opening Induced By 5-Aminolevulinic Acid In Apple Leaves

Stomata is the main channel for gas exchange between plants and atmosphere,controling the CO2 uptake and O2 release during photosynthesis,and it is the main channel of water loss in transpiration,playing an important role in plant water homestatics.A series of recent studies have shown that 5-aminolevulinic acid(ALA)can inhibit the stomata closure induced by dark,ABA,H2O2 and other factors,which has been attributed to flavonol biosynthesis,lower contents of H2O2 and cytoplasmic Ca2+in guard cells,but the further mechanism is unclear.Microtubules are arranged radial in guard cells.Studies have shown that they are sensitive to light,and associated with stomatal movement.In addition,protein reversible phosphorylation plays an important role in the cell signal transduction.And the PP2C protein phosphatase acts as a negative regulatory role in ABA signaling pathway,but our pretests show that PP2C does not participate in ALA inhibiting ABA-induced stomatal closure.Nevertheless,another protein phosphatase PP1/PP2A may participate in the process.Therefore,we used microtubule-specific drugs and phosphatase specific inhibitor to explore the relationship and mechanism between microtubules and PP1/PP2A in ALA-ABA regulating stomatal movement.In order to explain the mechanism of ALA regulation on stomatal movement of plants,it provides a theoretical basis for its application in agricultural production.The main results are as follows.1.We used the abaxial epidermis of detached leaves of apple(Malus × domestica Borkh.'Fuji'),and found that the regulatory effect of ALA and ABA on stomatal movement of apple leaves could be eliminated by vinblastine(VBT),a microtubule disassemble agent,or taxol,a microtubule stabilizer.Results of qRT-PCR showed that ALA reversed the inhibition of ABA on the expression of tubulin-related genes such as TUB1,MAP65-1 and MAP65-6.On the other hand,okadaic acid(OA),an activity inhibitor of PP1/PP2A protein phosphatase,caused stomatal closure and weakened ALA suppress the stomatal closure induced by ABA;however.Taxol and VBT alleviated or increased the inhibition of stomatal opening by OA,respectively.Furthermore,OA promoted the accumulation of H2O2 in guard cells,and with microtubules depolymerization at the downstream.The above results indicate that ALA may promote PP1/PP2A protein phosphatase activity of apple leaf guard cells,thereby inhibit ABA-induced H2O2 production,which up-regulates the expression of tubulin-related genes and promotes microtubule polymerization,and eventually inhibiting ABA-induced stomatal closure,thus opening the window for photosynthetic gas exchange of apple leaves.To verify this hypothesis,we use the model plant Arabidopsis thaliana as experimental material for the further research.2.In order to verify the working hypothesis.Using GFP-a-tubulin-6 transgenic arabidopsis thaliana cotyledons as a material,to explore the role of microtubules in ALA-ABA regulating stomatal movement.In light condition,ABA or VBT caused the majority of the microtubules to be assembled into small fragments and randomly distributed,the stomatal aperture decreased.However,ALA or Taxol inhibited stomatal closure induced by ABA or VBT.The above results indicates that depolymerization and polymerization of microtubules participate in stomatal movement regulated by ABA and ALA.In other words,ALA promotes microtubules polymerization of guard cell,thereby inhibiting ABA-induced stomatal closure.3.Using wild type,OE(PP2A-C2 overexpressed lines),pp2a-c2(T-DNA insertion mutant of PP2A-C2)and GFP-a-tubulin-6 with cotyledons of Arabidopsis thaliana as materials,studied the effect of ALA on PP1/PP2A activity and its interactions with the microtubules.The results showed that,(1)OA weakened the effect of ALA on stomatal opening,also weakened the inhibition of ALA to ABA-induced stomatal closure,it means that PP1/PP2A involves in the ALA-ABA regulating stomatal movement.(2)Exogenous ALA induced most of the gene expression enhancement of PP2A,and it reversed the inhibitory effect of ABA on the gene expression of PP2A-C2,PP2A-C5,PP2A-B'?,and PP2A-B'?.The results showed that ALA promotes PP2A activity by regulating the expression of catalytic and regulating subunit genes.(3)The effect of ALA inhibiting ABA-induced stomatal closure was significantly weakened in pp2a-c2,while the results were opposite in OE,showed that PP2A-C2 involves in the process of ALA-ABA regulating stomatal movement.(4)The microtubules were depolymerized and stomatal aperture was decreased after OA treatment.However,Taxol and VBT respectively alleviated and increased the microtubules depolymerization and stomatal closure induced by OA,it shows that microtubules play a role in the downstream of PP1/PP2A,and the inhibition of enzyme activity could lead to the depolymerization of microtubules.The above results show that the PP1/PP2A participates in the regulation of ALA-ABA of arabidopsis leaf stomatal movement,and ALA suppress the stomatal closure induced by ABA mainly through improving PP1/PP2A activity,then promotes the microtubules polymerization,and mainly up-regulating the expression of catalytic subunit genes and regulating subunit genes,such as PP2A-C2 and PP2A-B'?.4.The interaction of PP2A,ROS and microtubules in ALA-ABA regulation of stomatal movement was studied with wild type,OE,pp2a-c2 mutants and GFP-a-tubulin-6 cotyledons of Arabidopsis thaliana.Results show that,(1)Both OA treatment and in pp2a-c2 mutant induced to produce a large amount of H2O2 in guard cell,while the ability of ALA to scavenge the ABA-induced H2O2 level was significantly reduced,the results show that PP1/PP2A negatively regulates the production of H2O2 in guard cell.(2)ALA inhibited the depolymerization of microtubules and stomatal closure induced by exogenous H2O2.Moreover,H2O2 scavenger like AsA,CAT and NADPH oxidase inhibitors DPI also restored the inhibitory effect of ABA or exogenous H2O2 on microtubule polymerization and stomatal opening,suggesting that the reduction of H2O2 content will lead to microtubule polymerization.The results of these studies show that,PP1/PP2A promotes microtubules polymerization and stomatal opening by down-regulating the content of H2O2 in the guard cells during the ALA-ABA regulating stomatal movement.To sum up,using apple leaves and the model plant Arabidopsis thaliana,we proved that ALA may inhibited H2O2 content induced by ABA in guard cell by promoting the activity of PP1/PP2A,thereby promoted the microtubule polymerization,and then inhibiting ABA-induced stomatal closure.