The Study Of Actin-binding Protein SCAB1 On Regulating Stomatal Movement In Arabidopsis

Stomata serve as major gateway for gas exchange between plants and environment,and guard cells are widely taken as a model system in studies of plant signaling.Previous study from our lab identified a plant-specific actin-binding protein SCAB1(Stomatal Closure Related Actin Binding Proteinl),which is involved in the regulation of stomatal closure in response to ABA.SCAB1 contains a Pleckstrin homology(PH)domain known as a phospholipid-binding domain.Studies also suggest that phosphoinositides are required for regulating actin dynamics during stomatal movement.Therefore,the study of the mechanisms for SCAB 1 functioning in stomatal movement was carried out in this thesis.PIP-strip assay was employed to test the interaction between SCAB 1 and phosphoinositide(s).The results revealed that the central a-helical region(CHR,54-272 amino acid),but not the PH domain of SCAB1 specifically boaund to PI3P(Phosphatidylinositol 3-phosphate).To assess the PI3P-binding sites in CHR,four putative PI3P binding motifs(RXLR-dEER domain)were identified and mutated.PIP strip binding results showed that two RXLR-dEER domains are essential for the PI3P binding.Because CHR contains an actin-binding domain(ABD),PI3P might regulate actin dynamics through SCAB1.Observations showed that SCAB1 colocalized with PI3P in cells.Biochemical analysis showed that although PI3P did not affect the F-actin-binding and-bundling activity of SCAB1,this binding reduced the F-actin-stabilizing activity of SCAB1.In the scabl transgenic seedlings expressing SCAB1 mutant that abolished PI3P binding activity,actin filaments were insensitive to Lat A(Latrunculin A)treatment.After seedlings were treated with PI3K(Phosphatidylinositol 3-kinase)inhibitor to reduce PI3P level,F-actin became more stable.These results demonstrate that PI3P plays a role in regulating actin dynamics by regulating SCAB1 activity.The analysis of stomatal closure phenotypes showed that the phenotype of scab1 was complemented by expressing widetype SCAB1,but not the mutant SCAB1 lacking PI3P-binding activity.These results indicate that PI3P also plays a role in the regulation of stomatal movement.PI3P is an important factor in vesicle trafficking and may involve in vacuole fusion.Therefore,the relationship of PI3P,SCAB1,F-actin,and vacuoles were analyzed during stomatal closing.Observations showed that actin filaments reorganized on the sites where vacuoles deformed,and PI3P moved along actin filaments.Quantitative analysis showed that the vacuolar deformation slowed down when SCAB1 could not bind to PI3P or PI3P level decreased.These results uncover that PI3P regulates F-actin dynamics and vacuolar deformation by regulating SCAB1 activity.Previous results have shown that SCAB1 binds to IP3 and IP6.Both of them take part in calcium signaling.When scab1 mutant and wild type were treated with H2O2 or ABA,the free calcium level in scab1 mutant was higher than that in wild type,and the free calcium level in SCAB1 over-expression lines was lower than that of WT.ABA-insensitive phenotype and high calcium level of scab1 were not complemented by the SCAB1 mutants abolished IP3/IP6 binding activity.This evidence suggests that the interaction of SCAB1 and IP3/IP6 takes part in calcium signaling and stomatal movement.By measuring K+ current,the outward K+ channel activity of scab1 reduced comparing that in wild type when the plants were treated with H2O2.This suggests that SCAB1 affects the free IP3/IP6 level to control Ca2+ release,which may subsequently regulate the K+ channel and stomatal movement.In this study two mechanisms for SCAB1 functioning in stomatal movement are revealed:first,binding of PI3P to SCAB1 is required for vacuolar deformation,and second,binding of IP3/IP6 to SCAB1 is required for calcium signaling.This work provides novel findings and is important for the understanding of the regulation network in stomatal movement.