Molecular Mechanisms Of The Regulation Of β-Amylase 1(BAM1) By Sugar And Reactive Oxygen Signals

Stomatal pores,which are formed by pairs of guard cells,function as the main channel for gas exchange and water loss between plants and the atmosphere,which are essential for plant photosynthesis and transpiration.A variety of signaling pathway are involved in regulating stomatal movement,including starch-sugar pathway.Arabidopsis β-amylase 1(BAM1)specifically expressed in guard cells and plays important roles in regulating the content of guard cell starch.Here,in this thesis,we mainly focus on the molecular mechanism of BAM 1 regulation by reactive oxygen species(ROS)and sugars in plants.1.ROS regulate the guard cell starch by controlling the activity of BAM1:Competitive binding experiments show that with the increase of H2O2 concentration,the content of oxidatively modified BAM1 protein gradually increases,and the reduced BAM1 protein content gradually decreases,indicating that BAM1 protein has redox modification in vitro.Furthermore,biotin-switch experiments were performed with four purified proteins,BAM1,BAM1C32S,BAM1C470S,BAM1C32,470S,and proved that BAM1 protein could be modified by redox,and cysteines at positions 32 and 470 of the BAM1 protein were important for redox modification.By constructing BAM1 overexpression genetic material and BAM1C32,470S genetic material and treating them with different concentrations of H2O2,the starch content and stomata aparture were measured.The results show that H2O2 can regulate the change of starch content in guard cells and thus regulate stomata movement.Deletion mutations at the cysteine at the 32 and 470 sites were insensitive to the regulation of stomatal movement by hydrogen peroxide.Protein pull-down experiments and bimolecular fluorescence complementary(BiFC)experiments showed that BAM1 and Trx-fl interacted in vitro and in vivo.Analysis of trxfl-1 mutant stomata starch phenotype and stomata movement revealed that trxfl-1 stomata starch content is more than that of wild type,and stomatal aparture is smaller,which indicated that Trx-fl can coordinate with BAM1 to regulate starch metabolism in guard cells,and thus regulate stomatal movement.These results preliminarily analyzed the molecular mechanism of BAM 1 regulating stomatal movement in response to hydrogen peroxide signals2.The molecular mechanism of BAM1 regulation by sugar:Through sugar starvation experiments,it was showed that sugar starvation can reduce the BAM1 protein abundant,and the protein abundant of BAM 1 can gradually recover in pesent of the sucrose supply,indicating that sucrose signals are involved in regulating BAM1 protein degradation process.TOR kinase is a core element that regulates sugar signals.By application of TOR kinase inhibitor AZD8055,loss of TOR kinase function caused the BAM 1 protein to degrade more rapidly,indicating that sugar can play a role in stabilizing BAM1 protein,and the sugar stability of BAM1 protein requires TOR.Further experiments showed that sugar starvation caused the BAM1 protein to degrade.With sugar supplyment,its protein content gradually recovered However,its protein could not be recovered with AZD treatment,indicating that TOR kinase controls the BAM1 degradation process regulated by sugar signals.Further research shows that sugar starvation-induced degradation of BAM 1 protein depends on the ubiquitination degradation pathway.Through the observation of stomatal starch and opening degree of the estradiol-induced tor-es mutant,it was found that the content of tor-es stomatal starch was more than that of wild type,and the stomatal aparture was smaller than that of wild type,indicating that TOR is involved in regulating stomatal movement.These results show that sugar signal can regulate the stability of BAM 1 protein through TOR,and then regulate the change of starch content in stomata,and finally regulate stomata movement.