Study On The Binding Sites Of Arabidopsis SnRK2.6 And AFPs On The Transcription Factor ABI5

ABI5 subfamily members are a class of basic leucine zipper transcription factors that are important in ABA signal.They participate in the regulation of many physiological and biochemical processes such as seed development,germination,various biotic and abiotic stress responses.The stability and transcription activation activity of ABI5 are regulated by phosphorylation of various kinases,and the over-expressing ABI5 plants have no obvious phenotype.ABI5 multiple-site mimic phosphorylation mutants can activate transcriptional expression of downstream genes controlled by ABI5 without ABA.multi-site phosphorylation of ABI5 is mediate by Sn RK2 s,of which the more clearly studied is Sn RK2.6.Although there are many potential phosphorylation sites for Sn RK2 on ABI5,but it is unkonw whether all of these potential phosphorylation sites can be recognized and phosphorylated by Sn RK2.6.AFPs are a class of molecules protein that interact with ABI5.It is reported that AFPs promote the ubiquitination and degradation of ABI5 and then negatively regulate ABA signaling.However,neither the kinase activity nor the conserved structure of the corresponding frontal phosphatase was found on AFPs.And how does AFPs affect ABI5 activity and tability is remain unknow.In this study,yeast two-hybrid was performed to study the specific binding sites of AFPs on ABI5.In this study,multiple sequence alignments of nine members of the ABI5 subfamily were performed.Six potential phosphorylation sites of Sn RK2 were identified on ABI5.The bind nexus between potential phosphorylation sites of Sn RK2.6 on ABI5 and bind site of AFPs on ABI5 were studied by yeast two-hybrid.our results shows that:(1)Although there are multiple potential phosphorylation sites for Sn RK2 on ABI5,not all sites recognized by Sn RK2.6.Sn RK2.6 has some selectivity when binding to these sites of ABI5.(2)In yeast,Sn RK2.6 only recognize one of potential phosphorylation site,RQGS on ABI5,which is highly conserved among members of the ABI5 subfamily.(3)Through a series deletion mutants of ABI5,a precise site when AFPs bound to ABI5 was found,PTFGEMTLEDFLVK.(4)When retaining other domains on ABI5 and deleting PTFGEMTLEDFLVK,AFPs can still interactwith m ABI5,and AFPs have multiple binding sites on ABI5.(5)Yeast experiments of ABI5 1-135 aa and ABI5 202-213 aa point mutants showed that the Thr residues at positions 47 and 206 of ABI5 are important for the interaction between ABI5 1-135 aa and ABI5 202-213 aa and AFPs.T47 is mutated to A or E,AFP4 can not interact with ABI5 1-135 aa.T206 is mutated to A or E,AFP1/2/3/4 can not interact with ABI5202-213 aa.