Molecular Dissection Of Arabidopsis Tyrosylprotein Sulfotransferase In Regulation Of ABA Signaling

The plant hormone abscisic acid(ABA)plays vital roles during plant development and growth,such as seed dormancy and germination,post germination growth,and root growth.In particular,one of the most extensively studied functions of ABA is its role in regulating plant responses to abiotic stresses such as salinity,drought and cold.PYR/PYL/RCAR ABA receptors sense ABA and initiate a signaling cascade.PP2 Cs and Sn RK2 s are core components of ABA signaling pathway.The Arabidopsis Sn RK2 subfamily protein kinases Sn RK2.2,Sn RK2.3 and Sn RK2.6 are recognized as key regulators of ABA signaling.The post-translational modifications on components of ABA signaling play important roles in regulating the degree of ABA response and have been widely investigated recently,such as phosporylation,ubquitination,SUMOylation and redox-induced modification,which may have impacts on protein stability,localization,interaction,or activity.The protein tyrosine sulfation is a critical covalent posttranslational modification occurred on a wide range of peptides and proteins synthesized through the secretory pathway in multicellular eukaryotes,including higher plants.It is catalyzed by Golgi-localized transmembrane protein termed tyrosylprotein sulfotransferase(TPST),which catalyzes the transfer of sulfate from the universal sulfate donor PAPS to the hydroxyl group of a peptidyltyrosine residue.Only several secrete peptides have been identified as the substrates of TPST in Arabidopsis.The purpose of this study is to investigate whether tyrosine sulfation participates in ABA signaling pathway.Previous research had revealed that the zinc-finger transcription factor Zat12 responds to many different abiotic stresses,including drought and application of ABA.However,the components participate in regulating the expression of Zat12 in response to ABA or drought stress are not fully understood.To further dissect the activation of Zat12 in ABA signaling,we generated the transgenic plants pro Zat12:luciferase [WT(LUC)] expressing the reporter gene luciferase under the control of the Zat12 promoter and the transgenic plants were mutagenized by EMS.We conducted forward genetic screening to identify new components involved in ABA signaling and regulation of Zat12.The main results are as follows:(1)Isolation of mutation with enhanced bioluminescence.WT(LUC)were mutagenized by EMS and mutants were selected according to their aberrant LUC activities.We isolated one mutant allele with enhanced bioluminescence intensity under both normal growth conditions and after treatment with ABA compared with WT(LUC).The mutant had higher transcript level of LUC and Zat12 relative to WT(LUC)detected by q RT-PCR.The results suggest that the mutation negatively regulate the expression of Zat12.(2)Map-based cloning and whole genome re-sequencing revealed a premature stop codon of AT1G08030(TPST),thus we renamed the mutant as tpst-3.The genetic evidences of the two T-DNA insertion mutants tpst-1 and tpst-2,and the complementation line tpst-2com demonstrated that the TPST mutation is responsible for the mutant phenotypes.(3)The tpst mutants were hypersensitive to ABA compared with WT or WT(LUC).The complementation line tpst-2com rescued the phenotypes of the mutant.These genetic results suggested that TPST is involved in ABA responses and may act as a negative regulator in ABA signaling.Furthermore,the tpst mutants also exhibited hypersensitivity to sorbitol caused osmotic stress,which demonstrated that the TPST is also required for osmotic resistance.(4)The transcript levels of ABA responsive genes in tpst-1 were significantly higher after treatment with ABA,which also demonstrated that TPST acts as a negative regulator in ABA signaling.(5)Overexpressing of TPST did not influence the expression of Zat12 and the ABA response of transgenetic Arabidopsis.(6)The q RT-PCR and western blot revealed that the transcript level and protein level of TPST were upregulated by ABA treatment.Thus TPST responds to ABA.(7)LCI,Bi FC and pull-down experiments showed that TPST physically interacts with Sn RK2.2/2.3/2.6.The additional LCI experiments also demonstrated that TPST specifically interacts with the Sn RK2 subfamily.The interaction may occurred on Golgi,and this was supported by the subcellular expression of Sn RK2.2,which colocalized with the Golgi marker.(8)The phenotypes in response to ABA showed that the seedlings establishment rates and primary root length of tpst-1snrk2.2/2.3/2.6 were much higher than that of tpst-1,which indicated that TPST responds to ABA through Sn RK2.2/2.3/2.6.By contrast,TPST cannot be phosphorylated by Sn RK2.6.The above analyses suggested that TPST functions upstream of Sn RK2.2/2.3/2.6 in the ABA signaling pathway.(9)In vitro assays showed that Sn RK2.2/2.3/2.6 were tyrosine sulfated by TPST.(10)We used a cell-free system to investigate whether TPST affects the stability of Sn RK2.2/2.3/2.6.The protein levels of purified Sn RK2.2/2.3/2.6-GST incubated with proteins prepared from tpst-1 decreased much slower than proteins prepared from WT.The endogenous degradation rate of Sn RK2 s detected by anti-Sn RK2.2/2.3/2.4/2.6/2.9/2.10 antibody showed that Sn RKs were more stable in protein extracted from tpst-1 than that from WT.Moreover,the protein level of Sn RK2.2-GFP in pro Sn RK2.2:Sn RK2.2-GFP/WT was lower than that in pro Sn RK2.2:Sn RK2.2-GFP/tpst-1 after ABA treatment for 6 hours.These results indicated that TPST promotes the degradation of Sn RK2 s in vitro and in vivo,including ABA treatment.(11)The expression of Zat12 was induced by ABA treatment,which was apparently suppressed in snrk2.2/2.3/2.6.This demonstrated that the increased expression of Zat12 in tpst mutants was due to the high protein level of Sn RK2.2/2.3/2.6.In this study,we identified Sn RK2 s as new substrates of TPST in Arabidopsis,and TPST promotes the degradation of Sn RK2 s.Our work revealed the mechanism that TPST acts as a negative regulator in ABA signaling.