Mechanisms Of Brassinosteroids In Regulating Auxin Homeostasis Via ADS1 In Arabidopsis

Brassinosteroids(BRs)are a class of plant-specific steroidal hormones that play diverse roles in all plant tissues and organs.A large scale activation tagging genetic screen for suppressors of an Arabidopsis BR-deficient mutant,det2,led to the identification of ads1-D,activation tagged det-2 suppressor 1-Dominant.ads1-D partially recovered the defective phenotypes of det2-1 seedlings including short petioles and dwarf statues.ads1-D single mutant seedlings show elongated leaf petioles,bend-down leaf tips and elongated hypocotyls relative to wild type Col-0.The T-DNA insertion sites in ads1-D were determined using a TAIL-PCR and confirmed by an Arabidopsis re-sequencing method.The inserted T-DNA in the activation-tagged mutant strongly enhanced the expression of ADS1.Constitutive expression of ADS1 driven by a 35S promoter in the det2-1 and wild-type Col-0 recapitulated the ads1-D phenotype.These results confirmed that elevated expression of ADS1 caused the det2 suppression phenotype.Hypocotyl elongation analysis indicated that ads1-D seedlings were hypersensitive to exogenously applied BL.A CRISPR-Cas9 obtained loss-of-function mutant,ads1-cas9,showed reduced sensitivity to BL.Genetic analysis showed that ads1-D could partially suppress the defective phenotypes of bri1-9 and bin2-1.Therefore,ADS1 is likely a positive regulator regulating BR signaling pathway.ADS1is a C₂H₂-type zinc finger protein,localizes in the cytoplasm and nucleus,and possesses transcriptional activity.The expression patterns of ADS1 are similar to those of BIN2.Yeast two-hybrid,bimoleculer fluorescence complementation(BIFC)in tobacco,Co-immunoprecipitation analyses demonstrated that ADS1 can physically interact with BIN2 and its close homologs BIL1 and BIL2.In vitro and in vivo phosphorylation assays demonstrated that ADS1 can be phosphorylated by BIN2.Although adsl-D can rescue weak BR signaling mutants,it cannot rescue a null triple BR receptor mutant,bri1-701 brl1 brl3,indicating that the function of ADSl depends on the BR signaling.ADS1 expression levels were not significantly altered after BL treatment,suggesting that ADS1 may not be transcriptionally regulated by BR.Furthermore,we found that BL could induce the accumulation of the ADS1 protein in the nucleus.The leaf phenotypes of ADS1-OX are very similar to that of auxin accumulation mutants,such as yuc1-D,whereas ads1-cas9 also exhibits an auxin-related phenotype.We therefore tested the possible relationship of ADS1 with auxin.The hypocotyls of ads1-D were hypersensitive to an auxin polar transport inhibitor NPA and an auxin biosynthesis inhibitor yucasin.ADS1 is possibly related to auxin biosynthesis.We performed RNA-seq analyses and found that 466 out of 835(55%)total differentially expressed genes in ADS1-OX were also regulated by auxin.Auxin biosynthesis genes YUC3 and YUC7 were found to be significantly upregulated in ADS1-OX.Additional experiment indicated that YUC3 and YUC7 were significantly upregulated in BRI1-OX and bin2-3 bil1 bil2 and down-regulated in bri1-701 brl1 brl3.BL treatment can significantly up-regulate the expression of YUC3 and YUC7.In vivo chromatin immuno-precipitaiton(Ch IP)experiments demonstrated that ADS1 can interact with the promoter regions of YUC3 and YUC7.YUC3 and YUC7 were up-regulated after DEX-induced ADS1 expression.The accumulation of free IAA in ads1-D was significantly higher than that of wild-type plants,and exogenously application of BL can increase auxin content in wild type.Interestingly,we found that free IAA in ads1-cas9 was significantly higher than the wild type Col-0,and further analysis found that the auxin metabolism gene in ads1-cas9 was significantly down-regulated.These results demonstrated that ADS1 is involved in regulating Brassinosteroids-mediated auxin homeostasis.ADS1 could interact with members of the BZR1/BES1 family by yeast two-hybrid,Co-IP and BIFC.Promoter::Luciferase(LUC)reporter system anslysis and q RT-PCR results showed that BZR1/BES1 could also regulate auxin biosynthesis and metabolism genes.The relationship between ADS1 and BZR1/BES1 needs to be elucidated in the future.In summary,our results indicated that ADS1 is an important downstream regulatory component in the BR signaling pathway.ADS1 is one of the substrates of BIN2.BIN2can physically interact with and phosphorylate ADS1 and regulate its nucleus localication.It is likely that ADS1 regulates the biosynthesis of auxin in a BZR1/BES1dependent and independent way.