The Regulatory Mechanism Of Zinc Finger Protein ZFP36 With The Interacting Protein To Abscisic Acid-signaling

Water stress is a vital role affecting plant growth and production.Under water stress,plants acculumate plant hormone ABA(abscisic acid).ABA plays key roles in biotic and abiotic stresses.As a stress signal,ABA could enhance the generation of important signal molecule ROS(reactive oxygen species)and promote the activities of antioxidant defense enzymes thus improving the plant tolerance to water stress.Zinc finger proteins play important role in ABA-signaling.Previous study in our lab revealed zinc finger protein in rice ZFP36 played a key role in regulating the response of rice to drought and oxidative stress.ZFP36 is an important member in ABA-induced antioxidant defense,but the regulating mechanism remains unknown.Whether ZFP36 could interact with other proteins to transduct the signal or act as a DNA-binding cis-element to bind to the promoters of ABA-induced antioxidant defense enzymes remains to be sdudied.To further investigate the role of ZFP36 in ABA-induced antioxidant defense,here,using ZFP36 as a bait in yeast two-hybrid screening system,a protein interacting with ZFP36 named OsDIP1 was identified.GST(glutamine-S-transferase)Pull-down assay,BiFC analysis and Co-IP test confirmed that OsDIP1 interacts with ZFP36 in vitro and in vivo.OsDIP1 encodes 335 amino acid with a coding sequence of 1005 base pairs.OsDIP1,with unknown function,contains two R3H(R×××H)domains and one SUZ domain which possess single strand DNA(ssDNA),RNA binding ability.Subcellular localization and colocalization assay revealed that OsDIP1 is localized in the cell membrane,cytoplasm,and the nucleus,and ZFP36 is localized in the nucleus,the colocalization between ZFP36 and OsDIP1 is nucleus,which confirmed the interaction in space.There are ABRE(ABA response element)boxes in the promoter of OsDIP1,implying OsDIP1 is related to ABA.The expression of OsDIP1 was induced under different treatments like ABA,H2O2(hydrogen peroxide),CaC12(calcium chloride),polyethylene glycol(PEG),NaCl(sodium chloride).The tissue localization of OsDIP1 are almostly distributed in leaves,especially in young leaves.To gain insight into the functional significance of the interaction between OsDIPl and ZFP36 in ABA-induced signaling,we further explored the expression and activities of superoxide dismutase(SOD)and catalase(CAT)under overexpression of OsDIP1 or knockout of OsDIPl,the result showed OsDIPl is involved in ABA-induced up-regulation of the expression and the activities of SOD and CAT.To further investigate the relationship of OsDIPl with H2O2,the NADPH oxidase inhibitor,diphenylene iodonium(DPI),which blocks the ABA-induced H2O2 production,and the scavenger of H2O2,dimethylthiourea(DMTU)and catalase(CAT)were used.We treated rice plants with DPI or DMTU before ABA treatment.Results showed that pretreatment with DPI or DMTU significantly blocked ABA-induced OsDIPl expression.These datas suggest that H2O2 is required for ABA-induced OsDIP1 expression.To confirm whether OsDIPl was important in regulating drought or salt stress tolerance,we first tested OsDIP1 expression level in leaves of rice exposed to PEG or NaCl treatment using qRT-PCR and found that PEG or NaCl treatment strongly induced OsDIP1 expression,while pre-treatment with fluridon,which inhibits the biosynthesis of ABA,and then exposed to ABA treatment after PEG or NaCl treatment.Experimental results showed that pre-treatment with fluridon significantly blocked PEG or NaCl induced OsDIP1 expression,and additional exogenous ABA could almostly recover the expression of OsDIP1 under PEG or NaCl treatment which is inhibited by ABA inhibitor.It reveals the induced expression of OsDIP1 under PEG or NaCl treatment is dependent on the accumulation of ABA.The analyses of OsDIP1-overexpressed(35S:OsDIP1)and knockout by CRISPR-CAS9 transgenic rice plants)OsDIP1KO)showed that OsDIP1 is involved in ABA accumulation and the regulating the OsNCED1 and OsABA2 gene expression of ABA-biosynthesis genes.Overexpression of OsDIP1 in rice was found to accumulate the content of ABA,be sensitive to ABA in seed germination and root development,and enhance the tolerance of rice plants to drought and salt stresses.In contrast,the knock down mutant of OsDIP1 had lower content of ABA,less sensitive to ABA in seed germination and root development,and was more sensitive to drought stress and salinity stress.In maize,ZmDBF1 interacts with ZmDIP1,ZmDIP1 could elevate the binding activity of ZmDBF1 to the promoter of dehydrin ZMRAB1 7 resulting in enhancing cell water-retaining property.In rice,we also found that OsDBF1 interacts with OsDIP1 by combing yeast two-hybrid system,GST pull-down assay,BiFC analysis and Co-IP test in vitro and in vivo.Further study revealed that ZFP36,OsDIP1,and OsDBF1 could formed a trimer by combing yeast three-hybrid system,Co-IP test and GST Pull-down assay,and the trimer enhanced the DNA-binding affinity of OsDBF1 to the promoter of dehydrin RAB21.qRT-PCR assay reveals that ZFP36 acts upstream of OsDIP1,and OsDBFl acts downstream of OsDIP1 in ABA-signaling,so we hypothesis that OsDIP1 is a intermediate to transduct ABA-signal from ZFP36 to OsDBFl.OsDBFl also feedback regulates OsDIP1 by binding to the DRE2 box of the promoter of OsDIP1.The analysis of the protein structure of ZFP36 revealed the DNA-binding domain is between the two C2H2 structure,and bioinformational analysis of ZFP36 by KinasePhos 2.0 showed the potential phosphorylation site by MAPK(mitogen activated protein kinase)is also located between the two C2H2.Whether ZFP36 could act as a DNA-binding cis-element to bind to the promoters of ABA-induced antioxidant defense enzymes remains to be sdudied.In Arabidopsis,AtMPK3/6 phosphorylates ZAT10 which is a homolog of ZFP36,whether OsMPK1/5 which is the homologs of AtMPK3/6 could phosphorylates ZFP36 still remains unclear.In this study,we found that OsMPK5 could interact with ZFP36 by yeast two-hybrid system,BiFC,Co-IP and GST pull-down and the interacting domain is the DNA-binding domain of ZFP36,and OsMPK5 phosphorylates ZFP36T132,the phosphorylation site is the prediction site by KinasePhos 2.0.We hypothesis that the phosphorylated ZFP36 affects its DNA-binding affinity.Further in vitro EMSA confirmed it.Bioinformational analysis showed that there is a canonical palindromic sequence named EP2 motif in the promoter(-278 bp to+1 bp)of OsPRX24,which is the conservational binding motif of zinc finger protein.By combining transient expression system in Nicotiana benthamiana showing GUS or LUC activities,EMSA,yeast one-hybrid and ChIP RT-PCR,we confirmed that ZFP36 could directly bind to the promoter of OsPRX24.And EMSA revealed that OsMPK5-induced phosphorylation of ZFP36 enhanced the DNA-binding affinity,and the mutation of the phosphorylation site from T to A strongly reduced the DNA-binding affinity,suggesting that the loss of binding ability is mediated by phosphorylation of ZFP36 by OsMPK5 at T132.This work conclude that MAPK cascade transfers the signal to the transcriptional factor ZFP36,and ZFP36 could directly bind to the promoter of antioxidant defense enzyme OsPRX24 to modulate the activities of peroxidase,then to regulate the eliminattion of the second messenger H2O2 in ABA-signaling.In conclusion,we have identified a new partner of zinc finger protein ZFP36 which is also participating in ABA-induced antioxidant defense,named OsDIP1.Overexpression of OsDIP1 in rice plants was found to enhance the tolerance of rice plants to drought and salt stress.In contrast,the knockout(KO)mutants of OsDIPl was more sensitive to drought stress and salinity stress.ZFP36 could interact with OsDBF1 by OsDIP1,and ZFP36,OsDIP1,and OsDBF1 could form a trimer to enhance the binding affinity to the promoter RAB21 to the enhance the expression of RAB21 which is a dehydrin of retaing water,thus in improving the ability of retaing water in cells.Additionally,we also found OsMPK5-induced phosphorylation of ZFP36 enhanced the DNA-binding affinity to the promoter of peroxidase OsPRX24 to regulate H2O2 homeostasis.Our work expanded the mechanism of ABA-induced antioxidant defense,and provided reference for breeding excellent rice by genetic improvement.