Molecular Mechanism Of JAZ Proteins Modulating Seed Germination In Bread Wheat And Arabidopsis

The appropriate timing of seed dormancy and germination is critical for plants to adapt to various environmental conditions.Defective seed dormancy in grains of cereal crop,leads to seeds that are prone to preharvest sprouting(PHS),which usually causes substantial losses in both yield and quality.In addition,the promotion of seed germination especially for crops is crucial for sowing and subsequent postgerminative growth.As a result,regulating of seed germination of crops is crurial for agricutral production.Many plant hormone play roles in regulating seed germination,among which,abscisic acid(ABA)is the primary phytohormone in controlling seed dormancy and germination.The increase of ABA content in seeds promotes seed dormancy and activation of ABA biosynthesis and signaling pathways inhibites seed germination.In addition,mang plant hormone regulates seed germination by the crosstalk with ABA.Jasmonic Acid(JA)is another important plant hormone with a variety of biological functions and regulates plant development and abiotic stress response by the crosstalk with lots of plant hormone.But it is unclear that whether and how JA is involved in modulating seed germination.JAZ protein(Jasmonate ZIM-domain protein)is the key regulating factors in JA signaling pathways and play important roles in regulating plant development and abiotic stress response.In this study,we start exploring the crosstalk between JA and ABA in modulating seed germination though exploring the function of JAZ family member in plants,which provides theory basis in nderstanding the molecular mechanism of preharvest sprouting.The results are as follows:(1)Members of Ta JAZ family are identified at genome-wide level of wheat and their featuers are analyzed.Based on protein sequences of At JAZs in Arabidopsis,42 Ta JAZ famlily members are identified in wheat by using bioinformatics from database.Through analyzing conserved motif of amino acids,we find that all Ta JAZ1 family members both include ZIM and Jas domatin,and can be divided into 3 subfamilies: Group I?Group II and Group III.Besides,by analyzing the structures of intro and exon in Ta JAZ1 we show that the gene strctures of Ta JAZ famlily members in Group ? are simple with only one coding squence.And the most gene strctures of Ta JAZ famlily members in Group II and Group II are complex.Also though analyzing the expression level of Ta JAZ1 family members in different developmental stages of wheat,we found that not all of them showed same trends,implying that they may have different functions.Lastly,through analyzing the phylogenic relationship of Ta JAZ family members in wheat and At JAZ family members in Arabidopsis,we demonstrate that Ta JAZ are closeness to At JAZ3,implying that they may have similar functions.(2)Ta JAZ1 modulates seed germination through interacting with Ta ABI5.Comparing with wild type wheat varieties KN199,the visible sprouted kernels of over expression transgenic wheat is higher;the sensitivity to ABA is lower and the induction of the expression level of ABA maker genes is lower,which inllustrates that Ta JAZ1 functions as a positive regulator of PHS and a negative regulator of ABA signaling pathway.Consdering that ABI5 is the key element in ABA signaling pathway,we cloned Ta ABI5.Further luciferase complementation imaging assays(LCI)and Yeast two-hybrid(Y2H)assay demonstrate that Ta JAZ1 physically interacts with Ta ABI5 and the C-terminal part containing the Jas domain of Ta JAZ1 physically interacts with Ta ABI5.These results inllustrate that Ta JAZ1 may modulate seed germination by ABA signaling pathway.(3)At JAZ3 modulates seed germination through interacting with At ABI5.Similarly,Comparing with wild-type Col-0,the the sensitivity to ABA is lower,the induction of the expression level of ABA maker genes and the protein level of ABI5 is lower in overexpression lines 35S:JAZ3?jas-GFP.Further we find that 9 of 12 JAZ proteins(JAZ2,JAZ3,JAZ5,JAZ6,JAZ8,JAZ9,JAZ10,JAZ11,JAZ12)could interact with ABI5 using the luciferase complementation imaging assays.Choosing JAZ3 as a representative,we demonstrate that JAZ3 interacts with ABI5 in vivo and vitro.These results inllustrate that similar with Ta JAZ1,At JAZ3 may also modulate seed germination by ABA signaling pathway.(4)We demonstrate the mechanism of seed germination modulateing by the crosstalk between At JAZ3 and At ABI5.Dual-luciferase reporter sysyem and Ch IP-PCR assay demonstrates that JAZ3 represses the transcriptional activation activity of ABI5.Also JAZ enrichments in the promoter of EM1 could be observed and ABA application could abolish the enrichment of JAZ proteins in the gene promoter.Furthermore,we find that ABA treatment might promote the protein degradation of JAZ3 and phenidone(JA synthetic inhibitor)can blocked the protein degradation of JAZ3 by ABA treatment.Then RNA sequcence,q RT-PCR and hormone treatment assays demonstrate that ABA application could induce the expression of jasmonate(JA)biosynthetic genes(AOC2,ACX1,ACX2 and KAT5),and increase JA concentrations in Col-0.But the increasment of expression level of JA jasmonate(JA)biosynthetic genes and JA concentrations is lower in abi5-7.These results inllustate that the interation of JAZ3 and ABI5 leads to the crosstalk of ABA and JA,and further modulates seed germination.In conclusion,this study revealed the function of JAZ proteins in modulating seed germination through the interation between JAZ and ABI5,which provide evidence of the crosstalk between JA and ABA in modulating seed germination.This study is of significance of exploring the regulation mechanism of seed germination and resistance breeding for PHS in creals(such as wheat).