ZmCIPK32 Positively Regulates Germination Of Stressed Seeds Via Gibberellin Signal

Seed germination is mainly regulated by ABA-GA hormone homeostasis.The higher GA level or signaling pathway,the stronger the ability to antagonize ABA,and the more significant promotion of seed germination.CBL（Calcineurin B-like protein）,a Ca2+sensor,can sense Ca2+signal and bind to its target protein CIPK（CBL-interacting protein kinase）to form CBL-CIPK complexes,which is involved in plants development and stress response.In this paper,the systematic bioinformatics,subcellular localization,interaction and function of CBLs interacting protein kinase ZmCIPK32 were studied.It was found that ZmCIPK32 may participate in the medulating of seed germination by regulating GA signal.It provides new evidence and theoretical support for further elucidating the interaction between CBL-CIPK signal system and GA signal.The main results are as follows :（1）ZmCIPK32 is a typical CIPK family member.Sequence homology analysis showed that ZmCIPK32 had the highest homology with Os CIPK32 in rice,reaching 96.41%,followed by Os CIPK33 and At CIPK3,with homology of 85.91%and 76.13%,Respectively.Protein domain analysis showed that ZmCIPK32 had the N-terminal protein kinase domain and the C-terminal NAF domain unique to the CIPK family.It is speculated that this protein is a typical CIPK family member,and it is named as ZmCIPK32 because of its highest homology with rice Os CIPK32.（2）GA and PBZ treatment increased the expression of ZmCIPK32.Maize seeds were soaked in GA₃（Gibberlic acid 3）and PBZ（Paclobutrazol）solutions,and the expression patterns of ZmCIPK32 in different imbibition stages of maize seeds were analyzed by RT-q PCR.The results showed that the expression of ZmCIPK32 was the highest at 3 h after GA₃ treatment,which was 5.3 times that under normal conditions.The expression level was the highest at 6 h after PBZ treatment,which was 4 times that under normal conditions.It is speculated that ZmCIPK32 may respond to gibberellin signal transduction in the early stage of seed germination and participate in seed germination regulation.（3）ABA,Mannitol and Dark enhanced the activity of ZmCIPK32 promoter..In order to further study the function of ZmCIPK32,the expression characteristics of ZmCIPK32 promoter in early seedling development were analyzed by Agrobacterium-mediated leaf disc method and inflorescence infection methods.The results showed that the promoter was an inducible promoter,which was induced in seedlings treated with 10 μM ABA,375 m M Mannitol and Dark,and the activity was stronger at the base of leaves.（4）ZmCIPK32 is localized in the cell membrane and cytoplasm and interacts with ZmCBL1/9.On the premise of determining the expression of ZmCBL1 and ZmCBL9 proteins on the plasma membrane,the localization of ZmCIPK32 protein was further analyzed,and it was found that ZmCIPK32 was located in the cell membrane and cytoplasm.The interaction of ZmCIPK32 with ZmCBL1 and ZmCBL9 was confirmed by bimolecular fluorescence complementation.In order to find the interaction domain of ZmCIPK32 protein with ZmCBL1 and ZmCBL9 proteins,the ZmCIPK32 protein was truncated.Yeast two-hybrid analysis showed that the NAF domain was the key domain for the interaction of ZmCIPK32 with ZmCBL1 and ZmCBL9.（5）ZmCIPK32 promoted seed germination and early seedling growth of Arabidopsis under ABA,PBZ and Mannitol treatments.The effects of ZmCIPK32 on seed germination and early seedling growth of Arabidopsis thaliana under different stress treatments were analyzed.The results showed that ZmCIPK32 could promote seed germination under different concentrations of PBZ,Mannatiol and ABA.Under the treatment of 0.1 μM PBZ,200 m M Mannatiol and 0.5 μM ABA,the germination rate of Arabidopsis thaliana seeds increased by 13.35% ± 1.35,20.2% ± 2.5 and21.7% ± 3.8,respectively.The root length increased by 0.19 ± 0.03 cm,0.225 ± 0.075 cm and 0.375 ± 0.10 cm,respectively.The results showed that ZmCIPK32 could promote seed germination and early seedling growth under stress conditions.（6）ABA and Mannatiol treatment increased the content of GA₃ and GA₄ and decreased the content of ABA in ZmCIPK32 transgenic Arabidopsis.The effects of ZmCIPK32 on the content of ABA,GA₃ and GA₄ during seed germination under PBZ and Mannatiol treatments were analyzed by ESI-HPLC-MS/MS methods.The results showed that compared with WT,the ABA content of OE-1 and OE-2decreased under 0.1 μM PBZ treatment,and the content of GA₃ and GA₄ increased significantly,especially the content of GA₄.The ratio of GA₃/ABA and GA₄/ABA also increased significantly.Similar results were obtained under 200 m M Mannatiol treatment.The above results show that ZmCIPK32 may affect the level of GA/ABA by regulating the content of GA and ABA under stress conditions,and then promote seed germination under stress conditions.