Interaction Among Calcium,Abscisic Acid And MAPK Signaling Pathways In Maize

Plants have developed various sophisticated mechanisms and signaling pathways in response to environmental stresses.In plant cells,concentration of free cytosolic calcium(Ca²⁺)elevated in response to these stresses which is one of the key events in signal transduction and can lead to the possible alteration in biochemical processes.Temporal fluctuations in calcium concentration are sensed and deciphered by a bunch of calcium sensors or receptors.Among all of the sensors,calcium dependent protein kinase(CDPK)is the most predominant gene family that can sense and decode calcium signals into diverse responses in plant growth,development and variety of stresses.Apart from the calcium signaling,abscisic acid(ABA)and mitogen-activated protein kinase(MAPK)signaling mechanisms are proved to play pivotal roles in response to different stimuli.The induction of Ca²⁺signaling under different environmental conditions make CDPK an ideal candidate in the crosstalk between calcium,ABA and MAPK signal transduction pathways.Maize is one of the most important cereal crops worldwide having complex genome than rice and Arabidopsis due to existence of recombinant sequences and numerous duplicated genes.By doing homology search and conserved domain identification,we identified members of CDPKs in maize genome and performed extensive bioinformatics analysis including phylogeny,chromosomal location,gene structure and duplication,physical and chemical properties of protein and in-silico protein-protein interactional analysis.By performing real-time quantitative PCR(RTqPCR),fluorescent tagged subcellular localization assay,yeast two-hybrid(Y2H)and bimolecular fluorescence complementation(Bi FC),we analyzed gene expression pattern under different treatments,subcellular localization and physical interaction analysis to explore integration of(Ca²⁺,ABA and MAPK)signaling pathways.The main results are as follows:1.By homology and HMMER search,36 members of the CDPK family members in maize were obtained from Maize Genetics and Genomics Database(Maize GDB).The multiple alignment and phylogenetic analysis exhibited that the members of ZmCDPKs were divided into4 groups that were further subdivided into two sub-groups each.The members of the ZmCDPK family had conserved gene structure with 3-12 exons.The ZmCDPK proteins had conserved domains with molecular mass from 525 to 717 k Da.The distribution of the CDPK genes on maize chromosomes was not random as the genes were found in clustered form on different chromosomes except chromosome 9 And Ten segmental duplication events were observed.2.RT-qPCR analysis showed the expression variation of nine members from each phylogenetic group of the 36 ZmCDPK genes under ABA and CaCl₂ treatment.Two of them did not show variation in response to calcium treatment indicating their less calcium sensitivity,while other seven showed expressional variation under both ABA and CaCl₂ treatment.The gene pairs in the same phylogenetic branch shared similar expression pattern such ZmCPK26 and 28,and ZmCPK34 and 35.Some genes responded more to ABA/calcium treatment in leaves and some showed more expression in roots that could be depiction of their organ specific role.The expression of seven of the nine ZmCDPK genes were responsive to both ABA and CaCl₂treatment.3.The open reading frame(ORF)sequences of these nine members were cloned from maize inbred line‘B73',ligated with plant expression vector p CAMBIA2300-e GFP,and transferred into tobacco by using injection method for subcellular localization analysis.The Results showed that all of them were located on plasma membrane irrespective to the presence of N-myristoylation motif in the protein.4.After in silico prediction,the protein-protein interaction among the members of ZmPP2C,ZmCDPK,ZmMAPK and ZmSnRK2 were detected by Y2H and Bi FC.The results confirmed the interaction between some members of ZmCDPK and ZmSnRK2,indicating the activation effect of ZmCDPKs on both calcium and ABA signaling pathways,and the interaction between four ZmCDPK and thirteen ZmMAPK members in different combinations,explaining the direct crosstalk between calcium and MAPK signaling mechanisms,while no interaction was found between the nine ZmCDPK members and the eight ZmPP2C clade A members.In summary,ZmCDPKs have a role at the intersection point of calcium with ABA and MAPK signaling cascades in maize.