Genome-wide Identification Of PP2C Gene Family And Functional Characterization Of Group A PP2C Genes In Wheat(Triticum Aestivum L.)

Plant protein phosphatase 2Cs(PP2Cs)participate in many signaling pathways.Group A PP2 Cs are key regulators in ABA signaling pathway,while ABA has a significant impact on plant growth and stress responses.However,little research has been conducted on the PP2 C gene family,especially for group A PP2 C,in wheat(Triticum aestivum L.),one of the most important food crops.Moreover,common wheat is allohexaploid(AABBDD)with a large and complex genome,making it difficult for wheat gene function study.Nevertheless,the achievement of high-quality annotated reference genome of hexaploid wheat in recent years has accelerated the progress of wheat functional genomic research.In the present study,a genome-wide investigation of the PP2 C gene family in wheat was carried out,then the roles of group A TaPP2 Cs were studied,and the functions of TaPP2C-a10 and TaPP2C-a7 were further analyzed.The main research content and results are as follows:(1)After the genome-wide searching on the annotated reference genome of hexaploid wheat,a total of 95 TaPP2 C genes were identified,and 80% of TaPP2 C genes had three homoeologs from A,B,and D subgenomes.The phylogenetic analysis showed that TaPP2 C proteins were mainly divided into 13 groups(A-M).Expression patterns of TaPP2 C genes were analyzed using publicly available RNA-seq data,and the result revealed that several TaPP2 C genes expressed in particular tissues.Additionally,TaPP2 Cs could respond to abiotic stresses like drought,as well as biotic stresses like powdery mildew infection.Besides,the A,B and D homoeologs of some TaPP2 Cs were found to exhibit large differences in the expression levels under the same condition.(2)Expression analysis result of group A TaPP2 C genes showed that TaPP2C-a1,a3,a4,a5,a7,a8,a9 and a10 significantly responded to ABA,and most of them like TaPP2C-a7 and a10,also responded to GA,drought and salt stresses,suggesting that group A TaPP2 Cs might play roles in phytohormone signaling pathways like ABA signaling,as well as abiotic stresses like drought;this conclusion was also supported by the analysis results of cis-acting elements in the promoters of group A TaPP2 C genes.Several group A TaPP2 C genes were cloned from T.aestivum L.cv.Chinese Spring,and yeast two-hybrid assay was performed to analyze the interactions of group A TaPP2 Cs with TaSnRK2 s and TaPYL4,which are key regulators in ABA signaling pathway.The result showed that all these group A TaPP2 Cs interacted with subclass Ⅲ TaSnRK2 and TaSnRK2.1/2.2 from subclass Ⅱ,and some group A TaPP2 Cs like TaPP2C-a7 interacted with TaPYL4 in ABA-dependent way,indicating that group A TaPP2 Cs functioned in ABA signaling pathway.As some group A TaPP2 C genes abundantly expressed in grains,interactions between group A TaPP2 Cs and dormancy regulator TaDOG1 L were analyzed,and TaPP2C-a10 was found to interact with TaDOG1L1 and TaDOG1L4.(3)Subcellular localization analysis showed that TaPP2C-a10 localized in nuclei,and Bi FC assay confirmed that TaPP2C-a10 interacted with TaDOG1L4 in nuclei.Besides,the RT-qPCR analysis showed that TaPP2C-a10 abundantly expressed in grains at the later stage of seed development.These results suggested that TaPP2C-a10 might function to regulate seed dormancy and germination.To test this conjecture,TaPP2C-a10 was transformed into model plant Arabidopsis thaliana,and seed dormancy levels were reduced in the seeds of TaPP2C-a10 transgenic Arabidopsis.The transgenic Arabidopsis also displayed an ABA-insensitive phenotype: compared with the wild type,the transgenic lines had relative higher seed germination rates,post-germination growth and primary root growth under ABA treatment.Further study showed that the expression levels of some ABA-responsive genes in TaPP2C-a10 transgenic lines were reduced,suggesting that TaPP2C-a10 negatively regulated ABA signaling of transgenic Arabidopsis.Besides,Phylogenetic analysis result indicated that AHG-like genes including TaPP2C-a10 were conserved in angiosperm.(4)Further investigation on the function of TaPP2C-a7 was performed,and TaPP2C-a7 was also found to reduce the inhibition of ABA on seed germination,post-germination growth and primary root elongation by negatively regulating ABA signaling of transgenic Arabidopsis,while the dormancy levels of TaPP2C-a7 transgenic seeds were not affected.Additionally,TaPP2C-a7 transgenic lines was sensitive to drought stress.Expression analysis revealed that TaPP2C-a7 regulate drought stress response of transgenic Arabidopsis via both ABA-dependent and ABA-independent pathways.Yeast one-hybrid analysis result showed that transcription factors TaMyb1 D and TaMyb U could bind to the TaPP2C-a7 promoter fragment which contains MBS element,a MYB binding site involved in drought-inducibility.Yeast two-hybrid analysis result revealed that TaPP2C-a7/a10 also interacted with TaCBL3/9 and TaCIPK11,indicating that TaPP2C-a7/a10 might participate in regulating the activity of ion channel.(5)The phenotypic analysis on TaPP2C-a9 transgenic Arabidopsis indicated that TaPP2C-a9 also negatively regulated ABA signaling pathway.As TaPP2C-a7,a9 and a10 all belonged to the AHG1 subfamily of group A TaPP2 Cs,TaPP2 Cs in AHG1 subfamily might have conserved roles in ABA signaling pathway.These findings lay the foundation for the functional study of TaPP2 Cs,and provide important clues for the mechanism study of wheat ABA signaling pathway.