Polymorphism Among Members Of Abscisic Acid Receptor Family(ZmPYL)in Maize

Abscisic acid(ABA)regulates plant growth,development and abiotic tolerance.The Pyrabactin resistance 1-like(PYL)protein is direct receptor of ABA.However,there are functional polymorphism,functional redundancy,and even interaction with other signaling pathways among the PYL family members.In the present study,a yeast library of maize cDNA was constructed and used to screen for interacting proteins of the ZmPLY family members.After sequencing analysis and functional annotation,the screened interacting proteins were reverified by yeast two-hybrid(Y2H).Together with the phenotype of the Arabidopsis mutant of ABA abscisic acid receptor deletion,and its differential expression related genes under stress and induction,it was attempted to elucidate the sophisticated interaction mediated by the divergent functions among the ZmPYL members and their probable relation to abiotic tolerance.The main results are as follows:(1)The titer and volume of the primary library were 4.2 × 106 and 1.6 × 107 CFU/mL,respectively,with an average colony number of about 2000 per plate.The titer and volume of the secondary library were 3.3 × 106 and 1.2 × 107 CFU/mL,respectively,with an average colony number of about 1500 per plate.The titer and volume of the yeast library were both more than 3 × 107 CFU/mL,and all the 24 colonies randomly detected by PCR amplification were positive with amplified fragments more than 1000 bp.This result indicated that the yeast library of maize cDNA was qualified and could be used in subsequent experiments.(2)Based on the bioinformatics prediction information,the open reading frame sequences of 10(ZmPYL1/2/3/4/5/6/9/10/11/12)of the 13 ZmPYL gene family members were amplified and constructed into Y2H bait vector pGBKT7.Protein toxicity and autophosphorylation assays showed that the proteins encoded by the 10 amplified genes had not toxicity to yeast growth,and no autophosphorylation,and the bait vectors could be used for yeast library screening and Y2H.(3)Screening the yeast library by the constructed bait vectors,most of the mating efficiencies ranged from 3.4%to 4.8%,with two exceptions of 5.9%and 6.1%.After incubation on the quadruple-deficient medium,PCR identification,sequencing and redundancies removing,a total of 253 colonies were screened and identified as positive,and aligned to the sequences of registered genes.Their functional annotations were enriched into 11 cell components,8 molecular functions and 18 biological processes,suggesting that the ZmPYL members have polymorphic functions other than the ABA receptors.(4)The encoding genes of the proteins with high screened frequencies or annotated functions involved in signaling,growth and development,and abiotic tolerance were amplified,constructed into the Y2H capture vector pGADT7,and used for Y2H with the above bait vectors pGBKT7 to reverify the results of library screening.The interactions between the transcription factor ZmBES/BZRl-1(involved in light response mediated brassinosterid(BR)signaling]and ZmPYL2/3/9,between the transcription factor ZmTIFY10B(involved in JA-mediating abiotic tolerance)and ZmPYL10/11,between auxin transporter ZmPIN1 and ZmPYL1,between photosystem ? manganese-assisted oxygenation enhancer ZmOEE3 and ZmPYL10/9,and between ferredoxin ZmFDXl and 5,and ZmPYL4/6/10/12 and ZmPYL1/2/3/5/10/12,respectively,were reverified because all the three replicates of their co-transformed colonies grew on the quadruple-deficient medium and turned blue(5)The germination potential,taproot length and proline content of Arabidopsis quartet mutant phenotypic identification was performed in atpyrl/atpyll/atpyl2/atpyl4 were significantly decreased under alkaline stress of pH8.5 and induction of 100 ?mol/L methyl JA,while malondialdehyde content was significantly increased,comparing to the wild type.The dark morphogenesis characteristics of the mutant germinating in darkness were more obvious than the wild type,and expression differences of photomorphogenesis-related genes were also more significant than the wild type.Three light and two JA responsive cis-affecting elements were identified from the promoter sequences of the atpyll and atpyl2 genesAll the above results confirmed the functional redundancies and divergence among the ZmPYL members,which not only function as ABA receptors and transduce stress signals in response to internal and external environmental stimuli,but also interact with JA,BR,auxin and other signaling pathways at different levels of gene expression,and even directly regulates the activity of resistance-related proteins,to form a sophisticated signaling network.