Natural Variation And Functional Characterization Of ZmPYL And ZmPP2C-A Family Genes In Maize Abiotic Stress Response

Maize（Zea mays L.）is an important food sources for humans worldwide.Abiotic stresses,such as drought and salinity,cause dramatic yield loss of maize every year.Abscisic acid（ABA）is one of the major phytohormones that play critical roles in plant stress responses.PYR1/PYL/RCAR genes,which encode ABA receptors,play pivotal roles in ABA signaling.What’s more,as the core member for ABA signaling pathway,roles of clade A PP2C phosphatases（PP2C-A）in ABA-dependent stress responses have been well studied in Arabidopsis.However,the roles of PYL and PP2C-A members in abiotic stress responses and their natural variations remain largely unknown in maize.In this study,totally 13 members of ZmPYL（ZmPYL1-13）were identified based on the reference genomic sequence of maize,which located on all of the chromosomes except for chromosome 7,with 1 or 2 genes per chromosome.Phylogenetic analysis divided these ZmPYLs into subgroup I and II,and members from subgroup I had fewer introns than subgroup II.Real-time q PCR assays indicated that ZmPYLs played important roles in multiple abiotic stresses responses.Transgenic analysis showed that overexpression of ZmPYL3,ZmPYL9,ZmPYL10 and ZmPYL13 increased the sensitivity of transgenic plants to ABA significantly.The most important was that overexpression of ZmPYL8,ZmPYL9 and ZmPYL12 could enhance plant drought tolerance.Furthermore,the accumulation of proline and expression of drought-related marker genes were also enhanced in transgenic plants as compared with those in wild type plants.These results together demonstrated that ZmPYL8,ZmPYL9 and ZmPYL12had positive roles in plant drought tolerance.Association analyses based on a panel of368 maize inbred lines revealed that SNPs in ZmPYL8 and ZmPYL12 were significantly associated with drought resistance of maize.In a parallele study,we found that the express patterns of different ZmPP2C-A genes varied under ABA treatment,indicating that they played different roles in ABA responses.Additionally,expressions of ZmPP2C-A genes were also found to be significantly regulated by salt treatments,which suggested they may participate in maize salt stress responses.Thus,ZmPP2C-As were then over-expressed in Arabidopsis to characterize their function in abiotic stress responses,The results showed that,as compared with wild-type plants,plants overexpressing ZmPP2C-A2 or ZmPP2C-A6had lower survival rates,higher leaf water loss rates and less proline accumulation under drought treatment,indicating that these genes negatively regulated drought responses.Further association analysis between natural variation of ZmPP2C-As and drought tolerance also showed that ZmPP2C-A6 was significantly associated with drought resistance.As the ancestor of modern corn,Teosinte has a lot of advantages as compared with modern corn,including drought tolerance.To obtain more favorable alleles of PYL and PP2C-A family genes from wild maize germplasms,QTL mapping for drought tolerance were further conducted using a BC₂F₆ population derived from elite inbred line Zong3 and Teosinte（Zong3-teosinte,ZT）.Based on the published genotype data and the survival rates of the population under drought treatment in two seasons,a total of 39 QTLs were identified to be associated with drought tolerance of maize.On the other hand,25 drought responsive genes in the QTLs intervals were identified according to the transcriptomic data of 197 maize accessions under drought treatment,which maybe candidate genes for drought tolerance of Teosinte.However,there was no PYL or PP2C-A family genes in the 25 candidate genes.It was probably because that genetic variations of PYL and PP2C-A genes between Zong3 and Teosinte were limited.None the less,the ZT population and the candidate genes were useful for deep study of maize drought tolerance.In summary,the results here deepened our knowledge about the function of maize ZmPYLs and ZmPP2C-A genes in abiotic stresses and tolerance,and the natural variations identified in ZmPYLs and ZmPP2C-A genes could be used as potential molecular markers for breeding drought-resistant maize cultivars.