Functional Charaterization And Mechanism Elucidation Of Cold-responsive Transcription Factors,PtrERF9 And PtrZAT12,from Poncirus Trifoliata In Cold Resistance

Among the key environmental stresses,cold stress has the great impact on the geographic distribution,growth and development,and reproductive growth of citrus.Genetic engineering is an effective way to generate cold-resistant citrus germplasms.Understanding of the cold response mechanism and identification of key cold-responsive genes in citrus lay the foundation for the improvement of cold resilience.Poncirus trifoliata（L.）Raf.,a cold-hardy plant closely related to citrus,is considered as a good cold-resistant genetic resource.However,the genes of trifoliate orange with deriable cold tolerance function need to be further identified.Here,based on the previous transcriptome data,several cold-inducible genes in Poncirus trifoliata were identified,among which Ptr ERF9and Ptr ZAT12 were strongly induced by low temperature.In the current work the function and molecular mechanisms of these two genes in cold tolerance were characterized.Main results of this research are shown as below.1.The CDS of Ptr ERF9 gene is 696 bp,encoding 231 amino acids with an AP2domain at the N-terminal,indicating that Ptr ERF9 is a typical AP2/ERF transcription factor.Subcellular localization showed that Ptr ERF9 was located in the nucleus.Ptr ERF9 was induced by low temperature,dehydration,H₂O₂,and salt,with the largest induction under cold.In addition,the promoter activity assay indicated that cold treatment increased the activity of the Ptr ERF9 promoter.2.Based on Agrobacterium-mediated transformation and virus-mediated gene silencing（VIGS）technology,Ptr ERF9-overexpressing tobacco and lemon plants and TRV-Ptr ERF9 plants were obtained and used for cold tolerance assay.Overexpression of Ptr ERF9 enhanced the cold resistance of transgenic tobacco and lemon,as shown by lower electrolyte leakage,MDA content,and higher photosynthetic efficiency in the transgenic plants than in the wild type.By contrast,knockdown of Ptr ERF9 in trifoliate orange exhibited increased sensitivity to cold stress compared with control plants.These results indicated that Ptr ERF9 plays a positive role in the regulation of cold tolerance.3.Transcriptomic analysis showed that knockdown of Ptr ERF9 led to down-regulation of 2777 genes and up-regulation of 1087 genes in the VIGS line under normal growth conditions.These differentially expressed genes（DEGs）were involved in signal transduction,stress response,lipid metabolism,protein phosphorylation,sugar metabolism,and other biological pathways.These DEGs not only included some functional genes,such as FAD,PAL,and UDP,but also encoded some transcription factors such as NAC,b ZIP,and protein kinase genes（PP2C and CDPK）.Based on the transcriptomic data,we found that a glutathione S-transferase enzyme gene Ptr GSTU17 and an ACC synthesis enzyme gene Ptr ACS1 were significantly down-regulated.In-depth work with Y1H,EMSA and LUC assays indicated that Ptr ERF9 could bind to GCC-box in the Ptr GSTU17 and Ptr ACS1 promoter and activated their expression.These results revealed that Ptr GSTU17and Ptr ACS1 were two target genes of Ptr ERF9.4.After low temperature treatment,the expression level of Ptr GSTU17 and the GST activity in the Ptr ERF9-overexpressing lemon plants were higher than those in the wild type.Contents of H₂O₂ and O₂^·-in the transgenic lemon plants were lower than those in the wild type,while the TRV-Ptr ERF9 plants showed the opposite trend.These results demonstrated that Ptr ERF9 could activate Ptr GSTU17 expression,thereby regulating the balance of reactive oxygen species in the plants.5.Low temperature treatment could increase the endogenous ethylene production of plants.Application with ACC enhanced the cold tolerance of Poncirus trifoliate,which was otherwise decreased by exogenous AVG.Ptr ERF9 was up-regulated by ACC,but the cold-induced up-regulation of Ptr ERF9 was dependent upon ethylene synthsis.The abovementioned results indicated that Ptr ERF9 acts downstream of ethylene and modulates ethylene biosynthesis by regulating the expression of Ptr ACS1,forming a feedback regulation loop in trifoliate orange.6.Ptr ZAT12 contains 480 bp open reading frame and encodes 159 amino acids.The amino acid sequence analysis showed that Ptr ZAT12 has two QALGGH zinc finger domains and belongs to the C2H2 zinc finger protein family.Ptr ZAT12 is localized in the nucleus.Ptr ZAT12 was induced by cold,drought,and salt,but not by ABA.Overexpression of Ptr ZAT12 enhanced the cold tolerance of transgenic tobacco plants.The promoter of Ptr ZAT12 included two DRE/CRT motifs.Y1H and EMSA assays indicated that Ptr CBF1and Ptr CBF3 bound to the Ptr ZAT12 promoter.LUC assay showed that Ptr CBF1 positively regulated the expression of Ptr ZAT12,while Ptr CBF3 negatively regulated the expression of Ptr ZAT12.These results indicate that Ptr ZAT12 may participate in the cold response of Poncirus trifoliata through a CBF-dependent pathway.