Transcriptome Profiling For Low Temperature Response Of Poncirus Trifoliata And Dissection Of Function And Mechanisms Of PtrERF109 And PtrTZF1 In Cold Tolerance

The yield,quality and cultivation region of commercial citrus were seriously affected by low temperature.Genetic improvement based on genetic engineering is efficient for generating cold resistant citrus varieties,but the important prerequirement and basis are to characterize the mechanism of low temperature response and identify key genes involved in low temperature response Trifoliate orange(Poncirus trifoliata(L.)Raf.)is an closely related genus of citrus and widely used as a rootstock for citrus cultivation.After full cold acclimation,it can tolerate temperature lower than-26 ?.While understandings about its molecular mechanisms were limited.In this study,the expression profiles of trifoliate orange were constructed based on RNA-seq,and a number of cold-responsive genes were identified.Finally,functions and molecular mechanisms concerning for PtrERF109 and PtrTZF1 in cold tolerance were characterized.The main results are as belows:1.In this study,in order to obtain a general transcriptomic information of trifoliate orange,we significantly expand the breadth of tissue sampling(root,stem,leaf)under different stress treatment(4 ?,salt,dehydration).About 68 million(5.4 Gb)high-quality reads were obtained using RNA-seq paired-end sequencing technology.The clean reads were de novo assembled into a non-redundant set of 77,292(>100 bp)Unigenes with an average length of 1,112 bp(N50=1,778 bp).Sequence similarity search for Unigenes using BLASTX against non-redundant protein databases Nr and Swiss Prot,showed58001 and 36445 Unigenes have homologous sequences in the corresponding library.Of these,23,846 had significant sequence similarity to known genes,and they were assigned to 61 gene ontology(GO)categories and 25 clusters of orthologous groups(COG).In addition,26.3%(12031/45819)of Unigenes were distributed in the GO term of response to stimulus.KEGG analysis showed that these Unigenes might be involved in 128metabolic pathways.Then trifoliate orange seedlings were treated under 4 ?,and leaf samples at different time points(0 h,6 h,24 h,72 h)were collected for RNA isolation and library construction.About 7.2 million clean reads were obtained for each liabrary and mapped to the assembled transcriptome,resulting in the identification of 5549differentially expressed genes(DEGs).These comprised of 600(462 up-regulated,138down-regulated),2346(1631 up-regulated,715 down-regulated),and 5177(2702up-regulated,2475 down-regulated)genes from the cold-treated samples at 6,24 and 72 h,respectively.Among GO terms,‘response to chitin',‘response to stimulus',and‘response to stress'were the most significantly enriched categories of the DEGs,while‘plant hormone signal transduction',‘plant-pathogen interaction',and‘lipid metabolism'were the most significantly enriched metabolic pathways.A total of 60 transcription factors were shown to be cold responsive,among which the number of AP2/ERF transcription factors was the largest.In addition,a number of genes involved in the catabolism and signaling of hormones,such as abscisic acid,ethylene and gibberellin,were up-regulated by the cold stress.Meanwhile,levels of putrescine progressively increased under cold,which was consistent with up-regulation of an arginine decarboxylase gene.2.PtrERF109 is a typical AP2/ERF family transcription factor,belonging to the B-4subgroup of ERF subfamily.This gene is intronless and encodes 308 amino acids.PtrERF109 is located in the nucleus,has transcriptional activation activity,and the activation region is located at the C terminus.PtrERF109 was transiently and strongly induced by low temperature and ethylene,and also induced by dehydration,but not by ABA and salt treatment.Freezing tolerance of transgenic tobacco and lemon overexpressing of PtrERF109 was significantly enhanced.However,silencing of PtrERF109 by VIGS displayed hypersensitivity to freezing stress.These results suggested that PtrERF109 play a positive role in cold tolerance.RNA-seq analysis of the transgenic lemon demonstrated that PtrERF109 overexpression lead a comprehensive gene reprograming in the transgenic plants,in which,genes related to stress response,sugar metabolism,lipid metabolism,and energy metabolism were significantly enriched.Among them,the Prx1(class III peroxidase 1)encoding peroxidas is one of the genes with the highest fold change.The expression level of Prx1 in the overexpressing plants was increased with or without cold treatment,concurrent higher peroxidase activity and less H₂O₂ accumulation in PtrERF109-overexpression plants.By contrast,the VIGS plants showed lower Prx1 level and peroxidase activity,accompanied by increased H₂O₂accumulation relative to wild type.Furthermore,Yeat one-hybrid,EMSA,and dual luciferase assay proved that PtrERF109 could bind to GCC-box in the Prx1 promoter and activate its expression,thereby promoting the scavenging ROS in response to low temperature.3.PtrTZF1 encodes 655 amino acids without an intron,and contains two classical domains at the N terminus,including a tandem C3H(also called as TZF,Tandem CCCH Zinc Finger)and a tandem ANK domain.This protein belongs to the C3H type zinc finger protein,which was further categorized into the?subfamily(Arabidopsis)or the?subfamily(rice)of CCCH zinc finger protein.PtrTZF1 was quickly and strongly upregulated by low temperature,but continuously and slowly induced by dehydration.However,salt and ABA treatment did not cause dramatic alteration of the transcript.PtrTZF1 possessed a strong transcriptional activation and self-activation activity,and the C terminus was required for self-activation.PtrTZF1 was located in both nucleus and cytoplasm in tobacco epidermal cells.Overexpression of PtrTZF1 enhanced the cold tolerance in the transgenic tobacco and lemon,while silencing of this gene in trifoliate orange compromised cold tolerance,indicating that PtrTZF1 played a positive role in cold tolerance.Several stress related proteins,such as SPI(Serine protease inhibitor),b HLH(basic Helix-Loop-Helix),GRP(Glycine rich protein),GRP10(Glycine rich protein 10),and ZAT10(Cys2/His2-type zinc-finger protein 10),were confirmed as interacting proteins of PtrTZF1 by yeast two-hybrid screening system and BiFC.