Functional Characterization And Mechanism Elucidation Of AHL14/17-A/NINV7 Regulatory Module In Cold Stress Tolerance Of Poncirus Trifoliata

Cold stress is a detrimental abiotic factor that impairs plant metabolic and developmental processes,and thus restricts the distribution of citrus production to limited regions.Trifoliate orange(Poncirus trifoliata(L.)Raf.)is commonly invested citrus rootstock and essential germplasm in citrus breeding,which can tolerate freezing temperatures when it fully acclimated.Therefore,characterization of molecular mechanisms and identification of key genes that render freezing tolerance to P.trifoliata are essential for genetic improvement of citrus.Sugars,especially sucrose(Suc),accumulation was directly associated with plant tolerance to multiple abiotic stresses.Suc cannot be used directly,and it needs to be hydrolyzed before involvement in plant metabolic processes.Invertase(INV)is an enzyme that degrades irreversibly Suc to produce fructose(Frc)and glucose(Glc).However,the molecular mechanisms controlling sucrose degradation in response to low temperature is not fully unraveled.In this study,genome-wide identification and expression profile analysis of INV gene family in trifoliate orange were performed,and transcriptional regulation of coldresponsive alkaline/neutral INV gene,Ptr A/NINV7,during cold stress was investigated.Finally,functional characterization and molecular mechanisms of AT-hook Motif Containing Nuclear Localized transcription factors,Ptr AHL14 and Ptr AHL17,in cold tolerance were demonstrated.The key results are listed as below:1.Genome-wide identification of Ptr INV genes in P.trifoliata resulted in mining fourteen non-redundant Ptr INV family members,including seven alkaline/neutral INV genes(Ptr A/NINV1-7),two vacuolar INV genes(Ptr VINV1-2)and five cell wall INV genes(Ptr CWINV1-5).The comprehensive characterization of biochemical characteristics,phylogenetic relationship,chromosomal location,and exon–intron structures revealed the divergence and conservation of Ptr INV genes.The spatio-temporal expression pattern analysis of INV genes in different tissue types(leaves,stems and roots)of P.trifoliata during several abiotic stresses(dehydration,salt and cold)demonstrated the conserved induction of Ptr A/NINV7 among other Ptr INV genes.Furthermore,our insights revealed that the increased activity of soluble INV enzyme in response to low temperatures were directly associated with high accumulation of Suc,Glc,Frc and total sugar contents in the cold-tolerant Citrus sinensis,C.ichangensis,and P.trifoliata,illustrating the importance of soluble INV enzyme for cold tolerance.Besides,VIGS-mediated expression silencing of Ptr A/NINV7 impaired the freezing tolerance of P.trifoliata via reducing A/N-INV enzyme activity and abolishing total sugar accumulation,demonstrating the function of Ptr A/NINV7 for cold tolerance.This study emphasized the importance of INV enzyme pathway and elucidated the essential function of Ptr A/NINV7 in sucrose metabolism for freezing tolerance of Citrus.2.Promoter isolation of A/NINV7 gene from six Citrus species exhibited natural variation that reflects the fluctuation in the gene expression of A/NINV7 in response to cold stress.In addition to numerous SNPs,the alleles of A/NINV7 promoter have different MITE insertions.Promoter dissection of P.trifoliata Ptr A/NINV7 demonstrated that an A/T-rich genomic region(P2P3,-877 bp to-631 bp upstream of ATG),which has characteristics of the nuclear matrixattachment region(MAR),was indispensable for the cold induction.Accordingly,our insights deciphered a novel role of the genomic region(-877 bp to-631 bp upstream of ATG)in the transcriptional regulation of A/NINV7 in response to low-temperature.3.Two AT-Hook Motif Containing Nuclear Localized proteins of P.trifoliata(Ptr AHL14 and Ptr AHL17)were fished out via yeast one-hybrid(Y1H)screening of cold-induced P.trifoliata c DNA library with a bait of the Ptr A/NINV7 promoter region.Bioinformatics analysis exhibited that Ptr AHL17 belongs to the intronless group with type-I AT-hook DNA binding domain and type-A Plant and Prokaryote Conserved(PPC)functional domain,whereas Ptr AHL14 belongs to introns-containing group with type-II AT-hook DNA binding domain and type-B PPC domain.Subcellular localization,protein immune-detection,and transcriptional activation assay substantiated that Ptr AHL14 and Ptr AHL17 are nuclear-matrix transcriptional activators.Furthermore,molecular evidences using Y1 H,electrophoresis migration shift assay(EMSA),chromatin immunoprecipitation-q PCR(Ch IP-q PCR),GUS staining and dual luciferase assay demonstrated that Ptr AHL14 and Ptr AHL17 bind adjacently on A/T-rich motifs in the promoter and activate positively the transcription of Ptr A/NINV7.Gene expression analysis and protein immune-detection showed high induction of Ptr AHL14 and Ptr AHL17 in response to cold stress.Overexpression of Ptr AHL14 and Ptr AHL17 in Arabidopsis and lemon and VIGS-mediated gene silencing of Ptr AHL14 and Ptr AHL17 in P.trifoliata were performed for functional characterization.Our insights manifested that Ptr AHL14 and Ptr AHL17 modulate Ptr A/NINV7-mediated sucrose hydrolysis for the freezing tolerance of P.trifoliata.4.The physical interaction of Ptr AHL14 and Ptr AHL17 with themselves and with each other led to greater transcriptional activation of Ptr A/NINV7 and more robust Suc hydrolysis.Furthermore,Ptr AHL14 and Ptr AHL17 were demonstrated to interact with TBP-interacting histone acetyltransferases(HATs)(Ptr GCN5 and Ptr TAF1).Additionally,H3 ac and RNA POL II occupancy were revealed to enrich on the promoter of Ptr A/NINV7 in response to low temperatures.Thus,Ptr AHL14-Ptr AHL17 complex could recruit the H3 ac marking to switch on the MAR-containing chromatin in the promoter of Ptr A/NINV7 for RNA POL II occupancy,and finally increase the expression of Ptr A/NINV7.Overall,our finding deciphered a novel sugar-signaling module,composed of HATs-Ptr AHL14/17-Ptr A/NINV7-Glc,functions in coordinating the Suc catabolism for the cold tolerance.