Isolation,Function Characterization And Mechanism Elucidation Of Transcription Factor Upstream Of PtrBADH,A Cold Responsive Gene Of Poncirus Trifoliata

Cold stress is one of adverse environmental factors,which causes the changes of various metabolites in plants,affects their normal growth and development and thus restricts the productivity and yield of agricultural plants.Glycinebetaine(GB),or betaine for short,is widely distributed in plants and an effective and non-toxic small molecule osmotic regulator,which regulates the osmotic pressure balance in cells and plays an important role in plant response to stresses.It is well known that plant can accumulate GB under various stresses,such as cold,drought or salt stress.However,the associated mechanism and transcriptional regulation of this physiological response is poorly understood.Trifoliata orange(Poncirus trifoliata(L.)Raf.)belongs to Rutaceae and is widely used as a rootstock for citrus production.After full cold acclimation,it can survive at-26℃.Nevertheless,the molecular and physiological mechanisms also have been not clear.In our previous study,RNA-seq was used to construct transcriptome for cold response in trifoliata orange,and a number of low temperature-responsive genes were obtained.Based on those data,a cold-induced betaine aldehyde dehydrogenase gene(named PtrBADH)was identified,and its function in response to cold was characterized.In addition,the transcription factor MYC2,upstream of PtrBADH,was isolated,which elucidated the transcriptional regulation mechanism of GB accumulation under low temperature stress.The main results are as follows:1.PtrBADH is a cold responsive gene.Based on the RNA-Seq data in trifoliate orange under cold stress,we found three non-full-length genes,unigene0018527,unigene0018528 and unigene0018529,from the up-regulated differentially expressed genes,which were all significantly induced by low temperature.Through the BLAST-N in sweet orange database,those non-full-length genes had the closest homology with Cs9g02320(CsBADH).The fulllength gene,named PtrBADH,was cloned from poncirus by homologous sequence method,which contains an open reading frame of 1788 bp and encodes 595 amino acids.The predicted protein molecular mass is 65.605 kDa,the isoelectric point(pI)is 6.62,and the calculated value of protein instability index Ⅱ is 34.34,suggesting it is a stable protein.Subcellular localization analysis showed that PtrBADH was located in the endoplasmic reticulum.After low temperature treatment,PtrBADH expression,BADH activity and GB content were significantly increased.These results indicated that PtrBADH was indeed up-regulated by low temperature and involved in the accumulation of GB under cold stress.2.Functional evaluation of PtrBADH underlying cold resistance.Transgenic tobacco and lemon plants with overexpression of PtrBADHwere obtained by agrobacterium-mediated,and the PtrBADH silencing lines(abbreviated as TRV-PtrBADH)were gained by virus-induced gene silencing(VIGS).Before low temperature treatment,all the pl ants showed good growth condition and no significant difference in physiological indexes.After low temperature treatment,with overexpression of PtrBADH plants had a better phenotype,and the relative conductivity,malondialdehyde(MDA)content and the accumulation of reactive oxygen species(H2O2 and O2·-)were significantly lower than that of the wild type.At the same time,transgenic plants performed higher photosynthetic efficiency,BADH activity and GB content compared to the wild type.However,the TRV-PtrBADH seedlings were more injured and had the lower GB content than that of the control group under cold stress,thus displaying more sensitive to low temperature.These results indicate that PtrBADH plays a positive regulatory role in cold resistance of trifoliata orange.3.Isolation and identification of upstream regulators of PtrBADH.The PtrBADH promoter-deleted vector was constructed,and the transient expression experiment found that the promoter region(i.e.-786～-213)was necessary for the cold response of PtrBADH.Using the-786～-213 fragment of PtrBADH promoter as a bait,a bHLH transcription factor MYC2(named PtrMYC2)was captured by yeast one hybrid screening form the cDNA library of trifoliata orange leaves treated with low temperature.PtrMYC2 includes 2055 bp and encodes 684 amino acids,containing JAZs protein interaction domain(JID),transcriptional activation domain(AD)and typical bHLH conserved domain.PtrMYC2 is located in the nucleus and has transcriptional activation activity.Gene expression analysis showed that PtrMYC2 was significantly induced by low temperature,jasmonic acid and ethylene,but was not sensitive to salt stress.4.Functional evaluation of cold resistance of PtrMYC2.Transgenic tobacco plants overexpressing PtrMYC2 and VIGS lines(abbreviated as TRV-PtrMYC2)were obtained by Agrobacterium-mediated transformation.Cold resistance identification found that overexpression of PtrMYC2 in tobacco enhanced NtBADH expression,BADH activity and GB accumulation,and significantly improved cold resistance of transgenic plants.In contrast.when PtrMYC2 was silenced,the seedlings displayed decreased PtrBADH expression,lower BADH activity and GB content,leading to more sensitive to low temperature.These results indicate that PtrMYC2 may play an important role in poncirus under cold response by regulating GB accumulation.5.Mechanism of PtrMYC2 regulating GB accumulation under cold stress.Analysis of yeast one hybridization(Y1H),gel electrophoresis migration(EMSA)and chromatin immunoprecipitation(ChIP-PCR)showed that PtrMYC2 could specifically bind to G-box of the PtrBADH promoter.The detection of dual luciferase assay proved that PtrMYC2 was the transcriptional activator of PtrBADH promoter.Via agrobacterium-mediated transformation,the over-expression of PtrMYC2 in sweet orange callus and VIGS lines were obtained,gene expression of BADH was strongly increased under low temperature in the transgenic callus,while it was down-regulated in the VIGS line.These results illustrate that PtrMYC2 activates the PtrBADH expression by binding to the promoter of PtrBADH,thus promoting the GB synthesis at low temperature to improve the cold tolerance of plants.6.The synthesis of GB at low temperature depends on the PtrMYC2-regulated JA pathway.Low temperature could notably induce the JA synthetic genes expression and promote the increase of endogenous JA content in trifoliata orange;exogenous methyl jasmonate(MeJA)significantly enhanced the expression of PtrMYC2 and PtrBADH,as well as the accumulation of GB in plants under cold condition,while ibuprofen treatment(JA synthesis inhibitor,IBU)reduced the PtrMYC2 and PtrBADHtranscript level,the GB content and cold tolerance.Using PtrMYC2 overexpression callus and VIGS line analysis,it was found that JA-induced GB accumulation depended on PtrMYC2.The above results indicated that the accumulation of endogenous GB in trifoliata orange under cold stress depended on the JA signaling pathway mediated by PtrMYC2.