Functional Characterization Of Citrus Sinensis CBF1 And Poncirus Trifoliata NAC72 In Regulation Of Arginine Decarboxylase Gene Expression And Stress Tolerance

Citrus is one of the most widely cultivated fruit crop.Sweet orange(Citrus sinensis)takes the largest proportion and its annual yield is about 2/3 of the total citrus production.Sweet orange is a thermophilic specie,and low temperature is one of the fundamental environmental factors limiting its distribution and production.Polyamines are organic compounds that have two or more primary amino groups –NH2,and play important role in plant development,tissue differentiation,fruit ripening,senescence,and abiotic stress resistance.It is considerd that accumulation of polyamines at cellular level may increase abiotic stress tolerance in plants.Moreover,the increased expression level of polyamine biosynthesis genes lead to polyamine accumulation at cellular level.Therefore,it is important to study the regulation mechanism of polyamine to enhance low temperature tolerance in sweet orange.And recent studies have shed light on the importance of transcriptional regulation which is due to the interaction between transcription factors and their cis-acting regulatory elements in the promoter region of downstream target genes.Therefore,identification the transcriptional regulation of polyamine biosynthesis genes is an approach to increase abiotic stress tolerance of sweet orange.The main results are as follows:1.The identification of polyamine biosynthesis genes in sweet orange.In sweet orange,10 major polyamine biosynthesis genes were identified,including arginine decarboxylase(ADC),spermidine synthase(SPDS1,SPDS2),spermine synthase(SPMS),S-adenosylmethionine decarboxylase(SAMDC1,SAMDC2,SAMDC3,SAMDC4)and thermospermine synthase or ACAULIS5(ACL5-1,ACL5-2).They were located on 6 different chromosomes.The transcriptional analysis showed that most of these genes exhibit higher expression levels in roots and lower levels in peel and pulp.Most of them were down-regulated under putrescine treatment.The expression level of ADC,SPDS2 and SAMDC4 were up-regulated under spermidine treatment,and the others were down-regulated.However,the expression tendency during spermine treatment was quite the opposite.In citrus callus,SPDS1,SAMDC3 and SAMDC4 were induced under low temperature stress,SAMDC2,ACL5-2 respond to ABA treatment and other genes give respones to high salt treatment.However,the expression level of ADC and SAMDC4 were most obviously induced in citrus leaves under cold stress as compared to others.Polyamines content varies greatly in different tissues.Putrescine was significantly increased in citrus leaves,but spermine was the highest in citrus callus under cold stress.Overall,the ADC gene plays an important role in cold stress response among all these polyamine biosynthesis genes.2.CsCBF1 increased the ADC expression level and putrescine level under cold stressTranscription factor CBFs(C-repeat binding factor)play important role in respones to cold stress signaling.The Citrus CBFs expression level was increased under cold stress,especially CBF2 and CBF3.Only the expression level of CBF3 was increased under salt stress and CBF4 did not give response to ABA treatment.CsCBF1 bound to the promoter of ADC and regulated the ADC expression level among the CBFs.The ADC expression level was up-regulated and putrescine content was higher in the Citrus and Arabidopsis CsCBF1 overexpression lines,in addition,the Arabidopsis transgenic lines show better cold stress tolerance compared to the WT.The results indicated that the CsCBF1 plays a positive role in regulation of putrescine biosynthesis and cold stress resistance.3.PtrNAC72 decreased the ADC expression level and putrescine level under drought stressIn an earlier experiment,we isolated a NAM,ATAF1/2,and CUC(NAC)domain-containing transcription factor,PtrNAC72,from dehydration treated trifoliate orange(Poncirus trifoliata)by yeast one-hybrid screening.PtrNAC72,which was localized in the nucleus,binds specifically to the promoter of Pt ADC by recognition of the CCGAC cis-acting element and acts as a transcriptional repressor.PtrNAC72 expression was induced by cold,drought,and abscisic acid.The m RNA abundance of ADC gene and putrescine levels were increased in the Arabidopsis(Arabidopsis thaliana)t-DNA insertion mutant,nac72 compared with the wild type.However,transgenic tobacco lines overexpressing PtrNAC72 were more sensitive to drought that accompanied with more ROS accumulation after drought and dehydration treatments.By contrast,exogenous application of putrescine to the overexpression lines restored drought tolerance.Taken together,these results demonstrate that PtrNAC72 is a repressor of putrescine biosynthesis and may negatively regulate the drought stress response,at least in part,via the modulation of putrescine-associated reactive oxygen species homeostasis.In our research,we identified 10 different polyamine biosynthesis genes in Citrus sinensis database.ADC was the most important gene resonsed to abiotic stresses according to the results of gene expression level and polyamine content changes under stresses.Then we identified the transcription factors CsCBF1 and PtrNAC72 respetively in Citrus sinensis and Poncirus trifoliata which can increase or represse the ADC expression level and putrescine content in plants.Our research extends the transcriptional regulation network of polyamine accumulation under abiotic stress,which will enrich the gene resource in genetic improvement of Citrus.