Functional Identification And Transcriptional Regulatory Analysis Of Citrus Lycopene β-cyclase Genes

Carotenoids are a group of secondary metabolites that are important for plant biology and human nutrition.Although remarkable progress has been made towards understanding carotenoid biosynthesis,the mechanisms that regulate the transcription of carotenogenic genes remain poorly understood.Citrus fruit contains abundant carotenoids,and the carotenoid content and composition is a decisive factor for fruit appearance quality,nutritional property,and economic value.Therefore,it has much theoretical and practical significance to study the citrus carotenoid metabolism.Lycopene β-cyclases(LCYb)are critical enzymes located at the branch point of the carotenoid biosynthetic pathway.Numerous studies have shown that LCYb transcript levels are tightly correlated with carotenoid content and composition.However,the mechanisms regulating transcription of LCYb remain a mystery.No transcription factors have been demonstrated to directly regulate its expression in citrus or even in whole plant kingdoms.In this study,we used the citrus LCYb genes as entry point to perform detailed analysis of transcript levels,allelic sequence variations and enzymatic activities;then to conduct isolation and functional characterization of LCYb promoter sequences,expression activities and key cis-acting elements.Transcription factors that may interact with LCYb promoters were further obtained by using the yeast one-hybrid screening system.Lastly,we applied genetics,molecular biology and biochemistry methods for functional identification of candidate transcription factors that participate in transcriptional regulation of carotenoid metabolism.The main results were as follows: 1.Functional analysis of LCYb genes.There are two types of LCYb genes,each of which possesses two different alleles(LCYb1a,LCYb1 b,LCYb2a,and LCYb2b)in most citrus species.qRT-PCR analysis of transcript levels of two LCYb genes in ‘Anliu’ and ‘Hong Anliu’ sweet orange(Citrus sinensis)revealed that the CsLCYb1 had a constitutive expression pattern,while the CsLCYb2 possessed a tissue-specific expression pattern;and low transcript level of the chromoplast-specific CsLCYb2 gene may be associated with the massive lycopene accumulation in ‘Hong Anliu’ sweet orange.We further isolated LCYb2 genes from other citrus species and performed sequence polymorphism and phylogenetic analysis;the results showed that the allelic sequence polymorphisms of LCYb2 were highly associated with the evolutionary relationship among different citrus species.Functional analysis of LCYb2 alleles in engineering bacterial system showed that all isolated LCYb2 alleles,except the CsLCYb2 b,exhibited β-cyclase activities to mediate the conversion of red lycopene to orange β-carotene.Site-directed mutation analysis of CsLCYb2 a and CsLCYb2 b showed that two amino acids at positions 72 and 359 were critical sites determining enzymatic activities of LCYb2 alleles.2.Isolation and functional characterization of LCYb promoters.We isolated the 5’ upstream sequences of CsLCYb1 from citrus.The full-length CsLCYb1 promoter and a series of its 5’ deletions were fused to the GUS reporter gene and transferred into different plants(tomato,Arabidopsis and citrus callus)to test the promoter activities.The results of all transgenic species showed that the 1584 bp upstream region from the translational start site displayed maximal promoter activity,and the minimal promoter containing 746 bp upstream sequences was sufficient for strong basal promoter activity.Furthermore,the CsLCYb1 promoter activity was developmentally and tissue-specially regulated in transgenic Arabidopsis,and it was affected by multiple hormones and environmental cues in transgenic citrus callus under various treatments.Finer deletion analysis identified an enhancer element existing as a tandem repeat in the promoter region between-574 to-513 bp and conferring strong promoter activity.The copy numbers of the enhancer element differed among various citrus species,leading to the development of a derived simple sequence repeat marker to distinguish different species.We isolated two LCYb2 promoters from sweet orange.Bioinformatics analysis of promoter sequences revealed many common and exclusive cis-elements.Functional characterization of promoter activities by transient expression assay in tomato showed that both promoters were active in chromoplast-rich fruits.3.Isolation of LCYb promoter upstream regulators.We used the promoter sequences of LCYb and their key cis-elements as bait to search the citrus fruit cDNA library by using the yeast one-hybrid screening system.Some transcription factors that may be interacted with LCYb promoters were preliminarily obtained.4.Functional identification of candidate transcription factors CsMADS6,CsMADS5 and CsGARP4.According to the results of the yeast one-hybrid screening combined with previous reports,we selected three transcription factors that may be involved in carotenoid metabolism for further functional analysis.CsMADS6 belonged to the AGAMOUS-like subfamily of the MADS transcription factor family;its expression was strongly coordinated with fruit development and coloration.Acting as a nuclear-localized transcriptional activator,Cs MADS6 directly bound and activated the promoter of LCYb1.Overexpression of CsMADS6 in citrus calli increased the carotenoid content and induced the expression of LCYb1 and other carotenogenic genes,including PSY,PDS,and CCD1.Further experiments demonstrated that CsMADS6 up-regulated the expression of PSY,PDS,and CCD1 by directly binding to their promoters,which suggested the multi-targeted regulation of carotenoid metabolism by CsMADS6.In addition,the ectopic expression of CsMADS6 in tomato significantly increased the carotenoid content and the expression of carotenogenic genes.The transgenic sepals of CsMADS6-overexpressing lines exhibited dramatic changes in carotenoid profiles,which were accompanied by changes in plastid ultrastructure.Global transcriptome analysis of transgenic sepals revealed that CsMADS6 regulated a series of pathways,that promoted increases in flux through the carotenoid pathway.CsMADS5 belonged to the SQUAMOSA subfamily of the MADS transcription factor family and shared high sequence similarities with the Arabidopsis and tomato FUL proteins.Expression analysis showed that CsMADS5 transcript and protein levels were coordinately induced with fruit development and pigmentation.CsMADS5 protein was localized in the nucleus and functioned as a transcriptional activator.Biochemical assays indicated that CsMADS5 bound directly to the promoter of LCYb1 to activate it.Overexpression of CsMADS5 in citrus callus increased the α-carotenoid levels and induced the expression of LCYb1 and other carotenogenic genes,including PSY,PDS and CCD1.Further experiments demonstrated that CsMADS5 could also bind directly to activate the promoters of PSY,PDS and CCD1.In addition,the ectopic expression of CsMADS5 in tomato increased the carotenoid content and altered the expression of carotenogenic genes.Protein interaction analysis revealed that CsMADS5 and CsMADS6 physically interacted with each other in the nucleus.CsGARP4 belonged to the GARP transcription factor family and contained a Myb_DNA-binding and a Myb_CC_LHEQLE conserved domains.CsGARP4 expressed in all tissues with the highest levels in leaf and its expresson levels gradually increased with fruit development.Acting as a nuclear-localized transcriptional activator,CsMADS6 bound directly to the promoter of LCYb1 to activate it.Overexpression of CsGARP4 in tomato resulted in phosphate deficiency phenotype.qRT-PCR results showed that CsGARP4 highly expressed in transgenic lines,while the expression of its endogenous homologs was not changed.Physiological and biochemical analysis revealed that the carotenoid content and the expression of carotenogenic genes were significantly reduced.The contend of anthocyanin increased but that of chlorophyll decreased,accompanied by the increased expression of associated genes.No significant changes were observed in total phosphorus contents,but the expression of phosphate starvation-induced genes were significantly affected In conclusion,this study functionally identified three transcription factors in the transcriptional regulation of LCYb expression and carotenoid metabolism.CsMADS6 and CsMADS5 coordinately and positively modulate carotenoid metabolism by directly regulating LCYb1 and other carotenogenic genes.Moreover,CsMADS6 also has an extensively regulatory role,that is reprogramming the transcriptional networks to lead metabolic flux into the carotenoid pathway in particular.CsGARP4 may modulate the content of strigolactone(a kind of carotenoid derivative)by directly regulating LCYb1,thus affecting the plant responses to phosphate stress.