Cloning And Functional Characterization Of Carotenoid Cleavage Dioxygenases From Freesia Hybrida

Carotenoids are an important group of secondary metabolites in plants responsible for the photosynthesis.In general,carotenoids are C₄₀ isoprenoids,which are synthesised in plastids.A number of bioactive compounds found in plants are derived from carotenoid precursors.Carotenoids can be cleaved by carotenoid cleavage dioxygenases?CCDs?at manifold double bonds to generate a series of apocarotenoids in plants.The apocarotenoids have important contributions for the coloration and aroma of plants,and the regulation of plants growth and seeds germination as a precursor of plant hormones.Some apocarotenoids also have impressively medicinal and hygienical functions on numerous diseases with extensive application values.At present,numerous members of CCDs family have been characterized in plants.Freesia hybrida is a kind of bulbous flower plant,belonging to Iridaceae family.Some wild Freesia species were introduced into Europe in the 18^th century,and F.corymbosa and F.leichtlinii were employed as parent species for the breeding of the modern cultivars.In wild species and some cultivars of F.hybrida,?-ionone and dihydro-ionone were detected as the main compounds emitted from flowers.In this study,another apocarotenoid,safranal was mainly detected in the essential oil of Red River^?and less in Ambiance.In order to isolate the dominant genes responsible for the biosynthesis of these apocarotenoids in Freesia hybrida,FhCCD2,FhCCD1 and FhCCD4 genes were mined from the well-constructed transcriptome data of Red River^?and tentatively designated as the candidate genes.In order to clarify function of FhCCDs,the full-length cDNAs were obtained and then subjected to Escherichia coli transfection.This is the first time to our knowledge that CCD genes were isolated and functionally characterized from Freesia species.The main results in this study were as follows:Through GC-MS analysis,?-ionone and safranal were found in the essential oils of Red River^?.Safranal was also detected in essential oils of Ambiance but with lower content when compared with that in Red River^?.As for another zeaxanthin derived apocarotenoid,crocin-I was found in the extracts of Red River^?flowers through HPLC analysis,whereas it might be too lowly accumulated in Ambiance flowers to be detected.According to the information of Freesia transcriptomic database,four carotenoid cleavage dioxygenase genes were isolated from Red River^?flowers.Phylogenetic analysis demonstrated that the four members of CCDs family clustered together with CCD2,CCD1 and CCD4,respectively,and designated as FhCCD2,FhCCD1,FhCCD4a and FhCCD4c.The protein structure prediction of FhCCDs showed that they had the same characters with other known functional CCDs except FhCCD1.Briefly,seven?-folds made up a seven-bladed propeller structure.A ferrous ion was located at the axis of the propeller and coordinated by four conserved histidine residues.A molecular oxygen was then introduced into the structure by the ferrous ion to activate catalytic activity.The missing of one conserved histidine in FhCCD1resulted in the failing of ferrous ion to be bound.Subcelluar localization analysis showed that FhCCD2,FhCCD4a and FhCCD4c localized in chloroplasts,facilitating the direct utilization of carotenoids,which were also synthesized in the same organelle.In contrast,FhCCD1 was located in cytoplasm consistent with other CCD1 proteins characterized from other plants.Previous studies found that CCD2 genes have only been isolated from plants in Crocus genus.In contrast,CCD1 are widely distributed in plant lineages.In addition,amino acid sequences on both sides of intron insertion sites were highly homologous between CCD2 and CCD1.Therefore,it could be further postulated that CCD2 might have evolved from CCD1 or their common ancestor.During the evolutionary process of the CCDs,intron loss occurred,which led to the appearance of CCD2.Transcriptomic analysis indicated FhCCD2 was differently expressed in Red River^?and Ambiance,which was further validated by quantitative real-time PCR.The expression level of FhCCD2 increased gradually with the development of flowers in Red River^?.Moreover,FhCCD2 was differentially expressed in different tissues or organs,with highest expression levels detected in petals.Petals are the most proportional part of the organ in Freesia flowers,this expression profile was beneficial to the accumulation of safranal and crocin.To preliminarily unravel the molecular basis of the significantly differential expression levels of FhCCD2 between Red River^?and Ambiance,the promoter sequences of FhCCD2 from these two Freesia cultivars were cloned and named as ProFhCCD2-R and ProFhCCD2-A,respectively.Activation effects evaluated from either transcriptional level or translational level implied that there might be no obvious functional differences between the two promoters,as the expression of FhCCD2 could also be activated by ProFhCCD2-A in Red River^?,indicating that some unknown up-stream trans-regulators might be invalid in Ambiance,such as transcription factor genes.The spatio-temporal expression profiles of FhCCD4a in flowers of Red River^?was consistent with the emission of?-ionone.Biochemical analysis showed that the FhCCD2 cleaved zeaxanthin at the 7,8and 7',8'double bonds to generate intermediates prerequisite for the biosynthesis of safranal and crocin.In addition,the FhCCD4a exhibited?-carotene cleavage activity yielding?-ionone,whereas no cleavage activtity were observed in FhCCD1 and FhCCD4c using lycopene,?-carotene and zeaxanthin as substrates in in vitro assays.