Study On Metabolic Differences And Function Of Genes Related To Color Formation In Chrysanthemum Morifolium

Chrysanthemum morifolium cv.’Hangju’ is currently one of the most widely cultivated medicinal chrysanthemum types in China.As a traditional medicinal and edible chrysanthemum,Hangju has good effects of dispersing wind-heat,clearing heat and detoxifying,clearing liver and improving eyesight.It is not only used in Chinese herbal decoction pieces and proprietary Chinese medicines,but also widely used in herbal tea beverages and daily chemical products.Hangju is divided into "Hang Bai Ju"(with white petals)and "Hang Huang Ju"(with yellow petals),it is traditionally believed that yellow chrysanthemums are better at dissipating wind-heat,while white chrysanthemums are better at calming the liver and brightening the eyes.Based on the theory of "distinguishing the quality of Chinese medicinal materials based on character",the fixed characteristics,such as shape,color,smell,taste and texture,expressed in appearance have a certain relationship with their internal components.But so far,the difference and formation mechanisms of the active components between yellow and white chrysanthemums remain unclear.In this study,two types of Hangju with different flower colors,’Zaoxiaoyangju’(ZXYJ)and its bud mutation variety ’Jinjuyihao’(JJYH),were used as research materials to explore the change rule and formation mechanism of the internal components of Hangju caused by the difference of yellow and white flower colors,and screen the major genes causing the difference of flower color and verify their functions.The main research results are as follows:(1)In this study,a large-scale metabolite qualitative and quantitative analysis of ZXYJ and JJYH was performed using widely targeted metabolomics.The results showed that a total of 710 metabolites were detected and identified,including more than 20 classes.Through principal component analysis and orthogonal partial least square discriminant analysis,we found that ZXYJ and JJYH had significant differences in metabolic levels,mainly in flavonoids,lipids,phenylpropanoids and organic acids.There were only two anthocyanins in all the significantly changed metabolites,and both of them were low expression in JJYH.Through the analysis of flavonoids in the two kinds of chrysanthemum,we found that the content of chalcone and aurone did not change significantly,and the anthocyanin content in JJYH was low.Therefore,we preliminarily judged that anthocyanins and flavonoids were not the main reasons for the yellow color of JJYH.(2)Transcriptome sequencing of ZXYJ and JJYH showed that 706 030 transcripts were assembled,including 362 577 unigenes.Combined transcriptomic and metabolomic analysis revealed that the phenylpropanoid biosynthetic pathway and the phenylalanine metabolic pathway had the highest degree of differentially expressed genes and significantly changed metabolites enrichment,respectively.We found that altered phenylalanine metabolism in the two Hangju triggered differential accumulation of flavonoids,lignins,coumarins,quinic acids,and benzoic acids associated with the phenylpropanoid metabolite pathway.The redirection of metabolic flux may contribute to increased accumulation of total flavonoid levels in JJYH,which may begin as early as phenylalanine metabolism,namely the catalysis of PAL.(3)Based on the observation of the microstructure of ray floret of ZXYJ and JJYH,we found that the yellow pigment mainly accumulated in the upper and lower epidermis of ray floret,and there were glands on the abaxial surface of ray floret.The electronic tongue analysis showed that ZXYJ and JJYH had no significant difference in taste,such as sour,bitter,salty,fresh and astringent,but the electronic nose analysis found that there was a significant difference between the two,especially in the aromatic components.At the same time,with the detection of volatile organic compounds(VOCs)in ZXYJ and JJYH based on SPME-GC-MS/MS identified a total of 808 VOCs,among which terpenoids,esters and heterocyclic compounds accounted for more than half.We have strong evidence that could prove the VOCs in Hangju are complex and rich in content.In addition,through multivariate statistical analysis and discrimination,we found that there were significant differences between ZXYJ and JJYH in VOCs,and we screened 86 differentially accumulated VOCs,among which terpenes were the most abundant,and 15 of the 18 differentially accumulated terpenes were down regulated in JJYH,accounting for 83.3%.Based on LC-MS/MS,the qualitative and quantitative analysis of carotenoids in the ray floret of ZXYJ and JJYH at different growth stages was carried out,and a total of 14 carotenoids were identified.The results showed that lutein was the most abundant carotenoid component and the main pigment that caused the color difference.The total carotenoid content of ZXYJ was lower than that of JJYH in four growth periods.The carotenoid content of ZXYJ began to decrease during the period from S3 to S4,while the total carotenoid content of JJYH was always on the rise.Combined with the quantitative analysis of key enzyme genes and components in the carotenoid biosynthesis pathway,we found strong evidence that the carotenoid biosynthesis pathway in ZXYJ was not inhibited,so we speculated that the high expression of carotenoid cleavage dioxygenases(CCDs)genes were the reason for the decrease of carotenoid content in ZXYJ.(4)We cloned CmCCD1 and CmCCD4 a genes from the full-length transcriptome of Hangju and found that both of them had the RPE65 domain and seven-blade β-propeller structure through bioinformatics prediction.Through quantitative analysis,the expression levels of CCD1 and CCD4 a in both ZXYJ and JJYH were significantly different.We studied the selectivity and cleavage ability of CmCCD1 and CmCCD4 a to carotenoid substrates by using engineered bacteria that produced carotenoid.The results showed that both CmCCD1 and CmCCD4 a could cleavage β-carotene and zeaxanthin in E.coli,but their cleavage efficiency was different.The color changes and carotenoid content changes of the engineered bacteria showed that CmCCD4 a had a weaker cleavage ability for β-carotene than CmCCD1,which the binding energy data of molecular docking verified.In addition,CmCCD1 and CmCCD4 a showed cleavage ability to β-carotene,lutein and zeaxanthin in vitro.The detection of cleavage products by GC-MS showed that CmCCD1 and CmCCD4 a could cleave β-carotene and zeaxanthin to produce volatile β-ionone and trans-β-ionone,respectively,in engineered bacteria.After the oxidative cleavage reaction between CmCCD1 and zeaxanthin,in addition to trans-β-ionone,we also detected volatile components with norisoprene structure,such as 6-methyl-5-hepten-2-one,octyl formate and neryl acetate.A 1570 bp CmCCD4 a promoter sequence was amplified by FNPI-PCR,and two transcription factors interacting with the CmCCD4 a promoter,namely Cm ERF6 and Cm ERF061,were screened by yeast one-hybrid technique.The cellular localization preliminarily determined that the two transcription factors were localized in the nucleus.In conclusion,this study comprehensively analyzed the difference of metabolite levels in two colored Hangju,clarified the reorientation of phenylpropanoid metabolic flux in yellow and white Hangju,and identified the main carotenoid component causing color changes.We verified the functions of CmCCD1 and CmCCD4 a proteins responsible for the cleavage of carotenoid components and the production of volatile deactivated carotenoids.This study comprehensively evaluated the differences of active components in ZXYJ and JJYH,and preliminarily revealed the regulation mechanism of flower color and aroma formation of two chrysanthemum,providing new ideas for quality evaluation and further resource development of different flower color chrysanthemum.