| The red flesh citrus has high nutritional value and special sensory attractionto consumers.Citrus maxima cv.Chuhong is anelite local variety in Zhejiang province with strong flavor and deep red flesh and light red albedo caused by a large amount of carotenoids.C.maxima cv.Feicui is also an excellent local variety wih light flavor,and pale green flesh.In addition to flesh color,there were significant differences in other fruit quality such as aromatic tratit..They are not strictlya mutant and its wild-type,however,as for their same habitats,same pummelo genomic background while significantly different sensory quality,they are ideal experimental materials for coordinated fruit qualities research in citrus.Firstly,fruit quality-related metabolites in the fruits of two pummelos(carotenoids,primary metabolites,limonoids,ABA and aromatic compounds)were determined at the different developmental stages via HPLC,GC-MS and other technologies;secondly,carotenoid biosynthetic gene expression profiles were a nalyzed,and cloning and sequencing of genes involved in carotenoid biosynthetic pathway were also carried out;finally,the promoter sequences of various terpenoid metabolic pathway genes were analyzed to investigate changing of carotenoid profile during fruit development of Chuhong pummelo.Relationship between carotenoids metabolism and other fruit qualities was studied to lay theoretical foundation for mechanism of red flesh formation in Chuhong pummelo.The main results were as follows:1.Analysis of carotenoids content showed that at maturation stagey,albedo,segment membrane and juice sacs of Chuhong pummelo showed red color due to the over accumulation of lycopene and total carotenoids.Feicui pummelo had np lycopene in all fruit tissues,and its total carotenoids content was very low(≤8.02±0.72μg/g.DW)in albedo,segment membrane and juice sacs during fruit development.Before 150 DPA,lutein andβ-carotene were mainly accumulated in the flavedo of Chuhong and Feicui pummelo.With fruit development,the content of lycopene increased gradually and decreased sharply at 150 DPA in the albedo and segment membrane of Chuhong pummelo.The content of lycopene in the juice sacs of Chuhong pummelo had been continuously increasing and reached the highest level(426.98±35.80μg/g.DW)at 150 DPA.2.The primary metabolites in juice sacs of two pummelo cultivars were measured at four stages of 45 DPA,75DPA,100 DPA and 150 DPA when carotenoids varied largely.The results showed that among 20 primary metabolites,sucrose was main soluble sugar in juice sacs of two pummelos.At 150 DPA,sucrose content(92.30 mg/g DW)took up 61.70% of all detected sugar in juice sacs of Chuhong pumme lo while sucrose content(121.67 mg/g DW)took up 57.25% of all detected sugar in juice sacs of Feicui pummelo.During 100 DPA and 150 DPA,the content of sucrose,glucose,fructose,D-Galactose and lactulose showed no significant changes in juice sac of Chuhong pummelo while their contents had significantly increased in Feicui pummelo.And their contents in Feicui pummelo were significantly higher than that in Chuhong pummelo at 150 DPA.Detection of organic acid content showed that citric acid was the main one in two pummelos.The changes of citric acid content in two pummelos showed similar trends that it rose constantly at the early period and declined at maturity period.Throughoutfruit development stages,citric acid content in juice sacs of Chuhong pummelo was 5.8-21.6 times of that of Feicui pummelo.Five amino acids were detected in juice sacs of two pummelo cultivars.There was no significantly different content of 4-aminobutyric acid in juice sacs between two pummelo cultivars.The content of L-aspartic acid and asparagines in juice sacs of Chuhong pummelo were significantly higher than that in Feicui pummelo at 100 DPA and 150 DPA.3.Results showed that the during 100 DPA and150DPA there were significantly different contents of limonin and nomilin in the albedo,segment membrane and j uice sacs between Chuhong and Feicui pummelo.And the limonin and nomilin content changes in the segment membrane and juice sacs of Chuhong pummelo showed reverse trends to that of Feicui pummelo.At maturity period(150DPA)the content of limonin in segme nt membrane and juice sacs of Chuhong pummelo were 2.74 and 4.91 times that of Feicui pummelo,respectively while the content of nomilin in segment membrane and juice sacs of Chuhong pummelo were 3.16 and 4.62 times that of Feicui pummelo,respectively.4.At 100 DPA and 150 DPA,ABA content gradually decreased in juice sacs of Chuhong pummelo and at 150 DPA ABA content reached the lowest level(0.78μg/g DW).The ABA content gradually increased in juice sacs of Feicui pummelo,showing reverse trend to that of Chuhong pummelo.At 150 DPA ABA content reached the highest level(3.15μg/g DW)and was 4.04 times that of Chuhong pummelo.5.The results showed that 69 volatiles were detected in flavedo of two pummelo cultivars at maturation stage,and the mainly components was monoterpenes.The terpene substances accounted for 99.63% and 99.57 % of total amount of volatile substances in Chuhong pummelo and Feicui pummelo,respectively.Among volatile substances d-Limonene was the main one and accounted for 96.67% and 82.43% of total amount of volatile substances in the two pummelo cultivars.There were significant differences in the content of 52 kinds of substances in two pummelo cultivars.Geranyl acetate,one kind of monoterpene esters,showed greatest difference of content between two pummelo cultivars.The content of geranyl acetate in Feicui pummelo was 19.7 times that of Chuhong pummelo.6.At whole growth and development of fruit,it was found that 11 structure genes involved in carotenoids biosynthes in the flavedo,albedo,segment membrane and juice sacs of two pummelo cultivars showed significantly different expression.The partial correlation analysis showed that structure genes significantly correlated with the contents of lycopene and total carotenoid in the albedo,segment membrane and juice sacs of Chuhong pummelo varied.7.Eleven structure genes involved in carotenoids biosynthes in the juice sacs of two pummelo cultivars were cloned and aligned.The results showed that there were different transcripts of ten genes,except for ZISO.Six of the genes(DXR,PSY,CRTISO,LCYb2,LCYe and ZEP)had the main different transcripts in two pummelo varieties.These seven genes might be major contribution to the accumulation model of carotenoids in the juice sacs of two pummelo cultivars.Six transcripts of PSY gene were cloned in the flavedo,albedo,segment membrane and juice sacs of two pummel cultivars,and four in Chuhong pummel,three in Feicui pummelo.In all transcripts SNP polymorphism and the simple repetitive sequence(AAT)polymorphism were found.However,analysis of PSY gene sequences in other red flesh pummelo varieties and common pummelo showed that the AAT repetitive sequence had no direct relation with the lycopene accumulation.8.Bioinformatics analysis of promoters related to 30 genes in terpenoid metabolic pathways showed that there were ten transcripts including ATSOC1(MIKC-MADS family),F13F21.8(B3family),AIL2(AP2 family)and DOF1.7,DOF1.10,DOF2.2,DOF3.4,DOF4.7,DOF5.1 and DOF5.6(DOF family).They simultaneously regulated 5~11 MEP pathway genes,carotenoid biosynthesis or lyase genes,key genes of limonoids biosynthesis and other terpene synthase genes.It indicated that there were transcription factors in terpenoid metabolic pathways that could regulate several related genes simultaneously.In conclusion,the content of metabolites related to fruit quality--sugar,acid,limonines,ABA,aroma substances and carotenoids were significantly different between the fruits of the two pummelo cultivars;the transcripts of structural genes related to carotenoids in different tissues of the two pummelo cultivars showed a comprehensive effect of different expression,which ultimately led to the different accumulation of carotenoids in the fruits of two pummelos,no obvious structural gene mutation was found in the carotemoid metabolic pathway.The study provides a new theoretical foundation for the improvement of citrus comprehensive quality based on the visual analysis of carotenoids and othe related metabolic pathways. |
PDF Full Text Request