Functional Genomic Studies Of Carotenoid Biosynthesis In Sweet Orange(C.sinensis(L.)Osbeck)

Citrus,one of the most important crops and nutrition sources,which believed to originate in tropical and sub-tropical countries of Southeast Asia,is characterized by fragrant flowers and edible juicy fruit.The most important commercial varieties can be divided into four groups:sweet oranges(C.sinensis(L.)Osbeck),mandarins(C.reticulata(L.)),grapefruit(C.paradisi Macfad),lemons(C.limon(L.)Burm.)and limes(C.aurantifolia Swingle).In the world citrus market,there are two clearly differentiated markets:Fresh citrus fruits market with a predominance of oranges and processed citrus products market,mainly orange juice.The newest data reported by United States Department of Agriculture showed that world demand for citrus products will continue to expand.Citrus fruits have several health benefits to human being’s daily diet,playing important roles in supplying energy and nutrients.Citrus fruits supply mainly carbohydrates,additionally,vitamin C and dietary fiber.Moreover,citrus fruits are also a source of many phytochemicals,including carotenoids,flavonoids and limonoids.Especially,research found citrus is a complex of carotenoids with more than 115 carotenoids which is the largest number found in any fruit.Massive studies were carried out to investigate the genes on carotenoid biosynthetic pathway and the metabolic mechanisms.Cloning of genes encoding enzymes of carotenoid biosynthesis has been conducted extensively.Different citrus species and varieties have been employed to characterize carotenoid biosynthesis genes and their expression patterns to demonstrate their functions.For example,research found thatβ-cryptoxanthin and violaxanthin isomers were predominantly accumulated in Satsuma mandarin(C.unshiu)and sweet orange fruits,respectively.It was demonstrated that the expression balance between upstream synthesis genes(CitPSY,CitPDS,CitZDS,CitLCYB)and downstream synthesis genes(CitCHYB and CitZEP)might be the determinant factor on the ratios of P-cryptoxanthin and violaxanthin.The expression patterns were investigated in flavedo and juice sacs of different varieties,showing that carotenoid accumulation in both tissues was accompanied with a general increase in mRNA levels of a set of genes.Generally,the total amount of carotenoids was higher in flavedo compared to juice sacs,which was correlated with the higher gene expression levels in flavedo.Massive research indicates that transcriptional control of biosynthetic genes is the first level of regulation of carotenoid accumulation in citrus fruit.Several citrus mutants with fruit flesh color distinctly different from their wild types were employed to elucidate the possible different mechanisms in carotenoid biosynthetic pathway.For example,the lycopene accumulation and higher amounts of phytoene,phytofluene and β-carotene in the flesh of Cara Cara(C.sinensis)resulted from higher expression of MEP(Methylerythritol phosphate)-isoprenoid genes;by contrast,gene expression profiling indicated up-regulation of most carotenoid biosynthesis genes as the mechanism leading to lycopene accumulation in Hong Anliu juice sacs and peel.Further,two different alleles of LCYB2,LCYB2a and LCYB2b,were isolated from sweet orange and grapefruit;the protein from the latter was almost null in lycopene P-cyclase activity and preferentially expressed in red-fleshed grapefruit,leading to the accumulation of lycopene.All these studies demonstrated that different mechanisms of carotenoid biosynthesis involved in the mutant and wild types carotenoid accumulation,likewise,found that accumulation of fruit carotenoids is not always associated with the accumulation of mRNAs of carotenoid biosynthesis genes.Chromoplast,the non-photosynthetic plastids and site for carotenoid biosynthesis,was studied with many plants.Experimental evidence suggested that>95%of the plastid proteins detected are nuclear-encoded and imported into plastid,and only approximately 100 proteins were encoded by plastid genomes.The findings with chromoplasts from different fruits revealed the unique features of chromoplast proteomes.Previous studies on citrus chromoplast metabolism have been mainly focused on physiological aspects;very few studies were conducted to characterize the organelles and organelle-specific proteins at the subcellular level during citrus fruit ripening.Although massive studies were carried out to illustrate the mechanisms on carotenoid biosynthetic pathway,gene expression analysis could not always explain and fulfil the fragmentary view of the process and other still unknown genes,gene family members,or mechanisms may be operating.Moreover,19 novel gene members in the citrus carotenoid biosynthesis gene families were mined out from citrus genome databases using bioinformatics tools.Thus,the present study was focused on the expression levels and patterns of novel carotenoid biosynthetic pathway gene family members and their relationships with carotenoid accumulation in citrus,and the proteomic analysis of their active site chromoplast.These results should be useful for identification of regulators and potentially unknown mechanisms associated with the control of the carotenoid biosynthesis pathway in citrus.Experiment I.Investigate the expression levels of the novel carotenoid biosynthetic pathway gene family members in different tissues during citrus fruit maturationIn this study,comparative real time RT-PCR and HPLC(High Performance Liquid Chromatography)were employed to investigate the expression levels of carotenoid biosynthesis gene family members and the carotenoid accumulation during fruit maturation.15 fruits of Valencia orange were selected monthly from November 2011 through February 2012 and their diameters were measured using caliper;and then peel and juice colors were determined using a Minolta CR-330 colorimeter.Fifteen fully expanded and hardened leaves were selected in November 2011 from the most recent flush and likewise fifteen leaves from the previous growth flush were selected for processing,respectively.Juice soluble solid content(SSC),titratable acidity(TA),and maturity index(SSC/TA ratio)were determined respectively with a refractometer,by titration to pH 8.2 with 0.1 mol L-1 NaOH,and as the calculated SSC/TA ratio.The Fifteen fresh fruit and leaf samples were divided into three pools that served as biological replicates and two technical replicates of each of the three biological replicates were analyzed.The results presented in this work revealed the expression patterns of 23 carotenoid biosynthesis pathway gene family members in fruit(flavedo and juice sacs)and leaves(young and mature)of Valencia orange during fruit maturation,including 15 novel members.Their roles in the carotenoid biosynthesis pathway were suggested by comparing with previous studies in other plants.The results indicate that CCS-Contigl9 and CCS-Contigl6 might correspond to LCYB2a and LCYB2b,and that the 4 CHYB family members might belong to the BCH type.Global co-expression analysis of the complete transcriptome might reveal other carotenoid pathway genes that were co-expressed along with our pre-selected group of 23 genes;however,such an approach was beyond the scope of the current study.The correlations reported are solely among the genes for which we generated expression data.The relationship analysis between gene expression and carotenoids accumulation showed the expression of PSY-Contig22,ZDS-Contig23,CHYB-Contig25 and CHYB-Contig07 correlated well withβ,β-xanthophyll accumulation in flavedo,PSY appears to be the first bottleneck for carotenoid biosynthesis in non-photosynthetic tissue,and predominantly lutein accumulated in leaves accompanied with high expression of LCYB-Contig21 and low expression of CHYB family members.Taken together,these first-hand expression data will be useful to define the tissue-specific roles of each gene member in accumulation of different carotenoids in citrus leaves and maturing fruits.Experiment II.Investigate the expression patterns of these novel carotenoid biosynthetic pathway gene family members in Valencia and its deeply colored mutant Rohde Red ValenciaIn this research,carotenoid accumulation and biosynthetic gene expression levels during fruit maturation were compared between ordinary Valencia(VAL)and its more deeply colored mutant Rohde Red Valencia orange(RRV).Fruits of ordinary Valencia and Rohde Red Valencia orange were collected every other month from July through March in the 2012-2013 ripening season.The processing of all the samples was the same as in experiment I.Statistical analysis was performed by using JMP software.The results showed that although the expression of PSY was at a similar level in VAL and RRV during the marked accumulation of β,β-xanthophyll,the expression levels of PDS-Contigl7,ZDS and CCS-Contigl9 were higher in RRV than in VAL,which suggested a possibly higher supply of P-carotene in juice sacs of RRV However,only trace amounts of p-carotene were detected;and the ratio ofβ-cryptoxanthin/violaxanthin in matured RRV was 1.64,which was much lower than in Satsuma mandarin(5.17).There might be two reasons for these observations.First,because the expression levels of CHYB gene members were high,the conversion fromβ-cryptoxanthin to zeaxanthin was high.The other reason is ZEP also was expressed at higher levels,which led to the conversion zeaxanthin to violaxanthin.Thus,the high accumulation of β-cryptoxanthin in juice sacs of RRV might be caused by the high expression of PDS-Contigl 7,ZDS family members,and CCS-Contig19.In this study,the relationship between carotenoid accumulation and the expression of 23 carotenoid biosynthesis gene members in VAL and RRV during fruit development was investigated,especially,the possible mechanism accounting for high β-cryptoxanthin accumulation in juice sacs of RRV In flavedo,the carotenoid biosynthesis pathway shifted from α,β-xanthophylls synthesis to β,β-xanthophylls synthesis earlier in RRV than in VAL.The uncoordinated carotenoid accumulation and gene expression in RRV might relate to the expression of certain gene(s)in the MEP pathway.In juice sacs,LCYE might be the limiting step of carotenoid accumulation in the early development of VAL and RRV.The high expression of PDS-Contig17,eight ZDS family members,and CCS-Contig 19 seems responsible for the high accumulation of β-cryptoxanthin in juice sacs of RRV.In addition,further research related to post-transcriptional or translational control is needed.In particular,further research on other genes in the MEP pathway which are related to carotenoid metabolism is needed to elucidate carotenoid accumulation in citrus fruit.Experiment III.Proteomic analysis of chromoplasts from Valencia and Rohde Red ValenciaIn this study,juice of Valencia sweet orange and its deeply colored mutant Rohde Red Valencia were collected in December,and processed according to different purpose.Proteomic analysis was conducted with chromoplasts from the juice samples.The expression levels of 23 carotenoid biosynthetic pathway gene family members,carotenoid and volatile accumulation were also investigated with the same samples used for proteomic analysis.The protocol of chromoplasts isolation was according to Zeng et al.(Zeng et al.2011)with minor modification.Protein identification was performed by LC-MS/MS.Raw data was analyzed using Mascot(Matrix Science,London,UK;version 2.4)against NCBI-Greenplant database(ftp://ftp.ncbi.nlm.nih.gov/genbank/)assuming the digestion enzyme was trypsin.To predict the localization of the identified proteins,a local Blast search was carried out against Arabidopsis proteins(TAIR 10)and proteins with lowest expect value were chosen as Arabidopsis homologues,then the corresponding AT accession numbers were used to search against SUBA database(http://suba.plantenergy.uwa.edu.au).Those proteins were tentatively considered to be chromoplast proteins if they were predicted to localize in plastid by at least two of the web programs in SUBA database.The corresponding sequences of proteins predicted to localize in chromoplasts were used to perform Blast2GO and functionally classified according to Mapman(http://mapman.gabipd.org/web/guest/mapman)using annotations retrieved from databases.In the present study,a total of 238 and 199 proteins were putative plastid proteins in VAL and RRV,respectively.Functional classification using Blast2Go and Mapman bins revealed large numbers of proteins involved in carbohydrate metabolism,stress and redox,protein regulation and transport and amino acid metabolism in chromoplasts in both cultivars.Proteins involved in generating energy and precursors for the synthesis of metabolites are more active in chromoplasts of RRV than VAL;however,more amount of proteins involved in photosynthesis,proteins regulation and stress in VAL than RRV,which led to different metabolism in stress reaction and protein regulation in the two cultivars.It was surprised that none proteins on carotenoid biosynthetic pathway were detected,which implied that carotenoid metabolism was not active in December.306 proteins were identified as candidate proteins located in chromoplasts of VAL and RRV.A total of more than 400 chromoplast proteins have been reported in citrus(Zeng et al.2011).The reason may be the number of proteins probably varies according to the development stage of the plastids,and different sample and environmental conditions.In the present study,December may not be the striking season of proteins presence in the two late-mature cultivars.Still,several interesting findings have been revealed.Our study provided a very good overview of the relatively abundant proteins in the juice sacs of VAL and RRV,especially,compared the carotenoid biosynthesis related gene/protein/metabolic products(carotenoids and volatiles)between the two cultivars.This study revealed the common metabolic characteristics shared between the chromoplasts of two cultivars and showed some remarkable differences of some identified proteins in the chromoplasts of two cultivars.In conclusion,the present study would be useful to understand the mechanism for the citrus fruit color change at the genomic,transcriptomic and proteomic level,especially,carotenoid biosynthesis and its metabolism in sweet orange.