Identification Of Key Genes Regulating Citrus Essential Oil Biosynthesis

Plant essential oils are very important materials in food,beverage,cosmetics,pharmaceutical,and other industries.About 30%of plant essential oils are produced from citrus.Citrus essential oil is stored in oil glands of peel,petal,leaf and other tissues,of which the content in peel is the highest at more than 2%of fresh weight.The peel accounts for 20%of the citrus fruit weight.Increase of content of essential oil in the peel could significantly increase the added value of citrus processing.The capability of essential production is significantly varied among different citrus varieties,which is mainly controlled by genetic factors.However,molecular mechanism of essential oil production is rarely studied.In this study,the dynamic changes on oil glands development associated with essential oil accumulation,and volatile components during fruit development was systematically analyzed.Rongan kumquat and Huapi kumquat,the seedling mutant of Rongan with very low essential oil content,which have the similar genetic background were employed to screen differential expression genes related to essential oil synthesis through RNA-seq analysis.Several genes were selected as candidate genes for identification of their relationship with essential oil production with qRT-PCR analysis.Among them,the expression of FcMYC2 was found to be highly corresponded to the content of essential oil.It was cloned and characterized by bioinformatics analysis and expression analysis in different tissues and citrus species.Genetic transformation with sweet orange variety Jincheng was conducted to produce FcMYC2 overexpress,RNA-mediated interference and knock-out transgenic plants,in order to validate the function of FcMYC2 on biosynthesis of essential oil in citrus.The level of gene expression,number of oil glands,and contents of volatile components were investigated to identify the relationship of FcMYC2 with production of citrus essential oil.The major results achieved are as follows:1.Oil gland formed in the early stage of tissue initiation,the size of oil gland was affected by the accumulation of essential oilIn kumquat fruit,formation of oil gland began at fruit primordium stage.Expansion of oil gland follows the accumulation of oil in the secretory cavity during fruit development.Some of glands may cease the development or even degenerate,which could reduce the number of developed glands at maturity of the fruit.There is the large variation of size of oil glands at late stage of the fruit development.The increasing of the size of oil glands are faster at maturation than that of growing stage,also more obvious that,most of glands are expanding at same time at this stage.However,the accumulation of essential oil in the secretory cavity was not synchronized with the development of the fruit.At fruit growing stage,the rate of fruit increasing were much higher than that of in maturation stage,which is contradiction to the accumulation of essential oil.2.The proportions of volatile components in peel are in change during fruit development.The proportions of antimicrobial components decreased significantly at maturity.The proportion of terpenoid alcohols which have good antimicrobial activity,such as linalol,?-terpineol and geraniol,was decreased from 3.99%at 30 days after flowering to 0.95%at fruit ripening.Citral,carvone and camphor with good antimicrobial activity even could not be detected at fruit ripening stage.Some components with irritative odors,such as camphene,?-phellandrene,caryophyllene and?-bisabolol,which are commonly exist in the peel of other citrus species,were not detected in the kumquat at ripening stage.Camphor and 4-acetyl-1-methylcyclohexene?pungent?only existed in the peel 60 days post anthesis.This may be one of the reasons why the peel of kumquat is less irritating,edible and palatable.3.Accumulation of essential oil is mainly due to the production of monoterpenoids and sesquiterpenoidsThe contents of monoterpenoids increased continuously from 5272.03?g·g^-1?76.86%?to13587.04?g·g^-1?80.49%?,and sesquiterpenoids increased continuously from 976.07?g·g^-1?14.07%?to 2293.44?g·g^-1?14.15%?following the development of fruit.They increased by 1.58and 1.35 times higher at 150 DAF than 30 DAF,respectively.The content of non-terpenoid volatiles,which accounted for the very low content of essential oil,increased from 168.58?g·g^-1?2.54%?to 213.42?g·g^-1?1.19%?,only increased by 0.27 times,but the proportion decreased significantly.Throughout all the development stages,the monoterpenoids and sesquiterpenoids are always the highest proportions of the essential oil components.Therefore,the increase of oil content in kumquat fruit peel is the contribution of monoterpenoids and sesquiterpenoids production.Also,expansion of secretory cavities is the results of monoterpenoids and sesquiterpenoids accumulation.4.Selection of candidate genes related to the essential oil productionThe number of oil glands and the content of essential oil in Huapi?HP?kumquat were much lower than those in Rongan?RA?kumquat and other citrus species.398 commonly differentially expressed genes were identified by RNA-seq of peel and leaf samples between Huapi kumquat and Rongan kumquat.Annotation analysis indicated that these pathways involved in essential oil biosynthesis,such as terpenoid backbone biosynthesis?ko00900?,limonene and pinene degradation?ko00941?,monoterpenoid biosynthesis?ko00902?and sesquiterpenoid and triterpenoid biosynthesis?ko00909?.The key genes of MEP and MVA pathways in terpenoid backbone biosynthesis,several representative monoterpene,monoterpene alcohol and sesquiterpene synthase genes were selected for qRT-PCR analysis.The results showed that most of genes showed the different trends of expression between leaves and peels except a GGPPS gene in MEP and MVA pathways.But monoterpene synthase genes and linalol synthase genes which demonstrated same trend of expression in leaves and peels.We speculated that the expression level of the transcription factors shared by these monoterpene and sesquiterpene synthetase genes in HP may have changed.To narrow down the scope of screening,four transcriptional regulator genes cs2g14570,cs5g01450,cs7g08960 and cs4g03550 were found from 398 differentially expressed genes.Four transcription regulators were screened as candidate genes by qRT-PCR validation.Among them,the expression level of Cs2g14570?an AP2 transcription regulator?,Cs5g01450?a MYC2 transcription regulator?and Cs7g08960?an F-box protein?were significantly lower in HP leaves and peels than RA.The previous researches reported that these three types of transcriptional factors were involved in the regulation of secondary metabolites biosynthesis,particularly,MYC2 gene is involved in the regulation of sesquiterpenes,diterpenes and alkaloids biosynthesis in many species.Therefore,Cs5g01450?tentatively termed as FcMYC2?was chosen for further analysis to elucidate its function on biosynthesis of essential oil in citrus.5.FcMYC2 is a sequence-specific MYC2 type bHLH transcription factor family gene.FcMYC2 was successfully cloned from HP and RA.The full length of FcMYC2 was 1563 bp and GC content was 46%.It encoded 520 amino acids.The predicted molecular weight of FcMYC2protein was 58.35 KD and its isoelectric point was 6.83.Compared to its homologous gene Cs5g01450 in sweet orange genome?Citrus sinensis?,FcMYC2 has an insert of"CAT"and encodes one His at+805.This insertion is not in the conservative domain.The FcMYC2 gene has only one exon and no intron,same as Cs5g01450.There are two SNP loci in the FcMYC2 gene of RA,located at positions of+408 and+1320,respectively.There are two combinations of sequences of FcMYC2,G₄₀₈T₁₃₂₀ and T₄₀₈C₁₃₂₀,named as FcMYC2_gt and FcMYC2_tc,respectively.Those two FcMYC2 can be detected in both DNA and cDNA.The FcMYC2 gene of HP also has these two SNP loci,but formed four combinations sequences,G₄₀₈T₁₃₂₀,T₄₀₈C₁₃₂₀,T₄₀₈T₁₃₂₀ and G₄₀₈C₁₃₂₀,which can also be detected in HP DNA and cDNA.These SNP loci are located at the third place of genetic code,and did not change the encoded amino acid.The results of searching through the citrus genomic database?including citrus sinensis genomic database and citrus clementine genomic database?with both FcMYC2 base and amino acid sequences showed that only CS5g01450?orange1.1g010053m?in C.sinensis and Ciclev10019730m in C.clementine were highly consistent?E-value=0.0?with FcMYC2,and no other genes were found with high similarity.This suggested that those three genes are the same gens.Based on the uniqueness of FcMYC2 in citrus genome,its function may be irreplaceable.The sequence of FcMYC2 gene were greatly different from those of in other plant species.The bootstrap value is only 46%between FcMYC2 and the closest MYC2-like gene in Brassica.Although amino acids sequences of FcMYC2 are highly conservative in MYC-bHLH domain and HLH domain,but the homology of amino acids sequences in non-conservative domain is very low.This suggested that FcMYC2 is a MYC2 type bHLH transcription regulator family gene with high sequence specificity in citrus.Subcellular localization showed that FcMYC2 was located in the nucleus.The expression of FcMYC2 was induced by MeJA and reached the highest expression level at 6 hours after treatment with MeJA.6.The expression level of FcMYC2 is closely related to the content of essential oils.The expression level of FcMYC2 in HP stem epidermis,leaves and flower petals was similar to that in roots,which have no oil glands and essential oil.Relative high level of expression was only detected in peel,which has a few oil glands,but significantly lower than that of in RA stem,leaf and peel.Among different tissues of RA,expression of FcMYC2 was also at a very low level in the root,which was similar to that of HP root.Except in root tissue,FcMYC2 showed a high level of expression.The highest expression level was observed in the peel,which has the highest essential oil content,followed by the epidermis of leaves and stems.These results indicated that higher expression of FcMYC2 is significantly correlated with high essential oil content.The expression of FcMYC2 in the peel of HP and 7 different citrus cultivars was analyzed and the content of essential oil in the peel was titrated.The results showed that the expression level of FcMYC2 in density oil gland cultivars was significantly higher than that of HP.The level of FcMYC2 expression in Poncirus trifoliata,which was the lowest among those varieties was still 30times higher than that of HP.The results indicated that the low expression of FcMYC2 in HP was obvious genetically special.The results of essential oil content analysis showed that the essential oil content of HP peel was only 0.09 mg·g^-1,while that of in other citrus varieties was between4.49-18.23 mg·g^-1,significantly higher than that in HP.The correlation analysis showed that the expression level of FcMYC2 was significantly positively correlated with the content of essential oil in peel.7.FcMYC2 regulates the synthesis of monoterpenoids and sesquiterpenoids in citrusThe overexpression vector,RNA interference vectors and gene editing vectors of FcMYC2were constructed respectively.The epicotyls of Jincheng orange were transformed by Agrobacterium tumefaciens.Ninety-seven FcMYC2 transgenic plants were obtained,of which 38showed small secretory cavities or no visible secretory cavities by eye.Gene expression showed that the expression level of FcMYC2 in overexpressing plants was significantly increased,and the expression level of FcMYC2 in interfering plants was very low.Sequencing analysis of gene editing plants showed that FcMYC2 in some plants achieved completely edited,which resulted in code shifting mutation,early termination of translation,deletion of long segments and other mutations leading to loss of protein function.Volatile components analysis showed that there was no significant change in essential oil content,components and their proportions in the overexpression plants compared to control plants.The 10 major components were limonene,citronellal,citral,dimethyl anthranilate,?-citral,sabinene,?-ocimene,?-caryophyllene,?-3-carene and linalol,which accounted for 82.28%of the total volatiles of the control plants and 85.69%of the total volatile oils of overexpressed plants,respectively.In addition,monoterpenes,as the main components of citrus essential oil,have very similar ratios among overexpressing,interfering and control Jincheng plants,all at above 50%.The total essential oil content in leaves of interfered plants decreased to 12.65%of the control.The content of monoterpenoids and sesquiterpenoids also decreased significantly,especially the content of monoterpene oxides and the proportion of sesquiterpene oxides,but the proportion of monoterpene and sesquiterpene increased.The content of total essential oil in gene edited plants was very low at 1.52%of the control.Only a very small amount of limonene and?-Cyclocitral were detected,but not the sesquiterpenes.Results demonstrated that decreasing the level of FcMYC2 expression led to the decrease of monoterpene and sesquiterpene biosynthesis.In FcMYC2 knocked-out plants,the production of monoterpenoids and sesquiterpenoids was terminated.This study speculated that FcMYC2 is the key gene regulating the biosynthesis of monoterpenes and sesquiterpenes in citrus.