The Litsea Cubeba Genome And Functional Characterization Of Key Genes Involved In Monoterpene Biosynthesis Pathway

Litsea cubeba(Lour.)Person,belonging to the Lauraceae family,is widely distributed in the south of the Yangtze River in China and has been cultivated as an important woody oil tree for a long time since its fruit is rich in essential oil.The essential oil of L.cubeba is a clear light-yellow liquid with a lemon smell.More than 90% contents of essential oil in L.cubeba are the monoterpenes such as citral,eucalyptol,pinene and linalool,which endows the L.cubeba essential oil with aroma,rich in biological activity such as anti-oxidation,sterilization,anti-inflammatory and anti-virus.Therefore,it is used as a natural flavor and fragrance in the fields of chemical industry,food,medicine and ecological agriculture.After standardized cultivation and management,the fruit yield of L.cubeba has increased to 1000 kg yield per mu,and the content of essential oil can reach up to 12%.But with the increasing demand for natural essential oils,the essential oil of L.cubeba is still in short supply.Due to the long forest breeding cycle and the relatively weak research foundation,the synthesis mechanism of essential oils of L.cubeba and species in Lauraceae has not been solved,which has hindered the genetic improvement of L.cubeba and species in Lauraceae.In this study,genomics was used to construct the genomic chromosome map of L.cubeba,and the key genes of terpene synthesis pathway were functionally identified.The main results obtained are as follows:Phylogenetic sequencing and evolution analysis of L.cubeba.First,the diploid(2n = 24)genome was sequenced by Pac Bio sequencing platform,and chromosome assembly was further completed by Hi-C technology.1,514 scaffolds were obtained with a genome size of1,325.69 Mb and contig N50 of 607.34 kb.Phylogenetic analysis results supported the formation of sister groups of magnolias and dicotyledons after differentiation from a common ancestor with monocotyledons.Two whole-genome replication(WGD)events occurred in the L.cubeba genome,containing an older WGD event occurred before the differentiation of Lauraceae and Magnolia,and a subsequent WGD event occurred at the beginning of Lauraceae differentiation.Comparative genomic analysis revealed that the monoterpene synthase gene family expanded significantly in the Lauraceae,providing a genetic basis for the synthesis of its abundant monoterpene compounds.The key genes of the monoterpene biosynthesis pathway of L.cubeba were identified at the genomic level.Identification of the hydroxymethylglutaryl?CoA synthase(HMGS)and1-deoxy-D-xylulose 5-phosphate synthase(DXS),key genes in the MVA and MEP pathway respectively at the stage of terpene universal precursor synthesis,showed that there were three members of the Lcu HMGSs gene family in L.cubeba,among which Lcu HMGS1 was highly expressed in fruits,and Lcu HMGS3 was highly expressed in roots and fruits.There were 6members in the Lcu DXSs family of L.cubeba.Lcu DXS in Clade B expanded through chromosomal replication and tandem repeats,highly expressed specifically in the fruit,which may provide the genetic basis for the synthesis of rich monoterpene compounds in L.cubeba.Identification of the key geranyl diphosphate synthase(GPPS)gene catalyzing the synthesis of GPP,the direct precursor of monoterpenes,showed that there were four members in GPPS family.GPPS.SSU1 and GPPS.SSU2 were highly expressed in fruit 60 DAF and 90 DAF,consistent with the peak period of the synthesis of essential oil in the fruit of L.cubeba.At the stage of monoterpene final product synthesis,there were 54 family members in the terpene synthase(TPS)family,of which 27 members were involved in the monoterpene biosynthesis.As the final and most critical step in the monoterpene synthesis of L.cubeba,after a member of the monoterpene synthase gene family(mono-TPS)undergoed significant expansion through chromosomal replication and tandem repeats,it differentiated in sequence and expression pattern,which was tissue-specific and related to the stage of fruit development.Lcu HMGS1,a key gene of the MVA pathway,promoted terpene biosynthesis by increasing the supply of precursors.Lcu HMGS1 is a key gene of MVA pathway of terpene precursor synthesis pathway.Tissue-specific expression analysis found that Lcu HMGS1 was expressed in roots,stems,leaves and fruits,and was highly expressed in the early and middle stages of fruit development,indicating that Lcu HMGS1 may play an important role in the synthesis of monoterpenes in the fruit of L.cubeba.The types and contents of monoterpenes and sesquiterpenes of Lcu HMGS1 transgenic tobacco leaves increased significantly,and the yield of monoterpenes increased by 2.7-3.6 times.Transcription level expression analysis found that the gene expression of the terpene synthesis pathway of transgenic tobacco lines was significantly upregulated,indicating that overexpression of Lcu HMGS1 improved the metabolic flux of terpene synthesis.Meantime,Lcu HMGS1 over-expressed tobacco showed fast growth,enlarged vegetative organs,and promoted early flowering of tobacco by increasing GA3 content.The above results indicated that Lcu HMGS1 could promote the synthesis of plant monoterpenes,sesquiterpenes and diterpenes by regulating the precursor synthesis of terpenoids,thereby participating in the primary and secondary metabolism of plants.LcuGPPS.SSU1,a key gene for intermediate synthesis,regulated the organization,period,and yield of monoterpene synthesis.LcuGPPS.SSU1 was specifically and highly expressed in L.cubeba fruit,which could be induced by light and methyl jasmonate.Its promoter was highly and specifically expressed in the hair bodies of mature tobacco leaves,suggesting that LcuGPPS.SSU1 regulated the tissue and yield of monoterpene synthesis.Compared with the wild type,overexpression of LcuGPPS.SSU1 increased monoterpene yield and varieties in L.cubeba and tobacco leaves.At the same time,LcuGPPS.SSU1 could increase the expression of key genes in the precursor synthesis pathway,especially DXS,through feedback regulation mechanisms,so as to increase the metabolic flux of monoterpenes and other terpenoids.Due to the increased anabolic flow of terpenes,the expression levels of Nbe GGPPS2,Nbe GGPPS4 were increased to motivate the synthesis of primary terpenoid metabolites such as gibberellin,and further promoted the increase of transgenic tobacco biomass like leaf area,plant height,and number of fruitages.The functional differentiation of mono-TPSs,key genes for monoterpene skeleton synthesis in L.cubeba,determined the diversity of monoterpene compounds.LcuTPS22 was specifically high-expressed in the leaves of L.cubeba,and enzyme assays in vitro and transient transformation of tobacco leaves in vivo found that LcuTPS22 could catalyze GPP to produce?-pinene,?-pinene,camphene,eucalyptol and camphor,among which eucalyptol was the most abundant terpenoid in L.cubeba leaves,and camphor was also a representative terpene compound in Lauraceae leaves,which indicated that LcuTPS22 played a key role in the synthesis of monoterpenoids in Lauraceae leaves.LcuTPS19,LcuTPS20,LcuTPS25 and LcuTPS42 were highly expressed in different stages of fruit development.LcuTPS19,LcuTPS20 catalyzed the synthesis of thujaene,LcuTPS25 catalyzed the production of ?-pinene and linalool,and LcuTPS42 catalyzed the production of phlene,linalool and geraniol.Thereinto,geraniol was the direct precursor of citral,a key component of the essential oil in the L.cubeba fruit,with linalool and ?-pinene as the main monoterpene component.The monoterpene synthase gene family of L.cubeba had expanded significantly and differentiated in expression pattern and catalytic function,which provided a genetic basis for the rich monoterpene compounds of L.cubeba.