Polymethoxylated Flavones Profiling Of Various Citrus Germplasms In Combination With Mining And Characterization Of Biosynthesis-Related Genes

Citrus fruits are the main dietary source of polymethoxylated flavones(PMFs)for consumers.As a group of functional compounds with special biological activity,PMFs have significant impacts on consumer health,and play important roles in resisting stress during the development of citrus.Abundant of PMFs were accumulated in loose-skin mandarins'fruit flavedo.However,as a branch of flavonoid biosynthetic pathway,the molecular mechanisms and regulation mechanisms of PMF biosynthesis in citrus have not yet been systematically elucidated.Firstly,this study adopted reverse genetics methods by combining metabonomics with transcriptomic analysis to screen PMFs biosynthesis-related candidate O-methyltransferase genes(OMTs),and applied heterologous expression system in vitro and transgene-based gene function identification in vivo to characterize the function of candidate OMTs.Secondly,we used forward genetics ways to employ an artificial hybrid population and BSA-seq to locate key candidate genes involved in PMFs biosynthesis.Three major OMTs were identified and to preliminary construct the metabolic pathways for citrus PMFs biosynthesis.Finally,sequence polymorphisms and enzyme functions of the three cricial OMTs in different citrus germplasms were aligned and compared,to preliminarily reveal the molecular mechanism of the varied accumulation of PMFs in various citrus germplasms.The main results are shown as follows:1.Comparative profiling and natural variation of polymethoxylated flavones in various citrus germplasmsIn this study,eleven main PMFs were evaluated in the fruit flavedo or leaves of116 citrus accessions via UPLC–DAD–ESI-QTOF-MS/MS combined with HPLC analysis,which revealed significant species-specific and spatiotemporal characteristics.All Citrus reticulata and their natural or artificial hybrids investigate here accumulated detectable PMFs,especially in the fruit flavedo of the wild or early-cultivated mandarins at early fruit development stages.However,PMFs were not detected in citrons,pummelos,kumquats,trifoliata oranges,pepadas,Chinese box oranges and‘Mangshanyegan'.The results enlightened that PMF accumulation is a phenotype inherited from mandarin ancestors,which might be a dominant trait in the natural citrus germplasms.Meanwhile,the total content of PMFs tends to decrease during intraspecific and interspecific hybridization of loose-skinned mandarins.Therefore,researches keen on the biosynthetic pathway of PMFs is very important for citrus breeding.This study provides a comprehensive PMFs profiles in various citrus germplasms and will benefit future functional citrus breeding practices,thus helps us to design plants rich in total or specific PMFs for health benefits.The PMFs in fruit flavedo or leaves of the six citrus hybrid progeny populations were analyzed,and it was found that the phenotype of accumulated PMFs is segregated in the F₁ population of‘Red'tangerine(contains abundant of PMFs)×trifoliate orange(contains no PMFs),‘Shatian'pummelo(no accumulation of PMFs)בShatang'mandarin(contains abundant of PMFs).As a result,the ratio of F₁ progenies with detected PMFs is close to 1/8 or 1/9,thus,it seemed that the PMFs accumulation phenotype in citrus artificial F₁ hybrid population were a qualitative and recessive trait.In addition,no PMFs were detected in the crossing populations between?Chuhong?pummelo and?Feicui?pummelo,the result also verified the absence of PMFs biosynthetic pathway in pummelo.2.Mining and functional characterization of PMFs biosynthesis-related genesBased on the differential accumulation of PMFs in the fruit flavedo of different loose-skin mandarin tissues and different citrus germplasms,we combined transcriptome analysis with PMFs profiles to mine the highly expressed OMTs in the fruit flavedo of‘Red'tangerine,and adopted WGCNA correlation analysis among PMFs and gene expression patterns to mine candidate OMTs related to PMFs synthesis.By heterologous expression of candidate OMTs in Saccharomyces cerevisiae and Escherichia coli,yielding product were detected with HPLC after feeding with the flavonoid substrates,and 5 recombinant OMT enzymes were preliminarily determined to show O-methylation catalytic activities on flavones.After adding a variety of flavonoid substrates,the following products identification helped to preliminarily constructed the metabolic pathway of de novo synthesis of PMFs from apigenin in‘Red'tangerine,which can catalyze the formation of seven citrus-specific PMFs.Meanwhile,the biological functions of OMT3,OMT5 and OMT7 genes in plants were verified through overexpression in citrus callus,and transient injection the leaves of tobacco and citrus.Among them,OMT3 and OMT7 exhibited 7-and 4?-position conservative O-methylation catalytic activity.OMT4 and OMT5 were identified as multifunctional O-methyltransferases with catalytic activity on six sites.It is the first time to characterize flavonoid 5-O-methyltransferase in plants,and we initially construct a citrus-specific PMFs metabolic pathway,which provides theoretical base for improving citrus fruit quality,functional citrus breeding and factory production of PMFs.3.BSA analysis was applied to locate the key genes involved in citrus PMFs biosynthesisIn this study,the individual plants that with or without PMFs in the F1 progenies of‘Red'tangerine(accumulate PMFs)×Poncirus trifoliata(no PMFs)population were mixed in two separated extreme pools,respectively.SNPs generated by the BSA-DNA-seq were thus analyzed,a candidate interval in Scaffold 4 with a length of 7.58Mb in‘Clementine'genome was determined.Seven OMTs were tandem arranged in this candidate interval,and OMT3,OMT4,OMT5,OMT6 are highly similar genes.Among them,OMT4 and OMT5 were with extremely high expression levels in the fruit flavedo of several loose-skin mandarins and their function of recombinant enzymes showed O-methylation catalysis for flavonoids at six positions.Recombinant OMT3 only has the function of 7-and 4?-position O-methylation catalysis,while OMT6 has no O-methylation function for flavonoids.These results demonstrated that OMT3,OMT4 and OMT5 may be the cricial genes involoved in citrus PMFs biosynthesis.4.The molecular mechanism behind the varied PMFs accumulation patterns in various citrus germplasms and the screening of related transcription factorsAmplification and sequencing revealed the polymorphisms of the 3 cricial OMTs sequences in 22 representative citrus germplasms,while the following phylogenetic analysis from 110 citrus resequencing data generated with clear classifications.Some germplasms with no detectable PMFs were with prematurely terminated sequences of the 3 cricial OMTs,or only 7-and 6-site catalytic function(Ab OMT6),or no function at all(OMT6).In those germplasms accumulating PMFs,sequences the 3 key OMTs were intact or with highly similarities.However,the 3 key genes in‘Calamondin',‘Honghe'papeda and‘Bergarmo'are with the same or highly similar sequences,but still cannot accumulate PMFs,this might be attributed to the specific flavonoid metabolism type resulting in the absence of PMFs precursors.Therefore,it was speculated that the varied PMFs profiles in various citrus germplasms were closely related to the diversity of enzyme functions caused by the polymorphism of OMT3,OMT4 and OMT5 sequences.In addition,we also screened 18 transcription factors positively correlated with PMFs biosynthesis and one negatively correlated transcription factor.