Insights Into The Diversity And Variability Of Metabolites,Black Pigmentation,and The Genetic Basis Of Sesamin And Sesamolin In Sesame(Sesamum Indicum L.)

Sesame is a worldwide oilseed crop used in the food pharmacy.Several physiological functions are being associated with its leaves,flowers,seeds,and their derived products.However,the phytochemicals responsible for these various proprieties are not well understood.In addition,the seeds are an important source of clinically essential antioxidant lignans(principally sesamin and sesamolin),which genetic basis remains unclear.Thus,this study aimed to dissect the genetic basis of sesamin and sesamolin and reveal nutraceuticals’ variation in sesame.Our major findings are as follows:Firstly,we resequenced 410 sesame accessions and identified 5.38 and 1.16 million SNPs(single nucleotide polymorphisms)and InDels,respectively,by mapping against the sesame reference genome.Population genomic analyses revealed that sesame had evolved a geographic pattern in China,categorized into northern-,middle-,and southern-China groups,with origin in the south and subsequent introduction to the other regions.Selection sweep analyses unveiled 184 key genes associated with domestication and adaptation.KEGG annotation and enrichment analysis revealed that these genes are mainly involved in phenylpropanoid biosynthesis,glycolysis,gluconeogenesis,and carbon metabolism,indicating they might be useful for sesame environmental adaptation improvement.Genome-wide association study identified 17 and 72 significantly associated loci for sesamin and sesamolin,respectively.By integrating gene function annotation and expression analysis,we selected 11 candidate genes for future studies.The major pleiotropic SNP locus for lignans variation is located in the exon of the gene SiNST1.Overexpression of SiNST1 in sesame hairy roots induced a slight increase in lignin content and a significant increase in the content of major sesame lignans.WGCNA and qRT-PCR analyses revealed that SiNST1 might function in synergy with SiMYB58,SiMYB209,SiMYB134,and SiMYB276.We speculated that SiNST1 is a master switch in regulating lignan biosynthesis and seed coat thickness in sesame.Secondly,we analyzed the metabolome profile of different sesame tissues.In total,776 metabolites belonging to diverse classes were qualitatively and quantitatively identified.The different tissues exhibited obvious differences in metabolites composition.The majority of flavonoids predominantly accumulated in flowers.Amino acids and derivatives,and lipids were identified predominantly in fresh seeds followed by flowers.Many metabolites,including quinones,coumarins,tannins,vitamins,terpenoids,and some bioactive phenolic acids(acteoside,isoacteoside,verbascoside,plantamajoside,etc.)accumulated mostly in leaves.Lignans were principally detected in seeds.238 key significantly differential metabolites were filtered out.KEGG annotation enrichment analysis of the differential metabolites revealed that flavonoid biosynthesis,amino acids biosynthesis,and phenylpropanoid biosynthesis were the main differently regulated pathways.In addition to the tissue-specific accumulation of metabolites,we noticed a cooperative relationship between leaves,fresh carpels,and developing seeds in terms of metabolites transfer.Delphinidin-3-O-(6’’-O-p-coumaroyl)glucoside and most of the flavonols were up-regulated in the purple flowers indicating they might be responsible for the purple coloration.Thirdly,we explore the metabolic changes that occur during white sesame seed maturation through ultra-high-performance liquid chromatography-mass spectrometry(UPLC-MS/MS)-based widely targeted metabolomic analysis of developing and dormant seeds.In total,752 metabolites,including448 differential metabolites,were identified.The relative content of flavonoids,terpenoids,and tannins was insignificant.White sesame seed maturation was associated with a great alteration of metabolites,mostly amino acids,free fatty acids,organic acids,and sugars,indicating their efficient integration into seed-storage reserves.KEGG annotation and enrichment analysis of the differential metabolites suggested that amino acids and carbohydrates might play a critical role during sesame seed maturation.The phenylpropanoid biosynthesis pathway might be the most regulated during white sesame seed development.Fourthly,we acknowledge the diversity and variability of metabolites in sesame seeds of different colors and reveal key metabolites and pathways contributing to differences in antioxidant activities of black,brown,yellow,and white sesame seeds through LC-MS/MS-based widely targeted metabolomics analysis.Totally,671 metabolites were identified and chemically classified.The metabolic compounds varied significantly with the seed coat color and genotype.Many flavonoids,amino acids,and terpenoids were up-regulated in dark seeds.Sixty key differential metabolites were filtered out.Compared with light sesame seeds,the dark seeds exhibited higher relative content of apigenin,2’-hydroxygenistein,chrysoeriol-7-O-gentiobiose,galangin,genistein-7-O-galactoside,butin,hesperetin,luteolin,limocitrin,nepetin,phellamurin,tricin(flavonoids);histidine,isoleucine,leucine,lysine,methionine,valine(essential amino acids);echinacoside,isochlorogenic acid,acteoside,isoacteoside(phenolic acids);tocopherol(vitamin);martynoside,and myrianthic acid(terpenoids).Phenylpropanoid biosynthesis,amino acid biosynthesis,and tyrosine metabolism were the main differently regulated pathways.The DPPH,ABTS,and FRAP assays showed that the antioxidant activities of the seeds increased with the seed coat darkness.Therefore,the pharmacological proprieties of black seeds might be related to their high content of flavonoids and essential amino acids mostly.Finally,we report the isolation of melanin by the alkali method from the black and brown sesame seeds.Physicochemical methods,including scanning electron microscopy(SEM),solubility,precipitation,UV-Vis spectroscopy,Fourier transform infrared(FT-IR)spectroscopy,and thermogravimetric-differential scanning calorimetry(TG-DSC),were used to characterize the sesame melanins.The results clearly showed that the isolated pigments were similar to melanin from other sources.Both melanins were heat-stable and exhibited numerous characteristic absorption peaks.Through a comprehensible LC-MS/MS-based metabolome profiles analysis of Na OH and methanol extracts of black and white sesame seeds,caffeic,protocatechuic,indole-carboxylic,homogentisic,ferulic,vanillic,and benzoic acids were identified as the potential precursors of the sesame melanin.These findings expanded phytochemicals composition information in sesame and represent key resources for sesame biomass valorization and quality improvement.In addition,our results provide insights into the domestication history of sesame and will facilitate molecular breeding of elite varieties with a higher yield and oil and lignans contents,and marker-traits association studies.Furthermore,they widen our understanding of dark seeds’ pigmentation in sesame and show that black sesame seeds are promising sources of edible melanin for food and biotechnological applications.