Biochemical and Molecular Insights into Variation in Sesame Seed Antioxidant Capability as Revealed by Metabolomics and Transcriptomics Analysis

Sesame seeds are important resources for relieving oxidation stress-related diseases. Although a significant variation in seeds’ antioxidant capability is observed, the underlying biochemical and molecular basis remains elusive. Thus, this study aimed to reveal major seed components and key molecular mechanisms that drive the variability of seeds’ antioxidant activity (AOA) using a panel of 400 sesame accessions. The seeds’ AOA, total flavonoid, and phenolic contents varied from 2.03 to 78.5%, 0.072 to 3.104 mg CAE/g, and 2.717 to 21.98 mg GAE/g, respectively. Analyses revealed that flavonoids and phenolic acids are the main contributors to seeds’ AOA variation, irrespective of seed coat color. LC-MS-based polyphenol profiling of high (HA) and low (LA) antioxidant seeds uncovered 320 differentially accumulated phenolic compounds (DAPs), including 311 up-regulated in HA seeds. Tricin, persicoside, 5,7,4′,5′-tetrahydro-3′,6-dimethoxyflavone, 8-methoxyapigenin, and 6,7,8-tetrahydroxy-5-methoxyflavone were the top five up-regulated in HA. Comparative transcriptome analysis at three seed developmental stages identified 627~2357 DEGs and unveiled that differential regulation of flavonoid biosynthesis, phenylpropanoid biosynthesis, and stilbene biosynthesis were the key underlying mechanisms of seed antioxidant capacity variation. Major differentially regulated phenylpropanoid structural genes and transcription factors were identified. SINPZ0000571 (MYB), SINPZ0401118 (NAC), and SINPZ0500871 (C3H) were the most highly induced TFs in HA. Our findings may enhance quality breeding.


Introduction
Sesame (Sesamum indicum L.) is a vital industrial and oilseed crop in the Pedaliaceae family [1].Its high medicinal and nutritional values have raised interest in sesame products' consumption worldwide and use in various industries, including pharmaceutics, foods, biodiesel, cosmetics, etc. [2,3].Sesame seeds are abounding in nutraceuticals, including essential fatty acids, inherent lignans (sesamin, sesamolin, sesaminol, sesamol, sesamolinol, etc.), tocopherols, melatonin, phytosterols, and other essential antioxidants [3][4][5][6].Accordingly, sesame seed consumption is associated with enormous health benefits.For instance, clinical trials and in vivo and in vitro investigations showed that they have antioxidant, anti-diabetes, anti-hyperlipidemia, kidney and liver protection, anti-inflammatory, cardiovascular protection, anti-hypertension, antitumor, and anti-cancer properties [3][4][5]7].The high-antioxidant capacity of sesame seeds has led them to be used to improve the stability and quality and prevent autoxidation of countless foodstuffs [8,9].Unfortunately, although there is evidence of great variation in sesame seeds' antioxidant capacity, the underlying biochemical and molecular bases remain poorly elucidated, limiting efforts to develop novel sesame varieties with improved medicinal potentials.
Diverse factors, such as origin, seed coat color, and growing and processing conditions, impact the antioxidant capacity of sesame seeds [10][11][12].Early studies have demonstrated that black sesame seeds possess higher antioxidative ability than other colored seeds [10,13,14].However, these studies analyzed a small number of varieties, so it would be more appropriate to explore a vast population comprised high numbers of different colored sesame seeds before making a statement.Moreover, the huge beneficial health effects of sesame-specific lignans, including sesamin, sesamol, sesamolin, sesaminol, etc., result in the association of seeds' antioxidant activity variation to the difference in lignan content only [5,15,16].Nevertheless, some sesame seeds with low lignan content have shown higher antioxidant activities than those with high lignan content [10,17,18].Therefore, we hypothesized that changes in the composition of other antioxidants in the seeds may be the cause of the observed variability in antioxidant capacity.
A comparative analysis of the global metabolome of brown, white, black, and yellow sesame seeds disclosed that difference in the relative content of some phenolic compounds was the main causative factor of their antioxidant activity differences [14].We inferred that considerable differences might exist between the polyphenol profiles of low and high-antioxidant sesame seeds.Polyphenols, including flavonoids (flavanols, anthocyanidins, flavonols, flavanones, flavones, and chalcones), stilbene, tannins, phenolic acids, and saponin are members of plant secondary metabolites [19,20].They are excellent antioxidants and their daily consumption may lead to the relief of oxidative stress and the prevention of aging and several chronic and lifestyle diseases [19][20][21][22][23].Among the oilseed crops, sunflower, soybean, brassica, and olive have received more attention than sesame in terms of polyphenol composition [24,25].Therefore, a comprehensive characterization of polyphenol profile differences between HA and LA sesame seeds is of immense interest.It will enable the biochemical understanding of sesame seeds' antioxidant capability variation and essential resources for gene-metabolite network analyses that may serve in quality breeding.
In this study, we analyzed the AOA (antioxidant activity), TFC (total flavonoid content), and TPC (total phenolic content) of 400 sesame accessions.Furthermore, we conducted UPLC_MS/MS (ultra-performance liquid chromatography-mass spectroscopy)based widely targeted polyphenol profiling transcriptome analysis of HA and LA seeds.Our objectives were to identify major seed components governing variation in sesame seed antioxidant capacity and achieve insight into the associated molecular mechanisms.The results of this study provide biochemical and genetic indicators for the improvement in sesame seed antioxidants' composition.

Plant Materials and Growing Conditions
Four hundred sesame accessions were analyzed in this study (Supplementary Table S1).They were given by the Oil Crops Research Institute of the Chinese Academy of Agricultural Sciences (OCRI-CAAS), Wuhan, China.The sesame population included 45, 85, 32, and 234 black, brown, yellow, and white seeds, respectively.All seeds were cultivated under the same environmental conditions in Wuhan, China.All required agronomic practices for sesame were applied equally [3].During harvesting in September 2022, seed samples were collected in triplicate for all genotypes.Each replicate was a mixture of seeds from twelve individual plants.The seed samples were stored in the OCRI seeds room until the evaluation of the AOA, TPC, and TFC evaluations.
For the comparative polyphenol profiling and transcriptomics analysis, three high (HA; NS089, NS287, and NS148) and three low (LA; NS100, NS009, and NS120) antioxidant varieties were selected and cultivated similarly from June to September 2023 (Table S1).Developing seeds of NS089 (HA) NS100 (LA) were sampled at 10, 20, and 30 DPA (days postanthesis) for transcriptome sequencing.Seed samples of the six varieties were prepared (three replications) after harvest for the metabolomics analysis.All samples were directly frozen in liquid nitrogen and kept at −80 • C until used.

Assessment of Total Phenolic (TPC) and Flavonoid (TFC) Contents and Antioxidant Activity (AOA)
Seed extraction for AOA, TPC, and TFC evaluation was achieved following previously described methods [31,32].Briefly, for each replicate, 0.5 g of seeds were extracted for 4 h (constant shaking in darkness) with 5 mL 80% ethanol.Next, centrifugation (5000× g, 12 min) was followed by supernatant collection separately.All seed extracts were stored at −20 • C during the analyses.
The TFC and TPC were analyzed according to the methods of Choi et al. [32].Regarding the TPC, 400 µL dH 2 O and 100 µL Folin-Ciocalten reagent were added to 100 µL of seed extract, mixed well, and left for 6 min.Thereafter, 1 mL of Na 2 CO 3 (7% m/v) and 800 µL of dH 2 O were added subsequently.After 90 min of reaction at room temperature, the absorbance of the mixture was recorded at 760 nm (UV5200, Shanghai Metash Instruments Co., Ltd., Shanghai, China).In the blank, 80% ethanol was used in place of the extract.The TPC values were expressed as mg GAE/g (gallic acid equivalent per gram) of seeds (y = 1.971x − 0.0068, R 2 = 0.99).
Regarding the TFC, 1 mL of seed extract was mixed with 150 µL of NaNO 2 (5% m/v) and the mixture was kept for 6 min.Thereafter, 300 µL of AlCl 3 •6H 2 O (10% m/v) was added, followed by 1 mL of 1 M NaOH another 6 min later.Finally, 1.05 mL dH 2 O was added, followed by absorbance at 510 nm fifteen minutes later.The TFC was estimated using y = 3.253x + 0.1447 (R 2 = 0.9702) and expressed as mg CAE/g (catechin equivalent per gram) seeds.The AOA of the seeds was evaluated via DPPH assays, as recently reported [14,33].

Identification and Quantification of Phenolic Compounds
The spectrum information, mass spectra, and retention times were integrated to qualitatively identify the phenolic compounds.Specifically, the values of Q1 (precursor ions) and Q3 (product ion), retention times, fragmentation patterns, collision energy, and de-clustering potential were allied with standards when available (Sigma-Aldrich, St. Louis, MO, USA).When no standards were available, the compounds were structurally confirmed via the MWDB self-build database and verification in open databases (KNApSAcK, Mass-Bank, MoTo DB, HMDB, and METLIN) [34,35].The relative contents of the identified polyphenols were calculated via the triple quadrupole (QqQ) MS analysis (MRM modes) using the integrated SCIEX-OS software (version 1.4).

Data Analysis
All multivariate analyses were achieved in R (version 3.5.0)after quality validation and subsequent standardization of the data.The statistical packages pheatmap, MetaboAn-alystR, cor, and prcomp were used for hierarchical clustering analysis, orthogonal partial least squares discriminant analysis, correlation analysis, and principal component analysis.The variable importance of the projection (VIP) value of the phenolic compounds was extracted from the OPLS-DA results.Differentially accumulated metabolites (DAMs) were sorted out using the R-programming language ggplot2 program at thresholds of Log2FC > 1, p-value < 0.05, and VIP ≥ 1. KEGG functional analysis of DAMs was carried out by mapping http://www.kegg.jp/kegg/pathway.html(accessed on 17 November 2023) and subsequent metabolite sets for enrichment analysis.Excel 2021 software and GraphPad Prism (v9.0.01,La Jolla, CA, USA) were used for data processing and graph construction.SRplot was also used for PCA and correlation analyses [37].An ANOVA (analysis of variance) test was performed for multiple comparisons at p < 0.05.

Quantitative RT-PCR Analysis
The RNA was extracted from developing seed samples using a modified CTAB method [43].Reverse transcription (RT) was conducted with the Monad 1st Strand cDNA Synthesis Kit and the qRT-PCR analysis was achieved using Tb Green ® Premix Ex Taq™ II (Takara, Beijing, China) as previously described [44].All samples had three biological and technical replicates.The sesame histone gene (SiH3.3)was used as an internal control to normalize the expression levels of target genes via the 2 −∆∆CT method [45].The NCBI's primer designing tool, PRIMER-BLAST (Primer3), was used to design specific primers for each gene (Table S7).

Results and Discussion
3.1.Variation in Antioxidant Activity (AOA), Total Flavonoid (TFC), and Phenolic (TPC) Contents in the Sesame Population In order to thoroughly examine the variability of sesame seeds' antioxidant capability, we evaluated the AOA, TPC, and TFC of seeds from 400 diverse sesame accessions (Table S1).The frequency distribution of the three traits in the population is presented in Figure S1.In general, the AOA of the seeds varied from 2.03 to 78.5%, with a CV (coefficient of variation) of 45.12% (Table 1).Meanwhile, the TPC ranged from 2.717 to 21.98 mg GAE/g, with a CV of 44.52% (Table 1).The TFC varied from 0.072 to 3.104 mg CAE/g, with a CV of 42.29% (Table 1).These results show a significant variation in sesame seed antioxidant capacity and polyphenol profiles driven mainly by the genotypes.

Correlation between Seed Antioxidant Activity and Seed Phytochemicals
To identify the major seed phytochemicals that govern the variation in antioxidant capability, we carried out a correlation analysis.The same seeds were previously analyzed for fatty acid composition and oil content, sesamin and sesamolin (lignans) content, phytosterols content, melatonin content, and tocopherol (Vitamin E) content [44,[46][47][48].These previous data were taken into account for the correlation analysis.The analysis revealed a significantly high positive correlation between AOA and TPC (r = 0.8) and between AOA and TFC (r = 0.66) (Figures 1A and S2).The AOA showed significant low positive correlations with sesamin content (r = 0.18), total lignan (r = 0.18), and total phytosterol (r = 0.11) (Figures 1A and S2).There was a significant negative correlation between AOA and oil content and no significant correlations with other seed components (Figures 1A and S2).These results show that polyphenols are the main antioxidants in sesame seeds.Moreover, they indicate that lignans are not the sole major antioxidants in sesame seeds and that flavonoids, phenolic acids, and other phenolic compounds also significantly influence sesame seed antioxidant capability.Plant polyphenols, including flavonoids and non-flavonoids (stilbenes, phenolic acids, lignans, tannins, etc.), are important antioxidants with high pharmacological values [19,20,49].
We further performed PCA analysis to verify the correlation analysis results.As shown in Figure 1B, the PCA analysis results were supportive of observed correlations.The AOA and polyphenol components (TPC, TFC, sesamin, sesamolin, and total lignan) were projected closely on the PCA plot (Figure 1B).AOA and oil content were projected in opposite directions, confirming their negative correlations (Figure 1B).Overall, these findings denote that variation in polyphenol profiles of sesame seeds may be the key underlying factor of difference in seeds' antioxidant capabilities.In addition to its inherent lignans, sesame seeds may contain diverse other phenolic compounds with important antioxidant power.
shown in Figure 1B, the PCA analysis results were supportive of observed correlations.The AOA and polyphenol components (TPC, TFC, sesamin, sesamolin, and total lignan) were projected closely on the PCA plot (Figure 1B).AOA and oil content were projected in opposite directions, confirming their negative correlations (Figure 1B).Overall, these findings denote that variation in polyphenol profiles of sesame seeds may be the key underlying factor of difference in seeds' antioxidant capabilities.In addition to its inherent lignans, sesame seeds may contain diverse other phenolic compounds with important antioxidant power.

Influence of Seed Coat Colors on Sesame Seed Antioxidant Activity
Previous investigations on small numbers of sesame varieties revealed that black seeds possess higher antioxidant capability than other colored sesame seeds [10,13,14,50].To verify these reports, we compared the AOA, TFC, and TPC of black (BkS), yellow (YS), brown (BnS), and white (WS) sesame seeds (Figure 2).As shown in Figure 2A, most BkS had higher AOA than the majority of other colored seeds.However, the AOA of BkS and BnS were not statistically different (Figure 2A).The AOA of YS was significantly the lowest (Figure 2A).Although white seeds showed significantly lower AOA than black seeds and similarity to brown seeds, the lowest AOA values were recorded on some dark (black and brown) accessions (Figure 2A).These results denote that the AOA of sesame seeds is a complex trait, irrespective of seed coat color.Not all dark sesame seeds may possess high antioxidant capability.It is therefore required to dissect the molecular network regulating

Influence of Seed Coat Colors on Sesame Seed Antioxidant Activity
Previous investigations on small numbers of sesame varieties revealed that black seeds possess higher antioxidant capability than other colored sesame seeds [10,13,14,50].To verify these reports, we compared the AOA, TFC, and TPC of black (BkS), yellow (YS), brown (BnS), and white (WS) sesame seeds (Figure 2).As shown in Figure 2A, most BkS had higher AOA than the majority of other colored seeds.However, the AOA of BkS and BnS were not statistically different (Figure 2A).The AOA of YS was significantly the lowest (Figure 2A).Although white seeds showed significantly lower AOA than black seeds and similarity to brown seeds, the lowest AOA values were recorded on some dark (black and brown) accessions (Figure 2A).These results denote that the AOA of sesame seeds is a complex trait, irrespective of seed coat color.Not all dark sesame seeds may possess high antioxidant capability.It is therefore required to dissect the molecular network regulating the sesame seed antioxidants for exploitation in developing novel varieties with improved antioxidant capability.
Regarding the TPC and TFC, the white, brown, and black seeds exhibited statistically similar results (Figure 2B,C).The yellow seeds had the lowest TPC and TFC, as per the AOA (Figure 2B,C).Taken together, these findings infer that the antioxidant capacity of sesame seeds varies mostly upon the polyphenol profile and the variation characteristics of each phenolic compound in the different colored seeds.
Regarding the TPC and TFC, the white, brown, and black seeds exhibited statistically similar results (Figure 2B,C).The yellow seeds had the lowest TPC and TFC, as per the AOA (Figure 2B,C).Taken together, these findings infer that the antioxidant capacity of sesame seeds varies mostly upon the polyphenol profile and the variation characteristics of each phenolic compound in the different colored seeds.

Polyphenol Profiles of High and Low Antioxidant Sesame Seeds
To reveal the major phenolic compounds responsible for variation in sesame seeds' AOA, we performed comparative widely targeted polyphenol profiling of highantioxidant (HA) and low-antioxidant (LA) accessions [34,36].As shown in Figure S3, the detection of the metabolites was achieved in both electrospray ionization modes.The repeatability of the experiment was confirmed by the high correlations (r ≥ 0.99) recorded between QC samples (Figure S4).We structurally identified a total of 785 phenolic compounds in sesame seeds, including 41.53% flavonoids, 38.78% phenolic acids, 12.61% lignans, 6.24% coumarins, 0.51% tannins, and 0.38% stilbenes (Figure 3A, Table S3).This result shows that flavonoids and phenolic acids are the foremost phenolic compounds in sesame seeds.Accordingly, they may have a greater influence on seed AOA than lignans.It is reported that lignans are the primary antioxidant compounds in sesame seeds [5,51].Of the 326 identified flavonoids, flavones (42.94%), flavonols (23.93%), and flavanones (11.66%) were dominant (Figure 3B).Isoflavones and anthocyanidins accounted for 4.29 and 2.15%, respectively (Figure 3B).
To explore the variability in metabolites between HA and LA seeds, we conducted HCA and PCA analysis (Figures 3C and S5).As shown in Figure S5, the HCA revealed remarkable differences between the polyphenol profiles of HA and LA seeds.The majority of the phenolic compounds showed the highest relative content in HA compared to LA seeds (Figure S5).The PCA confirmed that the polyphenol profiles of HA and LA seeds were very different and could be discriminated by PC1 (50.15%) and PC2 (22.42%) (Figure 3C).These results represent support for the correlation between sesame seed AOA and polyphenol profile.

Polyphenol Profiles of High and Low Antioxidant Sesame Seeds
To reveal the major phenolic compounds responsible for variation in sesame seeds' AOA, we performed comparative widely targeted polyphenol profiling of high-antioxidant (HA) and low-antioxidant (LA) accessions [34,36].As shown in Figure S3, the detection of the metabolites was achieved in both electrospray ionization modes.The repeatability of the experiment was confirmed by the high correlations (r ≥ 0.99) recorded between QC samples (Figure S4).We structurally identified a total of 785 phenolic compounds in sesame seeds, including 41.53% flavonoids, 38.78% phenolic acids, 12.61% lignans, 6.24% coumarins, 0.51% tannins, and 0.38% stilbenes (Figure 3A, Table S3).This result shows that flavonoids and phenolic acids are the foremost phenolic compounds in sesame seeds.Accordingly, they may have a greater influence on seed AOA than lignans.It is reported that lignans are the primary antioxidant compounds in sesame seeds [5,51].Of the 326 identified flavonoids, flavones (42.94%), flavonols (23.93%), and flavanones (11.66%) were dominant (Figure 3B).Isoflavones and anthocyanidins accounted for 4.29 and 2.15%, respectively (Figure 3B).
To explore the variability in metabolites between HA and LA seeds, we conducted HCA and PCA analysis (Figures 3C and S5).As shown in Figure S5, the HCA revealed remarkable differences between the polyphenol profiles of HA and LA seeds.The majority of the phenolic compounds showed the highest relative content in HA compared to LA seeds (Figure S5).The PCA confirmed that the polyphenol profiles of HA and LA seeds were very different and could be discriminated by PC1 (50.15%) and PC2 (22.42%) (Figure 3C).These results represent support for the correlation between sesame seed AOA and polyphenol profile.

Differentially Accumulated Phenolic (DAPs) Compounds and KEGG Analysis
In order to uncover DAPs between HA and LA sesame seeds, we carried out OPLS-DA analysis.The score plot of the OPLS-DA confirmed the great difference in the polyphenol profiles between the two groups (Figure 4A).The R 2 Y and Q 2 of the pairwise comparison were 0.997 and 0.874, respectively, indicating the reliability of the model (Figure S6).We uncovered a total of 320 DAPs, including 311 highly accumulated in HA compared to LA seeds (Figure 4B, Table S4).The DAPs included 145 phenolic acids, 87 flavonoids, 64 lignans, 23 coumarins, and 1 tannin.Sesamolinol-glucoside and sesamolinol 4′-O-β-D-glucosyl (1→6)-O-β-D-glucoside were only two differentially accumulated sesame-specific lignans, supporting that the variation in seeds' AOA could not be attributed to differences in the content of specific lignans, such as sesamin, sesamolin, sesamolinol, etc., only.Phenolic acids, flavonoids, and other lignans are critical for the high AOA of sesame seeds.
To provide insights into differential molecular mechanisms between HA and LA seeds, we performed a KEEG analysis of DAPs (Figure 4C).The results showed that the main pathways differentially regulated between HA and LA were phenylalanine metabolism, biosynthesis of secondary metabolites, flavonoid biosynthesis, phenylpropanoid biosynthesis, and tyrosine metabolism (Figure 4C).Phenolic compounds are synthesized in plants from phenylalanine, tyrosine, and tryptophan, themselves occurring from chorismate (the ultimate product of the shikimate pathway) [52,53].Collectively, these findings infer that the antioxidant capacity of sesame seeds may be improved by inducing phenylalanine biosynthesis, phenylpropanoid biosynthesis, and flavonoid accumulation in developing seeds [54,55].Investigating gene-metabolite interactions in these pathways may offer crucial genetic resources for improving sesame seed antioxidant capability.

Differentially Accumulated Phenolic (DAPs) Compounds and KEGG Analysis
In order to uncover DAPs between HA and LA sesame seeds, we carried out OPLS-DA analysis.The score plot of the OPLS-DA confirmed the great difference in the polyphenol profiles between the two groups (Figure 4A).The R 2 Y and Q 2 of the pairwise comparison were 0.997 and 0.874, respectively, indicating the reliability of the model (Figure S6).We uncovered a total of 320 DAPs, including 311 highly accumulated in HA compared to LA seeds (Figure 4B, Table S4).The DAPs included 145 phenolic acids, 87 flavonoids, 64 lignans, 23 coumarins, and 1 tannin.Sesamolinol-glucoside and sesamolinol 4 ′ -O-β-D-glucosyl (1→6)-O-β-D-glucoside were only two differentially accumulated sesame-specific lignans, supporting that the variation in seeds' AOA could not be attributed to differences in the content of specific lignans, such as sesamin, sesamolin, sesamolinol, etc., only.Phenolic acids, flavonoids, and other lignans are critical for the high AOA of sesame seeds.
To provide insights into differential molecular mechanisms between HA and LA seeds, we performed a KEEG analysis of DAPs (Figure 4C).The results showed that the main pathways differentially regulated between HA and LA were phenylalanine metabolism, biosynthesis of secondary metabolites, flavonoid biosynthesis, phenylpropanoid biosynthesis, and tyrosine metabolism (Figure 4C).Phenolic compounds are synthesized in plants from phenylalanine, tyrosine, and tryptophan, themselves occurring from chorismate (the ultimate product of the shikimate pathway) [52,53].Collectively, these findings infer that the antioxidant capacity of sesame seeds may be improved by inducing phenylalanine biosynthesis, phenylpropanoid biosynthesis, and flavonoid accumulation in developing seeds [54,55].Investigating gene-metabolite interactions in these pathways may offer crucial genetic resources for improving sesame seed antioxidant capability.

Major Highly Accumulated Phenolic Compounds in High-Antioxidant Sesame Seeds
To reveal the major highly accumulated phenolic compounds in HA seeds, we filtered out the top 50 up-regulated metabolites in HA (Table 2).The top 50 up-regulated DAPs in HA included 29 flavonoids, 14 phenolic acids, 5 lignans, and 2 coumarins.It was worth noting that the top 20 highly accumulated phenolic compounds in HA seeds were all flavonoids (Figure S7).These major up-regulated DAPs in HA merit being investigated in future studies to better understand sesame seed bioactivities.For instance, tricin, the top DAP (|Log2FC| = 9.593), possesses diverse therapeutical potentials, including anti-cancer, anti-influenza, anti-angiogenic, and antioxidant effects [56][57][58].Diosmetin, peonidin, and apigenin have also recorded pharmacological attributes, such as anti-cancer, antioxidant, neuroprotective, etc. [59][60][61].Matairesinol has demonstrated antioxidant, anti-cancer, neuroprotective, and anti-inflammation abilities [62,63].In addition, the major DAPs could serve as key biomarkers for analyzing molecular networks regulating polyphenol biosynthesis during sesame seed development.As a support, correlation network analysis among DAPs revealed significant positive correlations between 22 phenolic acids, 17 lignans, and 10 flavonoids (Figure S8).

Major Highly Accumulated Phenolic Compounds in High-Antioxidant Sesame Seeds
To reveal the major highly accumulated phenolic compounds in HA seeds, we filtered out the top 50 up-regulated metabolites in HA (Table 2).The top 50 up-regulated DAPs in HA included 29 flavonoids, 14 phenolic acids, 5 lignans, and 2 coumarins.It was worth noting that the top 20 highly accumulated phenolic compounds in HA seeds were all flavonoids (Figure S7).These major up-regulated DAPs in HA merit being investigated in future studies to better understand sesame seed bioactivities.For instance, tricin, the top DAP (|Log2FC| = 9.593), possesses diverse therapeutical potentials, including anticancer, anti-influenza, anti-angiogenic, and antioxidant effects [56][57][58].Diosmetin, peonidin, and apigenin have also recorded pharmacological attributes, such as anticancer, antioxidant, neuroprotective, etc. [59][60][61].Matairesinol has demonstrated antioxidant, anti-cancer, neuroprotective, and anti-inflammation abilities [62,63].In addition, the major DAPs could serve as key biomarkers for analyzing molecular networks regulating polyphenol biosynthesis during sesame seed development.As a support, correlation network analysis among DAPs revealed significant positive correlations between 22 phenolic acids, 17 lignans, and 10 flavonoids (Figure S8).

Differentially Expressed Genes (DEGs) between HA and LA during Seed Development
To verify the implication of phenylpropanoid metabolism in variation in sesame seeds AOA, we carried out a comparative transcriptome analysis of HA and LA varieties at three seed developmental stages, including 10, 20, and 30 DPA (days post-anthesis).The summary of the high-throughput RNA sequencing data is presented in Table S5.The reliability of the RNA-seq data was confirmed through qRT-PCR analysis of eight randomly selected genes, with a consistency of R 2 of 0.91 (Figure S9).Analyses revealed 2357, 1597, and 627 DEGs between HA and LA at 10, 20, and 30 DPA, respectively (Figure 5A).Of these DEGs, 1114, 885, and 347 were up-regulated in HA at the respective developmental stages (Figure 5A).A Venn diagram showed that only 170 genes were differentially expressed between the two seed types along with the seed development (Figure 5B).randomly selected genes, with a consistency of R 2 of 0.91 (Figure S9).Analyses revealed 2357, 1597, and 627 DEGs between HA and LA at 10, 20, and 30 DPA, respectively (Figure 5A).Of these DEGs, 1114, 885, and 347 were up-regulated in HA at the respective developmental stages (Figure 5A).A Venn diagram showed that only 170 genes were differentially expressed between the two seed types along with the seed development (Figure 5B).GO (gene ontology) analysis revealed that the DEGs at 10 DPA were mostly enriched to the membrane and its components, oxidoreductase activity, lipid storage, and carbohydrate metabolic process (Figure S10A).Meanwhile, the main enriched GO terms at 20 DPA were intracellular non-membrane, ribosome, structural constituent of ribosome, structural molecule activity, and peptide and amide metabolic processes (Figure S10B).At 30 DPA, the most enriched GO terms were extracellular region, carbohydrate metabolic process, and iron ion binding (Figure S11).These results indicate different metabolism regulations during HA and LA seed developmental processes.KEGG enrichment analysis of DEGs revealed that phenylpropanoid biosynthesis, flavonoid biosynthesis, and stilbenoid biosynthesis were the most significantly differentially regulated pathways between HA and LA at early and late seed developmental stages (Figure 6A,B).Meanwhile, ribosome metabolic processes were the main processes differentially regulated at 20 DPA (Figure S12).Taken together, these results show that differences in the regulation of flavonoid and phenolic acid biosynthesis are the key driven mechanisms of variation in sesame seed AOA.GO (gene ontology) analysis revealed that the DEGs at 10 DPA were mostly enriched to the membrane and its components, oxidoreductase activity, lipid storage, and carbohydrate metabolic process (Figure S10A).Meanwhile, the main enriched GO terms at 20 DPA were intracellular non-membrane, ribosome, structural constituent of ribosome, structural molecule activity, and peptide and amide metabolic processes (Figure S10B).At 30 DPA, the most enriched GO terms were extracellular region, carbohydrate metabolic process, and iron ion binding (Figure S11).These results indicate different metabolism regulations during HA and LA seed developmental processes.KEGG enrichment analysis of DEGs revealed that phenylpropanoid biosynthesis, flavonoid biosynthesis, and stilbenoid biosynthesis were the most significantly differentially regulated pathways between HA and LA at early and late seed developmental stages (Figure 6A,B).Meanwhile, ribosome metabolic processes were the main processes differentially regulated at 20 DPA (Figure S12).Taken together, these results show that differences in the regulation of flavonoid and phenolic acid biosynthesis are the key driven mechanisms of variation in sesame seed AOA.

Expression Patterns of Phenylpropanoid Biosynthesis-Related DEGs
Based on the above results, we found it important to examine the expression patterns of phenylpropanoid pathway-related DEGs to identify potential target genes for modulating the sesame polyphenol profile.As shown in Figure 7, most phenylpropanoid structural genes, such as phenylalanine ammonia-lyase (SINPZ0401548 and SINPZ0501377), caffeoyl-CoA O-methyltransferase (SINPZ1000220 and SINPZ0500891),

Figure 2 .
Figure 2. Variation in antioxidant activity (A), total phenolic content (B), and total flavonoid content (C) among black (n = 45), brown (n = 85), yellow (n = 32), and white (n = 234) sesame seeds from different accessions.The red lines indicate the mean.Black dotted/dashed lines indicate quartiles.Different letters above the violin plots indicate statistical differences at p ˂ 0.05.

Figure 2 .
Figure 2. Variation in antioxidant activity (A), total phenolic content (B), and total flavonoid content (C) among black (n = 45), brown (n = 85), yellow (n = 32), and white (n = 234) sesame seeds from different accessions.The red lines indicate the mean.Black dotted/dashed lines indicate quartiles.Different letters above the violin plots indicate statistical differences at p < 0.05.

Figure 3 .
Figure 3. Variation in the polyphenol profiles of high (HA) and low (LA) antioxidant seeds.(A) Classification of the 785 identified phenolic compounds.(B) Sub-classification of flavonoids.(C) Principal component analysis (PCA).HA-1, HA-2, and HA-3 represent the three replications for HA.LA-1, LA-2, and LA-3 represent the three replications for LA.

Figure 3 .
Figure 3. Variation in the polyphenol profiles of high (HA) and low (LA) antioxidant seeds.(A) Classification of the 785 identified phenolic compounds.(B) Sub-classification of flavonoids.(C) Principal component analysis (PCA).HA-1, HA-2, and HA-3 represent the three replications for HA.LA-1, LA-2, and LA-3 represent the three replications for LA.

Figure 4 .
Figure 4. Differentially accumulated phenolic (DAP) compounds between high (HA) and low (LA) antioxidant seeds.(A) Score plot of the OPLS-DA analysis.(B) Volcano plot of the DAPs between HA and LA.Up-regulation and down-regulation indicate the metabolite has higher relative content in LA and HA, respectively.(C) KEGG annotation and enrichment results of DAPs.

Figure 4 .
Figure 4. Differentially accumulated phenolic (DAP) compounds between high (HA) and low (LA) antioxidant seeds.(A) Score plot of the OPLS-DA analysis.(B) Volcano plot of the DAPs between HA and LA.Up-regulation and down-regulation indicate the metabolite has higher relative content in LA and HA, respectively.(C) KEGG annotation and enrichment results of DAPs.

Figure 5 .
Figure 5. Differentially expressed genes (DEGs) between HA and LA.(A) Number of DEGs at the three seed developmental stages.(B) Venn diagram indicating the number of key DEGs.

Figure 5 .
Figure 5. Differentially expressed genes (DEGs) between HA and LA.(A) Number of DEGs at the three seed developmental stages.(B) Venn diagram indicating the number of key DEGs.

Figure 7 .
Figure 7. Expression patterns of phenylpropanoid pathway-related DEGs in HA and LA during seed development.The values 10, 20, and 30 indicate DPA.

Figure 7 .
Figure 7. Expression patterns of phenylpropanoid pathway-related DEGs in HA and LA during seed development.The values 10, 20, and 30 indicate DPA.

Table 1 .
Variation in antioxidant activity, total phenolic content, and total flavonoid content in 400 sesame seed accessions.

Table 2 .
List of the 50 top up-regulated phenolic compounds in high-antioxidant sesame seeds.
Note.VIP, value importance in projection; FDR, false discovery rate; FC, fold change.