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.

Integrating transcriptome and phytohormones analysis provided insights into plant height development in sesame.

Plant height is a key agronomic trait influencing crops yield. The height of sesame plants is important for yield performance, lodging resistance and plant architecture. Although plant height is significantly distinct among sesame varieties, the genetic basis of plant height remains largely unknown. In this study, in order to tackle genetic insights into the sesame plant height development, a comprehensive transcriptome analysis was conducted using the stem tips from two sesame varieties with distinct plant height, Zhongzhi13 and ZZM2748, at five time points by BGI MGIseq2000 sequencing platform. A total of 16,952 genes were differentially expressed between Zhongzhi13 and ZZM2748 at five time points. KEGG and MapMan enrichment analyses and quantitative analysis of phytohormones indicated that hormones biosynthesis and signaling pathways were associated with sesame plant height development. Plenty of candidate genes involved in biosynthesis and signaling of brassinosteroid (BR), cytokinin (CK) and gibberellin (GA) which were major differential hormones between two varieties were identified, suggesting their critical roles in plant height regulation. WGCNA revealed a module which was significantly positively associated with the plant height trait and founded SiSCL9 was the hub gene involved in plant height development in our network. Further overexpression in transgenic Arabidopsis validated the function of SiSCL9 in the increase of plant height by 26.86%. Collectively, these results increase our understanding of the regulatory network controlling the development of plant height and provide a valuable genetic resource for improvement of plant architecture in sesame.

Resequencing of 410 Sesame Accessions Identifies SINST1 as the Major Underlying Gene for Lignans Variation.

Sesame is a promising oilseed crop that produces specific lignans of clinical importance. Hence, a molecular description of the regulatory mechanisms of lignan biosynthesis is essential for crop improvement. Here, we resequence 410 sesame accessions and identify 5.38 and 1.16 million SNPs (single nucleotide polymorphisms) and InDels, respectively. Population genomic analyses reveal that sesame has evolved a geographic pattern categorized into northern (NC), middle (MC), and southern (SC) groups, with potential origin in the southern region and subsequent introduction to the other regions. Selective sweeps analysis uncovers 120 and 75 significant selected genomic regions in MC and NC groups, respectively. By screening these genomic regions, we unveiled 184 common genes positively selected in these subpopulations for exploitation in sesame improvement. Genome-wide association study identifies 17 and 72 SNP loci for sesamin and sesamolin variation, respectively, and 11 candidate causative genes. The major pleiotropic SNPC/A locus for lignans variation is located in the exon of the gene SiNST1. Further analyses revealed that this locus was positively selected in higher lignan content sesame accessions, and the "C" allele is favorable for a higher accumulation of lignans. Overexpression of SiNST1C in sesame hairy roots significantly up-regulated the expression of SiMYB58, SiMYB209, SiMYB134, SiMYB276, and most of the monolignol biosynthetic genes. Consequently, the lignans content was significantly increased, and the lignin content was slightly increased. Our findings provide insights into lignans and lignin regulation in sesame and will facilitate molecular breeding of elite varieties and marker-traits association studies.

Comparative Metabolomics Analysis of Different Perilla Varieties Provides Insights into Variation in Seed Metabolite Profiles and Antioxidant Activities.

Perilla seeds are essential functional foods and key ingredients in traditional medicine. Herein, we investigated the variation in phytochemical profiles and antioxidant activities of twelve different perilla seeds. The seeds showed significant variations in total phenolic and flavonoid contents ranging from 16.92 to 37.23 mg GAE/g (GAE, gallic acid equivalent) and 11.6 to 19.52 mg CAE/g (CAE, catechin equivalent), respectively. LC-QqQ-MS (liquid chromatography triple quadrupole tandem mass spectrometry)-based widely targeted metabolic profiling identified a total of 975 metabolites, including 68-269 differentially accumulated metabolites (DAMs). Multivariate analyses categorized the seeds into four groups based on the seed coat and leaf colors. Most key bioactive DAMs, including flavonoids (quercetin-3'-O-glucoside, prunin, naringenin, naringenin chalcone, butin, genistin, kaempferol-3-O-rutinoside, etc.), amino acids (valine, lysine, histidine, glutamine, threonine, etc.), and vitamins (B1, B3, B6, U, etc.) exhibited the highest relative content in PL3 (brown seed, purple leaf), PL1 (white seed, green-purple leaf), and PL4 (white seed, green leaf) groups, respectively. Meanwhile, key differentially accumulated phenolic acids showed a higher relative content in PL1 and PL4 than in other groups. Both seeds exhibited high antioxidant activities, although those of PL2 (brown seed, green leaf) group seeds were the lowest. Our results may facilitate the comprehensive use of perilla seeds in food and pharmaceutical industries.

Metabolome genome-wide association study provides biochemical and genetic insights into natural variation of primary metabolites in sesame.

Plants' primary metabolites are of great importance from the survival and nutritional perspectives. However, the genetic bases underlying the profiles of primary metabolites in oilseed crops remain largely unclear. As one of the main oilseed crops, sesame (Sesamum indicum L.) is a potential model plant for investigating oil metabolism in plants. Therefore, the objective of this study is to disclose the genetic variants associated with variation in the content of primary metabolites in sesame. We performed a comprehensive metabolomics analysis of primary metabolites in 412 diverse sesame accessions using gas chromatography-mass spectrometry and identified a total of 45 metabolites, including fatty acids, monoacylglycerols (MAGs), and amino acids. Genome-wide association study unveiled 433 significant single-nucleotide polymorphism loci associated with variation in primary metabolite contents in sesame. By integrating diverse genomic analyses, we identified 10 key candidate causative genes of variation in MAG, fatty acid, asparagine, and sucrose contents. Among them, SiDSEL was significantly associated with multiple traits. SiCAC3 and SiKASI were strongly associated with variation in oleic acid and linoleic acid contents. Overexpression of SiCAC3, SiKASI, SiLTPI.25, and SiLTPI.26 in transgenic Arabidopsis and Saccharomyces cerevisiae revealed that SiCAC3 is a potential target gene for improvement of unsaturated fatty acid levels in crops. Furthermore, we found that it may be possible to breed several quality traits in sesame simultaneously. Our results provide valuable genetic resources for improving sesame seed quality and our understanding of oilseed crops' primary metabolism.

CRISPR/Cas9-Mediated Efficient Targeted Mutagenesis in Sesame (Sesamum indicum L.).

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been widely utilized for targeted genome modification in a wide range of species. It is a powerful genome editing technology, providing significant benefits for gene functional research and molecular breeding. However, to date, no study has applied this genome editing tool to sesame (Sesamum indicum L.), one of the most ancient and important oil crops used widely in diverse industries such as food and medicine. Herein, the CRISPR/Cas9 system along with hairy root transformation was used to induce targeted mutagenesis in sesame. Two single guide RNAs (sgRNAs) were designed to target two sesame cytochrome P450 genes (CYP81Q1 and CYP92B14), which are the key biosynthetic gene of sesamin and sesamolin, respectively. Sequencing data illustrated the expected InDel mutations at the target sites, with 90.63 and 93.33% mutation frequency in CYP81Q1 and CYP92B14, respectively. The most common editing event was single nucleotide deletion and insertion. Sequencing of potential off-target sites of CYP92B14-sgRNA showed no off-target events in cases of three mismatches. High-performance liquid chromatography analysis showed that sesamin and sesamolin biosynthesis was effectively disrupted in the mutated hairy roots, confirming the crucial role of CYP81Q1 and CYP92B14 in sesame lignan biosynthesis. These results demonstrated that targeted mutagenesis was efficiently created by the CRISPR/Cas9 system, and CRISPR/Cas9 coupled with hairy root transformation is an effective tool for assessing gene functions in sesame.

Genome-wide characterization and identification of candidate ERF genes involved in various abiotic stress responses in sesame (Sesamum indicum L.).

BACKGROUND: The adverse effects of climate change on crop production are constraining breeders to develop high-quality environmentally stable varieties. Hence, efforts are being made to identify key genes that could be targeted for enhancing crop tolerance to environmental stresses. ERF transcription factors play an important role in various abiotic stresses in plants. However, the roles of the ERF family in abiotic stresses tolerance are still largely unknown in sesame, the "queen" of oilseed crops. RESULTS: In total, 114 sesame ERF genes (SiERFs) were identified and characterized. 96.49% of the SiERFs were distributed unevenly on the 16 linkage groups of the sesame genome. The phylogenetic analysis with the Arabidopsis ERFs (AtERFs) subdivided SiERF subfamily proteins into 11 subgroups (Groups I to X; and VI-L). Genes in the same subgroup exhibited similar structure and conserved motifs. Evolutionary analysis showed that the expansion of ERF genes in sesame was mainly induced by whole-genome duplication events. Moreover, cis-acting elements analysis showed that SiERFs are mostly involved in environmental responses. Gene expression profiles analysis revealed that 59 and 26 SiERFs are highly stimulated under drought and waterlogging stress, respectively. In addition, qRT-PCR analyses indicated that most of SiERFs are also significantly up-regulated under osmotic, submerge, ABA, and ACC stresses. Among them, SiERF23 and SiERF54 were the most induced by both the abiotic stresses, suggesting their potential for targeted improvement of sesame response to multiple abiotic stresses. CONCLUSION: This study provides a comprehensive understanding of the structure, classification, evolution, and abiotic stresses response of ERF genes in sesame. Moreover, it offers valuable gene resources for functional characterization towards enhancing sesame tolerance to multiple abiotic stresses.

Overexpression of sesame polyketide synthase A leads to abnormal pollen development in Arabidopsis.

BACKGROUND: Sesame is a great reservoir of bioactive constituents and unique antioxidant components. It is widely used for its nutritional and medicinal value. The expanding demand for sesame seeds is putting pressure on sesame breeders to develop high-yielding varieties. A hybrid breeding strategy based on male sterility is one of the most effective ways to increase the crop yield. To date, little is known about the genes and mechanism underlying sesame male fertility. Therefore, studies are being conducted to identify and functionally characterize key candidate genes involved in sesame pollen development. Polyketide synthases (PKSs) are critical enzymes involved in the biosynthesis of sporopollenin, the primary component of pollen exine. Their in planta functions are being investigated for applications in crop breeding. RESULTS: In this study, we cloned the sesame POLYKETIDE SYNTHASE A (SiPKSA) and examined its function in male sterility. SiPKSA was specifically expressed in sesame flower buds, and its expression was significantly higher in sterile sesame anthers than in fertile anthers during the tetrad and microspore development stages. Furthermore, overexpression of SiPKSA in Arabidopsis caused male sterility in transgenic plants. Ultrastructural observation showed that the pollen grains of SiPKSA-overexpressing plants contained few cytoplasmic inclusions and exhibited an abnormal pollen wall structure, with a thicker exine layer compared to the wild type. In agreement with this, the expression of a set of sporopollenin biosynthesis-related genes and the contents of their fatty acids and phenolics were significantly altered in anthers of SiPKSA-overexpressing plants compared with wild type during anther development. CONCLUSION: These findings highlighted that overexpression of SiPKSA in Arabidopsis might cause male sterility through defective pollen wall formation. Moreover, they suggested that SiPKSA modulates vibrant pollen development via sporopollenin biosynthesis, and a defect in its regulation may induce male sterility. Therefore, genetic manipulation of SiPKSA might promote hybrid breeding in sesame and other crop species.

The Dark Pigment in the Sesame (Sesamum indicum L.) Seed Coat: Isolation, Characterization, and Its Potential Precursors.

Sesame is a worldwide oilseed crop used in the food pharmacy. Its seed phenotypes determine the seed quality values. However, a thorough assessment of seed coat metabolites is lacking, and the dark pigment in the seed coat is not well-characterized. Herein, 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 NaOH 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. Our findings widen our understanding of dark seeds pigmentation in sesame. Furthermore, they show that black sesame seeds are promising sources of edible melanin for food and biotechnological applications.

Fine Mapping of a Major Pleiotropic QTL Associated with Sesamin and Sesamolin Variation in Sesame (Sesamum indicum L.).

Deciphering the genetic basis of quantitative agronomic traits is a prerequisite for their improvement. Herein, we identified loci governing the main sesame lignans, sesamin and sesamolin variation in a recombinant inbred lines (RILs, F8) population under two environments. The content of the two lignans in the seeds was investigated by HPLC. The sesamin and sesamolin contents ranged from 0.33 to 7.52 mg/g and 0.36 to 2.70 mg/g, respectively. In total, we revealed 26 QTLs on a linkage map comprising 424 SSR markers, including 16 and 10 loci associated with sesamin and sesamolin variation, respectively. Among them, qSmin_11.1 and qSmol_11.1 detected in both the two environments explained 67.69% and 46.05% of the phenotypic variation of sesamin and sesamolin, respectively. Notably, qSmin11-1 and qSmol11-1 were located in the same interval of 127-127.21cM on LG11 between markers ZMM1776 and ZM918 and acted as a pleiotropic locus. Furthermore, two potential candidate genes (SIN_1005755 and SIN_1005756) at the same locus were identified based on comparative transcriptome analysis. Our results suggest the existence of a single gene of large effect that controls expression, both of sesamin and sesamolin, and provide genetic information for further investigation of the regulation of lignan biosynthesis in sesame.

Comparative metabolomics analysis of different sesame (Sesamum indicum L.) tissues reveals a tissue-specific accumulation of metabolites.

BACKGROUND: Sesame (Sesamum indicum L.) leaves, flowers, especially seeds are used in traditional medicine to prevent or cure various diseases. Its seed's market is expanding. However, the other tissues are still underexploited due to the lack of information related to metabolites distribution and variability in the plant. Herein, the metabolite profiles of five sesame tissues (leaves, fresh seeds, white and purple flowers, and fresh carpels) have been investigated using ultra-high-performance liquid chromatography-mass spectrometry (UPLC-MS/MS)-based widely targeted metabolomics analysis platform. RESULTS: 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 and enrichment analyses 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. CONCLUSION: This study revealed that the metabolic processes in the sesame tissues are differently regulated. It offers valuable resources for investigating gene-metabolites interactions in sesame tissues and examining metabolic transports during seed development in sesame. Furthermore, our findings provide crucial knowledge that will facilitate sesame biomass valorization.

Genome-Wide Analysis of nsLTP Gene Family and Identification of SiLTPs Contributing to High Oil Accumulation in Sesame (Sesamum indicum L.).

The biosynthesis and storage of lipids in oil crop seeds involve many gene families, such as nonspecific lipid-transfer proteins (nsLTPs). nsLTPs are cysteine-rich small basic proteins essential for plant development and survival. However, in sesame, information related to nsLTPs was limited. Thus, the objectives of this study were to identify the Sesamum indicum nsLTPs (SiLTPs) and reveal their potential role in oil accumulation in sesame seeds. Genome-wide analysis revealed 52 SiLTPs, nonrandomly distributed on 10 chromosomes in the sesame variety Zhongzhi 13. Following recent classification methods, the SiLTPs were divided into nine types, among which types I and XI were the dominants. We found that the SiLTPs could interact with several transcription factors, including APETALA2 (AP2), DNA binding with one finger (Dof), etc. Transcriptome analysis showed a tissue-specific expression of some SiLTP genes. By integrating the SiLTPs expression profiles and the weighted gene co-expression network analysis (WGCNA) results of two contrasting oil content sesame varieties, we identified SiLTPI.23 and SiLTPI.28 as the candidate genes for high oil content in sesame seeds. The presumed functions of the candidate gene were validated through overexpression of SiLTPI.23 in Arabidopsis thaliana. These findings expand our knowledge on nsLTPs in sesame and provide resources for functional studies and genetic improvement of oil content in sesame seeds.

Transcriptome Dynamics during Black and White Sesame (Sesamum indicum L.) Seed Development and Identification of Candidate Genes Associated with Black Pigmentation.

Seed coat color is a crucial agronomic trait in sesame (Sesamum indicum L.) since it is strongly linked to seed oil, proteins, and lignans contents, and also influences consumer preferences. In East Asia, black sesame seed is used in the treatment and the prevention of various diseases. However, in sesame, little is known about the establishment of the seed coat color, and only one gene has been reported to control black pigmentation. This study provides an overview of developing seeds transcriptome of two varieties of sesame "Zhongfengzhi No.1" (white seed) and "Zhongzhi No.33" (black seed) and shed light on genes involving in black seed formation. Until eight days post-anthesis (DPA), both the seeds of the two varieties were white. The black sesame seed turned to yellow between 9 and 11 DPA and then black between 12 and 14 DPA. The black and white sesame showed similar trend-expressed genes with the numbers increased at the early stages of seed development. The differentially expressed genes (DEGs) number increased with seed development in the two sesame varieties. We examined the DEGs and uncovered that more were up-regulated at the early stages. The DEGs between the black and white sesame were mainly enriched in 37 metabolic pathways, among which the flavonoid biosynthesis and biosynthesis of secondary metabolites were dominants. Furthermore, we identified 20 candidate genes associated with pigment biosynthesis in black sesame seed, among which 10 were flavonoid biosynthesis and regulatory genes. These genes also include isochorismate and polyphenol oxidase genes. By comparing the phenotypes and genes expressions of the black and white sesame seed at different development stages, this work revealed the important role of 8-14 DPA in black pigment biosynthesis and accumulation. Moreover, it unfolded candidate genes associated with black pigmentation in sesame. These findings provide a vast transcriptome dataset and list of genes that will be targeted for functional studies related to the molecular mechanism involved in biosynthesis and regulation of seed coat color in sesame.