The seed coat is the external protective layer of seed and develops from the integument initially surrounding the ovule and is maternal in origin . It protects the embryo and endosperm from external factors such as mechanical injuries, desiccation and infections . Moreover, it helps developing seed to regulate its metabolism in response to changes in its external environment by transmitting environmental signals to the interior of the seed . In sesame, seed coat color is strongly associated with seed quality . Therefore, genetic resources on pigmentation mainly black seed coats will help to improve the sesame seed quality. In this study, RNA-seq was used to scrutinize transcriptome differences between “Zhongfengzhi No.1” (white seed) and “Zhongzhi No.33” (black seed) at different stages of seed development. DEGs differently regulated during seed coat development were screened, and candidate genes associate with black pigmentation were detected.
The key stage for black pigment biosynthesis and accumulation in black sesame
In this study, we analyzed seeds samples and observed that seeds started browning and changed to black from 11 DPA. A great difference was observed between the expression profiles of black and white sesame. The black sesame reached the maximum expressed gene number with 20,253 genes over 0.1 in FPKM at 11 DPA and more genes were up or down-regulated at the early stages before 17 DPA. These results are consistent with the findings of Wei et al. , who reported that the gene SIN_1016759/SiPPO was highly expressed in the seeds from 11 to 20 DPA. PPO encodes polyphenol oxidase and generates black pigments via the browning reaction in plant .
As in previous studies, our results confirmed the pivotal role of later stages in biosynthesis of nutrients (oil, protein, and lignans) in sesame . Indeed, we observed that more genes were active and up or down-regulated at the later stages from 23 to 30 DPA in the two varieties especially in black sesame. Our findings provide the support that in sesame developing seed, early stages (before 23 DPA) play an important role in pigments biosynthesis and substrates preparation for nutrients biosynthesis in the later stages. Taken altogether, we thus suggested that black pigment is biosynthesized and accumulates in seed at early stages of seed development that is from 8 to 17 DPA mainly.
Candidate genes controlling black pigment biosynthesis in sesame
Flavonoids, including anthocyanins and proanthocyanidins, lignin and melanin are secondary metabolites that influence seed color in plants . They are derived from the phenylpropanoid pathway and are controlled by a complex regulatory network with multiple transcription factors . This pathway involves many genes such as chalcone synthase (CHS), chalcone isomerase (CHI), flavonol 3-hydroxylase (F3H), flavonol 3′-hydroxylase (F3′H), dihydroflavonol-4-reductase (DFR), anthocyanidin synthase (ANS) anthocyanidin reductase (ANR) and laccase. Some of these genes have been cloned from Arabidopsis and many plants . Previous studies in sesame detected that two major genes with additive-dominant-epistatic effects plus polygenes with additive-dominant-epistatic effects control the seed coat color, and several major have been identified QTL . Besides, the gene SIN_1016759/SiPPO had been reported as the candidate gene for black sesame . Here, we studied the shared DEGs between black and white sesame and identified 17 candidate genes associated with black pigmentation in sesame. Among the 17 candidate genes, 5 SIN_1016759/PPO, SIN_1006242, SIN_1026689, SIN_1006025 and SIN_1025056 are located on chromosomes 4, 8 and 11. This finding is congruent with the work of Wang et al. . SIN_1016759/PPO encodes polyphenol oxidase; SIN_1006242 is a cytochrome P450 gene; SIN_1026689 is a WAT1-related protein; SIN_1006025 encodes isochorismate synthase, and SIN_1025056 encodes a beta-glucosidase isoform. In plants, isochorismate synthase converts chorismate the last product in the shikimate pathway into isochorismate, a precursor of phylloquinone (vitamin K1) and salicylic acid . In addition, the 17 candidate genes also included 2 chalcone synthase genes SIN_1018961 and SIN_1018959. Three chalcone synthase-like genes have been identified in A. thaliana . Chalcone synthase is the first committed enzyme in the biosynthesis of all flavonoids which function in the phenylpropanoid pathway . It catalyzes the reaction leading to naringenin chalcone formation from p-coumaroyl-CoA and three molecules of malonyl-CoA . All the 17 candidate genes will be targeted in the future for functional genomic study. We preferentially suggested that the genes SIN_1016759/SiPPO, SIN_1018961, and SIN_1018959 may play a major role in pigments biosynthesis especially black pigment in sesame.
The compound responsible for black seed color in sesame
In plants, browning reactions on seed coat pigments are often induced by the oxidation of phenolic compounds by polyphenol oxidases (PPO) such as laccases and tyrosinases and result in melanin formation mostly . Tyrosinase is the enzyme that catalyzes the first two steps in the melanin synthesis pathway and controls the rate and yield of melanin production . Xiaoli et al.  had reported that free tyrosine and polyphenols were needed for melanin biosynthesis and compared to anthocyanin, the melanin content in brown seed was higher than in yellow seed rapes. The metabolomic analysis demonstrated that phenylpropanoid biosynthesis, tyrosine metabolism, and riboflavin metabolism were the main pathways differentially activated between black and white sesame and were responsible for the color difference . Furthermore, the black pigments present in the skin of banana and sunflower have been assumed to be melanin . Wan et al.  detected that brown and dark peanut seed (mutants) had lower level of lignin, anthocyanin, proanthocyanidin content, and a higher level of melanin content compared to wild type with light color. Also, they reported that the expression of polyphenol oxidases was significantly activated in mutants developing seed. The above results indicated that PPO might be one of the dominant genes responsible for black pigment biosynthesis in sesame. Therefore, we suggested that the black pigment in sesame seed would be melanin. Future studies will help to confirm whether melanin is the black pigment or not.
Strategies to improve black sesame quality
Sesame is especially widely grown for its high-quality nutritional seeds . However, compared with white sesame, black sesame content less oil, protein, linoleic acid, sesamin, and sesamolin . Hence the necessity to improve black sesame quality. The study carried out by Wei et al.  revealed that in sesame, the genes SiPPO and SiNST1 (SIN_1005755) associated respectively with black pigmentation and lignification in the seed coat are strongly associated oil, protein, sesamin, and sesamolin content variation in seeds. Furthermore, in sesame aromatic amino acids L-phenylalanine (Phe) and L-tyrosine (Tyr) are needed for protein biosynthesis and served as precursors for numerous compounds including flavonoids, melanin, lignin, lignans, quinones, and condensed tannins . These amino acids are produced from chorismate, the final product of the shikimate pathway, which involves many genes . Here, we figured out 17 candidate genes associated with black pigment synthesis in sesame including the genes function for PPO, chalcone，isochorismate, and so on. Thus, functional analysis coupling with the genetic transformation of these genes simultaneously with other pivotal genes, may be sufficient to improve black sesame quality.