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Black nightshade (Solanum nigrum) belongs to the Solanaceae family and is used as a medicinal herb with health benefits. It has been reported that the black nightshade plant contains various phytochemicals that are associated with antitumor activities. Here we employed a genetic approach to study the effects of overexpression of Arabidopsis thaliana production of anthocyanin pigment 1 (AtPAP1) in black nightshade. Ectopic expression of AtPAP1 resulted in enhanced accumulation of anthocyanin pigments in vegetative and reproductive tissues of the transgenic plants. Analysis of anthocyanin revealed that delphinidin 3-O-rutinoside-5-O-glucoside, delphinidin 3,5-O-diglucoside, delphinidin 3-O-rutinoside, petunidin 3-O-rutinoside ( cis - p -coumaroyl)-5-O-glucoside, petunidin 3-(feruloyl)-rutinoside-5-glucoside, and malvidin 3-(feruloyl)-rutinoside-5-glucoside are highly induced in the leaves of AtPAP1 overexpression lines. Furthermore, ectopic expression of AtPAP1 evoked expression of early and late biosynthetic genes of the general phenylpropanoid and flavonoid pathways that include phenylalanine ammonia-lyase ( PAL ), cinnamate-4-hydroxylase ( C4H ), 4-coumarate CoA ligase ( 4CL ), chalcone isomerase ( CHI ), and quinate hydroxycinnamoyl transferase ( HCT ), which suggests these genes might be transcriptional targets of AtPAP1 in black nightshade. Concomitantly, the total content of anthocyanin in the transgenic black nightshade plants was higher compared to the control plants, which supports phenotypic changes in color. Our data demonstrate that a major anthocyanin biosynthetic regulator, AtPAP1, can induce accumulation of anthocyanins in the heterologous system of black nightshade through the conserved flavonoid biosynthesis pathway in plants., Competing Interests: The authors declare no competing financial interest.

Solanum nigrum fruits have been conventionally used in beverages due to their nutritional substances such as minerals, vitamins, amino acids, proteins, sugars, polyphenols, and anthocyanins. The characterization of components and regulatory mechanism of anthocyanins in S. nigrum fruits have rarely been reported. In this study, we determined that the peel and flesh of S. nigrum fruits shared similar HPLC profiles but different contents and total antioxidant activities for anthocyanins. After an efficient purification method, mainly including extraction with pH 1.0 distilled water and then desorption with pH 1.0 95% ethanol after a DM-130 resin adsorption step to obtain more pure anthocyanin extracts, the purity of anthocyanins extracted from S. nigrum fruits reached 56.1%. Moreover, eight anthocyanins from S. nigrum fruit were identified with HPLC-MS/MS for the first time. A typical R2R3-MYB transcription factor gene, SnMYB , was also cloned for the first time by rapid amplification of cDNA ends (RACE)-PCR from S. nigrum. Moreover, the contents of anthocyanins were shown to correlate well ( r = 0.93) with the expression levels of SnMYB gene during the fruit's developmental stages. Most significantly, SnMYB gene successfully produced high anthocyanin content (1.03 mg/g) when SnMYB gene was transiently expressed in tobacco leaves. Taken together, S. nigrum fruits are a promising resource for anthocyanin extraction, and SnMYB gene is an activator that positively regulates anthocyanin biosynthesis in S. nigrum., Competing Interests: The authors declare no conflict of interest.