Anthocyanin is one of the most important color-presenting materials in flavonoids, mainly existing in leaves, flowers, fruits of higher plants, formed by the combination of anthocyan and glycosyl . Cyanidin (Cy), delphinidin (Dp), pelargonidin (Pg), peonidin (Pn), petunidin (Pt), and malvidin (Mv) are six common anthocyans, and, common glycosyl includes glucose, galactose, sucrose, etc . Different types and amounts of glycosyls bound to different positions of anthocyans resulted in significant increase of anthocyanins compounds . Anthocyanins takes part in protecting plants from ultraviolet rays damage, attracting insect pollination and resisting to low temperature . As a kind of natural food colorants, anthocyanins have been widely used in food and cosmetics due to natural nontoxicity and no side effects . Additionally, numerous studies have demonstrated that anthocyanins have many promising benefits for human health, such as the prevention of cardiovascular disease, neuron disease, cancer, diabetes and inflammation [6–10]. Therefore, anthocyanins have risen to fame for high antioxidant capacity, as well as improving human health and extracting natural food pigments.
The anthocyanin biosynthesis pathways have been well characterized in some plants . Generally, phenylalanine is regarded as the initial of anthocyanin biosynthesis, which is composed of a series of enzymatic reactions, including phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), chalcone synthetase (CHS), chalcone isoenzyme (CHI), flavanone 3-hydroxylase (F3H), Flavonoid 3′,5′-Hydroxylase (F3’5’H), dihydroflavonol 4-reductase (DFR), anthocyanin synthetase (ANS), UDP glucose: flavonoid-3-O-glucosyltransferase (UFGT) and so on [12, 13].
Meanwhile, the structural genes in anthocyanin biosynthesis are regulated by multiple transcription factors, among which MYB transcription factors have been widely studied. Grape R2R3-MYB TFs VvMYBA1, VvMYBA6 and VvMYBA7 regulated anthocyanin biosynthesis by activating UFGT and 3AT expressions ; The overexpression of MdMYB3 increased the anthocyanin accumulation in tobaccos by activating the gene expressions of CHS, CHI, UFGT and FLS ; The overexpression of GhMYB1a in gerbera and tobacco (Nicotiana tabacum) decreased anthocyanin accumulation by upregulating the structural gene expressions of NtCHS, NtF3H, NtDFR, NtANS, and NtUFGT .
It was reported that MYB regulates anthocyanin biosynthesis by either alone or forming MBW complex (MYB-bHLH-WD40) [17, 18]. In MBW complex, bHLH and MYB transcription factors can specifically bind to the promoters of structural genes in anthocyanin biosynthesis pathway, while WD40 protein plays a stable role in MBW complex . The regulatory model of MBW complex included a ternary complex and a binary complex composed of MYB and bHLH. The ternary complex usually directly regulates the transcription of structural genes, but not synthesize intermediate regulators . For example, AcMYB123 and AcbHLH42 in Actinidia chinensis cv. Hongyang promote the anthocyanin accumulation by activating the promoters of AcANS and Ac3FGT1 . PyMYB10 and PyMYB114 co-transformed with PybHLH3 induced the promoters activity of PyDFR, PyANS, PyUFGT, PyGST and PyABC transporter . Therefore, the present study aimed to elucidate the regulation mechanism of main structural genes and transcription factors involving in the anthocyanin accumulation based on red and green walnuts.
Walnut (Juglans regia L.) is an ancient fruit tree from the Juglandaceae family, ranks the top four nuts in the world due to its higher economic value and its health benefits to humans because of rech in oleic acid, linoleic acid, linolenic acid, protein and many trace elements . In addition, walnuts are also considered to be an important ecological tree. The walnut industry has been included as the core point of industrial poverty alleviation in many areas, and plays an important role in helping targeted poverty alleviation and increasing farmers’ income. According to FAO statistics (http://faostat3.fao.org), China's walnut planting area and yield accounted for 48% and 56% of the world, respectively, both ranking the first in the world in 2019. However, phenotype traits of walnut varieties are close, with green leaves and yellow-brown or gray-yellow seed coat. Luckily, a red walnut accession ‘RW-1’ was found by our research group, with the red color leaf, pericarp, seed coat and xylem owing to high anthocyanin content , and several bHLH TFs and CHS structural genes were identified in anthocyanin biosynthesis [23, 24]. However, the molecular mechanism of anthocyanin biosynthesis has not yet been clearly elucidated in red walnut.
In recent years, the combination analysis of transcriptome and metabolome has widely used to clarify anthocyanin biosynthesis and accumulation in plants [25–31]. In the current study, the regulatory networks of anthocyanin biosynthesis in walnut were conducted using two ‘RW-1’ natural hybrid accessions, with red and green leaves respectively. The compared analysis of metabolomic and transcriptomics aimed to elucidate the pathway of anthocyanin metabolites and identify the different expressed genes (DEGs) in walnut anthocyanin biosynthesis.