Fruit color is an important economic trait. The color of red walnut cultivars is mainly attributed to anthocyanins. The aim of this study was to explore the differences in the molecular mechanism of leaf and peel color change between red and green walnut. A reference transcriptome of walnut was sequenced and annotated to identify genes related to fruit color at the ripening stage. More than 290 million high-quality reads were assembled into 39,411 genes using a combined assembly strategy. Using Illumina digital gene expression profiling, we identified 4568 differentially expressed genes (DEGs) between red and green walnut leaf and 3038 DEGs between red and green walnut peel at the ripening stage. We also identified some transcription factor families (MYB, bHLH, and WD40) involved in the control of anthocyanin biosynthesis. The trends in the expression levels of several genes encoding anthocyanin biosynthetic enzymes and transcription factors in the leaf and peel of red and green walnut were verified by quantitative real-time PCR. Together, our results identified the genes involved in anthocyanin accumulation in red walnut. These data provide a valuable resource for understanding the coloration of red walnut.

Background: Walnuts are among the most important dry fruit crops worldwide, typically exhibiting green leaves and yellow–brown or gray–yellow seed coats. A specific walnut accession with red leaves and seed coats, ‘RW-1’, was selected for study because of its high anthocyanin and proanthocyanidin (PA) contents. Anthocyanins and PAs are important secondary metabolites and play key roles in plant responses to biotic and abiotic stresses. However, few studies have focused on the molecular mechanism of anthocyanin biosynthesis in walnuts. Methods: In this study, we determined the anthocyanin and PA components and their contents in different color leaves of ‘RW-1’ natural hybrid progenies at various developmental stages. Integrated transcriptome and metabolome analyses were used to identify the differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs). We also performed conjoint analyses on DEGs and DAMs to ascertain the degree pathways, and explore the regulation of anthocyanin and PA biosynthesis. Results: The results of widely targeted metabolome profiling and anthocyanin detection revealed 395 substances, including four PAs and 26 anthocyanins, in red (SR) and green leaves (SG) of ‘RW-1’ natural hybrid progenies. From the research, the contents of all anthocyanin components in SR were higher than that in SG. Among them, the contents of delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-arabinoside and cyanidin 3-O-glucoside were significantly higher than others, and they were considered as the main types of anthocyanins. However, nine anthocyanins were detected only in SR. For PAs, the content of procyanidin C1 was higher in SR compared with SG, while procyanidin B1 and procyanidin B3 were higher in SR-1 and SR-3 but downregulated in SR-2 compared with the controls. Furthermore, transcriptome analysis revealed that the expressions of structural genes (C4H, F3H, F3′5′H, UFGT, LAR and ANR), three MYBs predicted as the activators of anthocyanin and PA biosynthesis, two MYBs predicted as the repressors of anthocyanin biosynthesis, and five WD40s in the anthocyanin and PA biosynthetic pathways were significantly higher in the SR walnuts. Gene-metabolite correlation analyses revealed a core set of 31 genes that were strongly correlated with four anthocyanins and one PA metabolites. The alteration of gene coding sequence altered the binding or regulation of regulatory factors to structural genes in different color leaves, resulting in the effective increase of anthocyanins and PAs accumulation in red walnut. Conclusions: This study provides valuable information on anthocyanin and PA metabolites and candidate genes for anthocyanin and PA biosynthesis, yielding new insights into anthocyanin and PA biosynthesis in walnuts.

BackgroundWalnuts are one of the most important dry fruit crops worldwide, typically exhibiting green leaves and yellow–brown or gray–yellow seed coats. A specific walnut type, red walnut ‘RW-1’, with red leaves and seed coats was selected as the plant material because of its higher anthocyanin and proanthocyanin (PA) contents. Anthocyanins and PAs coprise important secondary defense methods for plants to respond to biotic and abiotic stresses. However, few studies have focused on the molecular mechanism of anthocyanin biosynthesis in walnuts.ResultsFrom the results of widely targeted metabolome and anthocyanidin detection analysis, 395 substances, including 4 PAs and 26 anthocyanins, were identified from the red-leaf walnuts of RW-1 natural hybrid progenies (SR) and the green-leaf walnuts of RW-1 natural hybrid progenies (SG). Among these, all anthocyanin types in SR were significantly upregulated compared with SG. Additionally, delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-glucoside and cyanidin 3-O-glucoside were identified as the primary components of anthocyanidins because of their higher contents. Nine anthocyanidins, malvidin 3-O-galactoside, malvidin 3-O-arabinoside, cyanidin 3-O-(6-O-malonyl-beta-D-glucoside), delphinidin 3-O-glucoside, delphinidin 3,5-O-diglucoside (Delphin), peonidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-arabinoside and pelargonidin 3-O-(6-O-malonyl-beta-D-glucoside), were detected only in the SR walnuts. For PAs, proanthocyanin C1 was upregulated in SR compared with SG, while proanthocyanin B1 and proanthocyanin B3 were upregulated in SR-1 and SR-3 but downregulated in SR-2 compared with the controls. Furthermore, transcriptome analysis demonstrated that the expression of structural genes (C4H, F3H, F3’5’H, UFGTs, LAR and ANR), four MYBs and six WD40s in the anthocyanin and PA biosynthetic pathways were significantly higher in the SR walnut.ConclusionsOur results provide valuable information on anthocyanin and PA metabolites and candidate genes in anthocyanin and PA biosynthesis, which provides new insights into anthocyanin and PA biosynthesis in walnuts.

Background Walnut is one of the most important dry fruit crops worldwide, typically green leaves and yellow-brown or gray-yellow seed coats. A specific walnut type, red walnut ‘RW-1’ with red leaves and seed coats was selected as plant material because of higher anthocyanins contents. Anthocyanins are important colorants with strong antioxidant activity, especially, benefic for human health. However, few studies focused on the molecular mechanism of anthocyanin biosynthesis in walnut. Results From the results of Widely Targeted Metabolome and anthocyanidin detection analysis, 395 substances, including 4 procyanidins and 26 anthocyanins, were identified from the red-leaf walnuts of RW-1 natural hybrid progenies (SR) and the green-leaf walnuts of RW-1 natural hybrid progenies (SG). Among these, all the anthocyanins in SR were significantly up-accumulated comparing with SG. Also, delphinidin 3-O-galactoside, cyanidin 3-O-galactoside, delphinidin 3-O-glucoside and cyanidin 3-O-glucoside were identified to the primary components of anthocyanidins because of the higher contents. It was noted that 9 anthocyanins including malvidin 3-O-galactoside, malvidin 3-O-arabinoside, cyanidin 3-O-(6-O-malonyl-beta-D-glucoside), delphinidin 3-O-glucoside, delphinidin 3,5-O-diglucoside (Delphin), peonidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-(6-O-malonyl-beta-D-glucoside), petunidin 3-O-arabinoside and pelargonidin 3-O-(6-O-malonyl-beta-D-glucoside) were detected only in SR walnut. Furthermore, transcriptome analysis demonstrated that the expression of structural genes (C4H, F3H, F3’5’H and UFGTs), and four MYBs in anthocyanin biosynthetic pathway were significantly higher in SR walnut. Conclusions We identified the color formation of SR leaves is due to the accumulation of anthocyanins. And our results obtained the valuable information on the anthocyanin metabolites and candidate genes in anthocyanin biosynthesis, which provided new insights into the anthocyanin biosynthesis in walnuts.