Anthocyanins, which are colored pigments, have long been used as food and pharmaceutical ingredients due to their potential health benefits, but the intermediate signals through which environmental or developmental cues regulate anthocyanin biosynthesis remains poorly understood. Fleshy fruits have become a good system for studying the regulation of anthocyanin biosynthesis, and exploring the mechanism underlying pigment metabolism is valuable for controlling fruit ripening. The present study revealed that ABA accumulated during Lycium fruit ripening, and this accumulation was positively correlated with the anthocyanin contents and the LbNCED1 transcript levels. The application of exogenous ABA and of the ABA biosynthesis inhibitor fluridon increased and decreased the content of anthocyanins in Lycium fruit, respectively. This is the first report to show that ABA promotes the accumulation of anthocyanins in Lycium fruits. The variations in the anthocyanin content were consistent with the variations in the expression of the genes encoding the MYB-bHLH-WD40 transcription factor complex or anthocyanin biosynthesis-related enzymes. Virus-induced LbNCED1 gene silencing significantly slowed fruit coloration and decreased both anthocyanin and ABA accumulation during Lycium fruit ripening. An qRT-PCR analysis showed that LbNCED1 gene silencing clearly reduced the transcript levels of both structural and regulatory genes in the flavonoid biosynthetic pathway. Based on the results, a model of ABA-mediated development-dependent anthocyanin biosynthesis and fruit coloration during Lycium fruit maturation was proposed. In this model, the developmental cues transcriptionally activates LbNCED1 and thus enhances accumulation of the phytohormone ABA, and the accumulated ABA stimulates transcription of the MYB-bHLH-WD40 transcription factor complex to upregulate the expression of structural genes in the flavonoid biosynthetic pathway and thereby promoting anthocyanin production and fruit coloration. Our results provide a valuable strategy that could be used in practice to regulate the ripening and quality of fresh fruit in medicinal and edible plants by modifying the phytohormone ABA.

The R2R3-MYB is a large gene family involved in various plant functions, including carotenoid biosynthesis. However, this gene family lacks a comprehensive analysis in wolfberry (Lycium barbarum L.) and other Solanaceae species. The recent sequencing of the wolfberry genome provides an opportunity for investigating the organization and evolutionary characteristics of R2R3-MYB genes in wolfberry and other Solanaceae species. A total of 610 R2R3-MYB genes were identified in five Solanaceae species, including 137 in wolfberry. The LbaR2R3-MYB genes were grouped into 31 subgroups based on phylogenetic analysis, conserved gene structures, and motif composition. Five groups only of Solanaceae R2R3-MYB genes were functionally divergent during evolution. Dispersed and whole duplication events are critical for expanding the R2R3-MYB gene family. There were 287 orthologous gene pairs between wolfberry and the other four selected Solanaceae species. RNA-seq analysis identified the expression level of LbaR2R3-MYB differential gene expression (DEGs) and carotenoid biosynthesis genes (CBGs) in fruit development stages. The highly expressed LbaR2R3-MYB genes are co-expressed with CBGs during fruit development. A quantitative Real-Time (qRT)-PCR verified seven selected candidate genes. Thus, Lba11g0183 and Lba02g01219 are candidate genes regulating carotenoid biosynthesis in wolfberry. This study elucidates the evolution and function of R2R3-MYB genes in wolfberry and the four Solanaceae species.

The B-box proteins (BBXs) are a family of zinc-finger transcription factors with one/two B-Box domain(s) and play important roles in plant growth and development as well as stress responses. Wolfberry (Lycium barbarum L.) is an important traditional medicinal and food supplement in China, and its genome has recently been released. However, comprehensive studies of BBX genes in Lycium species are lacking. In this study, 28 LbaBBX genes were identified and classified into five clades by a phylogeny analysis with BBX proteins from Arabidopsis thaliana and the LbaBBXs have similar protein motifs and gene structures. Promoter cis-regulatory element prediction revealed that LbaBBXs might be highly responsive to light, phytohormone, and stress conditions. A synteny analysis indicated that 23, 20, 8, and 5 LbaBBX genes were orthologous to Solanum lycopersicum, Solanum melongena, Capsicum annuum, and Arabidopsis thaliana, respectively. The gene pairs encoding LbaBBX proteins evolved under strong purifying selection. In addition, the carotenoid content and expression patterns of selected LbaBBX genes were analyzed. LbaBBX2 and LbaBBX4 might play key roles in the regulation of zeaxanthin and antheraxanthin biosynthesis. Overall, this study improves our understanding of LbaBBX gene family characteristics and identifies genes involved in the regulation of carotenoid biosynthesis in wolfberry.

Lycium Linn. (Solanaceae) is a genus of economically important plants producing fruits and leaves with high nutritional value and medicinal benefits. However, genetic analysis of this plant and molecular breeding for quality improvement are limited by the lack of sufficient molecular markers. In this study, two parental strains, ‘Ningqi No. 1’ (Lycium barbarum L.) and ‘Yunnan Gouqi’ (Lycium yunnanense Kuang et A.M. Lu), and 200 F1 hybrid individuals were resequenced for genetic analysis. In total, 8,507 well-selected SNPs were developed, and a high-density genetic map (NY map) was constructed with a total genetic distance of 2,122.24 cM. A consensus genetic map was established by integrating the NY map and a previously published genetic map (NC map) containing 15,240 SNPs, with a total genetic distance of 3,058.19 cM and an average map distance of 0.21 cM. The 12 pseudochromosomes of the Lycium reference genome were anchored using this consensus genetic map, with an anchoring rate of 64.3%. Moreover, weak collinearities between the consensus map and the pepper, potato, and tomato genomes were observed. Twenty-five stable QTLs were identified for leaf- and fruit-related phenotypes, including fruit weight, fruit longitude, leaf length, the fruit index, and the leaf index; these stable QTLs were mapped to four different linkage groups, with LOD scores ranging from 2.51 to 19.37 and amounts of phenotypic variance explained from 6.2% to 51.9%. Finally, 82 out of 188 predicted genes underlying stable QTLs for fruit-related traits were differentially expressed according to RNA-seq analysis. A chromosome-level assembly can provide a foundation for further functional genomics research for wolfberry. The genomic regions of these stably expressed QTLs could be used as targets for further fine mapping and development of molecular markers for marker-assisted selection (MAS). The present study provided valuable information on saturated SNP markers and reliable QTLs for map-based cloning of functional genes related to yield and morphological traits in Lycium spp.

Black goji berry (Lycium ruthenicum Murray) contains a rich source of health-promoting anthocyanins which are used in herbal medicine and nutraceutical foods in China. A natural variant producing white berries allowed us to identify two key genes involved in the regulation of anthocyanin biosynthesis in goji berries: one encoding a MYB transcription factor (LrAN2-like) and one encoding a basic helix-loop-helix (bHLH) transcription factor (LrAN1b). We previously found that LrAN1b expression was lost in the white berry variant, but the molecular basis for this phenotype was unknown. Here, we identified the molecular mechanism for loss of anthocyanins in white goji berries. In white goji, the LrAN1b promoter region has a 229-bp deletion that removes 3 MYB-binding elements and 1 bHLH-binding element, which are key to its expression. Complementation of the white goji berry LrAN1b allele with the LrAN1b promoter restored pigmentation. Virus-induced gene silencing of LrAN1b in black goji berry reduced fruit anthocyanin biosynthesis. Molecular analyses showed that LrAN2-like and another bHLH transcription factor LrJAF13 can activate LrAN1b by binding directly to the MYB-recognizing element (MRE) and bHLH-recognizing element (BRE) of its promoter-deletion region. LrAN1b expression is enhanced by the interaction of LrAN2-like with LrJAF13 and the WD40 protein LrAN11. LrAN2-like and LrAN11 interact with either LrJAF13 or LrAN1b to form two MYB-bHLH-WD40 (MBW) complexes, which hierarchically regulate anthocyanin biosynthesis in black goji berry. This study on a natural variant builds a comprehensive anthocyanin regulatory network that may be manipulated to tailor goji berry traits.