The traditional Chinese medicinal plants Lycium barbarum L. and L. ruthenicum Murr. are valued for the abundance of bioactive carotenoids and anthocyanins in their fruits, respectively. However, the cellular and molecular mechanisms contributing to their species-specific bioactive profiles remain poorly understood. In this study, the red fruit (RF) of L. barbarum was found to accumulate high levels of carotenoids (primarily zeaxanthin), while they were undetectable in the black fruit (BF) of L. ruthenicum. Cytological and gene transcriptional analyses revealed that the chromoplast differentiation that occurs in the chloroplast during fruit ripening only occurs in RF, indicating that the lack of chromoplast biogenesis in BF leads to no sink for carotenoid storage and the failure to synthesize carotenoids. Similar enzyme activities of phytoene synthase 1 (PSY1), chromoplast-specific lycopene β-cyclase (CYC-B) and β-carotene hydroxylase 2 (CRTR-B2) were observed in both L. ruthenicum and L. barbarum, suggesting that the undetectable carotenoid levels in BF were not due to the inactivation of carotenoid biosynthetic enzymes. The transcript levels of the carotenoid biosynthetic genes, particularly PSY1, phytoene desaturase (PDS), β-carotene desaturase (ZDS), CYC-B and CRTR-B2, were greatly increased during RF ripening, indicating increased zeaxanthin biosynthesis. Additionally, carotenoid cleavage dioxygenase 4 (CCD4) was expressed at much higher levels in BF than in RF, suggesting continuous carotenoid degradation in BF. The failure of the chromoplast development in BF causes low carotenoid biosynthesis levels and continuous carotenoid degradation, which ultimately leads to undetectable carotenoid levels in ripe BF. In contrast, the successful chromoplast biogenesis in RF furnishes the sink necessary for carotenoid storage. Based on this observation, the abundant zeaxanthin accumulation in RF is primarily determined via both the large carotenoid biosynthesis levels and the lack of carotenoid degradation, which are regulated at the transcriptional level.

Herba epimedii (Epimedium), a traditional Chinese medicine, has been widely used as a kidney tonic and antirheumatic medicine for thousands of years. In Epimedium, flavonoids have been demonstrated to be the main bioactive components (BCs). However, the molecular biosynthetic and regulatory mechanisms of flavonoid-derived BCs remain obscure. In this study, we isolated twelve structural genes and two putative transcription factors (TFs) in the flavonoid pathway. Phytochemical analysis showed that the total content of four representative BCs (epimedin A, B, C and icariin) decreased slightly or dramatically in two lines of E. sagittatum during leaf development. Transcriptional analysis revealed that two R2R3-MYB TFs (EsMYBA1 and EsMYBF1), together with a bHLH TF (EsGL3) and WD40 protein (EsTTG1), were supposed to coordinately regulate the anthocyanin and flavonol-derived BCs biosynthesis in leaves. Overexpression of EsFLS (flavonol synthase) in tobacco resulted in increased flavonols content and decreased anthocyanins content in flowers. Moreover, EsMYB12 negatively correlated with the accumulation of the four BCs, and might act as a transcriptional repressor in the flavonoid pathway. Therefore, the anthocyanin pathway may coordinate with the flavonol-derived BCs pathway in Epimedium leaves. A better understanding of the flavonoid biosynthetic and regulatory mechanisms in E. sagittatum will facilitate functional characterization, metabolic engineering and molecular breeding studies of Epimedium species.

MicroRNAs (miRNAs) are master regulators of gene activity documented to play central roles in fruit ripening in model plant species, yet little is known of their roles in Lycium barbarum L. fruits. In this study, miRNA levels in L. barbarum fruit samples at four developmental stages, were assayed using Illumina HiSeqTM2000. This revealed the presence of 50 novel miRNAs and 38 known miRNAs in L. barbarum fruits. Of the novel miRNAs, 36 were specific to L. barbarum fruits compared with L. chinense. A number of stage-specific miRNAs were identified and GO terms were assigned to 194 unigenes targeted by miRNAs. The majority of GO terms of unigenes targeted by differentially expressed miRNAs are ‘intracellular organelle’, ‘binding’, ‘metabolic process’, ‘pigmentation’, and ‘biological regulation’. Enriched KEGG analysis indicated that nucleotide excision repair and ubiquitin mediated proteolysis were over-represented during the initial stage of ripening, with ABC transporters and sulfur metabolism pathways active during the middle stages and ABC transporters and spliceosome enriched in the final stages of ripening. Several miRNAs and their targets serving as potential regulators in L. barbarum fruit ripening were identified using quantitative reverse transcription polymerase chain reaction. The miRNA-target interactions were predicted for L. barbarum ripening regulators including miR156/157 with LbCNR and LbWRKY8, and miR171 with LbGRAS. Additionally, regulatory interactions potentially controlling fruit quality and nutritional value via sugar and secondary metabolite accumulation were identified. These include miR156 targeting of fructokinase and 1-deoxy-D-xylulose-5-phosphate synthase and miR164 targeting of beta-fructofuranosidase. In sum, valuable information revealed by small RNA sequencing in this study will provide a solid foundation for uncovering the miRNA-mediated mechanism of fruit ripening and quality in this nutritional food.

Epimedium sagittatum (Sieb. et Zucc.) Maxim, a popular traditional Chinese medicinal plant, has been widely used for treating sexual dysfunction and osteoporosis in China. The main bioactive components in herba epimedii are prenylated flavonol glycosides, which are end products of a branch of the flavonoid biosynthetic pathway. The MYB transcription factors (TF) act as activators or repressors to regulate the flavonoid pathway. In this study, 13 full-length cDNA clones of R2R3-MYB TFs from E. sagittatum (designated as EsMYB1 to EsMYB13) were isolated and characterized. Sequence similarity and phylogenetic analysis placed nine R2R3-MYB members of epimedii into five subgroups of the Arabidopsis R2R3-MYB family, while four members were not clustered into a defined subgroup. The number and length of introns from epimedii R2R3-MYB genes varied significantly, but intron positions and phases were well conserved. Expression patterns of epimedii R2R3-MYB genes in various tissues showed diverse. Finally, it is suggested that five epimedii R2R3-MYB genes may be involved in regulating the flavonoid pathway and could be used as valuable candidate genes for metabolic engineering studies in future. Sequence information of 13 R2R3-MYB genes discovered here will also provide an entry point into the overview of whole R2R3-MYB family in epimedii.