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.

The morphological identification of different Hippophae species (Shaji) was difficult. This study aims to discriminate between medicinal and non-medicinal Hippophae species by DNA barcodes, the ITS2, psbA-trnH, and a combination of ITS2 and psbA-trnH (ITS2 + psbA-trnH). DNA was extracted from the dried fruit samples. Primer pairs ITS2F/3R for ITS2 and psbAF/trnHR for psbA-trnH were used for PCR amplification. The purified PCR products were bidirectionally sequenced. Genetic distances were calculated according to the Kimura 2 parameter model and phylogenetic tree was constructed based on neighbor-joining (NJ) method, barcoding gap was also analyzed to assess identification efficiency. Amplification and sequencing efficiencies for both ITS2 and psbA-trnH were 100 %. Sequence data revealed that ITS2 + psbA-trnH was the most suitable candidate barcode at the species and subspecies level. The closely related Hippophae species were effectively differentiated in the NJ tree. The combination of the two loci, ITS2 + psbA-trnH is applicable to the identification of medicinal and non-medicinal Hippophae species.

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.

The Crabs Claw (CRC) YABBY gene is required for regulating carpel development in angiosperms and has played an important role in nectary evolution during core eudicot speciation. The function or expression of CRC-like genes has been explored in two basal eudicots, Eschscholzia californica and Aquilegia formosa. To further investigate the function of CRC orthologous genes related to evolution of carpel and nectary development in basal eudicots, a CRC ortholog, EsCRC, was isolated and characterized from Epimedium sagittatum (Sieb. and Zucc.) Maxim. A phylogenetic analysis of EsCRC and previously identified CRC-like genes placed EsCRC within the basal eudicot lineage. Gene expression results suggest that EsCRC is involved in the development of sepals and carpels, but not nectaries. Phenotypic complementation of the Arabidopsis mutant crc-1 was achieved by constitutive expression of EsCRC. In addition, over-expression of EsCRC in Arabidopsis and tobacco gave rise to abaxially curled leaves. Transgenic results together with the gene expression analysis suggest that EsCRC may maintain a conserved function in carpel development and also play a novel role related to sepal formation. Absence of EsCRC and ElCRC expression in nectaries further indicates that nectary development in non-core eudicots is unrelated to expression of CRC-like genes.

The genus Berchemia comprises important Chinese plants with considerable medicinal value; however, these plants are often misidentified in the herbal medicinal market. To differentiate the various morphotypes of Berchemia species, a proficient method employing the screening of universal DNA barcodes was used in this work. Three candidate barcoding loci, namely, psbA-trnH, rbcL, and the second internal transcribed spacer (ITS2), were used to identify an effective DNA barcode that can differentiate the various Berchemia species. Additionally, PCR amplification, efficient sequencing, intra- and inter-specific divergences, and DNA barcoding gaps were employed to assess the ability of each barcode to identify these diverse Berchemia plants authentically; the species were differentiated using the Kimura two-parameter and maximum composite likelihood methods. Sequence data analysis showed that the ITS2 region was the most suitable candidate barcode and exhibited the highest interspecific divergence among the three DNA-barcoding sequences. A clear differentiation was observed at the species level, in which a maximum distance of 0.264 was exhibited between dissimilar species. Clustal analysis also demonstrated that ITS2 clearly differentiated the test species in a more effective manner than that with the two other barcodes at both the hybrid and variety levels. Results indicate that DNA barcoding is ideal for species-level identification of Berchemia and provides a foundation for further identification at the molecular level of other Rhamnaceae medicinal plants.