CtACO1 Overexpression Resulted in the Alteration of the Flavonoids Profile of Safflower.

BACKGROUND: Flavonoids with various structures play a vital role in plant acclimatization to varying environments as well as in plant growth, development, and reproduction. Exogenous applications of ethylene and 1-aminocyclopropane carboxylic acid (ACC), could affect the accumulation of flavonoids. Very few attempts have been made to investigate the effect of 1-aminocyclopropane carboxylic acid oxidase (ACO), a unique enzyme that catalyzes ACC to ethylene, on genes and metabolites in the flavonoid biosynthetic pathway. In this study, two ACOs in safflower (CtACOs) were cloned, and then transgenic safflower with overexpressed CtACO1 was generated through the Agrobacterium-mediated floral dipping method. RESULTS: CtACO1 and CtACO2 were both characterized by the 2-oxoglutarate binding domain RxS and the ferrous iron binding site HxDxnH as ACOs from other plants. However, the transcript levels of CtACO1 in flowers at stages I, II, III, and IV were all higher than those of CtACO2. At the cellular level, by using electroporation transformation, CtACO1 was found to be localized at the cytomembrane in onion epidermal cells. CtACO1 overexpression had varying effects on genes involved in the ethylene and flavonoid biosynthetic pathways. The metabolites analysis showed that CtACO1 overexpression lines had a higher accumulation of quercetin and its glycosylated derivatives (quercetin 3-ß-d-glucoside and rutin). In contrast, the accumulation of quinochalcones (hydroxysafflor yellow A and carthamin), kaempferol glycosylated derivatives (kaempferol-3-O-ß-rutinoside and kaempferol-3-O-ß-d-glucoside), apigenin, and luteolin in CtACO1 overexpression lines were decreased. CONCLUSION: This study confirmed the feasibility of applying the floral dipping method to safflower and showed a novel regulatory effect of CtACO1 in the flavonoid biosynthetic pathway. It provides hypothetical and practical groundwork for further research on regulating the overall metabolic flux of flavonoids in safflower, particularly hydroxysafflor yellow A and other quinochalcones, by using appropriate genetic engineering strategies.

Expression Patterns of Three UGT Genes in Different Chemotype Safflower Lines and under MeJA Stimulus Revealed Their Potential Role in Flavonoid Biosynthesis.

Safflower (Carthamus tinctorius L.) has received a significant amount of attention as a medicinal plant in China. Flavonoids are the dominant active medical compounds. UDP-glycosyltransferase plays an essential role in the biosynthesis and storage of flavonoids in safflower. In this study, 45 UGT unigenes were screened from our transcriptomic database of safflower. Among them, 27 UGT unigenes were predicted to own a complete open reading frame with various pI and Mw. The phylogenetic tree showed that CtUGT3 and CtUGT16 were classified under the UGT71 subfamily involved in metabolite process, whereas CtUGT25 has high identities with PoUGT both catalyzing the glycosylation of flavonoids and belonging to the UGT90 subfamily. cDNA microarray exhibited that the three UGT genes displayed temporal difference in two chemotype safflower lines. To functionally characterize UGT in safflower, CtUGT3, CtUGT16 and CtUGT25 were cloned and analyzed. Subcellular localization suggested that the three UGTs might be located in the cell cytoplasm and chloroplast. The expression pattern showed that the three UGTs were all suppressed in two lines responsive to methyl jasmonate induction. The co-expression relation of expression pattern and metabolite accumulation demonstrated that CtUGT3 and CtUGT25 were positively related to kaempferol-3-O-ß-D-glucoside and CtUGT16 was positively related to quercetin-3-O-ß-D-glucoside in yellow line, whereas CtUGT3 and CtUGT25 were positively related to quercetin-3-O-ß-D-glucoside in white line. This study indicates that the three CtUGTs play a significant and multiple role in flavonoids biosynthesis with presenting different functional characterization in two safflower lines.

Molecular characterization of flavanone 3-hydroxylase gene and flavonoid accumulation in two chemotyped safflower lines in response to methyl jasmonate stimulation.

BACKGROUND: Among secondary metabolites, flavonoids are particularly crucial for plant growth, development, and reproduction, as well as beneficial for maintenance of human health. As a flowering plant, safflower has synthesized a striking variety of flavonoids with various pharmacologic properties. However, far less research has been carried out on the genes involved in the biosynthetic pathways that generate these amazing flavonoids, especially characterized quinochalcones. In this study, we first cloned and investigated the participation of a presumed flavanone 3-hydroxylase gene (F3H) from safflower (CtF3H) in a flavonoid biosynthetic pathway. RESULTS: Bioinformation analysis showed that CtF3H shared high conserved residues and confidence with F3H from other plants. Subcellular localization uncovered the nuclear and cytosol localization of CtF3H in onion epidermal cells. The functional expressions of CtF3H in Escherichia coli BL21(DE3)pLysS cells in the pMAL-C5x vector led to the production of dihydrokaempferol when naringenin was the substrate. Furthermore, the transcriptome expression of CtF3H showed a diametrically opposed expression pattern in a quinochalcone-type safflower line (with orange-yellow flowers) and a flavonol-type safflower line (with white flowers) under external stimulation by methyl jasmonate (MeJA), which has been identified as an elicitor of flavonoid metabolites. Further metabolite analysis showed the increasing tendency of quinochalcones and flavonols, such as hydroxysafflor yellow A, kaempferol-3-O-ß-D-glucoside, kaempferol-3-O-ß-rutinoside, rutin, carthamin, and luteolin, in the quinochalcone-type safflower line. Also, the accumulation of kaempferol-3-O-ß-rutinoside and kaempferol-3-O-ß-D-glucoside in flavonols-typed safflower line showed enhanced accumulation pattern after MeJA treatment. However, other flavonols, such as kaempferol, dihydrokaempferol and quercetin-3-O-ß-D-glucoside, in flavonols-typed safflower line presented down accumulation respond to MeJA stimulus. CONCLUSIONS: Our results showed that the high expression of CtF3H in quinochalcone-type safflower line was associated with the accumulation of both quinochalcones and flavonols, whereas its low expression did not affect the increased accumulation of glycosylated derivatives (kaempferol-3-O-ß-rutinoside and rutin) in flavonols-typed safflower line but affect the upstream precursors (D-phenylalanine, dihydrokaempferol, kaempferol), which partly revealed the function of CtF3H in different phenotypes and chemotypes of safflower lines.

Carthami flos: a review of its ethnopharmacology, pharmacology and clinical applications

ABSTRACTCarthami flos, the dried floret of Carthamus tinctorius L., Asteraceae (safflower), has been widely used in traditional Chinese medicine to treat a broad range of ailments, such as coronary heart disease, angina pectoris, gynecologic disease, stroke, and hypertension. However, although several studies on Carthami flos have been done consecutively, the results are usually scattered across various documents. This review aims to provide up-to-date information on the traditional uses, pharmacology, clinical applications, and toxicology of Carthami flos in China and thereby to provide a basis for further investigation of its use to treat dissimilar diseases. Various ethnomedical uses of Carthami flos have been documented in many ancient Chinese books. Crude extracts and isolated compounds from Carthami flos show a broad range of pharmacological properties, such as protective effects on brain tissue, on osteoblasts, and in myocardial ischemia, as well as anti-inflammatory, antithrombotic, antitumor, and antidiabetic activities. To date, safflower and safflor yellow injections have been used to treat coronary heart disease, chronic pulmonary heart disease, cerebrovascular diseases, orthopedic diseases, and diabetes mellitus. Regarding the toxicology of Carthami flos, among the side effects that have been observed are allergic reaction, spermatogenetic failure, fatty liver, and nephrotoxicity.

The medicinal uses of Callicarpa L. in traditional Chinese medicine: an ethnopharmacological, phytochemical and pharmacological review.

ETHNOPHARMACOLOGICAL RELEVANCE: Callicarpa L. (Verbenaceae) has been used for centuries in Traditional Chinese Medicine (TCM) for the prevention and treatment of a wide number of health disorders such as inflammation, rheumatism, hematuria, fracture, hematemesis, menoxenia, gastrointestinal bleeding, scrofula, etc. AIMS OF THE REVIEW: To assess the scientific evidence for therapeutic Callicarpa in TCM and to identify future research needs. METHODS: The available information on the ethnopharmacological uses in Chinese medicine, phytochemistry, pharmacology and clinical practice of Callicarpa species was collected via a library and electronic search (PubMed, ScienceDirect, Google Scholar and CNKI). RESULTS: A variety of ethnomedical use of Callicarpa has been recorded in many ancient Chinese books. Phytochemical investigation of this genus has resulted in identification of more than 200 chemical constituents, among which diterpenes, triterpenoids and flavonoids are the predominant groups. The isolates and crude extract have exhibited a wide spectrum of in vitro and in vivo pharmacological effects involving anti-inflammatory, hemostatic, neuroprotective, anti-amnesic, antitubercular, antioxidant, antimicrobial and analgesic activities. Preparations containing Callicarpa species exerted good efficacy on clinical applications of gynecological inflammation, internal and external hemorrhage as well as acne vulgaris and chronic pharyngitis, etc. From the toxicity perspective, only three Callicarpa species have been assessed. CONCLUSIONS: Pharmacological results have validated the use of Callicarpa species in the traditional medicine. As literature demonstrated, terpenoids and flavonoids are perhaps responsible for most of the activities shown by the plants of this genus. However, the detailed active compounds and the underlying mechanisms remain a work in progress. In addition, more attention should be paid to C. nudiflora as well as the domain of rheumatism.