Abstract
Carthami flos, the dried petal of safflower (Carthamus tinctorius L.) has been widely used in traditional Chinese medicine to treat cardiovascular and cerebrovascular diseases, in which quinochalcone glucosides such as hydrosafflower yellow A (HSYA), carthamin are uniquely present and have been identified as active compounds. In the present study, through sequencing of a safflower floret cDNA library and subsequent microarray analysis, we found 23 unigenes (5 PALs, 1 C4Hs, 5 4CLs, 6 CHSs, 2 CHIs, 2 DFRs, 2 FLSs) involved in flavonoid pathway, of which 4 were up-regulated differentially during quinochalcone glucosides accumulation with the floret developing stage. The up-regulated genes were verified by PCR methods. Considering chalcone synthase are entry enzyme in flavonoid biosynthesis, CHS1 was focused on target gene to verify its function furtherly. Bioinformation analysis showed that CHS1 shared 86.94% conserved residues with CHS from other plants. Subcellular localization showed that CtCHS1 was localized in cytoplasm in onion epidermal cells. The transgenic safflower plant with overexpression CtCHS1 by Agrobacterium-mediated pollen-tube pathway method was firstly generated. The results present that expression of PAL2, PAL3, CHS1, CHS4, CHS6 increased and expression of CHI1 and CHI2 decreased in the transgenic plant floret. Meanwhile, the accumulation of quinochalcone glucosides increased by ∼20–30% and accumulation of quercetin-3-β-D-glucoside and quercetin decreased by 48 and 63% in the transgenic plant floret. These results suggested that CtCHS1 played an important role in quinochalcone glucosides biosynthesis rather than flavonol biosynthesis. These results also demonstrated that the pollen-tube pathway method was an efficient method for gene transformation in safflower. Our study will provide a deep understanding of potential synthetic genes involved in quinochalcone biosynthetic pathway.
Highlights
Flavonoids with a variegated structure are ubiquitous plant secondary metabolites that their physiological and ecological role is to provide relief against a large amount of biotic and abiotic stresses in the plant kingdom, such as protection against light and temperature (WinkelShirley, 2002), flower coloration (Tanaka and Brugliera, 2013), defense against insect infestationFlavonoid Biosynthesis and Regulation in Safflower (El-Wakeil, 2013) and pathogen (Ortuño et al, 2006; Steinkellner et al, 2007)
We found that overexpression of CtCHS1 significantly increased the accumulation of quinochalcone glucosides in safflower, which provided a concrete evidence that CtCHS1 is solely involved in quinochalcone glucosides biosynthesis
The result reveals that CtCHS1 opens a tributary to quinochalcone biosynthetic pathway and block off CtCHIs that flow to downstream pathways to synthesize various flavonols aglycones and glycosides suggesting that CtCHS1 solely take part in quinochalcone biosynthetic pathway rather than flavonol (Figure 12). Quinochalcone glucosides such as hydrosafflower yellow A (HSYA), carthamin are uniquely present and have been identified as active compounds in safflower
Summary
Flavonoid Biosynthesis and Regulation in Safflower (El-Wakeil, 2013) and pathogen (Ortuño et al, 2006; Steinkellner et al, 2007) They are recognized as abundant resources for agents that promote and maintain health (Sankari et al, 2014). Because of their significance in genetic investigations and biomedicines, flavonoid biosynthesis has attracted considerable scientific attention over the years (Giannasi, 1978; Gutha et al, 2010). Numerous studies show that the main active compounds in safflower flower are flavonoids, such as quinochalcones (hydrosafflower yellow A, carthamin, tinctorimine, and cartorimine) and flavonols (kaempferol and its glucosides, and quercetin and its glucosides) (Tu et al, 2015). No previous studies have explored transgenic saflower plant via this approach
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