Molecular cloning and functional characterization of chalcone isomerase from Carthamus tinctorius

Abstract

Flavonoid is one of the widespread groups of plant secondary metabolites that provide several health benefits. However, the explicit mechanism of flavonoid biosynthesis in plants largely remains unclear. Chalcone isomerase an important class of enzyme presents crucial role during flavonoid metabolism in many plants. Here, we isolated the full-length cDNA (1161 bp) of a novel Chalcone Isomerase from safflower encoding 217 amino acid polypeptide using oligos from 5' and 3' ends. The result of Sanger sequencing and phylogenetic analysis revealed that CtCHI is highly homologous to other plants, including typical polyadenylation signals AATAA and Poly A tail. The transient expression in tobacco mesophyll cells using Green Fluorescent Protein tagging determined the subcellular localization of CtCHI in cell membrane and nucleus. The CtCHI ectopic expression in different safflower varieties at different flowering stages showed that CtCHI were found in abundance at the bud stage of Jihong No. 1. Further correlation analysis between CtCHI expression and flavonoid accumulation at various flowering phases suggested that CtCHI might play a potential role during flavonoid biosynthesis in safflower. In addition, the overexpression of pBASTA-CtCHI in transgenic Arabidopsis infiltrated with floral dip transformation showed relatively higher expression level and increased flavonoid accumulation than wild type. Moreover, the in vitro enzymatic activity and HPLC analysis of transgenic Arabidopsis confirmed the de novo biosynthesis of Rutin. Taken together, our findings laid the foundation of identifying an important gene that might influence flavonoid metabolism in safflower.

Keywords: Chalcone isomerase (CHI); Expression analysis; Flavonoids biosynthesis; HPLC; Safflower.

Fig. 1

The core metabolic pathway of flavonoids (anthocyanins) occurred in Arabidopsis thaliana (Jiang et al. 2015)

Fig. 2

Cloning and sequence analysis of the CtCHI gene. a cDNA of CtCHI. b Tertiary 3D structure of the CtCHI protein. c Alignment of the deduced amino acid sequence of CtCHI gene with other plant species. CtCHI: Carthamus tinctorius L.; SmCHI: Saussurea medusa; pl CHI: Paeonia lactiflora; Mh CHI: Malus hybrid; Pp CHI: Pyrus pyrifolia

Fig. 3

The content of yellow pigment of safflower accumulated during four different flowering periods in four different varieties. Safflower varieties were labeled as JHEM, JH1, JHS, and JH2. Different flowering periods were indicated as a bud, initial, full, and fading stage. Data are means ± SE (n = 3). Asterisks indicate the statistical significance levels according to Student’s t-test: *P < 0.05

Fig. 4

The correlation between expression of CtCHI gene and biosynthesis of flavonoids at various flowering stages of safflower. a Different flowering stages of safflower; b RT-qPCR transcript levels of CtCHI gene which are indicated in bars while the synthesis of safflower yellow pigment at various developmental stages are presented as red letter boxes. The 18s ribosomal RNA gene was used as an internal control. Error bars indicate the SE (n = 3)

Fig. 5

Subcellular localization of CtCHI-GFP in wild type tobacco mesophyll cells. GFP signals of CtCHI-pBASTA1302-GFP fusion construct localizes to both cytosol and nucleus. The pBASTA1302-GFP signals were detected dispersed in tobacco mesophyll cells. The GFP fluorescence exhibited signals were analyzed with a confocal laser scanning microscope

Fig. 6

Quantitative real time Expression levels of CtCHI gene and CHI activity in transgenic Arabidopsis lines. a Relative expression levels of CtCHI gene presented in four different transgenic lines in comparison with the wild type Arabidopsis. The data were normalized with 18s ribosomal RNA. b In vitro enzymatic activity of CHI using ELISA kit, RC9615, R&D Systems Inc., America). The color of the 3,3′,5,5′-tetramethylbenzidine was measured as absorbance (OD) using a spectrophotometer at 450 nm to calculate the concentration of CHI activity according to a standard regression curve. Data were presented as the mean ± SD. **P < 0.01 vs. the wild type group

Fig. 7

High performance liquid chromatography profile of Rutin in transgenic Arabidopsis lines purified through column Agilent zorbax SB-C 18 (4.6 mm × 150 mm, 5 μm); Mobile phase A as methanol–acetonitrile (V:V 1:10) and phase B as 0.4% phosphoric acid. a The peak labeled as Rutin was used as a reference in our study. b Chromatogram of high performance liquid chromatography of Rutin in transgenic Arabidopsis