Exploiting genes and functional diversity of chlorogenic acid and luteolin biosyntheses in Lonicera japonica and their substitutes

Abstract

Chlorogenic acids (CGAs) and luteolin are active compounds in Lonicera japonica, a plant of high medicinal value in traditional Chinese medicine. This study provides a comprehensive overview of gene families involved in chlorogenic acid and luteolin biosynthesis in L. japonica, as well as its substitutes Lonicera hypoglauca and Lonicera macranthoides. The gene sequence feature and gene expression patterns in various tissues and buds of the species were characterized. Bioinformatics analysis revealed that 14 chlorogenic acid and luteolin biosynthesis-related genes were identified from the L. japonica transcriptome assembly. Phylogenetic analyses suggested that the function of individual gene could be differentiation and induce active compound diversity. Their orthologous genes were also recognized in L. hypoglauca and L. macranthoides genomic datasets, except for LHCHS1 and LMC4H2. The expression patterns of these genes are different in the tissues of L. japonica, L. hypoglauca and L. macranthoides. Results also showed that CGAs were controlled in the first step of biosynthesis, whereas both steps controlled luteolin in the bud of L. japonica. The expression of LJFNS2 exhibited positive correlation with luteolin levels in L. japonica. This study provides significant information for understanding the functional diversity of gene families involved in chlorogenic acid and the luteolin biosynthesis, active compound diversity of L. japonica and its substitutes, and the different usages of the three species.

Keywords: Chlorogenic acid; DNA; DP; Gene expression; Honeysuckle; Luteolin; ORF; Othologous genes; PCR; RACE; RNA; Selaginella pulvinta; Sp; TPP; TPS; cDNA; complementary DNA; degenerate primer; deoxyribonucleic acids; open reading frame; polymerase chain reaction; raid amplification of cDNA ends; ribonucleic acids; trehalose-6-phosphate phosphatase; trehalose-6-phosphate synthase; trehalose-6-phosphate synthase gene.

Fig. 1

Biosynthesis of chlorogenic acid and lutelin. PAL, phenylalanine amononiar-lyase; 4CL, 4-coumarate CoA ligase; C4H,cinnamate 4-hydroxylase; CHS, chalcone synthase, CHI, chalcone isomerase FNS, flavone synthase; HQT, hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase. A, Phylogenetic relationship of plant PALs. The rooted Neighbor–Joining tree was constructed using the ClustalW program. PALs included 4 Arabidopsis thaliana, 7 rice, 15 wine grape and 3 Lonicera japonica PALs. B, Phylogenetic relationship of plant 4CLs. The rooted neighbor-joining tree was constructed using the ClustalW program. 4CLs included 4 Arabidopsis thaliana, 10 rice, 2 wine grape and 2 Lonicera japonica 4CLs. C, Phylogenetic relationship of plant CHSs. The rooted neighbor-joining tree was constructed using the ClustalW program. CHSs included 1 Arabidopsis thaliana, 12 rice, 5 wine grape and 2 Lonicera japonica CHSs. D, Phylogenetic relationship of plant CHIs. The rooted neighbor-joining tree was constructed using the ClustalW program. CHIs included 5 Arabidopsis thaliana, 7 rice, 4 wine grape and 2 Lonicera japonica CHIs. E, Phylogenetic relationship of plant HQTs. The rooted neighbor-joining tree was constructed using the ClustalW program. HQTs included 1 Arabidopsis thaliana, 6 rice, 2 wine grape and 2 Lonicera japonica HQTs. The genes in this study were indicated by underline.

Fig. 1

Biosynthesis of chlorogenic acid and lutelin. PAL, phenylalanine amononiar-lyase; 4CL, 4-coumarate CoA ligase; C4H,cinnamate 4-hydroxylase; CHS, chalcone synthase, CHI, chalcone isomerase FNS, flavone synthase; HQT, hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase. A, Phylogenetic relationship of plant PALs. The rooted Neighbor–Joining tree was constructed using the ClustalW program. PALs included 4 Arabidopsis thaliana, 7 rice, 15 wine grape and 3 Lonicera japonica PALs. B, Phylogenetic relationship of plant 4CLs. The rooted neighbor-joining tree was constructed using the ClustalW program. 4CLs included 4 Arabidopsis thaliana, 10 rice, 2 wine grape and 2 Lonicera japonica 4CLs. C, Phylogenetic relationship of plant CHSs. The rooted neighbor-joining tree was constructed using the ClustalW program. CHSs included 1 Arabidopsis thaliana, 12 rice, 5 wine grape and 2 Lonicera japonica CHSs. D, Phylogenetic relationship of plant CHIs. The rooted neighbor-joining tree was constructed using the ClustalW program. CHIs included 5 Arabidopsis thaliana, 7 rice, 4 wine grape and 2 Lonicera japonica CHIs. E, Phylogenetic relationship of plant HQTs. The rooted neighbor-joining tree was constructed using the ClustalW program. HQTs included 1 Arabidopsis thaliana, 6 rice, 2 wine grape and 2 Lonicera japonica HQTs. The genes in this study were indicated by underline.

Fig. 2

Content of chlorogenic acid and luteoloside in bud of Lonicera japonica Thunb (LJ), L. hypoglauca Miquel (LH), and L. macranthoides Hand.-Mazz (LM). The star means the significant difference at P < 0.05 level.

Fig. 3

Content of chlorogenic acid, luteolin and luteoloside in bud and leaves of Lonicera japonica.

Fig. 4

The transcript ratio of PAL gene families in buds and leaves of Lonicera japonica Thunb (LJ), L. hypoglauca Miquel (LH), and L. macranthoides Hand.-Mazz (LM).

Fig. 5

Enzymatic activity of PAL, C4H and 4CL in buds and leaves of Lonicera japonica Thunb (LJ), L. hypoglauca Miquel (LH), and L. macranthoides Hand.-Mazz (LM).

Fig. 6

Fold changes of PAL gene families in buds of Lonicera japonica Thunb (LJ), L. hypoglauca Miquel (LH), and L. macranthoides Hand.-Mazz (LM). The expression level of PAL genes in of Lonicera japonica Thunb was arbitrarily set to 1.