Evolutionary and functional analysis of mulberry type III polyketide synthases

Abstract

Background: Type III polyketide synthases are important for the biosynthesis of flavonoids and various plant polyphenols. Mulberry plants have abundant polyphenols, but very little is known about the mulberry type III polyketide synthase genes. An analysis of these genes may provide new targets for genetic improvement to increase relevant secondary metabolites and enhance the plant tolerance to biotic and abiotic stresses.

Results: Eighteen genes encoding type III polyketide synthases were identified, including six chalcone synthases (CHS), ten stilbene synthases (STS), and two polyketide synthases (PKS). Functional characterization of four genes representing most of the MnCHS and MnSTS genes by coexpression with 4-Coumaroyl-CoA ligase in Escherichia coli indicated that their products were able to catalyze p-coumaroyl-CoA and malonyl-CoA to generate naringenin and resveratrol, respectively. Microsynteny analysis within mulberry indicated that segmental and tandem duplication events contributed to the expansion of the MnCHS family, while tandem duplications were mainly responsible for the generation of the MnSTS genes. Combining the evolution and expression analysis results of the mulberry type III PKS genes indicated that MnCHS and MnSTS genes evolved mainly under purifying selection to maintain their original functions, but transcriptional subfunctionalization occurred during long-term species evolution. Moreover, mulberry leaves can rapidly accumulated oxyresveratrol after UV-C irradiation, suggesting that resveratrol was converted to oxyresveratrol.

Conclusions: Characterizing the functions and evolution of mulberry type III PKS genes is crucial for advancing our understanding of these genes and providing the basis for further studies on the biosynthesis of relevant secondary metabolites in mulberry plants.

Keywords: Chalcone synthase; Evolutionary analysis; Functional analysis; Gene expression; Mulberry; Stilbene synthase.

Fig. 1

Mulberry type III polyketide synthase superfamily. a Phylogenetic relationships among the type III PKS genes. Branch length is defined as the number of nucleotide substitutions per codon according to PAML. Different colors indicate higher posteriori probabilities of evolving under different selection regimens: red and blue correspond to positive and purifying selections, respectively, while black indicates the selection constraints of the branch are relaxed relative to the other branches of the same clade. The lowercase letters refer to the following: a, branch ancestral to the MnCHS and MnSTS families; b, branch ancestral to the MnSTS family clade; c, branch ancestral to the MnCHS family clade. *: genes without expression data (b) Intron/exon structure of the type III PKS genes. Boxes: exon; black line: intron; cyan boxes: N-terminal chalcone/stilbene synthase domain (IPR001099); yellow boxes: C-terminal chalcone/stilbene synthase domain (IPR012328)

Fig. 2

Phylogenetic analysis of mulberry type III polyketide synthases and other type III polyketide synthases. The indicated scale represents 0.1 amino acid substitutions per site. ALS, aloesone synthase; BBS, bibenzyl synthase; BPS, benzophenone synthase; STCS, stilbenecarboxylate synthase; VPS, valerophenone synthase; OKS, octaketide synthase; 2PS, 2-pyrone synthase; ORS, 2′- oxoalkylresorcinol synthase. The asterisk indicates the branch that is the most recent common ancestor to the plant CHS family. The β-ketoacyl carrier protein synthase III (FABH) of Escherichia coli was used as an outgroup

Fig. 3

Evolutionary relationships among mulberry type III polyketide synthases. a Genes represented by a series of triangles of the same color are from the same genomic fragment. Genes from the type III PKS superfamily are indicated by black triangles. Triangles also indicate gene orientation on strands. The colinear homologous genes are linked by gray lines. b Evolutionary relationships were determined to clarify the order of duplication events for the type III polyketide synthase superfamily. The black vertical line indicates tandem duplication. Numbers indicate when (million years ago, MYA) the segmental duplication events occurred. c Organization of the mulberry type III PKS gene clusters. The long terminal repeat retrotransposons are presented in yellow. Right and left arrows indicate whether a retrotransposon is located on the + or − strand, respectively. RLG: retrotransposon belongs to the Gypsy superfamily; RLL: retrotransposon belongs to the Lard superfamily. Numbers represent the sequence ID

Fig. 4

Conserved amino acid sites on a three-dimensional model of a typical STS enzyme. a Three dimensional STS dimer structure indicating the conserved amino acid sites. Positive selection and type II divergence sites are shown in purple (numbering is based on MnSTS). Catalytic triads are shown in gold (numbering is based on Medicago sativa CHS). b Evolutionarily conserved residues in the STS enzyme. All sites are labeled according to their conservation scale. The asterisk indicates the site that experienced positive selection pressure. The remaining six sites underwent positive selection and type II divergence

Fig. 5

In vivo characterization of CHS and STS by coexpression with 4-coumaroyl-CoA ligase in Escherichia coli. a In vivo assay of CHS. 4-Coumaric acid (1) and naringenin (2) were used as standard compounds. Mn4CL: E. coli-expressed Mn4CL. Mn4CL + MnCHS2: E. coli-coexpressed Mn4CL and MnCHS2. Mn4CL + MnCHS6: E. coli-coexpressed Mn4CL and MnCHS6. Selected ion chromatograms generated during liquid chromatography-electrospray ionization mass spectrometry analyses of the compounds are provided in the small panels on the right: 4-coumaric acid, m/z = 163.1; naringenin, m/z = 271.1. b In vivo assay of STS. Resveratrol (3) was used as the standard compound. Mn4CL: E. coli-expressed Mn4CL. Mn4CL + MnSTS7: E. coli-coexpressed Mn4CL and MnSTS7. Mn4CL + MnSTS8: E. coli-coexpressed Mn4CL and MnSTS8. Selected ion chromatograms of the compound are provided in the small panels on the right: resveratrol, m/z = 227.1

Fig. 6

Heat maps of hierarchical clustering of mulberry type III polyketide synthase superfamily genes. Data were adjusted by log transformation and the mean center method. Hierarchical clustering with average linkages was used to calculate K-medians with five clusters

Fig. 7

Effect of UV-C irradiation on type III PKS gene expression and the biosynthesis of compounds. a The MnSTS2 real-time PCR primers could anneal to the other three STS genes (i.e., MnSTS3, MnSTS8, and MnSTS9), although all primers were designed to match the most variable STS regions, including the 3′ untranslated regions. Relative gene expression levels were normalized against a mulberry actin gene (MnACTIN3). Data are provided as the mean + standard deviation. b Variability among compounds following UV-C irradiation. Data are provided as the mean ± standard deviation