Functional and Structural Investigation of Chalcone Synthases Based on Integrated Metabolomics and Transcriptome Analysis on Flavonoids and Anthocyanins Biosynthesis of the Fern Cyclosorus parasiticus

Abstract

The biosynthesis of flavonoids and anthocyanidins has been exclusively investigated in angiosperms but largely unknown in ferns. This study integrated metabolomics and transcriptome to analyze the fronds from different development stages (S1 without spores and S2 with brown spores) of Cyclosorus parasiticus. About 221 flavonoid and anthocyanin metabolites were identified between S1 and S2. Transcriptome analysis revealed several genes encoding the key enzymes involved in the biosynthesis of flavonoids, and anthocyanins were upregulated in S2, which were validated by qRT-PCR. Functional characterization of two chalcone synthases (CpCHS1 and CpCHS2) indicated that CpCHS1 can catalyze the formation of pinocembrin, naringenin, and eriodictyol, respectively; however, CpCHS2 was inactive. The crystallization investigation of CpCHS1 indicated that it has a highly similar conformation and shares a similar general catalytic mechanism to other plants CHSs. And by site-directed mutagenesis, we found seven residues, especially Leu199 and Thr203 that are critical to the catalytic activity for CpCHS1.

Molecular and structural characterization of a promiscuous chalcone synthase from the fern species Stenoloma chusanum

“…The purified recombinant proteins encoded by the empty pET‐32a vector were used as the negative control. The enzyme assay process and HPLC parameters were consistent with the previously reported studies (Niu et al, 2021). To analyze the effect of the reaction temperature on ScCHS1 activity, the reaction was screened using the different temperatures (20–60°C) at phosphate buffer (pH 6.5).…”

“…Then CHSs from ferns and seed plants appeared and clustered into a large branch. ScCHS1 was grouped together with two CHS from ferns LoCHS1 (MK216767) (Ni et al, 2020) and CpCHS1 (OK136250) (Niu et al, 2021), and located in the root of the CHS/STS (stilbene synthase) sub-clades from seed plants (Figure S2). These results suggested that the evolution of CHS was consistent with the evolutionary status of the different species.…”

“…For the protein crystallization, ScCHS1 was amplified via PCR and ligated into the pETDuet‐1 vector digested by Bam H I and Not I, and then transformed into E. coli Rosetta (DE3) strain (the corresponding primers have been listed in Table S5). The procedures employed for protein purification have been described previously (Niu et al, 2021). The purified protein was collected and concentrated for the subsequent structural and biochemical studies.…”

“…After verification, the mutated plasmids were transformed into E. coli BL21 (DE3). The protein expression, purification, and enzyme activity determination of the mutants were carried out by the same procedure as mentioned above (Niu et al, 2021).…”

“…CHS is a homodimer, comprising two distinct 40–45 kDa monomer units and each monomer commonly maintains a catalytic triad (Cys‐164, His‐303, and Asn‐336) buried deep inside with the two functionally independent active sites (Ferrer et al, 1999). At present, the crystal structure of CHS has been clarified from several seed plants (Pandith et al, 2020), whereas relatively few studies have focused on the crystal structure of CHS in spore plants, especially in ferns (Liou et al, 2018; Niu et al, 2021).…”