Aixia Cheng’s research while affiliated with Shandong University and other places

Main conclusion The evolutionary conservation of type III polyketide synthases (PKS) in Selaginella has been elucidated, and the critical amino acid residues of the anther-specific chalcone synthase-like enzyme (SmASCL) have been identified. Abstract Selaginella species are the oldest known vascular plants and a valuable resource for the study of metabolic evolution in land plants. Polyketides, especially flavonoids and sporopollenin precursors, are essential prerequisites for plant land colonization. Although type III polyketide synthases (PKS) are widely studied in seed plants, the related enzymes in Selaginella remain poorly characterized. Here, eight type III PKSs were identified in the Selaginella moellendorffii genome and classified into three clusters. Two PKSs were selected for further research based on their phylogenetic relationships and protein sequence similarity. Functional studies revealed that they were chalcone synthase (SmCHS) and anther-specific CHS-like enzyme (SmASCL). These enzymes are involved in the biosynthesis of flavonoids and sporopollenin, respectively. Their sequence information and enzymatic activity are similar to the orthologs in other plants. Phylogenetic analysis revealed that the ASCL and CHS enzymes were separated into two clades from the Bryophyta. These results suggest that CHS and ASCL emerged in the first land plants and then remained conserved during plant evolution. To study the structural basis of the enzymatic function of SmASCL, a series of mutants were constructed. The number of condensation reactions catalyzed by the P210L/Y211D and I200V/G201T double mutants exceeds that of the wild-type enzyme. Our study provides insight into the characteristics and functions of type III PKSs in S. moellendorffii. It also offers clues for a deeper understanding of the relationship between active sites and the enzymatic function of ASCLs.

Plagiochasma appendiculatum, a thalloid liverwort, contains high levels of bisbibenzyls, aromatic compounds with potent antitumor as well as antifungal activities. In the present study, rapid growth callus was induced from the thallus of P. appendiculatum, and optimal culture conditions, including medium, temperature, pH, and plant growth regulators for callus production were evaluated. Under optimal culture conditions, the biomass of the callus doubled with a sigmoidal growth curve after 15 days. Differentiation and plant regeneration were studied on a medium supplemented with different plant hormones (α-naphthaleneacetic acid [NAA], 6-benzyladenine [6-BA], and 2,4-dichlorophenoxyacetic acid [2,4-D]). NAA and 6-BA stimulated rhizoid and thallus differentiation, respectively, whereas 2,4-D inhibited the differentiation of thallus and rhizoid. Different metabolic profiles of callus, differentiated thallus, and thallus in the soil were studied by high-performance liquid chromatography. The results showed that both the callus and thallus could synthesize bisbibenzyls. In addition, the kinds and content of bisbibenzyl differed significantly between the callus and thallus. In conclusion, P. appendiculatum thallus cultured in vitro possesses the ability to biosynthesize bisbibenzyl, and it may be utilized for the mass production of specific bisbibenzyls in an appropriate growth environment.

During the course of evolution of land plants, different classes of flavonoids, including flavonols and anthocyanins, sequentially emerged, facilitating adaptation to the harsh terrestrial environment. Flavanone 3β-hydroxylase (F3H), an enzyme functioning in flavonol and anthocyanin biosynthesis and a member of the 2-oxoglutarate dependent dioxygenase (2-ODD) family, catalyzes the hydroxylation of (2S)-flavanones to dihydroflavonols, but its origin and evolution remains elusive. Here we demonstrate that functional flavone synthase Is (FNS Is) are widely distributed in the primitive land plants liverworts and evolutionarily connected to seed plant F3Hs. We identified and characterized a set of 2-ODD enzymes from several liverwort species and plants in various evolutionary clades of the plant kingdom. The bifunctional enzyme FNS I/F2H emerged in liverworts, and FNS I/F3H evolved in Physcomitrium (Physcomitrella) patens and Selaginella moellendorffii, suggesting they represent the functional transition forms between canonical FNS Is and F3Hs. The functional transition from FNS Is to F3Hs provides a molecular basis for the chemical evolution of flavones to flavonols and anthocyanins, which contributes to the acquisition of a broader spectrum of flavonoids in seed plants and facilitates their adaptation to the terrestrial ecosystem.