Dataset Information

Characterization of phenylalanine ammonia-lyase genes facilitating flavonoid biosynthesis from two species of medicinal plant Anoectochilus

ABSTRACT:

Background

Anoectochilus roxburghii and Anoectochilus formosanus, belong to the Anoectochilus genus, have been used for Chinese herbal drugs as well as health food. Phenylalanine ammonia-lyase (PAL), the key enzyme in primary metabolism and phenylpropanoid metabolism, produces secondary metabolites (flavonoids) in plants, which are beneficial for the biosynthesis of phenylpropanoid metabolites.

Methods

The PAL genes were cloned from A. formosanus and A. roxburghii according to our previous transcriptomic analysis. The PALs were introduced into pCAMBIA2300-35S-PAL-eGFP to generate 35S-PAL-eGFP. The constructs were further used for subcellular localization and transgenic Arabidopsis. The expression of AfPAL and ArPAL under precursor substance (L-Phe), NaCl, UV, and red-light were analyzed by real-time quantitative PCR (RT-qPCR).

Results

AfPAL and ArPAL , encoding 2,148 base pairs, were cloned from A. formosanus and A. roxburghii. The subcellular localization showed that the ArPAL and AfPAL were both localized in the nucleus with GPF. Quantitative RT-PCR analysis indicated that the ArPAL and AfPAL genes function in the phenylalanine pathway as well as response to induced conditions. Overexpression of the AfPAL and ArPAL could increase flavonoids and anthocyanin content in the transgenic Arabidopsis.

Discussion

The results suggest that AfPAL and ArPAL play a crucial role in the flavonoid biosynthesis in Anoectochilus. Also, our study provides new insights into the enrichment of secondary metabolites of traditional Chinese medicines A. formosanus and A. roxburghii, which can improve their medicinal active ingredients and be used for drug discovery in plants.

SUBMITTER: Yang L

PROVIDER: S-EPMC9270878 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND: Phenylalanine ammonia lyase (PAL) is a key enzyme of the phenylpropanoid pathway that catalyzes the deamination of phenylalanine to trans-cinnamic acid, a precursor for the lignin and flavonoid biosynthetic pathways. To date, PAL genes have been less extensively studied in gymnosperms than in angiosperms. Our interest in PAL genes stems from their potential role in the defense responses of Pinus taeda, especially with respect to lignification and production of low molecular weight phenolic compounds under various biotic and abiotic stimuli. In contrast to all angiosperms for which reference genome sequences are available, P. taeda has previously been characterized as having only a single PAL gene. Our objective was to re-evaluate this finding, assess the evolutionary history of PAL genes across major angiosperm and gymnosperm lineages, and characterize PAL gene expression patterns in Pinus taeda. METHODS: We compiled a large set of PAL genes from the largest transcript dataset available for P. taeda and other conifers. The transcript assemblies for P. taeda were validated through sequencing of PCR products amplified using gene-specific primers based on the putative PAL gene assemblies. Verified PAL gene sequences were aligned and a gene tree was estimated. The resulting gene tree was reconciled with a known species tree and the time points for gene duplication events were inferred relative to the divergence of major plant lineages. RESULTS: In contrast to angiosperms, gymnosperms have retained a diverse set of PAL genes distributed among three major clades that arose from gene duplication events predating the divergence of these two seed plant lineages. Whereas multiple PAL genes have been identified in sequenced angiosperm genomes, all characterized angiosperm PAL genes form a single clade in the gene PAL tree, suggesting they are derived from a single gene in an ancestral angiosperm genome. The five distinct PAL genes detected and verified in P. taeda were derived from a combination of duplication events predating and postdating the divergence of angiosperms and gymnosperms. CONCLUSIONS: Gymnosperms have a more phylogenetically diverse set of PAL genes than angiosperms. This inference has contrasting implications for the evolution of PAL gene function in gymnosperms and angiosperms.