Systematic characterization of gene families and functional analysis of PvRAS3 and PvRAS4 involved in rosmarinic acid biosynthesis in Prunella vulgaris

is an important material for Chinese medicines with rosmarinic acid (RA) as its index component. Based on the chromosome-level genome assembly we obtained recently, 51 RA biosynthesis-related genes were identified. Sequence feature, gene expression pattern and phylogenetic relationship analyses show...

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Published inFrontiers in plant science Vol. 15; p. 1374912
Main Authors Yan, Chao, Li, Caili, Jiang, Maochang, Xu, Yayun, Zhang, Sixuan, Hu, Xiangling, Chen, Yuhang, Lu, Shanfa
Format Journal Article
LanguageEnglish
Published Switzerland Frontiers Media S.A 01.05.2024
Subjects
biosynthetic pathway
CRISPR/Cas9
in vitro enzymatic activity assay
Plant Science
Prunella vulgaris
PvRAS3
PvRAS4
CRISPR/Cas9
biosynthetic pathway
Prunella vulgaris
rosmarinic acid synthase
in vitro enzymatic activity assay
rosmarinic acid
PvRAS3
PvRAS4
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Summary:is an important material for Chinese medicines with rosmarinic acid (RA) as its index component. Based on the chromosome-level genome assembly we obtained recently, 51 RA biosynthesis-related genes were identified. Sequence feature, gene expression pattern and phylogenetic relationship analyses showed that 17 of them could be involved in RA biosynthesis. enzymatic assay showed that PvRAS3 catalyzed the condensation of -coumaroyl-CoA and caffeoyl-CoA with pHPL and DHPL. Its affinity toward -coumaroyl-CoA was higher than caffeoyl-CoA. PvRAS4 catalyzed the condensation of -coumaroyl-CoA with pHPL and DHPL. Its affinity toward -coumaroyl-CoA was lower than PvRAS3. UPLC and LC-MS/MS analyses showed the existence of RA, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid and caffeoyl-4'-hydroxyphenyllactic acid in . Generation and analysis of homozygous mutants showed significant decrease of RA, 4-coumaroyl-3',4'-dihydroxyphenyllactic acid, 4-coumaroyl-4'-hydroxyphenyllactic acid and caffeoyl-4'-hydroxyphenyllactic acid and significant increase of DHPL and pHPL. It suggests that PvRAS3 is the main enzyme catalyzing the condensation of acyl donors and acceptors during RA biosynthesis. The role of PvRAS4 appears minor. The results provide significant information for quality control of medicinal materials.
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Sibongile Mafu, University of Massachusetts Amherst, United States
These authors have contributed equally to this work
Edited by: Naoki Kitaoka, Hokkaido University, Japan
Reviewed by: Takafumi Shimizu, Research Institute of Innovative Technology for the Earth (RITE), Japan
ISSN:1664-462X
1664-462X
DOI:10.3389/fpls.2024.1374912