Mass Spectrometry Imaging‐Assisted Discovery of Gallotannin Biosynthetic Genes in the Root of Paeonia suffruticosa

A highly efficient gene screening strategy for discovering enzymes responsible for gallotannin biosynthesis in the root of Paeonia suffruticosa was developed by integrating matrix‐assisted laser desorption/ionization mass spectrometry imaging (MALDI ‐ MSI), spatial transcriptome sequencing, and phylogenetic analysis. Through this strategy, one UGT gene ( PsUGT84A ) and eight serine carboxypeptidase‐like (SCPL) genes ( PsSCPL311 , PsSCPL272 , PsSCPL155 , PsSCPL406 , PsSCPL886 , PsSCPL531 , PsSCPL799 , and PsSCPL979 ) were first identified and characterized. The key UDP‐glycosyltransferase (UGT) PsUGT84A involved in the initial step of gallotannin biosynthesis can catalyze the synthesis of β ‐galloylglucose ( β G) from gallic acid. Subsequently, a series of SCPL acyltransferases enable the sequential acylation of the 6‐OH, 2‐OH, 3‐OH, and 4‐OH at the central glucose molecule of β G to ultimately synthesize the fully galloylated derivative, pentagalloylglucose (PGG). Furthermore, heterologous reconstruction of the gallotannin biosynthetic pathway in Nicotiana benthamiana was achieved. This study represents the first comprehensive elucidation of gallotannin biosynthesis in P. suffruticosa , providing essential enzymatic tools for the efficient production of hydrolysable tannins.