Cong Guo

Gymnosperms represent an ancient lineage with diverse chemical profiles. Their terpenoids constitute a major class notable for substantial structural diversity. Terpene synthases (TPSs) are the driving force of terpene diversity. TPS-d is a gymnosperm-specific TPS subfamily, yet the evolution and functional diversity of TPS-d are still understudied. In this review, we analyze 134 functionally characterized gymnosperm TPSs to elucidate the evolutionary history and functional diversity of the predominant TPS-d subfamily. Phylogenetic analysis revealed substantial lineage-specific expansion and divergence within the TPS-d subfamily post-speciation. Crucially, we highlight the shifts in domain architecture during TPS-d evolution, providing structural evidence for the progressive attrition and eventual loss of the ancestral γ domain in TPS-d2 and TPS-d1 clades. Furthermore, we summarize the functional diversity of TPS-d enzymes in specialized metabolites, emphasizing their ecological roles in plant defense. Overall, this review provides a comprehensive overview of the evolutionary and functional landscape of the gymnosperm-specific TPS-d subfamily, serving as a valuable reference for future studies on TPSs in gymnosperms.

As a group of the most important phytohormone, auxin homeostasis is regulated in a complex manner. Generally, auxin conjugations especially IAA glucosides are dominant on high auxin level conditions. Former terminal glucosylation researches mainly focus on O -position, while IAA- N -glucoside or IAA-Asp- N -glucoside has been neglected since their found in 2001. In our study, IAA-Asp- N -glucoside was firstly found specifically abundant (as high as 4.13 mg/g) in ginkgo seeds of 58 cultivars from Ginkgo Resource Nursery built in 1990. Furthermore, a novel N -glucosyltransferase GbNGT1, which could catalyze IAA-Asp and IAA to form their corresponding N -glucoside, was identified through differential transcriptome analysis and in vitro enzymatic test. The enzyme was demonstrated to possess specific catalyze capacity toward the N -position of IAA-amino acid or IAA among 52 substrates, and was typical of acid tolerance, metal ion independence and high temperature sensitivity. Docking and site-directed mutagenesis of this enzyme confirmed that E15G mutant could almost abolish enzyme catalytic activity towards IAA-Asp and IAA in vitro and in vivo . The IAA modification of GbNGT1 and GbGH3.5 was verified by transient expression assay in Nicotiana benthamiana . In conclusion, our results complement the terminal metabolic pathway of auxin, and the specific catalytic function of GbNGT1 towards IAA-amino acid provide a new way to biosynthesis indole-amide compounds. Highlight: The N-glucosylation of IAA or IAA-amino acids in auxin metabolism had been neglected over decades, our work for GbNGT1 redeems the missing chain of auxin metabolic pathway.