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QS-21 is a potent vaccine adjuvant currently sourced by extraction from the Chilean soapbark tree. It is a key component of human vaccines for shingles, malaria, coronavirus disease 2019 and others under development. The structure of QS-21 consists of a glycosylated triterpene scaffold coupled to a complex glycosylated 18-carbon acyl chain that is critical for immunostimulant activity. We previously identified the early pathway steps needed to make the triterpene glycoside scaffold; however, the biosynthetic route to the acyl chain, which is needed for stimulation of T cell proliferation, was unknown. Here, we report the biogenic origin of the acyl chain, characterize the series of enzymes required for its synthesis and addition and reconstitute the entire 20-step pathway in tobacco, thereby demonstrating the production of QS-21 in a heterologous expression system. This advance opens up unprecedented opportunities for bioengineering of vaccine adjuvants, investigating structure-activity relationships and understanding the mechanisms by which these compounds promote the human immune response.

Root growth in Arabidopsis is inhibited by exogenous auxin–amino acid conjugates, and mutants resistant to one such conjugate [indole-3-acetic acid (IAA)–Ala] map to a gene (AtIAR1) that is a member of a metal transporter family. Here, we test the hypothesis that AtIAR1 controls the hydrolysis of stored conjugated auxin to free auxin through zinc transport. AtIAR1 complements a yeast mutant sensitive to zinc, but not manganese- or iron-sensitive mutants, and the transporter is predicted to be localized to the endoplasmic reticulum/Golgi in plants. A previously identified Atiar1 mutant and a non-expressed T-DNA mutant both exhibit altered auxin metabolism, including decreased IAA–glucose conjugate levels in zinc-deficient conditions and insensitivity to the growth effect of exogenous IAA–Ala conjugates. At a high concentration of zinc, wild-type plants show a novel enhanced response to root growth inhibition by exogenous IAA–Ala which is disrupted in both Atiar1 mutants. Furthermore, both Atiar1 mutants show changes in auxin-related phenotypes, including lateral root density and hypocotyl length. The findings therefore suggest a role for AtIAR1 in controlling zinc release from the secretory system, where zinc homeostasis plays a key role in regulation of auxin metabolism and plant growth regulation. [ABSTRACT FROM AUTHOR]

The most abundant phenolic compound in Solanaceous plants is chlorogenic acid (CGA), which possesses protective properties such as antimicrobial and antioxidant activities. These properties are particularly relevant when plants are under adverse conditions, such as pathogen attack, excess light, or extreme temperatures that cause oxidative stress. Additionally, CGA has been shown to absorb UV-B light. In tomato and potato, CGA is mainly produced through the HQT pathway mediated by the enzyme hydroxycinnamoyl-CoA:quinate hydroxycinnamoyl transferase. However, the absence of natural or induced mutants of this gene has made it unclear whether other pathways contribute to CGA production and accumulation. To address this question, we used CRISPR technology to generate multiple knock-out mutant lines in the tomato HQT gene. The resulting slhqt plants did not accumulate CGA or other caffeoylquinic acids (CQAs) in various parts of the plant, indicating that CQA biosynthesis depends almost entirely on the HQT pathway in tomato and, likely, other Solanaceous crops. We also found that the lack of CGA in slhqt plants led to higher levels of hydroxycinnamoyl-glucose and flavonoids compared to wild-type plants. Gene expression analysis revealed that this metabolic reorganization was partly due to flux redirection, but also involved modulation of important transcription factor genes that regulate secondary metabolism and sense environmental conditions. Finally, we investigated the physiological role of CGA in tomato and found that it accumulates in the upper epidermis where it acts as a protector against UV-B irradiation., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling editor RB declared a shared affiliation with the authors FD’O, MP, GM at the time of review., (Copyright © 2023 D’Orso, Hill, Appelhagen, Lawrenson, Possenti, Li, Harwood, Morelli and Martin.)