Lars H. Kruse

The plant‐derived specialized metabolite montbretin A (MbA) is under development as an improved treatment for Type 2 diabetes, a worldwide epidemic. MbA is an acylated flavonol glycoside so far only found in small amounts in montbretia ( Crocosmia × crocosmiiflora ) corms. The compound's structural complexity and low natural abundance present major challenges for large‐scale production. To enable the development of a scalable MbA production system, we have previously engineered Nicotiana benthamiana to produce MbA. In addition to low levels of MbA, N. benthamiana also produces the pharmaceutically inactive analogues montbretin B and montbretin C, differing from MbA only in their acyl moieties. Follow‐up research focusing on precursor engineering has improved MbA yield, but further yield optimization is required to establish N. benthamiana as a scalable MbA production system. Here, we report evidence that transport likely constrains heterologous montbretin production in N. benthamiana and that transporters can be leveraged as bioengineering tools to enhance yield. Specifically, co‐expression of tonoplast localized CcABCC4 transporters with montbretin biosynthetic genes in N. benthamiana increased montbretin levels, including those of MbA. Yield increase is likely due to vacuolar sequestration and relies on the presence of an acyl group on the molecule. Eight out of fifteen ABCC4/14 transporters from plant species not known to produce montbretins also enhanced montbretin accumulation in N. benthamiana . These observations suggest that ABCC transporters might be versatile tools for bioengineering.

Terpenoids are major contributors to grapevine berry and wine aroma, with mono- and sesquiterpenoids exhibiting substantial variation among cultivars. However, the genetic basis of cultivar-specific terpenoid profiles remains only partially understood due to gene duplications that expanded the terpenoid synthase (TPS) gene family across different cultivars. Here, we compared haplotype-resolved genome assemblies for seven cultivars to analyze copy number variation and expression profiles of terpenoid biosynthesis genes and terpenoid accumulation throughout flower and berry development. These assemblies resolved duplicated TPS loci at the haplotype level, revealing extensive copy number variation across cultivars and between haplotypes within a cultivar, with up to 44 mono-TPS genes on a single haplotype. We identified eight specific monoterpenoids as key drivers of cultivar-specific profiles in ripe berries. Total TPS transcript abundance correlated strongly with total terpenoid accumulation over time. In contrast, TPS copy number was largely decoupled from both expression and terpenoid accumulation, indicating that expression regulation, rather than gene dosage, underlies aroma differences among cultivars. Transcript-metabolite network analysis revealed both expected correlations between specific TPS genes and putative products as well as novel associations. We functionally validated selected candidate TPS in Nicotiana benthamiana, characterizing α-farnesene, β-ocimene, and multi-product α-terpineol synthases. The in vivo characterization demonstrated that minor sequence variations can alter catalytic activity and product profile. Together, these findings show that the extensive duplication of TPS genes in grapevine does not itself determine terpenoid output; instead, cultivar-specific aroma arises from how these genes are expressed and from sequence differences that shape enzyme activity.

The co-occurrence of polysaccharides and proanthocyanidins in the aqueous extracts of western redcedar (Thuja plicata Donn; WRC) bark limits their commercial utilization. To better understand their association, proanthocyanidins and polysaccharides were extracted with cold water (3.4% w/w bark) and isolated as an alcohol-insoluble residue (AIR, 1.0% w/w bark). The polysaccharide content (∼30% w/w AIR) was analyzed by acidic and enzymatic depolymerization, revealing the presence of pectins, xyloglucans, and xylans. NMR spectroscopy identified features, such as acetylation and methyl esterification. Thiolysis followed by HPLC-DAD revealed that proanthocyanidins (1.46% w/w AIR) exhibit a mean degree of polymerization of 5.3, a cis/trans ratio of 0.40, and a procyanidin/prodelphinidin ratio of 3.90. This study provides a detailed structural characterization of proanthocyanidins and polysaccharides in the AIR of WRC bark. The findings highlight their strong association, which may contribute to distinctive properties that warrant further exploration, particularly in efforts to valorize bark residues.

The metabolic diversity of plants, comprising over a million different metabolites across the plant kingdom, harbors enormous potential for pharmaceutical and biotechnological applications. Resin glycoside (RG) acylsugars from the Convolvulaceae are of interest due to their medicinal and agricultural potential. However, understanding the biological relevance of RGs is challenging as they exhibit a high lineage-specific structural diversity. Liquid chromatography–tandem mass spectrometry (LC-MS/MS) coupled with computational peak annotation can provide insights into this diversity. Here, we present a comprehensive protocol for the characterization of RG diversity using a sensitive LC-MS/MS instrument, a knowledge-based computational pipeline, and a web tool for peak annotation. The described experimental approach provides a step-by-step guide for RG sampling, extraction, purification for downstream analyses such as bioassays, and structural annotation using LC-MS/MS and computational metabolomics. The protocol focuses on qualitative analysis for putative annotation (Annotation Level 2 as defined by the Metabolomics Standards Initiative) of RGs and can serve as a valuable template for researchers exploring plant metabolic diversity beyond RGs and acylsugars.

SUMMARY: Pyrrolizidine alkaloids (PAs) are toxic specialized metabolites produced in several plant species and frequently contaminate herbal teas or livestock feed. In comfrey ( Symphytum officinale , Boraginaceae), they are produced in two different organs of the plant, the root and young leaves. In this study, we demonstrate that homospermidine oxidase (HSO), a copper‐containing amine oxidase (CuAO) responsible for catalyzing the formation of the distinctive pyrrolizidine ring in PAs, is encoded by two individual genes. Specifically, So CuAO1 is expressed in young leaves, while So CuAO5 is expressed in roots. CRISPR/Cas9‐mediated knockout of socuao5 resulted in hairy roots (HRs) unable to produce PAs, supporting its function as HSO in roots. Plants regenerated from socuao5 knockout HRs remained completely PA‐free until the plants began to develop inflorescences, indicating the presence of another HSO that is expressed only during flower development. Stable expression of So CuAO1 in socuao5 knockout HRs rescued the ability to produce PAs. In vitro assays of both enzymes transiently expressed in Nicotiana benthamiana confirmed their HSO activity and revealed the ability of HSO to control the stereospecific cyclization of the pyrrolizidine backbone. The observation that the first specific step of PA biosynthesis catalyzed by homospermidine synthase requires only one gene copy, while two independent paralogs are recruited for the subsequent homospermidine oxidation in different tissues of the plant, suggests a complex regulation of the pathway. This adds a new level of complexity to PA biosynthesis, a system already characterized by species‐specific, tight spatio‐temporal regulation, and independent evolutionary origins in multiple plant lineages.

SUMMARY: The plant‐specialized metabolite montbretin A (MbA) is being developed as a new treatment option for type‐2 diabetes, which is among the ten leading causes of premature death and disability worldwide. MbA is a complex acylated flavonoid glycoside produced in small amounts in below‐ground organs of the perennial plant Montbretia (Crocosmia × crocosmiiflora). The lack of a scalable production system limits the development and potential application of MbA as a pharmaceutical or nutraceutical. Previous efforts to reconstruct montbretin biosynthesis in Nicotiana benthamiana (Nb) resulted in low yields of MbA and higher levels of montbretin B (MbB) and montbretin C (MbC). MbA, MbB, and MbC are nearly identical metabolites differing only in their acyl moieties, derived from caffeoyl‐CoA, coumaroyl‐CoA, and feruloyl‐CoA, respectively. In contrast to MbA, MbB and MbC are not pharmaceutically active. To utilize the montbretia caffeoyl‐CoA biosynthesis for improved MbA engineering in Nb, we cloned and characterized enzymes of the shikimate shunt of the general phenylpropanoid pathway, specifically hydroxycinnamoyl‐CoA: shikimate hydroxycinnamoyl transferase (CcHCT), p‐coumaroylshikimate 3′‐hydroxylase (CcC3′H), and caffeoylshikimate esterase (CcCSE). Gene expression patterns suggest that CcCSE enables the predominant formation of MbA, relative to MbB and MbC, in montbretia. This observation is supported by results from in vitro characterization of CcCSE and reconstruction of the shikimate shunt in yeast. Using CcHCT together with montbretin biosynthetic genes in multigene constructs resulted in a 30‐fold increase of MbA in Nb. This work advances our understanding of the phenylpropanoid pathway and features a critical step towards improved MbA production in bioengineered Nb.

Plants’ ability to chemically modify core structures of specialized metabolites is the main reason why the plant kingdom contains such a wide and rich array of diverse compounds. One of the most important types of chemical modifications of small molecules is the addition of an acyl moiety to produce esters and amides. Large-scale phylogenomics analyses have shown that the enzymes that perform acyl transfer reactions on the myriad small molecules synthesized by plants belong to only a few gene families. This review is focused on describing the biochemistry, evolutionary origins, and chemical ecology implications of one of these families—the BAHD acyltransferases. The growth of advanced metabolomic studies coupled with next-generation sequencing of diverse plant species has confirmed that the BAHD family plays critical roles in modifying nearly all known classes of specialized metabolites. The current and future outlook for research on BAHDs includes expanding their roles in synthetic biology and metabolic engineering.

SUMMARY: Plant responses to environmental change are mediated via changes in cellular metabolomes. However, Brachypodium distachyon (Poaceae) under 17 organ–condition combinations, including copper deficiency, heat stress, low phosphate, and arbuscular mycorrhizal symbiosis. We found that both leaf and root metabolomes were significantly affected by the growth medium. Leaf metabolomes were more diverse than root metabolomes, but the latter were more specialized and more responsive to environmental change. We found that 1 week of copper deficiency shielded the root, but not the leaf metabolome, from perturbation due to heat stress. Machine learning (ML)‐based analysis annotated approximately 81% of the fragmented peaks versus approximately 6% using spectral matches alone. We performed one of the most extensive validations of ML‐based peak annotations in plants using thousands of authentic standards, and analyzed approximately 37% of the annotated peaks based on these assessments. Analyzing responsiveness of each predicted metabolite class to environmental change revealed significant perturbations of glycerophospholipids, sphingolipids, and flavonoids. Co‐accumulation analysis further identified condition‐specific biomarkers. To make these results accessible, we developed a visualization platform on the Bio‐Analytic Resource for Plant Biology website (https://bar.utoronto.ca/efp_brachypodium_metabolites/cgi‐bin/efpWeb.cgi), where perturbed metabolite classes can be readily visualized. Overall, our study illustrates how emerging chemoinformatic methods can be applied to reveal novel insights into the dynamic plant metabolome and stress adaptation.

SUMMARY: Large enzyme families catalyze metabolic diversification by virtue of their ability to use diverse chemical scaffolds. How enzyme families attain such functional diversity is not clear. Furthermore, duplication and promiscuity in such enzyme families limits their functional prediction, which has produced a burgeoning set of incompletely annotated genes in plant genomes. Here, we address these challenges using BAHD acyltransferases as a model. This fast‐evolving family expanded drastically in land plants, increasing from one to five copies in algae to approximately 100 copies in diploid angiosperm genomes. Compilation of >160 published activities helped visualize the chemical space occupied by this family and define eight different classes based on structural similarities between acceptor substrates. Using orthologous groups (OGs) across 52 sequenced plant genomes, we developed a method to predict BAHD acceptor substrate class utilization as well as origins of individual BAHD OGs in plant evolution. This method was validated using six novel and 28 previously characterized enzymes and helped improve putative substrate class predictions for BAHDs in the tomato genome. Our results also revealed that while cuticular wax and lignin biosynthetic activities were more ancient, anthocyanin acylation activity was fixed in BAHDs later near the origin of angiosperms. The OG‐based analysis enabled identification of signature motifs in anthocyanin‐acylating BAHDs, whose importance was validated via molecular dynamic simulations, site‐directed mutagenesis and kinetic assays. Our results not only describe how BAHDs contributed to evolution of multiple chemical phenotypes in the plant world but also propose a biocuration‐enabled approach for improved functional annotation of plant enzyme families.

Plant responses to environmental change are mediated via changes in cellular metabolomes. However, Brachypodium distachyon , a model Poaceae species, under 17 different organ-condition combinations, including copper deficiency, heat stress, low phosphate and arbuscular mycorrhizal symbiosis (AMS). We used a combination of information theory-based metrics and machine learning-based identification of metabolite structural classes to assess metabolomic changes. Both leaf and root metabolomes were significantly affected by the growth medium. Leaf metabolomes were more diverse than root metabolomes, but the latter were more specialized and more responsive to environmental change. We also found that one week of copper deficiency shielded the root metabolome, but not the leaf metabolome, from perturbation due to heat stress. Using a recently published deep learning based method for metabolite class predictions, we analyzed the responsiveness of each metabolite class to environmental change, which revealed significant perturbations of various lipid classes and phenylpropanoids such as cinnamic acids and flavonoids. Co-accumulation analysis further identified condition-specific metabolic biomarkers. Finally, to make these results publicly accessible, we developed a novel visualization platform on the Bioanalytical Resource website, where significantly perturbed metabolic classes can be readily visualized. Overall, our study illustrates how emerging chemoinformatic methods can be applied to reveal novel insights into the dynamic plant metabolome and plant stress adaptation.