The airborne herbivore‐induced plant volatile indole is converted to benzoxazinoid defense compounds in maize plants

New Phytologist · 2025-02-25 · 9 citations

Herbivore-induced plant volatiles act as danger signals to prime defense responses in neighboring plants, yet in many cases the mechanism behind this priming is not known. Volatile signals may be recognized directly by receptors and/or converted into other active compounds. Here we investigate the metabolic fate of volatile indole, a known priming signal in maize (Zea mays), to determine if its conversion to other compounds could play a role in its priming of defenses. We identified benzoxazinoids as major products from volatile indole using heavy isotope-labeled volatile indole and Pathway of Origin Determination in Untargeted Metabolomics (PODIUM) analysis. We then used benzoxazinoid biosynthesis maize mutants to investigate their role in indole-mediated priming. Labeled volatile indole was converted into DIMBOA-glucoside in a bx2 (benzoxazinone synthesis2)-dependent manner. The bx2 mutant plants showed elevated green leaf volatile (GLV) production in response to wounding and Spodoptera frugiperda regurgitant irrespective of indole exposure. Thus, volatile indole is converted into benzoxazinoids, and part of its priming mechanism may be due to the enhanced production of these phytoanticipins. However, indole-mediated enhanced GLV production does not rely on the conversion of indole to benzoxazinoids, so indole also has other signaling functions.

Exploring the amino acid-derived metabolomes of Arabidopsis and their associated natural variation using isotope labeling and mGWAS

PLANT PHYSIOLOGY · 2025-08-27

In this study, we used stable isotope labeling coupled with reversed-phase HPLC-MS to annotate the origin of metabolite features in Arabidopsis (Arabidopsis thaliana) (Columbia-0) seedling rosettes and stems. Using this strategy, a total of 1,240 metabolite features were shown to be derived from 15 amino acids, and these represented 10% to 30% of the total ion counts detected by untargeted LC-MS. The amino acid-derived metabolomes (AADMs) of rosettes and stems exhibited differing patterns of accumulation. Precursor-of-origin annotations (POA) revealed that some metabolites were generated solely from individual amino acids, whereas others were derived from multiple sources. Amino acid feeding altered the abundance of their corresponding AADMs as well as the levels of features derived from other amino acids. These data suggest that the accumulation of amino acid-derived features (AADFs) is restricted by availability of their amino acid precursors and that perturbation of amino acid metabolic networks can lead to long distance changes in end-product accumulation. The alignment of annotated AADFs with features from a previous metabolic genome-wide association study (mGWAS) led to the identification of 87,820 and 61,618 metabolite feature-single nucleotide polymorphism (SNP) associations (P < 10-4) in leaves and stems, respectively. Genes associated with AADF accumulation, including METHYLTHIOALKYLMALATE SYNTHASE 1 (MAM1) and ᴅ-AMINO ACID RACEMASE 1 (DAAR1), were retrieved from this analysis, demonstrating that the integration of isotope labeling and mGWAS can contribute to the identification of genes involved in plant metabolite accumulation.

Model-guided metabolic engineering of 2-phenylethanol in Arabidopsis

Metabolic Engineering · 2025-11-21

A semidominant point mutation of Mediator tail subunit MED5b in Arabidopsis leads to altered enrichment of H3K27me3 and reduced expression of targets of MYC2

G3 Genes Genomes Genetics · 2025-02-14

The Mediator complex coordinates regulatory input for transcription driven by RNA polymerase II in eukaryotes. reduced epidermal fluorescence4-3 (ref4-3) is a semidominant mutation that results in a single amino acid substitution in the Mediator tail subunit Med5b. Previous characterization of ref4-3 revealed altered expression of a variety of loci in Arabidopsis, including those contributing to phenylpropanoid biosynthesis. Examination of existing RNA-seq data indicated that loci enriched for the transcriptionally repressive chromatin modification H3K27me3 are overrepresented among genes that are misregulated in ref4-3. We used ChIP-seq and RNA-seq to examine the possibility that perturbation of H3K27me3 homeostasis in ref4-3 plants contributed to altered transcript levels. We observed that ref4-3 results in a modest global reduction of H3K27me3 at enriched loci and that this reduction is not dependent on gene expression; however, altered H3K27me3 was not strongly predictive of altered expression in ref4-3 plants. Instead, our analyses revealed a substantial enrichment of targets of the MYC2 transcriptional regulator among genes that exhibit decreased expression in ref4-3. Consistent with previous characterization of ref4-3, we observed that ref4-3-dependent decreased expression of MYC2 targets can be suppressed by loss of another Mediator tail subunit, MED25. This observation is consistent with previous biochemical characterization of MYC2. Our data highlight the diverse and distinct impacts that a single amino acid change in the tail subunit of Mediator can have on transcriptional circuits and raise the prospect that Mediator directly contributes to H3K27me3 homeostasis in plants.

Final Technical Report for Coupling Metabolic Source Isotopic Pair Labeling and Genome Wide Association for Metabolite and Gene Annotation in Plants

2024-12-11

Genome‐wide association identifies a BAHD acyltransferase activity that assembles an ester of glucuronosylglycerol and phenylacetic acid

The Plant Journal · 2024-04-01 · 4 citations

Genome-wide association studies (GWAS) are an effective approach to identify new specialized metabolites and the genes involved in their biosynthesis and regulation. In this study, GWAS of Arabidopsis thaliana soluble leaf and stem metabolites identified alleles of an uncharacterized BAHD-family acyltransferase (AT5G57840) associated with natural variation in three structurally related metabolites. These metabolites were esters of glucuronosylglycerol, with one metabolite containing phenylacetic acid as the acyl component of the ester. Knockout and overexpression of AT5G57840 in Arabidopsis and heterologous overexpression in Nicotiana benthamiana and Escherichia coli demonstrated that it is capable of utilizing phenylacetyl-CoA as an acyl donor and glucuronosylglycerol as an acyl acceptor. We, thus, named the protein Glucuronosylglycerol Ester Synthase (GGES). Additionally, phenylacetyl glucuronosylglycerol increased in Arabidopsis CYP79A2 mutants that overproduce phenylacetic acid and was lost in knockout mutants of UDP-sulfoquinovosyl: diacylglycerol sulfoquinovosyl transferase, an enzyme required for glucuronosylglycerol biosynthesis and associated with glycerolipid metabolism under phosphate-starvation stress. GGES is a member of a well-supported clade of BAHD family acyltransferases that arose by duplication and neofunctionalized during the evolution of the Brassicales within a larger clade that includes HCT as well as enzymes that synthesize other plant-specialized metabolites. Together, this work extends our understanding of the catalytic diversity of BAHD acyltransferases and uncovers a pathway that involves contributions from both phenylalanine and lipid metabolism.

Method for regulation of plant lignin composition

OSTI OAI (U.S. Department of Energy Office of Scientific and Technical Information) · 2023-01-23

A method is disclosed for the regulation of lignin composition in plant tissue. Plants are transformed with a gene encoding an active F5H gene. The expression of the F5H gene results in increased levels of syringyl monomer providing a lignin composition more easily degraded with chemicals and enzymes.

Pinoresinol rescues developmental phenotypes of Arabidopsis phenylpropanoid mutants overexpressing <i>FERULATE 5-HYDROXYLASE</i>

Proceedings of the National Academy of Sciences · 2023-07-24 · 9 citations

Most phenylpropanoid pathway flux is directed toward the production of monolignols, but this pathway also generates multiple bioactive metabolites. The monolignols coniferyl and sinapyl alcohol polymerize to form guaiacyl (G) and syringyl (S) units in lignin, components that are characteristic of plant secondary cell walls. Lignin negatively impacts the saccharification potential of lignocellulosic biomass. Although manipulation of its content and composition through genetic engineering has reduced biomass recalcitrance, in some cases, these genetic manipulations lead to impaired growth. The reduced-growth phenotype is often attributed to poor water transport due to xylem collapse in low-lignin mutants, but alternative models suggest that it could be caused by the hyper- or hypoaccumulation of phenylpropanoid intermediates. In Arabidopsis thaliana , overexpression of FERULATE 5-HYDROXYLASE ( F5H ) shifts the normal G/S lignin ratio to nearly pure S lignin and does not result in substantial changes to plant growth. In contrast, when we overexpressed F5H in the low-lignin mutants cinnamyl dehydrogenase c and d ( cadc cadd ), cinnamoyl-CoA reductase 1 , and reduced epidermal fluorescence 3 , plant growth was severely compromised. In addition, cadc cadd plants overexpressing F5H exhibited defects in lateral root development. Exogenous coniferyl alcohol (CA) and its dimeric coupling product, pinoresinol, rescue these phenotypes. These data suggest that mutations in the phenylpropanoid pathway limit the biosynthesis of pinoresinol, and this effect is exacerbated by overexpression of F5H , which further draws down cellular pools of its precursor, CA. Overall, these genetic manipulations appear to restrict the synthesis of pinoresinol or a downstream metabolite that is necessary for plant growth.

<scp>UGT76F1</scp> glycosylates an isomer of the <scp>C7</scp>‐necic acid component of pyrrolizidine alkaloids in <i>Arabidopsis thaliana</i>

The Plant Journal · 2023-03-30 · 1 citations

Identification of unknown metabolites and their biosynthetic genes is an active research area in plant specialized metabolism. By following a gene-metabolite association from a genome-wide association study of Arabidopsis stem metabolites, we report a previously unknown metabolite, 2-hydroxy-2-(1-hydroxyethyl)pentanoic acid glucoside, and demonstrated that UGT76F1 is responsible for its production in Arabidopsis. The chemical structure of the glucoside was determined by a series of analyses, including tandem MS, acid and base hydrolysis, and NMR spectrometry. T-DNA knockout mutants of UGT76F1 are devoid of the glucoside but accumulate increased levels of the aglycone. 2-hydroxy-2-(1-hydroxyethyl)pentanoic acid is structurally related to the C7-necic acid component of lycopsamine-type pyrrolizidine alkaloids such as trachelantic acid and viridifloric acid. Feeding norvaline greatly enhances the accumulation of 2-hydroxy-2-(1-hydroxyethyl)pentanoic acid glucoside in wild-type but not the UGT76F1 knockout mutant plants, providing evidence for an orthologous C7-necic acid biosynthetic pathway in Arabidopsis despite the apparent lack of pyrrolizidine alkaloids.

Manipulation of Lignin Monomer Composition Combined with the Introduction of Monolignol Conjugate Biosynthesis Leads to Synergistic Changes in Lignin Structure

Plant and Cell Physiology · 2022-03-11 · 26 citations

The complexity of lignin structure impedes efficient cell wall digestibility. Native lignin is composed of a mixture of three dominant monomers, coupled together through a variety of linkages. Work over the past few decades has demonstrated that lignin composition can be altered through a variety of mutational and transgenic approaches such that the polymer is derived almost entirely from a single monomer. In this study, we investigated changes to lignin structure and digestibility in Arabidopsis thaliana in near-single-monolignol transgenics and mutants and determined whether novel monolignol conjugates, produced by a FERULOYL-CoA MONOLIGNOL TRANSFERASE (FMT) or a p-COUMAROYL-CoA MONOLIGNOL TRANSFERASE (PMT), could be integrated into these novel polymers to further improve saccharification efficiency. Monolignol conjugates, including a new conjugate of interest, p-coumaryl p-coumarate, were successfully integrated into high-H, high-G and high-S lignins in A. thaliana. Regardless of lignin composition, FMT- and PMT-expressing plants produced monolignol ferulates and monolignol p-coumarates, respectively, and incorporated them into their lignin. Through the production and incorporation of monolignol conjugates into near-single-monolignol lignins, we demonstrated that substrate availability, rather than monolignol transferase substrate preference, is the most important determining factor in the production of monolignol conjugates, and lignin composition helps dictate cell wall digestibility.