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Red alder (Alnus rubra Bong.) is an ecologically significant and important fast-growing commercial tree species native to western coastal and riparian regions of North America, having highly desirable wood, pigment, and medicinal properties. We have sequenced the genome of a rapidly growing clone. The assembly is nearly complete, containing the full complement of expected genes. This supports our objectives of identifying and studying genes and pathways involved in nitrogen-fixing symbiosis and those related to secondary metabolites that underlie red alder's many interesting defense, pigmentation, and wood quality traits. We established that this clone is most likely diploid and identified a set of SNPs that will have utility in future breeding and selection endeavors, as well as in ongoing population studies. We have added a well-characterized genome to others from the order Fagales. In particular, it improves significantly upon the only other published alder genome sequence, that of Alnus glutinosa. Our work initiated a detailed comparative analysis of members of the order Fagales and established some similarities with previous reports in this clade, suggesting a biased retention of certain gene functions in the vestiges of an ancient genome duplication when compared with more recent tandem duplications.

Chlorogenic acid (CGA) and guaiacyl/syringyl (G/S) lignin formation involves hydroxycinnamoyl ester intermediacy, the latter formed via hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT) and hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) activities. HQT and HCT RNAi silencing of a commercial tobacco (Nicotiana tabacum) K326 line was examined herein. NtHQT gene silencing gave relatively normal plant phenotypes, with CGA levels reduced (down to 1% of wild type) with no effects on lignin. RNAi NtHCT silencing had markedly adverse phenotypes (e.g., stunted, multiple stems, delayed flowering, with senescence delayed by several months). Lignin contents were partially lowered, with a small increase in cleavable p-hydroxyphenyl (H) monomers; those plants had no detectable CGA level differences relative to wild type. In vitro NtHCT kinetic parameters revealed preferential p-coumaroyl CoA and shikimate esterification, as compared to other structurally related potential acyl group donors and acceptors. In the presence of coenzyme A, NtHCT catalyzed the reverse reaction. Site-directed mutagenesis of NtHCT (His153Ala) abolished enzymatic activity. NtHQT, by comparison, catalyzed preferential conversion of p-coumaroyl CoA and quinic acid to form p-coumaroyl quinate, the presumed CGA precursor. In sum, metabolic pathways to CGA and lignins appear to be fully independent, and previous conflicting reports of substrate versatilities and metabolic cross-talk are resolved.

The biochemical activities of dirigent proteins (DPs) give rise to distinct complex classes of plant phenolics. DPs apparently began to emerge during the aquatic-to-land transition, with phylogenetic analyses revealing the presence of numerous DP subfamilies in the plant kingdom. The vast majority (>95%) of DPs in these large multigene families still await discovery of their biochemical functions. Here, we elucidated the 3D structures of two pterocarpan-forming proteins with dirigent-like domains. Both proteins stereospecifically convert distinct diastereomeric chiral isoflavonoid precursors to the chiral pterocarpans, (–)- and (+)-medicarpin, respectively. Their 3D structures enabled comparisons with stereoselective lignan– and aromatic terpenoid–forming DP orthologs. Each protein provides entry into diverse plant natural products classes, and our experiments suggest a common biochemical mechanism in binding and stabilizing distinct plant phenol–derived mono- and bis-quinone methide intermediates during different C–C and C–O bond–forming processes. These observations provide key insights into both their appearance and functional diversification of DPs during land plant evolution/adaptation. The proposed biochemical mechanisms based on our findings provide important clues to how additional physiological roles for DPs and proteins harboring dirigent-like domains can now be rationally and systematically identified.

Two western red cedar pinoresinol-lariciresinol reductase (PLR) homologues were studied to determine their enantioselective, substrate versatility, and kinetic properties. PLRs are downstream of dirigent protein engendered, coniferyl alcohol derived, stereoselective coupling to afford entry into the 8- and 8'-linked furofuran lignan, pinoresinol. Our investigations showed that each PLR homolog can enantiospecifically metabolize different furofuran lignans with modified aromatic ring substituents, but where phenolic groups at both C4/C4' are essential for catalysis. These results are consistent with quinone methide intermediate formation in the PLR active site. Site-directed mutagenesis and kinetic measurements provided additional insight into factors affecting enantioselectivity and kinetic properties. From these data, PLRs can be envisaged to allow for the biotechnological potential of generation of various lignan skeleta, that could be differentially "decorated" on their aromatic ring substituents, via the action of upstream dirigent proteins.

The discovery of dirigent proteins (DPs) and their functions in plant phenol biochemistry was made over two decades ago with Forsythia × intermedia. Stereo-selective, DP-guided, monolignol-derived radical coupling in vitro was then reported to afford the optically active lignan, (+)-pinoresinol from coniferyl alcohol, provided one-electron oxidase/oxidant capacity was present. It later became evident that DPs have several distinct sub-families. In vascular plants, DPs hypothetically function, along with other essential enzymes/proteins (e.g. oxidases), as part of lignin/lignan forming complexes (LFCs). Herein, we used an integrated bottom-up, top-down, and native mass spectrometry approach to detect potential interacting proteins in a DP-enriched solubilized protein fraction from Forsythia × intermedia, via adaptation of our initial report of DP solubilization and purification. Because this hybrid species lacks a published genome, de novo sequencing was performed using publicly available transcriptome and genomic data from closely related species. We detected and identified two new DP homologs, which appear to form hetero-trimers. Molecular dynamics simulations suggest that similar hetero-trimers were possible between Arabidopsis DP homologs with comparable sequence similarity. Other identified proteins in the DP-enriched preparation were putatively associated with DP function or the cell wall. Although their co-occurrence after extraction and chromatographic separation is suggestive for components of a protein complex in vivo, none were found to form stable complexes with DPs under the specific experimental conditions we have explored. Nevertheless, our integrated mass spectrometry method development helps prepare for future investigations directed to detect hypothetical LFCs and other related complexes isolated from plant biomass fractionation.

Chlorogenic acid (CGA) and guaiacyl/syringyl (G/S) lignin formation involves hydroxycinnamoyl ester intermediacy, the latter formed via hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferase (HCT) and hydroxycinnamoyl CoA:quinate hydroxycinnamoyl transferase (HQT) activities.

Summary With the development of transcriptomic technologies, we are able to quantify precise changes in gene expression profiles from astronauts and other organisms exposed to spaceflight. Members of NASA GeneLab and GeneLab-associated analysis working groups (AWGs) have developed a consensus pipeline for analyzing short-read RNA-sequencing data from spaceflight-associated experiments. The pipeline includes quality control, read trimming, mapping, and gene quantification steps, culminating in the detection of differentially expressed genes. This data analysis pipeline and the results of its execution using data submitted to GeneLab are now all publicly available through the GeneLab database. We present here the full details and rationale for the construction of this pipeline in order to promote transparency, reproducibility, and reusability of pipeline data; to provide a template for data processing of future spaceflight-relevant datasets; and to encourage cross-analysis of data from other databases with the data available in GeneLab., Graphical abstract, Highlights • Analysis of omics data from different spaceflight studies presents unique challenges • A standardized pipeline for RNA-seq analysis eliminates data processing variation • The GeneLab RNA-seq pipeline includes QC, trimming, mapping, quantification, and DGE • Space-relevant data processed with this pipeline are available at genelab.nasa.gov, Omics; Space Sciences

A series of Wrightia hanleyi extracts was screened for activity against Mycobacterium tuberculosis H37Rv. One active fraction contained a compound that initially appeared to be either the isoflavonoid wrightiadione or the alkaloid tryptanthrin, both of which have been previously reported in other Wrightia species. Characterization by NMR and MS, as well as evaluation of the literature describing these compounds, led to the conclusion that wrightiadione (1) was misidentified in the first report of its isolation from W. tomentosa in 1992 and again in 2015 when reported in W. pubescens and W. religiosa. Instead, the molecule described in these reports and in the present work is almost certainly the isobaric (same nominal mass) and isosteric (same number of atoms, valency, and shape) tryptanthrin (2), a well-known quinazolinone alkaloid found in a variety of plants including Wrightia species. Tryptanthrin (2) is also accessible synthetically via several routes and has been thoroughly characterized. Wrightiadione (1) has been synthesized and characterized and may have useful biological activity; however, this compound can no longer be said to be known to exist in Nature. To our knowledge, this misidentification of wrightiadione (1) has heretofore been unrecognized.

Summary A foundational study assessed effects of biochemical pathway introduction into poplar to produce eugenol, chavicol, p‐anol, isoeugenol and their sequestered storage products, from potentially available substrates, coniferyl and p‐coumaryl alcohols. At the onset, it was unknown whether significant carbon flux to monolignols vs. other phenylpropanoid (acetate) pathway metabolites would be kinetically favoured. Various transgenic poplar lines generated eugenol and chavicol glucosides in ca. 0.45% (~0.35 and ~0.1%, respectively) of dry weight foliage tissue in field trials, as well as their corresponding aglycones in trace amounts. There were only traces of any of these metabolites in branch tissues, even after ~4‐year field trials. Levels of bioproduct accumulation in foliage plateaued, even at the lowest introduced gene expression levels, suggesting limited monolignol substrate availability. Nevertheless, this level still allows foliage collection for platform chemical production, with the remaining (stem) biomass available for wood, pulp/paper and bioenergy product purposes. Several transformed lines displayed unexpected precocious flowering after 4‐year field trial growth. This necessitated terminating (felling) these particular plants, as USDA APHIS prohibits the possibility of their interacting (cross‐pollination, etc.) with wild‐type (native plant) lines. In future, additional biotechnological approaches can be employed (e.g. gene editing) to produce sterile plant lines, to avoid such complications. While increased gene expression did not increase target bioproduct accumulation, the exciting possibility now exists of significantly increasing their amounts (e.g. 10‐ to 40‐fold plus) in foliage and stems via systematic deployment of numerous ‘omics’, systems biology, synthetic biology and metabolic flux modelling approaches.

International audience; This contribution primarily addresses formation of the plant-protecting and human health-protecting lignans found in flax seed coat tissue. Yet, the fundamentals of how plants in general biochemically produce their protective seed coats (protecting the embryo until germination) are very poorly understood at the molecular level, in spite of its massive evolutionary importance. Flax is no exception.Herein, we address the natural product molecular species produced from the “mixed” biochemical pathways to both the flax seed lignans and other metabolite classes associated with same, as well as what is known about their localization in situ and their biosynthesis (proteins and enzymes involved). The biosynthetic pathway known to this point to the plant defense lignans in leaf and stem tissue is also discussed.Discovery of the biochemical steps leading to flax seed coat lignan biosynthesis is described, with a particular emphasis on the genes, dirigent proteins (DPs), downstream pinoresinol-lariciresinol reductases, and secoisolariciresinol dehydrogenase. Advances made thus far from X-ray structural biology investigations, as well as MALDI TOF MS/MS and Ion Mobility metabolite imaging, of these biochemical processes to the lignan components in situ are also summarized.While the predominant health-protecting flax seed coat lignans are secoisolariciresinol diglucoside (SDG) derived, they are essentially not present as such in this tissue. Instead, SDG exists in covalently linked form to other “mixed” biochemical pathway metabolites, of which there are currently eight other distinct natural product entities considered to be involved. The biosynthesis of the other components, such as herbacetin diglucoside and 3-hydroxy-3-methylglutaric acid, is also briefly mentioned. Moreover, our knowledge of the macromolecular structure(s) of these lignan-containing mixed biochemical pathway metabolites during seed coat maturation is briefly summarized.Flaxseed, however, also contains other lignans, such as pinoresinol, pinoresinol diglucoside, isolariciresinol, and matairesinol, albeit in tiny amount, which are released on alkali (base) treatment of flax seed or preparations thereof.The lignan yatein, by comparison, accumulates in flax leaf and stem tissue and appears to be involved in plant defense. What is known about its biochemical pathway and function is summarized.Interestingly, flax DPs are encoded by a 44-membered multi-gene family, of which only a few are of known biochemical/physiological function. Future research is urgently needed to ascertain the in vivo functions of this DP family, including probing the evolutionary ramifications of same and their relevance to successful evolutionary transition of aquatic plants to terrestrial vascular plant form.

International audience; Identification of DIR encoding genes in flax genome. Analysis of phylogeny, gene/protein structures and evolution. Identification of new conserved motifs linked to biochemical functions. Investigation of spatio-temporal gene expression and response to stress. Dirigent proteins (DIRs) were discovered during 8-8′ lignan biosynthesis studies, through identification of stereoselective coupling to afford either (+)- or (−)-pinoresinols from E-coniferyl alcohol. DIRs are also involved or potentially involved in terpenoid, allyl/propenyl phenol lignan, pterocarpan and lignin biosynthesis. DIRs have very large multigene families in different vascular plants including flax, with most still of unknown function. DIR studies typically focus on a small subset of genes and identification of biochemical/physiological functions. Herein, a genome-wide analysis and characterization of the predicted flax DIR 44-membered multigene family was performed, this species being a rich natural grain source of 8-8′ linked secoisolariciresinol-derived lignan oligomers. All predicted DIR sequences, including their promoters, were analyzed together with their public gene expression datasets. Expression patterns of selected DIRs were examined using qPCR, as well as through clustering analysis of DIR gene expression. These analyses further implicated roles for specific DIRs in (−)-pinoresinol formation in seed-coats, as well as (+)-pinoresinol in vegetative organs and/or specific responses to stress. Phylogeny and gene expression analysis segregated flax DIRs into six distinct clusters with new cluster-specific motifs identified. We propose that these findings can serve as a foundation to further systematically determine functions of DIRs, i.e. other than those already known in lignan biosynthesis in flax and other species. Given the differential expression profiles and inducibility of the flax DIR family, we provisionally propose that some DIR genes of unknown function could be involved in different aspects of secondary cell wall biosynthesis and plant defense.

Background Rice Os9BGlu31 is a transglucosidase that can transfer glucose to phenolic acids, flavonoids, and phytohormones. Os9BGlu31 displays a broad specificity with phenolic 1-O-β- d -glucose esters acting as better glucose donors than glucosides, whereas the free phenolic acids of these esters are also excellent acceptor substrates. Methods Based on homology modeling of this enzyme, we made single point mutations of residues surrounding the acceptor binding region of the Os9BGlu31 active site. Products of the wild type and mutant enzymes in transglycosylation of phenolic acceptors from 4-nitrophenyl β- d -glucopyranoside donor were identified and measured by UPLC and negative ion electrospray ionization tandem mass spectrometry (LCMSMS). Results The most active variant produced was W243N, while I172T and L183Q mutations decreased the activity, and other mutations at W243 (A, D, M, N, F and Y) had variable effects, depending on the acceptor substrate. The Os9BGlu31 W243N mutant activity was higher than that of wild type on phenolic acids and kaempferol, a flavonol containing 4 hydroxyl groups, and the wild type Os9BGlu31 produced only a single product from each of these acceptors in significant amounts, while W243 variants produced multiple glucoconjugates. Fragmentation analysis provisionally identified the kaempferol transglycosylation products as kaempferol 3-O, 7-O, and 4'-O glucosides and 3,7-O, 4′,7-O, and 3,4′-O bis-O-glucosides. The Os9BGlu31 W243 mutants were also better able to use kaempferol 3-O-glucoside as a donor substrate. General significance The W243 residue was found to be critical to the substrate and product specificity of Os9BGlu31 transglucosidase and mutation of this residue allows production of a range of glucoconjugates.

Continually exposed to potential pathogens, vascular plants have evolved intricate defense mechanisms to recognize encroaching threats and defend themselves. They do so by inducing a set of defense responses that can help defeat and/or limit effects of invading pathogens, of which the non-host disease resistance response is the most common. In this regard, pea (Pisum sativum) pod tissue, when exposed to Fusarium solani f. sp. phaseoli spores, undergoes an inducible transcriptional activation of pathogenesis-related genes, and also produces (+)-pisatin, its major phytoalexin. One of the inducible pathogenesis-related genes is Disease Resistance Response-206 (DRR206), whose role in vivo was unknown. DRR206 is, however, related to the dirigent protein (DP) family. In this study, its biochemical function was investigated in planta, with the metabolite associated with its gene induction being pinoresinol monoglucoside. Interestingly, both pinoresinol monoglucoside and (+)-pisatin were co-localized in pea pod endocarp epidermal cells, as demonstrated using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging. In addition, endocarp epidermal cells are also the site for both chalcone synthase and DRR206 gene expression. Taken together, these data indicate that both (+)-pisatin and pinoresinol monoglucoside function in the overall phytoalexin responses.

Background: Dirigent protein (DP) discovery gave new paradigm for monolignol-derived coupling in planta. Results: (+)-Pinoresinol-forming DP (PsDRR206) three-dimensional structure was obtained at 1.95 Å resolution. Conclusion: The tightly packed trimeric DP has three putative substrate binding sites spatially far apart, suggesting that each site involves monomer coupling directly. Significance: New insights into monolignol radical-radical coupling in planta., Control over phenoxy radical-radical coupling reactions in vivo in vascular plants was enigmatic until our discovery of dirigent proteins (DPs, from the Latin dirigere, to guide or align). The first three-dimensional structure of a DP ((+)-pinoresinol-forming DP, 1.95 Å resolution, rhombohedral space group H32)) is reported herein. It has a tightly packed trimeric structure with an eight-stranded β-barrel topology for each DP monomer. Each putative substrate binding and orientation coupling site is located on the trimer surface but too far apart for intermolecular coupling between sites. It is proposed that each site enables stereoselective coupling (using either two coniferyl alcohol radicals or a radical and a monolignol). Interestingly, there are six differentially conserved residues in DPs affording either the (+)- or (−)-antipodes in the vicinity of the putative binding site and region known to control stereoselectivity. DPs are involved in lignan biosynthesis, whereas dirigent domains/sites have been implicated in lignin deposition.

A Gordonia species was cultured from soil of a red alder (Alnus rubra) plant. Here we present the assembled and annotated genome sequence to aid investigations into the potential of this organism as a symbiont and comparative studies of the genus Gordonia.

A Gordonia species was cultured from soil of a red alder ( Alnus rubra ) plant. Here we present the assembled and annotated genome sequence to aid investigations into the potential of this organism as a symbiont and comparative studies of the genus Gordonia .

Podophyllum hexandrum and, to a much lesser extent P. peltatum, are sources of podophyllotoxin, extensively used as a chemical scaffold for various anti-cancer drugs. In this study, integrated omics technologies (including advanced mass spectrometry/metabolomics, transcriptome sequencing/gene assemblies, and bioinformatics) gave unequivocal evidence that both plant species possess a hitherto unknown aporphine alkaloid metabolic pathway. Specifically, RNA-seq transcriptome sequencing and bioinformatics guided gene assemblies/analyses in silico suggested presence of transcripts homologous to genes encoding all known steps in aporphine alkaloid biosynthesis. A comprehensive metabolomics analysis, including UPLC-TOF-MS and MALDI-MS imaging in situ, then enabled detection, identification, localization and quantification of the aporphine alkaloids, magnoflorine, corytuberine and muricinine, in the underground and aerial tissues. Interestingly, the purported presence of alkaloids in Podophyllum species has been enigmatic since the 19th century, remaining unresolved until now. The evolutionary and phylogenetic ramifications of this discovery are discussed.

The creosote bush (Larrea tridentata) harbors members of the monolignol acyltransferase, allylphenol synthase, and propenylphenol synthase gene families, whose products together are able to catalyze distinct regiospecific conversions of various monolignols into their corresponding allyl- and propenyl-phenols, respectively. In this study, co-expression of a monolignol acyltransferase with either substrate versatile allylphenol or propenylphenol synthases in Escherichia coli established that various monolignol substrates were efficiently converted into their corresponding allyl/propenyl phenols, as well as providing proof of concept for efficacious conversion in a bacterial platform. This capability thus potentially provides an alternate source to these important plant phytochemicals, whether for flavor/fragrance and fine chemicals, or ultimately as commodities, e.g., for renewable energy or other intermediate chemical purposes. Previous reports had indicated that specific and highly conserved amino acid residues 84 (Phe or Val) and 87 (Ile or Tyr) of two highly homologous allyl/propenyl phenol synthases (circa 96% identity) from a Clarkia species mainly dictate their distinct regiospecific catalyzed conversions to afford either allyl- or propenyl-phenols, respectively. However, several other allyl/propenyl phenol synthase homologs isolated by us have established that the two corresponding amino acid 84 and 87 residues are not, in fact, conserved.

We investigate the performance and scalability of the randomized CX low-rank matrix factorization and demonstrate its applicability through the analysis of a 1TB mass spectrometry imaging (MSI) dataset, using Apache Spark on an Amazon EC2 cluster, a Cray XC40 system, and an experimental Cray cluster. We implemented this factorization both as a parallelized C implementation with hand-tuned optimizations and in Scala using the Apache Spark high-level cluster computing framework. We obtained consistent performance across the three platforms: using Spark we were able to process the 1TB size dataset in under 30 minutes with 960 cores on all systems, with the fastest times obtained on the experimental Cray cluster. In comparison, the C implementation processed the 1TB size dataset 21X faster on the Amazon EC2 system, due to careful cache optimizations, bandwidth-friendly access of matrices and vector computation using SIMD units. We report these results and their implications on the hardware and software issues arising in supporting data-centric workloads in parallel and distributed environments.

Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula × Populus alba), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below approximately 50% of the nontransgenic control. Relatively small lignin reductions of approximately 10% were associated with reduced productivity, decreased wood syringyl/guaiacyl lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than approximately 50% 4CL RNA expression were characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees.

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had k(cat)/Km values of 1050, 7650, and 1560 M(-1) S(-1) for arogenate versus 38, 240, and 16 M(-1) S(-1) for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k(cat)/Km values of 1140, 490, and 620 M(-1) S(-1), with prephenate not serving as a substrate unless excess recombinant protein (>150 microg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6.

© 2015 The American Chemical Society and American Society of Pharmacognosy. An integrated omics approach using genomics, transcriptomics, metabolomics (MALDI mass spectrometry imaging, MSI), and bioinformatics was employed to study spatiotemporal formation and deposition of health-protecting polymeric lignans and plant defense cyanogenic glucosides. Intact flax (Linum usitatissimum) capsules and seed tissues at different development stages were analyzed. Transcriptome analyses indicated distinct expression patterns of dirigent protein (DP) gene family members encoding (-)- and (+)-pinoresinol-forming DPs and their associated downstream metabolic processes, respectively, with the former expressed at early seed coat development stages. Genes encoding (+)-pinoresinol-forming DPs were, in contrast, expressed at later development stages. Recombinant DP expression and DP assays also unequivocally established their distinct stereoselective biochemical functions. Using MALDI MSI and ion mobility separation analyses, the pinoresinol downstream derivatives, secoisolariciresinol diglucoside (SDG) and SDG hydroxymethylglutaryl ester, were localized and detectable only in early seed coat development stages. SDG derivatives were then converted into higher molecular weight phenolics during seed coat maturation. By contrast, the plant defense cyanogenic glucosides, the monoglucosides linamarin/lotaustralin, were detected throughout the flax capsule, whereas diglucosides linustatin/neolinustatin only accumulated in endosperm and embryo tissues. A putative biosynthetic pathway to the cyanogens is proposed on the basis of transcriptome coexpression data. Localization of all metabolites was at ca. 20 μm resolution, with the web based tool OpenMSI enabling not only resolution enhancement but also an interactive system for real-time searching for any ion in the tissue under analysis.