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Two pentacyclic triterpenes, D-friedomadeir-14-en-3β-yl acetate and D:C-friedomadeir-7-en-3β-yl acetate, named madeiranyl acetate and isomadeiranyl acetate, respectively, were isolated from leaves of Euphorbia stygiana, together with the two known madeiranes, D-friedomadeir-14-en-3-one and D:C-friedomadeir-7-en-3-one, which were obtained from the stem bark. In addition, four known lupane and taraxerane-type triterpenes, namely lupenyl acetate, lupenone, taraxeryl acetate and taraxerone, were also isolated from the same source. Structures were elucidated by physical, chemical and spectroscopic methods (1H NMR, 13C NMR, IR and mass spectra) and by comparison with literature data, and in the case of D:C-friedomadeir-7-en-3β-yl acetate by X-ray analysis as well. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

How stereoselective monolignol-derived phenoxy radicalradical coupling reactions are differentially biochemically orchestrated in planta, whereby for example they afford (+)- and (-)-pinoresinols, respectively, is both a fascinating mechanistic and evolutionary question. In earlier work, biochemical control of (+)-pinoresinol formation had been established to be engendered by a (+)-pinoresinol-forming dirigent protein in Forsythia intermedia, whereas the presence of a (-)-pinoresinol- forming dirigent protein was indirectly deduced based on the enantiospecificity of downstream pinoresinol reductases (AtPrRs) in Arabidopsis thaliana root tissue. In this study of 16 putative dirigent protein homologs in Arabidopsis, AtDIR6, AtDIR10, and AtDIR13 were established to be root-specific using a β-glucuronidase reporter gene strategy. Of these three, in vitro analyses established that only recombinant AtDIR6 was a (-)-pinoresinol-forming dirigent protein, whose physiological role was further confirmed using overexpression and RNAi strategies in vivo. Interestingly, its closest homolog, AtDIR5, was also established to be a (-)-pinoresinol-forming dirigent protein based on in vitro biochemical analyses. Both of these were compared in terms of properties with a (+)-pinoresinolforming dirigent protein from Schizandra chinensis. In this context, sequence analyses, site-directed mutagenesis, and region swapping resulted in identification of putative substrate binding sites/regions and candidate residues controlling distinct stereoselectivities of coupling modes. [ABSTRACT FROM AUTHOR]

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 Arabiclopsis, 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 ADTI (Atlgl1790), ADT2 (At3g07630), and ADT6 (Atlg08250), more efficiently utilized arogeuate than prephenate, whereas the remaining three, ADT3 (At2927820), ADT4 (At3944720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had kcat/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 kcat/Km values of 1140, 490, and 620 M-1 s-1, with prephenate not serving as a substrate unless excess recombinant protein (>150 μg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6. [ABSTRACT FROM AUTHOR]

In this study, we determined the crystal structures of the apo-form, binary, and ternary complexes of the Arabidopsis alkenal double bond reductase encoded by AtSg16970. This protein, one of 11 homologues in Arabidopsis thaliana, is most closely related to the Pinus taeda phenyipropenal double bond reductase, involved in, for example, heartwood formation. Both enzymes also have essential roles in plant defense, and can function by catalyzing the reduction of the 7-8-double bond of phenylpropanal substrates, such as p-coumaryl and coniferyl aldehydes in vitro. At5g16970 is also capable of reducing toxic substrates with the same alkenal functionality, such as 4-hydroxy-(2E)-nonenal. The overall fold of At5g16970 is similar to that of the zinc-independent medium chain dehydrogenase/reductase superfamily, the members of which have two domains and are dimeric in nature, i.e. in contrast to their original classification as being zinc-containing oxidoreductases. As provisionally anticipated from the kinetic data, the shape of the binding pocket can readily accommodate p-coumaryl aldehyde, coniferyl aldehyde, 4-hydroxy-(2E)-nonenal, and 2-alkenals. However, the enzyme kinetic data among these potential substrates differ, favoring p-coumaryl aldehyde. Tyr-260 is provisionally proposed to function as a general acid/base for hydride transfer. A catalytic mechanism for this reduction, and its applicability to related important detoxification mammalian proteins, is also proposed. [ABSTRACT FROM AUTHOR]

Abstract: A recent in silico analysis revealed that the Arabidopsis genome has 14 genes annotated as putative 4-coumarate:CoA ligase isoforms or homologues. Of these, 11 were selected for detailed functional analysis in vitro, using all known possible phenylpropanoid pathway intermediates (p-coumaric, caffeic, ferulic, 5-hydroxyferulic and sinapic acids), as well as cinnamic acid. Of the 11 recombinant proteins so obtained, four were catalytically active in vitro, with fairly broad substrate specificities, confirming that the 4CL gene family in Arabidopsis has only four members. This finding is in agreement with our previous phylogenetic analyses, and again illustrates the need for comprehensive characterization of all putative 4CLs, rather than piecemeal analysis of selected gene members. All 11 proteins were expressed with a C-terminal His6-tag and functionally characterized, with one, At4CL1, expressed in native form for kinetic property comparisons. Of the 11 putative His6-tagged 4CLs, isoform At4CL1 best utilized p-coumaric, caffeic, ferulic and 5-hydroxyferulic acids as substrates, whereas At4CL2 readily transformed p-coumaric and caffeic acids into the corresponding CoA esters, while ferulic and 5-hydroxyferulic acids were converted quite poorly. At4CL3 also displayed broad substrate specificity efficiently converting p-coumaric, caffeic and ferulic acids into their CoA esters, whereas 5-hydroxyferulic acid was not as effectively utilized. By contrast, while At4CL5 is the only isoform capable of ligating sinapic acid, the two preferred substrates were 5-hydroxyferulic and caffeic acids. Indeed, both At4CL1 and At4CL5 most effectively utilized 5-hydroxyferulic acid with k enz ∼10-fold higher than that for At4CL2 and At4CL3. The remaining seven 4CL-like homologues had no measurable catalytic activity (at ∼100μg protein concentrations), again bringing into sharp focus both the advantages to, and the limitations of, current database annotations, and the need to unambiguously demonstrate true enzyme function. Lastly, although At4CL5 is able to convert both 5-hydroxyferulic and sinapic acids into the corresponding CoA esters, the physiological significance of the latter observation in vitro was in question, i.e. particularly since other 4CL isoforms can effectively convert 5-hydroxyferulic acid into 5-hydroxyferuloyl CoA. Hence, homozygous lines containing T-DNA or enhancer trap inserts (knockouts) for 4cl5 were selected by screening, with Arabidopsis stem sections from each mutant line subjected to detailed analyses for both lignin monomeric compositions and contents, and sinapate/sinapyl alcohol derivative formation, at different stages of growth and development until maturation. The data so obtained revealed that this “knockout” had no significant effect on either lignin content or monomeric composition, or on the accumulation of sinapate/sinapyl alcohol derivatives. The results from the present study indicate that formation of syringyl lignins and sinapate/sinapyl alcohol derivatives result primarily from methylation of 5-hydroxyferuloyl CoA or derivatives thereof rather than sinapic acid ligation. That is, no specific physiological role for At4CL5 in direct sinapic acid CoA ligation could be identified. How the putative overlapping 4CL metabolic networks are in fact organized in planta at various stages of growth and development will be the subject of future inquiry. [Copyright &y& Elsevier]

Pea phytoalexins (-)-maackiain and (+)-pisatin have opposite C6a/C11a configurations, but biosynthetically how this occurs is unknown. Pea dirigent-protein (DP) PsPTS2 generates 7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene (DMDIF), and stereoselectivity toward four possible 7,2'-dihydroxy-4',5'-methylenedioxyisoflavan-4-ol (DMDI) stereoisomers was investigated. Stereoisomer configurations were determined using NMR spectroscopy, electronic circular dichroism, and molecular orbital analyses. PsPTS2 efficiently converted cis-(3R,4R)-DMDI into DMDIF 20-fold faster than the trans-(3R,4S)-isomer. The 4R-configured substrate's near β-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4S-isomer's α-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4R-configured β-axial OH was closest to Asp 51 , whereas 4S-isomer's α-equatorial OH was further away. Neither cis-(3S,4S)- nor trans-(3S,4R)-DMDIs were substrates, even with the former having C3/C4 stereochemistry as in (+)-pisatin. PsPTS2 used cis-(3R,4R)-7,2'-dihydroxy-4'-methoxyisoflavan-4-ol [cis-(3R,4R)-DMI] and C3/C4 stereoisomers to give 2',7-dihydroxy-4'-methoxyisoflav-3-ene (DMIF). DP homologs may exist in licorice (Glycyrrhiza pallidiflora) and tree legume Bolusanthus speciosus, as DMIF occurs in both species. PsPTS1 utilized cis-(3R,4R)-DMDI to give (-)-maackiain 2200-fold more efficiently than with cis-(3R,4R)-DMI to give (-)-medicarpin. PsPTS1 also slowly converted trans-(3S,4R)-DMDI into (+)-maackiain, reflecting the better 4R configured OH leaving group. PsPTS2 and PsPTS1 provisionally provide the means to enable differing C6a and C11a configurations in (+)-pisatin and (-)-maackiain, via identical DP-engendered mono-QM bound intermediate generation, which PsPTS2 either re-aromatizes to give DMDIF or PsPTS1 intramolecularly cyclizes to afford (-)-maackiain. Substrate docking simulations using PsPTS2 and PsPTS1 indicate cis-(3R,4R)-DMDI binds in the anti-configuration in PsPTS2 to afford DMDIF, and the syn-configuration in PsPTS1 to give maackiain., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: Conflicts of interest N.G.L. is the president of Ealasid, Inc. which has propagated red alder Clone 639 through a licensing agreement with WSU. All other authors declare no competing financial interests., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Spaceflight presents a multifaceted environment for plants, combining the effects on growth of many stressors and factors including altered gravity, the influence of experiment hardware, and increased radiation exposure. To help understand the plant response to this complex suite of factors this study compared transcriptomic analysis of 15 Arabidopsis thaliana spaceflight experiments deposited in the National Aeronautics and Space Administration's GeneLab data repository. These data were reanalyzed for genes showing significant differential expression in spaceflight versus ground controls using a single common computational pipeline for either the microarray or the RNA-seq datasets. Such a standardized approach to analysis should greatly increase the robustness of comparisons made between datasets. This analysis was coupled with extensive cross-referencing to a curated matrix of metadata associated with these experiments. Our study reveals that factors such as analysis type (i.e., microarray versus RNA-seq) or environmental and hardware conditions have important confounding effects on comparisons seeking to define plant reactions to spaceflight. The metadata matrix allows selection of studies with high similarity scores, i.e., that share multiple elements of experimental design, such as plant age or flight hardware. Comparisons between these studies then helps reduce the complexity in drawing conclusions arising from comparisons made between experiments with very different designs., (© 2023. The Author(s).)

Aquatic plant transition to land, and subsequent terrestrial plant species diversification, was accompanied by the emergence and massive elaboration of plant phenol chemo-diversity. Concomitantly, dirigent protein (DP) and dirigent-like protein subfamilies, derived from large multigene families, emerged and became extensively diversified. DP biochemical functions as gateway entry points into new and diverse plant phenol skeletal types then markedly expanded. DPs have at least eight non-uniformly distributed subfamilies, with different DP subfamily members of known biochemical/physiological function now implicated as gateway entries to lignan, lignin, aromatic diterpenoid, pterocarpan and isoflavene pathways. While some other DP subfamily members have jacalin domains, both these and indeed the majority of DPs throughout the plant kingdom await discovery of their biochemical roles. Methods and approaches were developed to discover DP biochemical function as gateway entry points to distinct plant phenol skeletal types in land plants. Various DP 3D X-ray structural determinations enabled structure-based comparative sequence analysis and modeling to understand similarities and differences among the different DP subfamilies. We consider that the core DP β-barrel fold and associated characteristics are likely common to all DPs, with several residues conserved and nearly invariant. There is also considerable variation in residue composition and topography of the putative substrate binding pockets, as well as substantial differences in several loops, such as the β1-β2 loop. All DPs likely bind and stabilize quinone methide intermediates, while guiding distinctive regio- and/or stereo-chemical entry into Nature's chemo-diverse land plant phenol metabolic classes., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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, presumably with different functions. Some known DPs require other essential enzymes/proteins ( e.g. oxidases) for their functions. However, the lack of a fully sequenced genome for Forsythia × intermedia made it difficult to profile other components co-purified with the (+)-pinoresinol forming DP. Herein, we used an integrated bottom-up, top-down, and native mass spectrometry (MS) approach to de novo sequence the extracted proteins via adaptation of our initial report of DP solubilization and purification. Using publicly available transcriptome and genomic data from closely related species, we identified 14 proteins that were putatively associated with either DP function or the cell wall. Although their co-occurrence after extraction and chromatographic separation is suggestive for potential protein-protein interactions, none were found to form stable protein complexes with DPs in native MS under the specific experimental conditions we have explored. Interestingly, two new DP homologs were found and they formed hetero-trimers. Molecular dynamics simulations suggested that similar hetero-trimers were possible between Arabidopsis DP homologs with comparable sequence similarities. Nevertheless, our integrated mass spectrometry method development helped prepare for future investigations directed to the discovery of novel proteins and protein-protein interactions. These advantages can be highly beneficial for plant and microbial research where fully sequenced genomes may not be readily available.

Multiple Arabidopsis arogenate dehydratase ( ADT ) knock-out (KO) mutants, with phenotypes having variable lignin levels (up to circa 70% reduction), were studied to investigate how differential reductions in ADTs perturb its overall plant systems biology. Integrated "omics" analyses (metabolome, transcriptome, and proteome) of wild type (WT), single and multiple ADT KO lines were conducted. Transcriptome and proteome data were collapsed into gene ortholog (GO) data, with this allowing for enzymatic reaction and metabolome cross-comparisons to uncover dominant or likely metabolic biosynthesis reactions affected. Network analysis of enzymes-highly correlated to stem lignin levels-deduced the involvement of novel putative lignin related proteins or processes. These included those associated with ribosomes, the spliceosome, mRNA transport, aminoacyl tRNA biosynthesis, and phosphorylation. While prior work helped explain lignin biosynthesis regulation at the transcriptional level, our data here provide support for a new hypothesis that there are additional post-transcriptional and translational level processes that need to be considered. These findings are anticipated to lead to development of more accurate depictions of lignin/phenylpropanoid biosynthesis models in situ , with new protein targets identified for further biochemical analysis and/or plant bioengineering. Additionally, using KEGG defined functional categorization of proteomics and transcriptomics analyses, we detected significant changes to glucosinolate, α-linolenic acid, nitrogen, carotenoid, aromatic amino acid, phenylpropanoid, and photosynthesis-related metabolic pathways in ADT KO mutants. Metabolomics results also revealed that putative carotenoid and galactolipid levels were generally increased in amount, whereas many glucosinolates and phenylpropanoids (including flavonoids and lignans) were decreased in the KO mutants., 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., (Copyright © 2021 Hixson, Marques, Wendler, McDermott, Weitz, Clauss, Monroe, Moore, Brown, Lipton, Bell, Paša-Tolić, Davin and Lewis.)

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 RNA i 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. RNA i 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.

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., Competing Interests: The authors declare no competing interests.

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., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Meng et al.)

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., (© 2020 Wiley Periodicals, Inc.)

Key Message: 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.

Arogenate dehydratase (ADT) catalyzes the final step of phenylalanine (Phe) biosynthesis. Previous work showed that ADT-deficient Arabidopsis ( Arabidopsis thaliana ) mutants had significantly reduced lignin contents, with stronger reductions in lines that had deficiencies in more ADT isoforms. Here, by analyzing Arabidopsis ADT mutants using our phenomics facility and ultra-performance liquid chromatography-mass spectrometry-based metabolomics, we describe the effects of the modulation of ADT on photosynthetic parameters and secondary metabolism. Our data indicate that a reduced carbon flux into Phe biosynthesis in ADT mutants impairs the consumption of photosynthetically produced ATP, leading to an increased ATP/ADP ratio, the overaccumulation of transitory starch, and lower electron transport rates. The effect on electron transport rates is caused by an increase in proton motive force across the thylakoid membrane that down-regulates photosystem II activity by the high-energy quenching mechanism. Furthermore, quantitation of secondary metabolites in ADT mutants revealed reduced flavonoid, phenylpropanoid, lignan, and glucosinolate contents, including glucosinolates that are not derived from aromatic amino acids, and significantly increased contents of putative galactolipids and apocarotenoids. Additionally, we used real-time atmospheric monitoring mass spectrometry to compare respiration and carbon fixation rates between the wild type and adt3/4/5/6 , our most extreme ADT knockout mutant, which revealed no significant difference in both night- and day-adapted plants. Overall, these data reveal the profound effects of altered ADT activity and Phe metabolism on secondary metabolites and photosynthesis with implications for plant improvement., (© 2018 American Society of Plant Biologists. All Rights Reserved.)

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.

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., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We report the development and testing of an accurate mass-time (AMT) tag approach for the LC/MS-based identification of plant natural products (PNPs) in complex extracts. An AMT tag library was developed for approximately 500 PNPs with diverse chemical structures, detected in electrospray and atmospheric pressure chemical ionization modes (both positive and negative polarities). In addition, to enable peak annotations with high confidence, MS/MS spectra were acquired with three different fragmentation energies. The LC/MS and MS/MS data sets were integrated into online spectral search tools and repositories (Spektraris and MassBank), thus allowing users to interrogate their own data sets for the potential presence of PNPs. The utility of the AMT tag library approach is demonstrated by the detection and annotation of active principles in 27 different medicinal plant species with diverse chemical constituents., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Phe is formed from arogenate in planta through the action of arogenate dehydratase (ADT), and there are six ADT isoenzymes in the "model" vascular plant species Arabidopsis thaliana. This raised the possibility that specific ADTs may be differentially regulated so as to control Phe biosynthesis for protein synthesis vs its much more massive deployment for phenylpropanoid metabolism. In our previous reverse genetics study using 25 single/multiple ADT knockout (KO) lines, a subset of these knockouts was differentially reduced in their lignin contents. In the current investigation, it was hypothesized that Phe pool sizes might correlate well with reduction in lignin contents in the affected KO lines. The free amino acid contents of these KO lines were thus comprehensively analyzed in stem, leaf and root tissues, over a growth/developmental time course from 3 to 8 weeks until senescence. The data obtained were then compared to, and contrasted with, the differential extent of lignin deposition occurring in the various lines. Relative changes in pool sizes were also analyzed by performing a pairwise confirmatory factor analysis for Phe:Tyr, Phe:Trp and Tyr:Trp, following determination of the deviation from the mean for Phe, Tyr and Trp in each plant line. It was found that the Phe pool sizes measured were differentially reduced only in lignin-deficient lines, and in tissues and at time points where lignin biosynthesis was constitutively highly active (in wild type lines) under the growth conditions employed. In contrast, this trend was not evident across all ADT KO lines, possibly due to maintenance of Phe pools by non-targeted isoenzymes, or by feedback mechanisms known to be in place., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Single and multiple T-DNA knockouts of genes encoding arogenate dehydratases (ADTs) in Arabidopsis were obtained in homozygous form. These were analyzed for potential differences in lignin contents and compositions, as well as for distinct phenotypes over growth and development. Of these different lines, distinct reductions in lignin contents were obtained, with those having different G:S ratios depending upon the combination of ADT genes being knocked out. Results from pyrolysis GC/MS analyses indicated that differential carbon flux occurred into the vascular bundles (vb) and interfascicular fibers (if). These results provide additional new insight into factors controlling lignin heterogeneity and configuration.

While laccases, multi-copper glycoprotein oxidases, are often able to catalyze oxidation of a broad range of substrates, such as phenols and amines in vitro, their precise physiological/biochemical roles in higher plants remain largely unclear, e.g., Arabidopsis thaliana contains 17 laccases with only 1 having a known physiological function. To begin to explore their roles in planta, spatial and temporal expression patterns of Arabidopsis laccases were compared and contrasted in different tissues at various development stages using RT-PCR and promoter-GUS fusions. Various cell-specific expressions were noted where specific laccases were uniquely expressed, such as LAC4 in interfascicular fibers and seed coat columella, LAC7 in hydathodes and root hairs, LAC8 in pollen grains and phloem, and LAC15 in seed coat cell walls. Such specific cell-type expression patterns provide new leads and/or strategies into determining their precise physiological/biochemical roles. In addition, there was an apparent redundancy of gene expression patterns for several laccases across a wide variety of tissues, lignified and non-lignified, perhaps indicative of overlapping function(s). Preliminary evidence, based on bioinformatics analyses, suggests that most laccases may also be tightly regulated at both transcriptional (antisense transcripts, histone and DNA methylation) and posttranscriptional (microRNAs) levels of gene expression.

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.

Western red cedar (Thuja plicata) heartwood contains abundant amounts of structurally complex plicatic acid-derived lignans that help confer protective properties and longevity to this tissue type. Although the lignan biochemical entry point is dirigent protein-mediated, the formation of heartwood and its associated lignans in some species remains poorly understood due to technical difficulties of working with the former. To begin to address such questions, this study therefore focused on the anatomical localization of dirigent protein and 18s rRNA (control) gene transcripts within recalcitrant woody tissues, including heartwood. This in situ mRNA hybridization approach enabled detection of dirigent protein transcripts in cork cambia, vascular cambia and ray parenchyma cells of the sapwood, but not the heartwood under the conditions employed. By contrast, the hybridization of the 18s rRNA (control) transcript resulted in its detection in all tissue types, including radial parenchyma cells of apparently preformed heartwood. Application of in situ hybridization to such recalcitrant tissues thus demonstrates the utility of this technique in identifying specific cell types involved in heartwood formation, as well as the relationship of dirigent protein localization to that of heartwood metabolite generation.

The lignin deficient double mutant of cinnamyl alcohol dehydrogenase (CAD, cad-4, cad-5 or cad-c, cad-d) in Arabidopsis thaliana [Sibout, R., Eudes, A., Mouille, G., Pollet, B., Lapierre, C., Jouanin, L., Séguin, A., 2005. Cinnamyl alcohol dehydrogenase-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell 17, 2059-2076], was comprehensively examined for effects on disruption of native lignin macromolecular configuration; the two genes encode the catalytically most active CAD's for monolignol/lignin formation [Kim, S.-J., Kim, M.-R., Bedgar, D.L., Moinuddin, S.G.A., Cardenas, C.L., Davin, L.B., Kang, C., Lewis, N.G., 2004. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc. Natl. Acad. Sci., USA 101, 1455-1460]. The inflorescence stems of the double mutant presented a prostrate phenotype with dynamic modulus properties greatly reduced relative to that of the wild type (WT) line due to severe reductions in macromolecular lignin content. Interestingly, initially the overall pattern of phenolic deposition in the mutant was apparently very similar to WT, indicative of comparable assembly processes attempting to be duplicated. However, shortly into the stage involving (monomer cleavable) 8-O-4' linkage formation, deposition was aborted. At this final stage, the double mutant had retained a very limited ability to biosynthesize monolignols as evidenced by cleavage and release of ca. 4% of the monolignol-derived moieties relative to the lignin of the WT line. In addition, while small amounts of cleavable p-hydroxycinnamaldehyde-derived moieties were released, the overall frequency of (monomer cleavable) 8-O-4' inter-unit linkages closely approximated that of WT for the equivalent level of lignin deposition, in spite of the differences in monomer composition. Additionally, 8-5' linked inter-unit structures were clearly evident, albeit as fully aromatized phenylcoumaran-like substructures. The data are interpreted as a small amount of p-hydroxycinnamaldehydes being utilized in highly restricted attempts to preserve native lignin configuration, i.e. through very limited monomer degeneracy during template polymerization which would otherwise afford lignins proper in the cell wall from their precursor monolignols. The defects introduced (e.g. in the vascular integrity) provide important insight as to why p-hydroxycinnamaldehydes never evolved as lignin precursors in the 350,000 or so extant vascular plant species. It is yet unknown at present, however, as to what levels of lignin reduction can be attained in order to maintain the requisite properties for successful agronomic/forestry cultivation. Nor is it known to what extent, if any, such deleterious modulations potentially compromise plant defenses. Finally, prior to investigating lignin primary structure proper, it is essential to initially define the fundamental characteristics of the biopolymer(s) being formed, such as inter-unit frequency and lignin content, in order to design approaches to determine overall sequences of linkages.

A major goal currently in Arabidopsis research is determination of the (biochemical) function of each of its approximately 27,000 genes. To date, however, 12% of its genes actually have known biochemical roles. In this study, we considered it instructive to identify the gene expression patterns of nine (so-called AtCAD1-9) of 17 genes originally annotated by The Arabidopsis Information Resource (TAIR) as cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) homologues [see Costa, M.A., Collins, R.E., Anterola, A.M., Cochrane, F.C., Davin, L.B., Lewis N.G., 2003. An in silico assessment of gene function and organization of the phenylpropanoid pathway metabolic networks in Arabidopsis thaliana and limitations thereof. Phytochemistry 64, 1097-1112.]. In agreement with our biochemical studies in vitro [Kim, S.-J., Kim, M.-R., Bedgar, D.L., Moinuddin, S.G.A., Cardenas, C.L., Davin, L.B., Kang, C.-H., Lewis, N.G., 2004. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc. Natl. Acad. Sci. USA 101, 1455-1460.], and analysis of a double mutant [Sibout, R., Eudes, A., Mouille, G., Pollet, B., Lapierre, C., Jouanin, L., Séguin A., 2005. Cinnamyl Alcohol Dehydrogenase-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell 17, 2059-2076.], both AtCAD5 (At4g34230) and AtCAD4 (At3g19450) were found to have expression patterns consistent with development/formation of different forms of the lignified vascular apparatus, e.g. lignifying stem tissues, bases of trichomes, hydathodes, abscission zones of siliques, etc. Expression was also observed in various non-lignifying zones (e.g. root caps) indicative of, perhaps, a role in plant defense. In addition, expression patterns of the four CAD-like homologues were investigated, i.e. AtCAD2 (At2g21730), AtCAD3 (At2g21890), AtCAD7 (At4g37980) and AtCAD8 (At4g37990), each of which previously had been demonstrated to have low CAD enzymatic activity in vitro (relative to AtCAD4/5) [Kim, S.-J., Kim, M.-R., Bedgar, D.L., Moinuddin, S.G.A., Cardenas, C.L., Davin, L.B., Kang, C.-H., Lewis, N.G., 2004. Functional reclassification of the putative cinnamyl alcohol dehydrogenase multigene family in Arabidopsis. Proc. Natl. Acad. Sci. USA 101, 1455-1460.]. Neither AtCAD2 nor AtCAD3, however, were expressed in lignifying tissues, with the latter being found mainly in the meristematic region and non-lignifying root tips, i.e. indicative of involvement in biochemical processes unrelated to lignin formation. By contrast, AtCAD7 and AtCAD8 [surprisingly now currently TAIR-annotated as probable mannitol dehydrogenases, but for which there is still no biochemical or other evidence for same] displayed gene expression patterns largely resembling those of AtCAD4/5, i.e. indicative perhaps of a quite minor role in monolignol/lignin formation. Lastly, AtCAD1 (At1g72680), AtCAD6 (At4g37970) and AtCAD9 (At4g39330), which lacked detectable CAD catalytic activities in vitro, were also expressed predominantly in vascular (lignin-forming) tissues. While their actual biochemical roles remain unknown, definition of their expression patterns, nevertheless, now begins to provide useful insights into potential biochemical/physiological functions, as well as the cell types in which they are expressed. These data thus indicate that the CAD metabolic network is composed primarily of AtCAD4/5 and may provisionally, to a lesser extent, involve AtCAD7/8 based on in vitro catalytic properties and (promoter regions selected to obtain) representative gene expression patterns. This analysis has, therefore, enabled us to systematically map out bona fide CAD gene involvement in both the assembly and differential emergence of the various component parts of the lignified vascular apparatus in Arabidopsis, as well as those having other (e.g. putative plant defense) functions. The data obtained also further underscore the ongoing difficulties and challenges as regards current limitations in gene annotations versus actual determination of gene function. This is exemplified by the annotation of AtCAD2, 3 and 6-9 as purported mannitol dehydrogenases, when, for example, no in vitro studies have been carried out to establish such a function biochemically. Such annotations should thus be discontinued in the absence of reliable biochemical and/or other physiological confirmation. In particular, AtCAD2, 3, 6 and 9 should be designated as dehydrogenases of unknown function. Just as importantly, the different patterns of gene expression noted during distinct phases of growth and development in specific cells/tissues gives insight into the study of the roles that these promoters have.

To our knowledge, xylary reaction tissue has never been reported in a forage crop species. Here we report the discovery of reaction tissue in a transgenic line of Medicago sativa (pC3H, for the gene for p-coumarate-3-hydroxylase) with reduced lignin content and in the wild-type (WT) line. Based on microscopy and biomechanical testing of internodal alfalfa branch sections, the transgenic (pC3H-I) line, relative to the WT (1) apparently formed more reaction tissue containing gelatinous fibers with adjacent thick-walled fibers (presumed to be "intermediate" tissue) more rapidly, (2) had more xylem tissue, and (3) had comparable tensile dynamic modulus properties. These findings thus establish the (limited) ability of this perennial angiosperm to form (inducible) reaction tissue in a manner somewhat analogous to that of woody arborescent angiosperms. The potential of effectuating reductions in lignin amounts in (woody) angiosperms with increased formation of reaction (tension wood) tissue is discussed because reaction tissues are often viewed as a deleterious trait in processing for many agronomic/industrial applications, especially with the current interest in biofuels.

Previous studies have indicated that the Arabidopsis thalianairregular xylem 4 (irx4) mutant is severely lignin-deficient, forming abnormal lignin from aberrant monomers. Studies of lignin structure in dwarfed cinnamoyl CoA reductase (CCR)-downregulated tobacco were also previously reported to incorporate feruloyl tyramine derivatives. The lignin in the Arabidopsis irx4 mutant was re-investigated at 6 weeks and at maturation (9 weeks). Application of (1)H, (13)C, 2D Heteronuclear Multiple Quantum Coherence and 2D Heteronuclear Multiple Bond Coherence spectroscopic analyses to the lignin-enriched isolates from both Arabidopsis wild-type (Ler) and the CCR-irx4 mutant at both developmental stages revealed that only typical guaiacyl/syringyl lignins were formed. For the irx4 mutant, the syringyl content at 6 weeks growth was lower, in accordance with a delayed but coherent program of lignification. At maturation, however, the syringyl/guaiacyl ratio of the irx4 mutant approached that of wild-type. There was no evidence for feruloyl tyramines, or homologues thereof, accumulating as a chemical signature in lignins resulting from CCR mutation. Nor were there any noticeable increases in other phenolic components, such as hydroxycinnamic acids. These findings were further confirmed by application of thioacidolysis, alkaline nitrobenzene oxidation and acetyl bromide analyses. Moreover, in the case of CCR downregulation in tobacco, there were no NMR spectroscopic correlations that demonstrated feruloyl tyramines being incorporated into the lignin biopolymers. This study thus found no evidence that abnormal lignin formation occurs when CCR activity is modulated.

Propenyl- and allyl-phenols, such as methylchavicol, p-anol and eugenol, have gained importance as flavoring agents and also as putative precursors in the biosynthesis of 9,9'-deoxygenated lignans, many of which have potential medicinal applications. In spite of several decades of investigation, however, the complete biosynthetic pathway to a propenyl/allylphenol had not yet been reported. We have subjected a Thai basil variety accumulating relatively large amounts of the simplest volatile allylphenol, methylchavicol, to in vivo administration of radiolabeled precursors and assays of protein preparations in vitro. Through these experiments, the biosynthesis of chavicol was shown to occur via the phenylpropanoid pathway to p-coumaryl alcohol. Various possibilities leading to deoxygenation of the latter were examined, including reduction of the side-chain double bond to form p-dihydrocoumaryl alcohol, followed by dehydration to afford chavicol, as well as formation of p-methoxycinnamyl alcohol, with further side-chain modification to afford methylchavicol. A third possibility studied was activation of the side-chain alcohol of p-coumaryl alcohol, e.g.via esterification, to form a more facile leaving group via reductive elimination. The latter was shown to be the case using p-coumaryl esters as potential substrates for a NAD(P)H-dependent reductase to afford chavicol, which is then O-methylated to afford methylchavicol.

Secoisolariciresinol dehydrogenase (SDH) catalyzes the NAD+ dependent enantiospecific conversion of secoisolariciresinol into matairesinol. In Podophyllum species, (-)-matairesinol is metabolized into the antiviral compound, podophyllotoxin, which can be semi-synthetically converted into the anticancer agents, etoposide, teniposide and Etopophos. Matairesinol is also a precursor of the cancer-preventative "mammalian" lignan, enterolactone, formed in the gut following ingestion of, for example, various high fiber dietary foods, as well as being an intermediate to numerous defense compounds in vascular plants. This study investigated the mode of enantiospecific Podophyllum SDH catalysis, the order of binding, and the stereospecificity of hydride abstraction/transfer from secoisolariciresinol to NAD+. SDH contains a highly conserved catalytic triad (Ser153, Tyr167 and Lys171), whose activity was abolished with site-directed mutagenesis of Tyr167Ala and Lys171Ala, whereas mutagenesis of Ser153Ala only resulted in a much reduced catalytic activity. Isothermal titration calorimetry measurements indicated that NAD+ binds first followed by the substrate, (-)-secoisolariciresinol. Additionally, for hydride transfer, the incoming hydride abstracted from the substrate takes up the pro-S position in the NADH formed. Taken together, a catalytic mechanism for the overall enantiospecific conversion of (-)-secoisolariciresinol into (-)-matairesinol is proposed.

The beta-glucuronidase (GUS) gene is used extensively in plant biology studies; this analysis summarizes its advantages and limitations. With the advances in genomic sequencing and computational analyses (including bioinformatics), its application in the study of plant gene expression is now an integral component of modern day plant science. This chapter focuses on the detailed challenges of carrying out GUS studies for both qualitative and quantitative analyses, including the increasing employment of GUS from Bacillus strains, rather than E. coli; the Bacillus GUS genes encode proteins with enhanced properties, such as both increased thermostability and stability in the presence of crosslinking fixatives.

Of 17 genes annotated in the Arabidopsis genome database as cinnamyl alcohol dehydrogenase (CAD) homologues, an in silico analysis revealed that 8 genes were misannotated. Of the remaining nine, six were catalytically competent for NADPH-dependent reduction of p-coumaryl, caffeyl, coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes, whereas three displayed very low activity and only at very high substrate concentrations. Of the nine putative CADs, two (AtCAD5 and AtCAD4) had the highest activity and homology (approximately 83% similarity) relative to bona fide CADs from other species. AtCAD5 used all five substrates effectively, whereas AtCAD4 (of lower overall catalytic capacity) poorly used sinapyl aldehyde; the corresponding 270-fold decrease in k(enz) resulted from higher K(m) and lower k(cat) values, respectively. No CAD homologue displayed a specific requirement for sinapyl aldehyde, which was in direct contrast with unfounded claims for a so-called sinapyl alcohol dehydrogenase in angiosperms. AtCAD2, 3, as well as AtCAD7 and 8 (highest homology to sinapyl alcohol dehydrogenase) were catalytically less active overall by at least an order of magnitude, due to increased K(m) and lower k(cat) values. Accordingly, alternative and/or bifunctional metabolic roles of these proteins in plant defense cannot be ruled out. Comprehensive analyses of lignified tissues of various Arabidopsis knockout mutants (for AtCAD5, 6, and 9) at different stages of growth/development indicated the presence of functionally redundant CAD metabolic networks. Moreover, disruption of AtCAD5 expression had only a small effect on either overall lignin amounts deposited, or on syringyl-guaiacyl compositions, despite being the most catalytically active form in vitro.

An enantio-specific polyphenol oxidase (PPO) was purified approximately 1,700-fold to apparent homogeneity from the creosote bush (Larrea tridentata), and its encoding gene was cloned. The posttranslationally processed PPO ( approximately 43 kDa) has a central role in the biosynthesis of the creosote bush 8-8' linked lignans, whose representatives, such as nordihydroguaiaretic acid and its congeners, have potent antiviral, anticancer, and antioxidant properties. The PPO primarily engenders the enantio-specific conversion of (+)-larreatricin into (+)-3'-hydroxylarreatricin, with the regiochemistry of catalysis being unambiguously established by different NMR spectroscopic analyses; the corresponding (-)-enantiomer did not serve as a substrate. This enantio-specificity for a PPO, a representative of a widespread class of enzymes, provides additional insight into their actual physiological roles that hitherto have been difficult to determine.

The creosote bush (Larrea tridentata) lignans are linked via 8-8' bonds, with the simplest apparently being E-p-anol derived. Of the latter, four of the six theoretically possible diastereoisomers were isolated, namely (-)-larreatricin, (-)-8'-epi-larreatricin, meso-3,3'-didemethoxynectandrin B and the new compounds, (+)- and (-)-3,3'-didemethoxyverrucosins. Following synthesis of each in either racemic or meso form, and chiral HPLC separation of the antipodes of the racemates, it was established that naturally occurring (-)-larreatricin and (-)-8'-epi-larreatricin were present in 92 and 98% enantiomeric excess, respectively, whereas 3,3'-didemethoxyverrucosin was essentially racemic and 3,3'-didemethoxynectandrin B was in the meso-form. The evidence suggests that formation of these lignans occurs under regiospecific, rather than stereoselective, coupling control. This contrasts with laccase-catalyzed "random" coupling of E-p-anol in vitro which generates the corresponding racemic 8-8', 8-3' and 8-O-4' linked dimeric moieties.

The isolation and characterization of a multigene family of the first class of dirigent proteins (namely that mainly involved in 8-8' coupling leading to (+)-pinoresinol in this case) is reported, this comprising of nine western red cedar (Thuja plicata) DIRIGENT genes (DIR1-9) of 72-99.5% identity to each other. Their corresponding cDNA clones had coding regions for 180-183 amino acids with each having a predicted molecular mass of ca. 20 kDa including the signal peptide. Real time-PCR established that the DIRIGENT isovariants were differentially expressed during growth and development of T. plicata (P < 0.05). The phylogenetic relationships and the rates and patterns of nucleotide substitution suggest that the DIRIGENT gene may have evolved via paralogous expansion at an early stage of vascular plant diversification. Thereafter, western red cedar paralogues have maintained an high homogeneity presumably via a concerted evolutionary mode. This, in turn, is assumed to be the driving force for the differential formation of 8-8'-linked pinoresinol derived (poly)lignans in the needles, stems, bark and branches, as well as for massive accumulation of 8-8'-linked plicatic acid-derived (poly)lignans in heartwood.