Dirigent isoflavene-forming PsPTS2: 3D structure, stereochemical, and kinetic characterization comparison with pterocarpan-forming PsPTS1 homolog in pea.

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 ß-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 ß-axial OH was closest to Asp51, 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.

Pterocarpan synthase (PTS) structures suggest a common quinone methide-stabilizing function in dirigent proteins and proteins with dirigent-like domains.

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.

De novo sequencing and native mass spectrometry revealed hetero-association of dirigent protein homologs and potential interacting proteins in Forsythia × intermedia.

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.

RNAi Modulation of Chlorogenic Acid and Lignin Deposition in Nicotiana tabacum and Insufficient Compensatory Metabolic Cross-Talk.

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.

Pinoresinol-lariciresinol reductase: Substrate versatility, enantiospecificity, and kinetic properties.

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.

Reduced Arogenate Dehydratase Expression: Ramifications for Photosynthesis and Metabolism.

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.

A genome-wide analysis of the flax (Linum usitatissimum L.) dirigent protein family: from gene identification and evolution to differential regulation.

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.

Eugenol specialty chemical production in transgenic poplar (Populus tremula × P. alba) field trials.

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.

Trimeric structure of (+)-pinoresinol-forming dirigent protein at 1.95 Å resolution with three isolated active sites.

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 ß-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.

Active site cleft mutants of Os9BGlu31 transglucosidase modify acceptor substrate specificity and allow production of multiple kaempferol glycosides.

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-ß-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 ß-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.

Dirigent Protein-Mediated Lignan and Cyanogenic Glucoside Formation in Flax Seed: Integrated Omics and MALDI Mass Spectrometry Imaging.

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.

Next generation sequencing in predicting gene function in podophyllotoxin biosynthesis.

Podophyllum species are sources of (-)-podophyllotoxin, an aryltetralin lignan used for semi-synthesis of various powerful and extensively employed cancer-treating drugs. Its biosynthetic pathway, however, remains largely unknown, with the last unequivocally demonstrated intermediate being (-)-matairesinol. Herein, massively parallel sequencing of Podophyllum hexandrum and Podophyllum peltatum transcriptomes and subsequent bioinformatics analyses of the corresponding assemblies were carried out. Validation of the assembly process was first achieved through confirmation of assembled sequences with those of various genes previously established as involved in podophyllotoxin biosynthesis as well as other candidate biosynthetic pathway genes. This contribution describes characterization of two of the latter, namely the cytochrome P450s, CYP719A23 from P. hexandrum and CYP719A24 from P. peltatum. Both enzymes were capable of converting (-)-matairesinol into (-)-pluviatolide by catalyzing methylenedioxy bridge formation and did not act on other possible substrates tested. Interestingly, the enzymes described herein were highly similar to methylenedioxy bridge-forming enzymes from alkaloid biosynthesis, whereas candidates more similar to lignan biosynthetic enzymes were catalytically inactive with the substrates employed. This overall strategy has thus enabled facile further identification of enzymes putatively involved in (-)-podophyllotoxin biosynthesis and underscores the deductive power of next generation sequencing and bioinformatics to probe and deduce medicinal plant biosynthetic pathways.

Allyl/propenyl phenol synthases from the creosote bush and engineering production of specialty/commodity chemicals, eugenol/isoeugenol, in Escherichia coli.

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.

Arogenate dehydratase isoenzymes profoundly and differentially modulate carbon flux into lignins.

How carbon flux differentially occurs in vascular plants following photosynthesis for protein formation, phenylpropanoid metabolism (i.e. lignins), and other metabolic processes is not well understood. Our previous discovery/deduction that a six-membered arogenate dehydratase (ADT1-6) gene family encodes the final step in Phe biosynthesis in Arabidopsis thaliana raised the fascinating question whether individual ADT isoenzymes (or combinations thereof) differentially modulated carbon flux to lignins, proteins, etc. If so, unlike all other lignin pathway manipulations that target cell wall/cytosolic processes, this would be the first example of a plastid (chloroplast)-associated metabolic process influencing cell wall formation. Homozygous T-DNA insertion lines were thus obtained for five of the six ADTs and used to generate double, triple, and quadruple knockouts (KOs) in different combinations. The various mutants so obtained gave phenotypes with profound but distinct reductions in lignin amounts, encompassing a range spanning from near wild type levels to reductions of up to ∼68%. In the various KOs, there were also marked changes in guaiacyl:syringyl ratios ranging from ∼3:1 to 1:1, respectively; these changes were attributed to differential carbon flux into vascular bundles versus that into fiber cells. Laser microscope dissection/pyrolysis GC/MS, histochemical staining/lignin analyses, and pADT::GUS localization indicated that ADT5 preferentially affects carbon flux into the vascular bundles, whereas the adt3456 knock-out additionally greatly reduced carbon flux into fiber cells. This plastid-localized metabolic step can thus profoundly differentially affect carbon flux into lignins in distinct anatomical regions and provides incisive new insight into different factors affecting guaiacyl:syringyl ratios and lignin primary structure.

Opposite stereoselectivities of dirigent proteins in Arabidopsis and schizandra species.

How stereoselective monolignol-derived phenoxy radical-radical 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 ß-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 (+)-pinoresinol-forming 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.

Assessment of a putative proton relay in Arabidopsis cinnamyl alcohol dehydrogenase catalysis.

Extended proton relay systems have been proposed for various alcohol dehydrogenases, including the Arabidopsis thaliana cinnamyl alcohol dehydrogenases (AtCADs). Following a previous structural biology investigation of AtCAD5, the potential roles of three amino acid residues in a putative proton relay system, namely Thr49, His52 and Asp57, in AtCAD5, were investigated herein. Using site-directed mutagenesis, kinetic and isothermal titration calorimetry (ITC) analyses, it was established that the Thr49 residue was essential for overall catalytic conversion, whereas His52 and Asp57 residues were not. Mutation of the Thr49 residue to Ala resulted in near abolition of catalysis, with thermodynamic data indicating a negative enthalpic change (ΔH), as well as a significant decrease in binding affinity with NADPH, in contrast to wild type AtCAD5. Mutation of His52 and Asp57 residues by Ala did not significantly change either catalytic efficiency or thermodynamic parameters. Therefore, only the Thr49 residue is demonstrably essential for catalytic function. ITC analyses also suggested that for AtCAD5 catalysis, NADPH was bound first followed by p-coumaryl aldehyde.

Annotated genome sequence of a fast-growing diploid clone of red alder (Alnus rubra Bong.).

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.

Dirigent protein subfamily function and structure in terrestrial plant phenol metabolism.

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 ß-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 ß1-ß2 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.

Meta-analysis of the space flight and microgravity response of the Arabidopsis plant transcriptome.

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.

The laccase multigene family in Arabidopsis thaliana: towards addressing the mystery of their gene function(s).

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.