A bacterial pinoresinol/lariciresinol reductase (PLR) homolog named NrPinZ was obtained from a Novosphingobium rhizosphaerae sp. LY bacterial strain, with NrPinZ being part of its 5-step biochemical system catabolizing pinoresinol into coniferyl aldehyde and vanillin. Recombinant NrPinZ reduces racemic 8-8' furanofuran lignans [(±)-pinoresinols, medioresinols, and syringaresinols] with similar overall catalytic efficiencies. In those reductions, only one of the two furan ring systems is reduced. Two other bacterial PLR homologs, NaPinZ and SlPinZ, from N. aromaticivorans F199 and Sphingobium lignivorans SYK-6, respectively, had comparable substrate versatilities and catalytic efficacies. Plant PLR homologs, by comparison, are either enantiospecific, enantioselective, or variants thereof, being able to reduce either one or both furan rings. For example, a recombinant enantioselective PLR (PLR_Tp2) from western red cedar (Thuja plicata) preferentially reduces both (+)-pinoresinol furan rings to afford (-)-secoisolariciresinol. BoltZ-2 modeling of NrPinZ and PLR_Tp2, together with substrate docking of (+)- and (-)-pinoresinols, medioresinols, and syringaresinols, was very instructive. The NrPinZ active site P1/P2 sub-pockets allow for both racemic forms to be catabolized. Conversely, the smaller P1 pocket in PLR_Tp2 preferentially positions (+)-pinoresinol for downstream metabolism into (-)-secoisolariciresinol, thereby providing a biochemical explanation for the different stereochemical outcomes. NrPinZ, NaPinZ, and SlPinZ, catalyzing substrate versatile catabolism of both racemic forms, may have important ramifications for gymnosperm and angiosperm lignin and lignan biodegradation, including its evolutionary significance and potential in enzyme engineering.

Following the down-selection of 14 Arabidopsis thaliana arogenate dehydratase (ADT) knockout and over-expression (OE) genotypes, the most highly contrasting quadruple knockout adt3/4/5/6 and ADT OE genotypes were subjected to proteomics, metabolomics, and scanning electron microscopy (SEM) analyses as needed, with results compared to Columbia wild-type (WT). The basal adt3/4/5/6 stem cross-sections, ∼70% lignin content reduced, exhibited buckled vessel cell walls and partially detached xylary fibers, in contrast to WT and ADT4m/5 m OE genotypes that did not. Anatomical defects primarily resulted from guaiacyl lignin level reductions in vessels with concomitant increased stem syringyl:guaiacyl (S/G) ratios.Phenylpropanoid and various upstream shikimate-chorismate pathway enzyme abundances, as well as specific monolignol oxidases (laccases/peroxidases), generally increased in adt3/4/5/6 at different stem and rosette leaf growth/development stages, relative to WT. Opposite effects were largely observed with the ADT5m OE genotype. By contrast, flavonoid and glucosinolate pathway enzyme amounts varied. Such enzyme abundance increases were overall unproductive as adt3/4/5/6 was unable to restore WT, ADT4 OE, ADT5 OE, ADT5m OE, and ADT4m/5 m OE secondary metabolite (lignin, phenylpropanoid, lignan, flavonoid, phenolic acid, and glucosinolate) levels. Conversely, ADT OE genotypes did not significantly increase programmed lignin levels or alter S/G compositions.In sum, proteomics analyses of adt3/4/5/6 and adt5 ‘perceived’ that lignin and low molecular weight secondary metabolite amounts were not at ‘programmed’ levels as for WT and ADT OE genotypes but observed increases in relevant pathway protein abundances were futile. Notably though, proteomics analyses did not lead to predicting that lignin and associated biochemical pathways would have reduced metabolite levels, relative to WT and ADT OE genotypes. Genotype adt3/4/5/6, possibly the highest lignin level reduced genotype reported, did not utilize other phenolics to compensate. By contrast, the differential temporal and spatial deposition of cell wall oxidases again indicate the exquisite control over lignin deposition, and our lack of knowledge of precise lignin structure and assembly in subcellular regions of the lignified cell walls.

Monolignol-derived dimers containing β-β linkages are synthesized by vascular plants and can be released during lignin depolymerization. In this work, we isolated a bacterium, Novosphingobium rhizosphaerae LY, that grows with the β-β lignan (+)-pinoresinol as a sole growth substrate. Sequence analysis suggested that this strain encodes a broad range of pathways for assimilation of aromatic monomers as well as one enzyme implicated in pinoresinol catabolism but lacks other known pathways for aromatic dimer catabolism. We constructed a genome-wide barcoded transposon library and identified genes required for pinoresinol catabolism. Using feeding studies, compound isolation, targeted synthesis, and analysis of purified enzymes, we elucidated the biochemical intermediates and reaction pathway involved in pinoresinol catabolism. We demonstrated that the first enzymatic reaction is the reductive cleavage of a furan ring in (±)-pinoresinol with retention of configuration to yield lariciresinol. We additionally confirmed that the final pathway enzyme, PinU, is related to lignostilbene dioxygenases and oxidatively cleaves a diguaiacylbutadiene intermediate to yield vanillin and coniferaldehyde. Finally, based on the enzyme characterization, we demonstrated that the strain can grow with a second β-β lignan, (-)-syringaresinol, as a sole growth substrate. In combination, these results demonstrate a new biocatalytic route for transforming a widely occurring group of plant phenylpropanoid natural products.IMPORTANCEPlants synthesize a variety of aromatic phenylpropanoid compounds containing β-β linkages, including lignin, a major structural polymeric component of the vascular plant cell wall, and lignans, biochemically related secondary metabolites with a wide range of bioactivities. Although microbial catabolic pathways have been described for dimeric phenylpropanoids featuring other interunit linkages, relatively little is known about pathways for catabolism of β-β-linked compounds. In this work, we isolated a Novosphingobium strain capable of degrading the β-β-linked lignan (+)-pinoresinol and elucidated the catabolic pathway. Understanding how bacteria catabolize β-β-linked compounds provides a basis for new biocatalytic transformations of lignans and oligolignols and has the potential to improve bacterial lignin valorization.

There have been frequent reports of more than one strain of the nitrogen-fixing symbiont, Frankia, in the same root nodule of plants in the genus Alnus, but quantitative assessments of their relative contributions have not been made to date. Neither has the diversity of other microbes, having potential functional roles in symbiosis, been systematically evaluated. Alnus rubra root nodule microbiota were studied using Illumina short read sequencing and kmer-based read classification. Single end 76 bp sequencing was done to a median depth of 96 million reads per sample. Reads were assigned to taxa using KrakenUniq, with taxon abundances being estimated using its companion program Bracken. This was the first high resolution study of Alnus root nodules using next generation sequencing (NGS), quantifying multiple Cluster 1 A Frankia strains in single nodules, and in some cases, a Cluster 4 strain. Root nodules were found to contain diverse bacteria, including several genera containing species known to have growth-promoting effects. Evidence was found for partitioning of some bacterial strains in older versus younger lobes.

Red alder (Alnus rubra) has highly desirable wood, dye pigment, and (traditional) medicinal properties which have been capitalized on for thousands of years, including by Pacific West Coast Native Americans. A rapidly growing tree species native to North American western coastal and riparian regions, it undergoes symbiosis with actinobacterium Frankia via their nitrogen-fixing root nodules. Red alder’s desirable properties are, however, largely attributed to its bioactive plant phenol metabolites, including for plant defense, for its attractive wood and bark coloration, and various beneficial medicinal properties. Integrated transcriptome and metabolome data analyses were carried out using buds, leaves, stems, roots, and root nodules from greenhouse grown red alder saplings with samples collected during different time-points (Spring, Summer, and Fall) of the growing season. Pollen and catkins were collected from field grown mature trees. Overall plant phenol biochemical pathways operative in red alder were determined, with a particular emphasis on potentially identifying candidates for the long unknown gateway entry points to the proanthocyanidin (PA) and ellagitannin metabolic classes, as well as in gaining better understanding of the biochemical basis of diarylheptanoid formation, i.e. that help define red alder’s varied medicinal uses, and its extensive wood and dye usage.

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 beta-axial OH orientation significantly enhanced its leaving group abilities in generating A-ring mono-quinone methide (QM), whereas 4S- isomer's alpha-equatorial-OH was a poorer leaving group. Docking simulations indicated that the 4R-configured beta-axial OH was closest to Asp51, whereas 4S-isomer's alpha-equatorial OH was further away. Neither cis-(3 S ,4 S )- nor trans-(3 S ,4 R )-DMDIs were substrates, even with the former having C3/C4 stereo- chemistry as in (+)-pisatin. PsPTS2 used cis-(3 R ,4 R )-7,2 0-dihy- droxy-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-(3 R ,4 R )-DMDI to give (-)-maackiain 2200-fold more efficiently than with cis-(3 R ,4 R )- DMI to give (-)-medicarpin. PsPTS1 also slowly converted trans-(3 S ,4 R )-DMDI into (+)-maackiain, reflecting the better 4R configured OH leaving group. PsPTS2 and PsPTS1 provisionally provide the means to enable differing C6a and C11a confi gurations 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-con fi guration in PsPTS1 to give maackiain.

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.

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.

This chapter addresses DP gateway entry points to distinct vascular plant phenol classes, while focusing on DPs involved in lignan biosynthesis. Structurally diverse lignans are typically phenylpropanoid dimers and higher oligomers, with physiological roles mainly in vascular plant defense. Many lignans are optically active, with diverse coupling modes, although the inter-unit linkage. Such biochemical processes were also of critical evolutionary importance, as they ultimately led to emergence of terrestrial-based plant life (from aquatic forerunners), including initially the successful colonization of land which, in turn, led to the circa 391,000 vascular plant species in existence today. Dirigent Protein (DP) involvement in Casparian strip lignification is considered to be part of a hypothesized lignin-forming protein complex containing a Casparian strip domain protein scaffold, DPs, various oxidative enzymes, and other enzymes affording cofactors, such as hydrogen peroxide. The range of heterogeneity that can be accommodated in the second radical substrate, including whether it needs to be a propenylphenol radical.

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

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

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

Podophyllum 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.

Fast growing hybrid poplar offers the means for sustainable production of specialty and commodity chemicals, in addition to rapid biomass production for lignocellulosic deconstruction. Herein we describe transformation of fast-growing transgenic hybrid poplar lines to produce 2-phenylethanol, this being an important fragrance, flavor, aroma, and commodity chemical. It is also readily converted into styrene or ethyl benzene, the latter being an important commodity aviation fuel component. Introducing this biochemical pathway into hybrid poplars marks the beginnings of developing a platform for a sustainable chemical delivery system to afford this and other valuable specialty/commodity chemicals at the scale and cost needed. These modified plant lines mainly sequester 2-phenylethanol via carbohydrate and other covalently linked derivatives, thereby providing an additional advantage of effective storage until needed. The future potential of this technology is discussed. MALDI metabolite tissue imaging also established localization of these metabolites in the leaf vasculature.

Background: How vascular plants control phenoxy radical coupling is enigmatic. Results: Two dirigents engendered (−)-pinoresinol formation in Arabidopsis. Coupling stereoselectivity was reversed from (+)- to (−)-pinoresinol through swapping identical regions. Conclusion: Novel insights into stereoselective control over phenoxy radical coupling were obtained. Significance: This is the first report of dirigent-mediated phenoxy radical coupling control leading to opposite stereoselectivities and identification of protein regions involved. 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 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 (+)-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.

Qubits, Atoms, Photons, Entanglements, Quantum Mechanics and Diamonds meet the Information Age. Current research indicates that diamonds may hold the key to a cryptanalyst's nightmare, 'The unbreakable cipher'. This paper introduces the basic theory and principles behind this innovative technology, reports on the research that has led to present day breakthroughs, investigates those that hold promise and looks at the possibilities for future research in this field. This report is presented as a primer to the 'World of Quantum Cryptography' and its impact on Network Security and Management. Consequently, it does not provide any mathematical proofs behind the theoretical models which relate to Quantum Mechanics. It does, however, make reference to and direct those who require this depth of knowledge to the sources if this information is desired or is the basis for further research.