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.
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.
In
Arabidopsis thaliana, four genes have been annotated as provisionally encoding phenylalanine ammonia lyase (PAL). In this study, recombinant native AtPAL1, 2, and 4 were demonstrated to be ...catalytically competent for
l-phenylalanine deamination (
K
m values between 64 and 71 μM), whereas AtPAL3 was of very low specific activity in its N-terminal His-tagged form.
In
Arabidopsis thaliana, four genes have been annotated as provisionally encoding PAL. In this study, recombinant native AtPAL1, 2, and 4 were demonstrated to be catalytically competent for
l-phenylalanine deamination, whereas AtPAL3, obtained as a N-terminal His-tagged protein, was of very low activity and only detectable at high substrate concentrations. All four PALs displayed similar pH optima, but not temperature optima; AtPAL3 had a lower temperature optimum than the other three isoforms. AtPAL1, 2 and 4 had similar
K
m values (64–71 μM) for
l-Phe, with AtPAL2 apparently being slightly more catalytically efficacious due to decreased
K
m and higher
k
cat values, relative to the others. As anticipated, PAL activities with
l-tyrosine were either low (AtPAL1, 2, and 4) or undetectable (AtPAL3), thereby establishing that
l-Phe is the true physiological substrate. This detailed knowledge of the kinetic and functional properties of the various PAL isoforms now provides the necessary biochemical foundation required for the systematic investigation and dissection of the organization of the PAL metabolic network/gene circuitry involved in numerous aspects of phenylpropanoid metabolism in
A. thaliana spanning various cell types, tissues and organs.
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.
Background: Biosynthetic pathways to structurally complex plant medicinals are incomplete or unknown.
Results: Next generation sequencing/bioinformatics and metabolomics analysis of Podophyllum tissues gave putative unknown genes in podophyllotoxin biosynthesis.
Conclusion: Regio-specific methylenedioxy bridge-forming CyP450s were identified catalyzing pluviatolide formation.
Significance: Database of several medicinal plant transcriptome assemblies and metabolic profiling are made available for scientific community.
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.
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.
Dirigent protein homolog DRR206 is involved in phytoalexin (lignan) biosynthesis, and the encoding gene is induced upon fungal exposure. MALDI mass spectrometry imaging established that both the ...monoglucoside derived from (+)-pinoresinol, and the isoflavonoid pisatin, were co-localized in infected endocarp epidermal cells where pathway genes were also detected. Display omitted
•The gene encoding DRR206, a dirigent protein homolog, was induced upon fungal exposure of pea pod.•Recombinant DRR206 engendered stereoselective formation of (+)-pinoresinol.•Pinoresinol monoglucoside was detected as a phytoalexin response. MALDI MS imaging established that pinoresinol monoglucoside and pisatin were co-localized. Co-localization was in endocarp epidermal cells.
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.
This comprehensive review describes the current status and knowledge of biochemical and molecular processes involved in allyl/propenyl phenol, lignan, norlignan and lignin biosynthesis. Recent ...advances made over the last decade are critically discussed, and placed in context with earlier studies largely dating back to the 1950s. Beginning with the recently established formation of phenylalanine in plants, each downstream biochemical conversion is described from the perspective of the mechanistic details known to this point. Particular emphasis is placed upon proteinaceous control of monolignol-derived radical-radical coupling processes, leading to lignans and lignins, as well as apparently related processes affording the various ellagitannins and phenolic terpenoids. The evidence for non-random macromolecular lignin assembly is discussed in detail, this being in contrast to earlier notions that such processes were random. The latter assumptions have largely resulted from a lack of robust analytical procedures and rigorous quantification, as well as a lack of incisive experimental design. In addition, the often-noted severe effects of modulating lignin compositions and contents on plant vascular tissue properties (i.e. in terms of compromised biophysical properties) are described herein, as well as the severe limitations as regards recent claims of compensatory 'combinatorial chemistry' lignin formation. Much of the latter confusion has also resulted from the serious deficiencies in current lignin analytical protocols and quantification, as well as in the general lack of experimental approaches/design to probe lignin primary structure(s).
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 (≈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 kenzresulted from higher Kmand lower kcatvalues, respectively. No CAD homologue displayed a specific requirement for sinapyl aldehyde, which was in direct contrast with un-founded 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 Kmand lower kcatvalues. Accordingly, alternative and/or bifunctional metabolic roles of these proteins in plant defense cannot be ruled out. Comprehensive anylses of lignified tissues of various Arabidopsis knock-out 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.
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