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Keywords: lignans; metabolic engineering; analytical method; biological activity; lignans metabolism EN lignans metabolic engineering analytical method biological activity lignans metabolism N.PAG N.PAG 3 01/15/21 20210112 NES 210112 Lignans constitute a multifaceted group of phytochemicals widely distributed in terrestrial plant lineages (Ayres and Loike, [2]; Vassão et al., [11]). Critical analysis of the lignan research literature by Yeung et al. also revealed important features about trends in lignan research. In-depth phytochemical study using UPLC-HRMS confirmed the high lignan and neolignan accumulation potential of this species, including 7 neolignans newly described, and their potential use in cosmetic applications. [Extracted from the article]

Abstract: 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 8weeks 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 &y& Elsevier]

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]

Abstract: A major goal currently in Arabidopsis research is determination of the (biochemical) function of each of its ∼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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]