Syringyl Moieties Research Articles

Chemotaxonomy of fossil woods from the Lower Rhine Embayment, Germany

In the Lower Rhine Embayment (western Germany), the Neogene lignite bearing sequence is rich in large fossil wood trunks. Woods collected from sand-filled channels of a meandering river system (Pliocene: Reuver series) and from coal seams (Miocene: seams Garzweiler, Frimmersdorf and Morken) were investigated by Curie-point pyrolysis coupled with gas chromatography–mass spectrometry in order to determine their palaeobotanical classification. In comparison with reference data of individual pyrograms of modern woods, it became possible to classify 15 fossil wood trunks chemotaxonomically as follows: eleven samples were identified as Taxodioxylon sp. by reference Sequoia sempervirens, three samples were identified as Populoxylon sp. by reference Populus nigra and one sample was identified as Sciadopityoxylon sp. by two references of Sciadopitys verticillata. Microanatomical crosschecks describe the equivalent samples taxonomically as Taxodioxylon gypsaceum (GÖPPERT) KRÄUSEL, Taxodioxylon germanicum (GREGUSS) VAN DER BURGH, Populus nigra L. and Sciadopityoxylon wettsteinii JURASKY, and confirm the chemical classifications. For both fossil and reference woods, our relative quantifications of guaicyl and syringyl moieties are in precise accordance with discriminations of gymnosperms versus angiosperms. This means that not only unknown recent woods can chemotaxonomically accurately be determined by Curie-point pyrolysis gas chromatography–mass spectrometry and their equivalent modern wood reference from our data base, but also the taxonomy of botanically related wood types reaching back to the geological past up to a Miocene age at least.

Hydrocracking of Organosolv Lignin in Subcritical Water to Useful Phenols Employing Various Raney Nickel Catalysts

Hydrothermal conversion of organosolv lignin has been conducted under a reductive hydrogen atmosphere near the critical point of water. Several Raney nickel catalysts were tested with respect to degree of liquefaction, yields of phenols, and influence on the product distribution. The application of Raney nickel catalysts is crucial for the hydrothermal liquefaction of lignin since the liquefaction rate increased approximately 2.5 times compared to hydrothermal conversion without catalysts. In addition, Raney nickel caused complete demethoxylation of guaiacyl and syringyl moieties resulting in an oil composed mainly of six highly reactive simple phenols (phenol and monoalkylated derivatives). Depending on the catalyst type, yields of these phenols were 1.3–8.0 wt % (≈2.6%–16% theoretical yield). Flash pressure release with subsequent condensation of the hot vapors proved to be a fast and easy approach to obtain a liquid fraction with a high content (25%) of phenol and para- or ortho-alkylated derivatives, ...

The Characteristics and Reactivity of Acid-Labile Aryl Ether Units in Wood Lignin

Wood lignin contains significant amounts of acid-labile aryl ether units, which play a significant role in lignin modification or delignification processes. We have evaluated the rate and reaction kinetics on the acid-catalyzed cleavages of aryl ether structures for wood lignin in situ based on the formation of phenolic hydroxyl groups. The content of acid-labile aryl ether units was quite uniform for a variety of softwood wood lignins (∼4% per C9 unit) and it varied appreciably among hardwood species, ranging from 4% for aspen to 9% for beech wood lignin. These variations, however, appear to be related to the content of syringyl units in wood lignin. The reactivity of these reactive aryl ether structures was noticeably higher for the spruce than for the aspen wood lignin. This difference in reactivity, based on the behavior of lignin model compound reactions, can be attributed to the influence of syringyl moieties in aspen wood lignin. It appears that most of the acid-labile aryl ether units in hardwood were associated with the syringy moiety being present as a benzyl unit, which is much less reactive than the corresponding guaiacyl moiety.

Investigation into the deterioration process of archaeological bamboo strips of China from four different periods by chemical and anatomical analysis

The chemical and the anatomical features of the archeological bamboo strips of China from four different periods were compared with the non-degraded fresh bamboo to understand the deterioration process. Unprocessed bamboo strips, isolated hemicelluloses and lignin fractions were analyzed with wet chemical analysis, GC, FTIR, UV and XRD to investigate the chemical degradation. It was found that the crystalline cellulose and the polyxylose degraded with increasing age, leaving the amorphous cellulose and the lignin with syringyl moieties modified as the main residual cell wall materials. Serious degradation of p-coumaric acid and ferulic acid were detected and indicated the cleavage of the linkage between the lignin and the carbohydrate, which might facilitate the degradation of carbohydrate. The anatomical analysis with SEM and TEM revealed the vanishing of the parenchymatous ground tissue and vessels and the cellulose microfibrils in the multilayered secondary wall. The differences in lignin content and in tissue density (cell thickness/cell size) between the parenchyma cells and the sclerenchymatous fiber cells fundamentally led to the formation of the unique anatomical structure of the archeological bamboo strips which was composed of parallel solid bundles. The effects of the chemical and the anatomical degradation on the mechanical properties were also discussed.

From Zinnia to Arabidopsis: approaching the involvement of peroxidases in lignification

Zinnia elegans constitutes one of the most useful model systems for studying xylem differentiation, which simultaneously involves secondary cell wall synthesis, cell wall lignification, and programmed cell death. Likewise, the in vitro culture system of Z. elegans has been the best characterized as the differentiation of mesophyll cells into tracheary elements allows study of the biochemistry and physiology of xylogenesis free from the complexity that heterogeneous plant tissues impose. Moreover, Z. elegans has emerged as an excellent plant model to study the involvement of peroxidases in cell wall lignification. This is due to the simplicity and duality of the lignification pattern shown by the stems and hypocotyls, and to the basic nature of the peroxidase isoenzyme. This protein is expressed not only in hypocotyls and stems but also in mesophyll cells transdifferentiating into tracheary elements. Therefore, not only does this peroxidase fulfil all the catalytic requirements to be involved in lignification overcoming all restrictions imposed by the polymerization step, but also its expression is inherent in lignification. In fact, its basic nature is not exceptional since basic peroxidases are differentially expressed during lignification in other model systems, showing unusual and unique biochemical properties such as oxidation of syringyl moieties. This review focuses on the experiments which led to a better understanding of the lignification process in Zinnia, starting with the basic knowledge about the lignin pattern in this plant, how lignification takes place, and how a sole basic peroxidase with unusual catalytic properties is involved and regulated by hormones, H2O2, and nitric oxide.

Laccase catalysed modification of lignin subunits and coupling to p-aminobenzoic acid

Laccase catalysed oxidation of syringyl and guaiacyl subunits of lignin and their modification with an aromatic amine, p-aminobenzoic acid (PABA) were investigated. Laccase from Galerina sp. HC1 isolated earlier by us was used as the main catalyst, and Trametes versicolor laccase was used for comparison. Among the syringyl compounds, syringic acid and syringaldehyde were oxidised to 2,6-dimethoxy-1,4-benzoquinone, and in the presence of PABA yielded a cross-coupling imine product. The reaction with methyl syringol resulted in several products whose structures were determined. The possible oxidative coupling pathways were proposed for the formation of the identified products. Oxidation of syringol and the guaiacyl compounds resulted mainly in homooligomers by free radical mechanism, with a negligible tendency of reaction with the nucleophilic group of PABA. Similar treatment of Eucalyptus Kraft lignin, which is rich in syringyl moieties, showed the presence of identical products obtained with syringic acid and syringaldehyde. (C) 2013 Elsevier B.V. All rights reserved. (Less)

Antihypertensive Evaluation of Lignin Related High-Added-Value 4-Aryl-Hexahydroquinolines

4-Aryl-hexahydroquinolines prepared by Hantzsch reaction from aromatic aldehydes obtained from lignin had been proved to be excellent antioxidant property. In view of polyhydroquinoline containing similarities in structure to the biologically important compound, 1,4-dihydropyridine, their antihypertensive activities were herein determined in spontaneously hypertensive rats (SHRs) during different time. The results show that those compounds possessing excellent antioxidant activity exhibit good antihypertensive property, and the compound containing syringyl moiety exhibit better antihypertensive activity than that containing guaiacyl moiety. This study suggests that those compounds may serve as promising agents for cure hypertension and free radical-related diseases.

Source and transformations of lignin in Carex-dominated peat

We identified the effects of vegetation changes, and aerobic and anaerobic decay on the lignin composition in the Penido Vello peat record (Galicia, Spain). The ombrotrophic part of this peat record was dominated by graminoids and has significant contributions of ericoids at some depths. The organic matter (OM) of different peat fractions (bulk, NaOH-extractable fraction, and non-extractable residues) of 15 samples from the upper meter was analysed with pyrolysis-gas chromatography/mass spectrometry (pyrolysis-GC/MS). In addition, the dominant plant species were analysed, including Carex durieui, Agrostis curtisii, Molinia caerulea, Deschampsia flexuosa, Festuca rubra, Eriophorum angustifolium, Erica mackaiana and Calluna vulgaris, and their lignin composition compared to that of the peat OM. The high abundance of guaiacol and 4-formylguaiacol in fresh plant tissue compared to peat OM suggests that in addition to p-coumaric and ferulic acid (which are abundant in graminoids), other non-lignin phenolic monomers are contributed by graminoid species. For the non-lignin phenolics, graminoids differed from ericoids in the high abundance of ferulic acid (4-vinylguaiacol), while p-coumaric acid (4-vinylphenol) showed high and similar abundances in ericoids and graminoids. This result suggests that ratios between p-hydroxyphenyl (or p-coumaric acid) and other lignin moieties in (pyrolysates of) peat cannot be used as source indicator. Comparison of plant and peat fractions using factor analysis allowed a distinction between the effects of source (plant identity) and decay on the lignin composition of the Penido Vello peat, and different stages of decomposition were identified. Preferential decay of guaiacyl over syringyl moieties was found for the first stage of decay. This preferential decay is probably related to the large abundance of guaiacyl moieties in easily degradable non-lignin phenolics. Preferential decay of syringyl moieties occurred during subsequent aerobic decay.

Synthesis and bioactivity evaluation of lignin related high-added-value 1,4-dihydropyridines and polyhydroacridines

A series of 4-aryl-1,4-dihydroquinolines and 9-aryl-3,4,6,7,9,10-hexahydroacridine-1,8(2H,5H)-diones were prepared by Hantzsch reaction using aromatic aldehydes obtained from lignin. Their antioxidant properties were evaluated by scavenging effect on 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical and 2,2′-azino-bis (3-ethylenzothiazoline-sulphonic acid) diammonium salt (ABTS+) radical. Antihypertensive activities were also determined in spontaneously hypertensive rats (SHRs) during different time. The results show that the compounds containing syringyl moiety exhibit better antioxidant and antihypertensive activities than those containing guaiacyl and p-hydroxyphenyl moieties.

Purification and kinetic characterization of two peroxidases of Selaginella martensii Spring. involved in lignification

Two cationic peroxidases from Selaginella martensii Spring. (SmaPrx2 and SmaPrx3) were purified using a three-step protocol which includes ammonium sulfate precipitation, adsorption chromatography on phenyl sepharose and cationic exchange chromatography on SP sepharose. The molecular mass for SmaPrx2 and SmaPrx3 was calculated to be 36.3kDa and 45.6kDa, respectively, according to MALDI-TOF/TOF. The isoelectric points were estimated in 9.2 and 9.5 for SmaPrx2 and SmaPrx3, respectively, according to isoelectrofocusing. Both enzymes show a typical peroxidase UV-visible spectrum with a Soret peak at 403nm for SmaPrx2 and 404nm for SmaPrx3. The specific activities showed against several substrates and the kinetic parameters suggest SmaPrx2 and SmaPrx3 have specific roles in cell wall formation and especially in lignin biosynthesis. Several peptides from tryptic digestion of both peroxidases were identified through MALDI-TOF MS/MS. The presence in these peptides of structural determinants typical of syringyl peroxidases indicates these proteins show no structural restrictions to oxidize syringyl moieties. These data, along with the invitro capacity of using sinapyl alcohol as substrate and the low K(m) in the μM range suggest these two peroxidases may be responsible for the oxidation of syringyl monolignols that leads to syringyl lignins biosynthesis.

Regulatory System of the Protocatechuate 4,5-Cleavage Pathway Genes Essential for Lignin Downstream Catabolism

Sphingobium sp. strain SYK-6 converts various lignin-derived biaryls with guaiacyl (4-hydroxy-3-methoxyphenyl) and syringyl (4-hydroxy-3,5-dimethoxyphenyl) moieties to vanillate and syringate. These compounds are further catabolized through the protocatechuate (PCA) 4,5-cleavage (PCA45) pathway. In this article, the regulatory system of the PCA45 pathway is described. A LysR-type transcriptional regulator (LTTR), LigR, activated the transcription of the ligK-orf1-ligI-lsdA and ligJABC operons in the presence of PCA or gallate (GA), which is an intermediate metabolite of vanillate or syringate, respectively, and repressed transcription of its own gene. LigR bound to the positions -77 to -51 and -80 to -48 of the ligK and ligJ promoters, respectively, and induced DNA bending. In the presence of PCA or GA, DNA bending on both promoters was enhanced. The LigR-binding regions of the ligK and ligJ promoters in the presence of inducer molecules were extended and shortened, respectively. The LTTR consensus sequences (Box-K and Box-J) in the ligK and ligJ promoters were essential for the binding of LigR and transcriptional activation of both operons. In addition, the regions between the LigR binding boxes and the -35 regions were required for the enhancement of DNA bending, although the binding of LigR to the -35 region of the ligJ promoter was not observed in DNase I footprinting experiments. This study shows the binding features of LigR on the ligK and ligJ promoters and explains how the PCA45 pathway genes are expressed during degradation of lignin-derived biaryls by this bacterium.

Gas Chromatography/Mass Spectrometry of the Lignans in Resin of Callitris preissii

The total extract of resin from Callitris preissii was analyzed by gas chromatography/mass spectrometry and 34 lignans, underivatized and as trimethylsilyl ether derivatives, were identified by interpretation of the mass spectra. The lignans in the resin include 13 new lignans, i.e., four secoisolariciresinol derivatives, four lariciresinol derivatives, 3′,4′-methylenedioxy-7′-hydroxylariciresinol, 3,4-methylenedioxypinoresinol isomer, and two matairesinol derivatives. Furthermore, 17 lignans of the resin are characterized by methylenedioxy substitution on either or both guaiacylpropyl units or the syringylpropyl unit: i.e., five secoisolariciresinol derivatives, three lariciresinol derivatives, 3′,4′-methylenedioxy-7′-hydroxylariciresinol, three pinoresinol derivatives, and five matairesinol derivatives, have methylenedioxy structures. Eleven lignans, including two secoisolariciresinol derivatives, four lariciresinol derivatives, two pinoresinol derivatives, and three matairesinol derivatives, contain a syringyl moiety.

RNAi-mediated suppression of p -coumaroyl-CoA 3′-hydroxylase in hybrid poplar impacts lignin deposition and soluble secondary metabolism

p-Coumaroyl-CoA 3'-hydroxylase (C3'H) is a cytochrome P450-dependent monooxygenase that catalyzes the 3'-hydroxylation of p-coumaroyl shikimate and p-coumaroyl quinate. We used RNA interference to generate transgenic hybrid poplar suppressed in C3'H expression and analyzed them with respect to transcript abundance, cell wall structure and chemical composition, and soluble metabolite levels. RT-PCR expression profiles confirmed the down-regulation of C3'H in a number of lines, which generally correlated very well with reduced total cell wall lignin content. The most strongly repressed line was chosen for further analysis and compared with the wild-type trees. In-depth characterization revealed that along with the significant decrease in total lignin content, a significant shift in lignin monomer composition was observed, favoring the generation of p-hydroxyphenyl units at the expense of guaiacyl units while the proportion of syringyl moieties remained constant. Suppression of C3'H also resulted in the accumulation of substantial pools of 1-O-p-coumaroyl-beta-d-glucoside and other phenylpropanoid glycosides, and p-coumaroyl shikimate, providing further insight into the role of C3'H in the lignin biosynthetic pathway. The data presented indicate that when down-regulated, C3'H becomes a rate-limiting step in lignin biosynthesis and further support the involvement of hydroxycinnamic acid shikimate esters in the lignin biosynthetic pathway.

アブラヤシの化学成分組成分析

As the worldwide production of palm oil is increasing, concomitant wastes of unutilized parts of the oil palm is also increasing. Thus, effective utilization of these wastes is expected. In this paper, therefore, the chemical composition of cellulose, hemicellulose, lignin and other minor cell wall components was studied for six different parts of the oil palm such as trunk, frond, mesocarp, shell, kernel cake and empty fruit bunch (EFB). As a result, it was shown that cellulose is in a range between 20-40wt% with hemicellulose being 10-35wt%, whereas lignin in a range between 23-52wt%. More in detail, the shell contained the highest lignin about 52wt% but the kernel cake no lignin, while the rest in a range between 23-35wt%, being composed of guaiacyl and syringyl moieties without p-hydroxyphenyl residue. This is very similar to the hardwood-type lignin, rather than softwood-type lignin. On hemicellulose, mannan was rich in kernel cake, while glucuronoxylan rich in the other parts, with 1.8-8.5 xylose units per one uronic acid. These lines of information are useful for the efficient utilization of the whole parts of the oil palm which is necessary for the sustainable development of the biomass resources.

Over-expression of UDP-glucose pyrophosphorylase in hybrid poplar affects carbon allocation

The effects of the over-expression of the Acetobacter xylinum UDP-glucose pyrophosphorylase (UGPase) under the control of the tandem repeat Cauliflower Mosaic Virus promoter (2x35S) on plant metabolism and growth were investigated in hybrid poplar (Populus albaxgrandidentata). Transcript levels, enzyme activity, growth parameters, leaf morphology, structural and soluble carbohydrates, and soluble metabolite levels were quantified in both transgenic and wild-type trees. Transgenic 2x35S::UGPase poplar showed impaired growth rates, displaying reduced height growth and stem diameter. Morphologically, 2x35S::UGPase trees had elongated axial shoots, and leaves that were substantially smaller in size when compared with wild-type trees at equivalent developmental stages. Biochemical analysis revealed significant increases in soluble sugar, starch, and cellulose contents, and concurrent decreases in lignin content. Lignin monomer composition was altered in favour of syringyl moieties. Detailed soluble metabolite analysis revealed that 2x35S::UGPase trees had as much as a 270-fold increase in the salicylic acid 2-O-beta-D-glucoside (SAG), a compound typically associated with the stress response. These data suggest that while it is possible to alter the allocation of carbon in favour of cellulose biosynthesis, whole plant changes result in unexpected decreases in growth and an increase in defence metabolites.

Metal Ion Uptake from Aqueous Solution by Olive Stones: A Carbon‐13 Solid‐State Nuclear Magnetic Resonance and Potentiometric Study

The use of biomasses that result from the agriculture and food industries in removing heavy metals from wastewaters is attracting increasing interest. We present a joined potentiometric and cross polarization magic angle spinning (CP-MAS) carbon-13 (13C) nuclear magnetic resonance (NMR) study on the interaction of olive stones with copper(II), nickel(II), and cadmium(II). The potentiometric measurements allow both to distinguish two kind of basic sites in the olive stones and to postulate the coordination models for the three studied metals. The NMR spectral analysis allows the attribution of the different signals to the components of the olive stone matrix. A comparison of CP-MAS 13C NMR spectra of the samples after metal treatment suggests a specific complexation between metal ions and hydroxyl groups on guaiacyl and syringyl moieties.

Structural Motifs of Syringyl Peroxidases Are Conserved during Angiosperm Evolution

The most distinctive variation in the monomer composition of lignins in vascular land plants is that between the two main groups of seed plants. Thus, whereas gymnosperm (softwood) lignins are typically composed of guaiacyl (G) units, angiosperm (hardwood) lignins are largely composed of similar levels of G and syringyl (S) units. However, there are some studies that suggest that certain angiosperm peroxidases are unable to oxidize sinapyl alcohol, and a coniferyl alcohol shuttle has been proposed for oxidizing S units during the biosynthesis of lignins. With this in mind, a screening of the presence of S peroxidases in angiosperms (including woody species and forages) was performed. Contrarily to what might be expected, the intercellular washing fluids from lignifying tissues of 25 woody, herbaceous, and shrub species, belonging to both monocots and dicotyledons, all showed both S peroxidase activities and basic peroxidase isoenzymes analogous, with regard the isoelectric point, to the Zinnia elegans basic peroxidase isoenzyme, the only S peroxidase that has been fully characterized. These results led to the protein database in the search for homologies between angiosperm peroxidases and a true eudicot S peroxidase, the Z. elegans peroxidase. The findings showed that certain structural motifs of S peroxidases are conserved within the first 15 million years of angiosperm history, because they are found in peroxidases from the two major lineages of flowering plants, eumagnoliids and eudicotyledons, of note being the presence of these peroxidases in Amborella and Nymphaeales, which represent the first stages of angiosperm evolution. These phylogenetic studies also suggest that guaiacyl peroxidases apparently constitute the most "evolved state" of the plant peroxidase family evolution.

Quantitative Characterization of a Hardwood Milled Wood Lignin by Nuclear Magnetic Resonance Spectroscopy

The structure of Eucalyptus grandis milled wood lignin (MWL) was investigated by 2D 1H-13C HSQC, HMQC, and 1H-1H TOCSY correlation NMR techniques and by quantitative 13C NMR as well as by the permanganate oxidation degradation technique. The combination of 2D NMR and quantitative 13C NMR spectroscopy of nonacetylated and acetylated lignin preparations allowed reliable identification and calculation of the amount of different lignin structures. About 85% of side-chain moieties were estimated on the structural level. This information was substantiated by data on the quantity of various functional groups and interunit linkages as a whole. A modified method for calculation of the h:g:s ratio has been suggested and compared with previously suggested approaches. E. grandis MWL has been determined to have an h:g:s ratio of 2:36:62. The amounts of various phenolic/etherified noncondensed/condensed guaiacyl and syringyl moieties were approximately estimated. E. grandis MWL contained approximately 0.60/Ar of beta-O-4 moieties along with small amounts of other structural units such as pino/syringyresinol (0.03/Ar), phenylcoumaran (0.03/Ar), and spirodienone (0.05/Ar). The degree of condensation was estimated at approximately 21%; the main condensed structures are 4-O-5 moieties (approximately 0.09/Ar). The structure of E. grandis MWL was compared with those of other lignin preparations isolated from various hardwoods.

Molecular Composition of Leaves and Stems of Genetically Modified Bt and Near‐Isogenic Non‐Bt Maize—Characterization of Lignin Patterns

Transformation of crops, including maize (Zea mays L.), with the cry1Ab gene from Bacillus thuringiensis to combat lepidopteran pests results in pleiotropic effects regarding lignin biosynthesis. Lignin patterns in stems and leaves of two genetically modified Bt-maize varieties (Novelis T and Valmont T) were studied along with their non-Bt near-isolines (Nobilis and Prelude, respectively). Molecular-level based thermochemolysis using tetramethylammonium hydroxide (TMAH) in combination with gas chromatography-mass spectrometry (GC-MS) was used to quantitate the total lignin contents and to identify monomeric lignin subunits including p-hydroxyphenyl (P), guaiacyl (G), and syringyl (S) moieties. The results were supplemented and confirmed by cupric oxide oxidation. The stems of the transgenic lines had higher concentrations of total lignin than the respective isogenic lines: Valmont T/Prelude by 18% and Novelis T/Nobilis by 28%. In contrast, differences in the total lignin concentration of leaves between the transgenic and the respective near-isogenic lines were marginal. There were significant modifications in the ratio of p-hydroxyphenyl/guaiacyl/syringyl molecular marker units of stem lignin between transgenic and isogenic lines. The guaiacyl units (in particular the G18 marker) accounted chiefly for the higher total lignin contents in the transgenic lines. The leaf lignin patterns did not show significant differences in molecular markers between isogenic and transgenic lines. TMAH-induced thermochemolysis--conducted in both the on-line and off-line modes--provided detailed information on the molecular composition of lignin, thus proving superior to the established "wet chemistry" methods of lignin determination.

Reassessment of effects on lignification and vascular development in the irx4 Arabidopsis mutant

The Arabidopsis thaliana irregular xylem4 ( irx4) cinnamoyl-CoA reductase 1 (CCR1) mutant was reassessed for its purported exclusive rate-limiting or key effects on lignification. Analyses of gross growth characteristics and stem cross-section anatomy, from seedling emergence to senescence, revealed that stunted irx4 mutant lines were developmentally delayed, which in turn indirectly but predictably led to modest reductions (ca. 10–15%) in overall lignin amounts. Such developmental changes are not generally observed in suppression of other monolignol pathway forming enzymes (e.g., 4-coumarate CoA ligase) even when accompanied by significant reductions in lignin amounts. With the greatly arrested development of the irx4 mutant, formation of the lignin-derived syringyl moieties was also predictably delayed (by about 1–2 weeks), although at maturation the final guaiacyl:syringyl ratios were essentially identical to wild-type. No evidence was obtained for so-called abnormal lignin precursors being incorporated into the lignin, as shown by solid-state 13C NMR spectroscopic analysis in contrast to a claim to the contrary [Jones, L., Ennos, A.R., Turner, S.R., 2001. Cloning and characterization of irregular xylem4 ( irx4): a severely lignin-deficient mutant of Arabidopsis. Plant J. 26, 205–216]. A previous claim of an “abnormal” lignin present in stunted CCR downregulated tobacco was also not substantiated, with only trace differences being noted in the presumed cell-wall constituent levels. More importantly, a linear correlation between total lignin amounts and lignin-derived fragmentation products was observed at all stages of Arabidopsis growth/development in both wild-type and irx4 mutant lines, regardless of lignin content, i.e., in harmony with an exquisitely controlled and predictable macromolecular assembly process. Recombinant CCR1 displayed fairly broad substrate versatility for all phenylpropanoid CoA substrates, with both feruloyl and 5-hydroxyferuloyl CoA being the best substrates. Taken together, these data indicate that other CCR isoforms are apparently capable of generating monolignol-derived lignified elements in irx4 when CCR1 is impaired, i.e., indicative of a functionally redundant CCR metabolic network operative in Arabidopsis. Other dwarfed phenotypes have also been observed following downregulation/disruption of unrelated metabolic processes but which also involve CoA ester metabolism, i.e., with hydroxymethylglutaryl CoA reductases in Arabidopsis and a bacterial enoyl CoA hydratase/lyase overexpressed in tobacco. Although the reasons for dwarfing in each case are unknown, a common mechanism for the various pleiotropic effects is proposed through perturbation of CoASH pool levels. Finally, this study demonstrates the need for progressive analyses over the lifespan of an organism, rather than at a single time point which cannot reveal the progressive developmental changes occurring.