Phenolic profiling of caffeic acid O-methyltransferase-deficient poplar reveals novel benzodioxane oligolignols

Caffeic acid O-methyltransferase (COMT) catalyzes preferentially the methylation of 5-hydroxyconiferaldehyde to sinapaldehyde in monolignol biosynthesis. Here, we have compared HPLC profiles of the methanol-soluble phenolics fraction of xylem tissue from COMT-deficient and control poplars (Populus spp.), using statistical analysis of the peak heights. COMT down-regulation results in significant concentration differences for 25 of the 91 analyzed peaks. Eight peaks were exclusively detected in COMT-deficient poplar, of which four could be purified for further identification using mass spectrometry/mass spectrometry, nuclear magnetic resonance, and spiking of synthesized reference compounds. These new compounds were derived from 5-hydroxyconiferyl alcohol or 5-hydroxyconiferaldehyde and were characterized by benzodioxane moieties, a structural type that is also increased in the lignins of COMT-deficient plants. One of these four benzodioxanes amounted to the most abundant oligolignol in the HPLC profile. Furthermore, all of the differentially accumulating oligolignols involving sinapyl units were either reduced in abundance or undetectable. The concentration levels of all identified oligolignols were in agreement with the relative supply of monolignols and with their chemical coupling propensities, which supports the random coupling hypothesis. Chiral HPLC analysis of the most abundant benzodioxane dimer revealed the presence of both enantiomers in equal amounts, indicating that they were formed by radical coupling reactions under simple chemical control rather than guided by dirigent proteins.     

Mathematical modeling of monolignol biosynthesis in Populus xylem

Recalcitrance of lignocellulosic biomass to sugar release is a central issue in the production of biofuel as an economically viable energy source. Among all contributing factors, variations in lignin content and its syringyl-guaiacyl monomer composition have been directly linked with the yield of fermentable sugars. While recent advances in genomics and metabolite profiling have significantly broadened our understanding of lignin biosynthesis, its regulation at the pathway level is yet poorly understood. During the past decade, computational and mathematical methods of systems biology have become effective tools for deciphering the structure and regulation of complex metabolic networks. As increasing amounts of data from various organizational levels are being published, the application of these methods to studying lignin biosynthesis appears to be very beneficial for the future development of genetically engineered crops with reduced recalcitrance. Here, we use techniques from flux balance analysis and nonlinear dynamic modeling to construct a mathematical model of monolignol biosynthesis in Populus xylem. Various types of experimental data from the literature are used to identify the statistically most significant parameters and to estimate their values through an ensemble approach. The thus generated ensemble of models yields results that are quantitatively consistent with several transgenic experiments, including two experiments not used in the model construction. Additional model results not only reveal probable substrate saturation at steps leading to the synthesis of sinapyl alcohol, but also suggest that the ratio of syringyl to guaiacyl monomers might not be affected by genetic modulations prior to the reactions involving coniferaldehyde. This latter model prediction is directly supported by data from transgenic experiments. Finally, we demonstrate the applicability of the model in metabolic engineering, where the pathway is to be optimized toward a higher yield of xylose through modification of the relative amounts of the two major monolignols. The results generated by our preliminary model of in vivo lignin biosynthesis are encouraging and demonstrate that mathematical modeling is poised to become an effective and predictive complement to traditional biotechnological and transgenic approaches, not just in microorganisms but also in plants.     

Phenylcoumaran benzylic ether reductase, a prominent poplar xylem protein, is strongly associated with phenylpropanoid biosynthesis in lignifying cells

 It has previously been shown (D.R. Gang et al., 1999, J Biol Chem 274: 7516–7527) that the most abundant protein in the secondary xylem of poplar (Populus trichocarpa cv. `Trichobel') is a phenylcoumaran benzylic ether reductase (PCBER), an enzyme involved in lignan synthesis. Here, the distribution and abundance of PCBER in poplar was studied at both the RNA and protein level. The cellular expression pattern was determined by immunolocalization of greenhouse-grown plants as well as of a field-grown poplar. Compared to other poplar tissues, PCBER is preferentially produced in the secondary xylem of stems and roots and is associated with the active growth period. The protein is present in all cells of the young differentiating xylem, corresponding to the zone of active phenylpropanoid metabolism and lignification. In addition, PCBER is located in young differentiating phloem fibers, in xylem ray parenchyma, and in xylem parenchyma cells at the growth-ring border. Essentially the same expression pattern was observed in poplars grown in greenhouses and in the field. The synthesis of PCBER in phenylpropanoid-synthesizing tissues was confirmed in a bending experiment. Induction of PCBER was observed in the pith of mechanically bent poplar stems, where phenylpropanoid metabolism is induced. These results indicate that the products of PCBER activity are synthesized mainly in lignifying tissues, suggesting a role in wood development. Received: 28 September 1999 / Accepted: 15 March 2000     

In situ characterization of a NO-sensitive peroxidase in the lignifying xylem of Zinnia elegans

The lignifying xylem from Zinnia elegans stems gives an intense reaction with 3,3',5,5'-tetramethylbenzidine (TMB), a reagent previously reported to be specific for peroxidase/H2O2. However, the staining of lignifying xylem cells with TMB is apparently the result of two independent mechanisms: one, the catalase-sensitive (H2O2-dependent) peroxidase-mediated oxidation of TMB, and the other, the catalase-insensitive oxidation of TMB, probably mediated by xylem oxidases which are specific from lignifying tissues. The catalase-insensitive oxidation of TMB by the Z. elegans xylem was sensitive to sodium nitroprusside (SNP), a nitric oxide (NO)-releasing compound that, when used at 5.0 mM, is capable of sustaining NO concentrations of 6.1 &mgr;M in the aqueous phase. This effect of SNP was totally reversed by 150 &mgr;M 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl-3-oxide (PTIO), an efficient NO scavenger in biological systems, so the above-mentioned effect must be ascribed to NO, and not to other nitrogen oxides. This response of the catalase-insensitive TMB-oxidase activity of the lignifying Z. elegans xylem was similar to that shown by a basic peroxidase isolated from the intercellular washing fluid, which showed TMB-oxidase activity, and which was also inhibited by 5 mM SNP, the effect of SNP also being reversed by 150 &mgr;M PTIO. These results suggest that peroxidase was the enzyme responsible for the NO-sensitive catalase-insensitive TMB-oxidase activity of the lignifying Z. elegans xylem. Further support for this statement was obtained from competitive inhibitor-dissected histochemistry, which showed that this stain responded to peroxidase-selective competitive inhibitors, such as ferulic acid and ferrocyanide, in a similar way to the Z. elegans basic peroxidase. From these results, we conclude that this NO-sensitive catalase-insensitive oxidation of TMB is apparently performed by the Z. elegans basic peroxidase, and that the regulation of this enzyme by NO may constitute an intrinsically programmed event during the differentiation and death of the xylem.     

Proteomic analysis of hybrid poplar xylem sap

Xylem sap collected from Populus trichocarpa × Populus deltoides using root pressure was estimated to contain more than 100 proteins. Ninety-seven of these proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These proteins were classified into 10 functional categories including metabolism, signaling, stress response and cell wall functions. The majority of xylem sap proteins were metabolic enzymes involved in processes including translation, proteolysis, and glycolysis. Stress-related proteins were also prevalent. In contrast to xylem sap proteins collected from annual plants, the majority of poplar xylem sap proteins do not appear to be classically secreted since only 33 proteins were predicted to have an N-terminal signal peptide targeting them to the secretory pathway. Of the remaining 64 proteins, 27 were predicted to be secreted non-classically. While a number of proteins identified here have been previously reported in xylem sap proteomes of annual plants, many xylem sap proteins were identified in poplar which may reflect functions specific to perennial plants.     

Isolation and characterization of thioredoxin h from poplar xylem

Redox-dependent regulation based on disulphide/dithiol exchange reactions has been extensively studied in herbaceous plants, but up to now, there is no information concerning these systems in trees. Based on existing ESTs, a cDNA coding for a thioredoxin h has been isolated from a xylem poplar cDNA library. The nucleotidic sequence of poplar thioredoxin h displays significant homology to other thioredoxins h isolated from plants. It shows a variation in the active site with the sequence WCPPC instead of the more canonical WCGPC sequence found in most thioredoxins. The cDNA sequence has been introduced in an expression plasmid (pET3d) in order to express the corresponding recombinant polypeptide. The protein has been expressed to a high level and purified from Escherichia coli cells with a very high yield. Several of the physical and kinetic characteristics of this redox protein are described and found to be similar to other thioredoxin h. On the other hand, its stability to heat denaturation, is very different from those of other thioredoxins h characterized so far.     

PtxtPME1 and homogalacturonans influence xylem hydraulic properties in poplar

While the xylem hydraulic properties, such as vulnerability to cavitation (VC), are of paramount importance in drought resistance, their genetic determinants remain unexplored. There is evidence that pectins and their methylation pattern are involved, but the detail of their involvement and the corresponding genes need to be clarified. We analyzed the hydraulic properties of the 35S::PME1 transgenic aspen that ectopically under‐ or over‐express a xylem‐abundant pectin methyl esterase, PtxtPME1. We also produced and analyzed 4CL1::PGII transgenic poplars expressing a fungal polygalacturonase, AnPGII, under the control of the Ptxa4CL1 promoter that is active in the developing xylem after xylem cell expansion. Both the 35S::PME1 under‐ and over‐expressing aspen lines developed xylem with lower‐specific hydraulic conductivity and lower VC, while the 4CL1::PGII plants developed xylem with a higher VC. These xylem hydraulic changes were associated with modifications in xylem structure or in intervessel pit structure that can result in changes in mechanical behavior of the pit membrane. This study shows that homogalacturonans and their methylation pattern influence xylem hydraulic properties, through its effect on xylem cell expansion and on intervessel pit properties and it show a role for PtxtPME1 in the xylem hydraulic properties.     

Vessel- and ray-specific monolignol biosynthesis as an approach to engineer fiber-hypolignification and enhanced saccharification in poplar

Lignin is one of the main factors determining recalcitrance to processing of lignocellulosic biomass towards bio-based materials and fuels. Consequently, wood of plants engineered for low lignin content is typically more amenable to processing. However, lignin-modified plants often exhibit collapsed vessels and associated growth defects. Vessel-specific reintroduction of lignin biosynthesis in dwarfed low-lignin cinnamoyl-CoA reductase1 (ccr1) Arabidopsis mutants using the ProSNBE:AtCCR1 construct overcame the yield penalty while maintaining high saccharification yields, and showed that monolignols can be transported between the different xylem cells acting as ‘good neighbors’ in Arabidopsis. Here, we translated this research into the bio-energy crop poplar. By expressing ProSNBE:AtCCR1 into CRISPR/Cas9-generated ccr2 poplars, we aimed for vessel-specific lignin biosynthesis to: (i) achieve growth restoration while maintaining high saccharification yields; and (ii) study the existence of ‘good neighbors’ in poplar wood. Analyzing the resulting ccr2 ProSNBE:AtCCR1 poplars showed that vessels and rays act as good neighbors for lignification in poplar. If sufficient monolignols are produced by these cells, monolignols migrate over multiple cell layers, resulting in a restoration of the lignin amount to wild-type levels. If the supply of monolignols is limited, the monolignols are incorporated into the cell walls of the vessels and rays producing them and their adjoining cells resulting in fiber hypolignification. One such fiber-hypolignified line had 18% less lignin and, despite its small yield penalty, had an increase of up to 71% in sugar release on a plant base upon saccharification.     

Identification and partial purification of an oxidase from lignifying xylem of Leyland cypress (X Cupressocyparis leylandii)

 Oxidase activity was exclusively present in lignifying cells of developing xylem of Leyland cypress. The oxidase was enriched in 200 mM CaCl₂ extracts of crude cell walls and seems to be ionically associated with the cell walls. Oxidase activity was selected and concentrated using affinity chromatography on Concanavalin-A Sepharose which suggests that it is a high-mannose type glycoprotein. A subsequent purification step using gel permeation chromatography on Sephadex GF-150 partially separated the oxidase activity from peroxidase activity. An oxidase band of apparent Mr 92 kD capable of oxidising N, N, N′, N′ - tetramethyl phenylene diamine/α-naphthol was identified after non-denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis. The 92 kD oxidase band was enriched in the oxidase-rich fraction and absent from the peroxidase-rich fraction from the gel permeation step. In addition, the 92 kD oxidase band could be differentiated from peroxidase bands because it was not intensified by the addition of hydrogen peroxide. The partially purified oxidase effectively oxidised and polymerised coniferyl alcohol to form insoluble material that yielded a Fourier transform infra-red spectrum similar to dehydrogenation polymers of coniferyl alcohol. This coniferyl alcohol oxidase appears to be specific to lignifying xylem cells and may participate in lignin deposition but further studies are required to fully define this oxidase and its possible homology with other oxidases identified in the lignifying xylem of different species of trees. Received: 20 May 1997 / Accepted: 7 August 1997     

Exogenous chalcone synthase expression in developing poplar xylem incorporates naringenin into lignins

Lignin, a polyphenolic polymer, is a major chemical constituent of the cell walls of terrestrial plants. The biosynthesis of lignin is a highly plastic process, as highlighted by an increasing number of noncanonical monomers that have been successfully identified in an array of plants. Here, we engineered hybrid poplar (Populus alba x grandidentata) to express chalcone synthase 3 (MdCHS3) derived from apple (Malus domestica) in lignifying xylem. Transgenic trees displayed an accumulation of the flavonoid naringenin in xylem methanolic extracts not inherently observed in wild-type trees. Nuclear magnetic resonance analysis revealed the presence of naringenin in the extract-free, cellulase-treated xylem lignin of MdCHS3-poplar, indicating the incorporation of this flavonoid-derived compound into poplar secondary cell wall lignins. The transgenic trees also displayed lower total cell wall lignin content and increased cell wall carbohydrate content and performed significantly better in limited saccharification assays than their wild-type counterparts.

Expressing exogenous, apple-derived chalcone synthase in actively lignifying poplar xylem tissue results in less total lignin, improved saccharification rates, and incorporation of naringenin into lignins.     

Immunolocalization of phenylalanine ammonia-lyase and cinnamate-4-hydroxylase in differentiating xylem of poplar

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) and cinnamate-4-hydroxylase (C4H; EC 1.14.13.11) are pivotal enzymes involved in lignification. We synthesized peptides as the epitopes according to the amino acid sequences of these enzymes, coupled them with hemocyanin, and injected them into mice. The antiserums against peptides of PAL and C4H specifically detected PAL and C4H in the crude enzymes extracted from differentiating xylem of poplar, respectively. PAL and C4H were localized in differentiating xylem of poplar. PAL labeling was mainly localized in the cytosol, and somewhat localized on the rough-endoplasmic reticulum (r-ER) and the Golgi apparatus. In contrast, C4H was mainly observed on r-ER and the Golgi apparatus. These findings suggest that conversion of phenylalanine to cinnamic acid occurs in the cytosol and the following reaction occurs near the membrane of r-ER and the Golgi apparatus. The possibility of coordinated localization of PAL and C4H is discussed.     

Identification and partial characterization of a coniferyl alcohol oxidase from lignifying xylem of Sitka spruce (Picea sitchensis)

Oxidase activity in the developing xylem of branches of Sitka spruce [Picea sitchensis] (Bong) Carr. was expressed in synchrony with the deposition of lignin. The activity was closely associated with the cell wall but it could be extracted by elution with salt solutions such as 1 M NaCl or CaCl₂. A number of different oxidase isoforms with isoelectric points in the range 8–5 were present in these cell wall extracts. These enzymes displayed a marked preference for the oxidation of coniferyl alcohol and efficiently initiated polymerization of coniferyl alcohol into insoluble, lignin-like polymers. They also had a substrate preference and profile of sensitivity to inhibitors that was dissimilar to those reported for classical catechol oxidase or laccase-type polyphenol oxidases. A novel procedure that combines extraction and affinity chromatography on Concanavalin-A to select high-mannose-type glycoproteins provided oxidase activity at higher purity and yield than previously used methods. A single band of oxidase activity (apparent M_r approx. 84 kDa) which was capable of oxidizing α-naphthol/N,N,N′N′-tetramethyl p-phenylene diamine in the absence of added hydrogen peroxide was detected in these cell wall extracts using non-denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The addition of hydrogen peroxide did not intensify the staining of this band but it confirmed the presence of a true peroxidase band of apparent M_r approx. 40 kDa. The properties of this coniferyl alcohol oxidase are different from those of laccase-type polyphenol oxidases (EC 1.10.3.2) previously implicated in lignin deposition in tree species, and their possible roles in this process are discussed. Received: 9 January 1997 / Accepted: 14 March 1997     

Cloning, characterization and localization of three novel class III peroxidases in lignifying xylem of Norway spruce (Picea abies)

Plant class III peroxidases (POXs) take part in the formation of lignin and maturation of plant cell walls. However, only a few examples of such peroxidases from gymnosperm tree species with highly lignified xylem tracheids have been implicated so far. We report here cDNA cloning of three xylem-expressed class III peroxidase encoding genes from Norway spruce (Picea abies). The translated proteins, PX1, PX2 and PX3, contain the conserved amino acids required for heme-binding and peroxidase catalysis. They all begin with putative secretion signal propeptide sequences but diverge substantially at phylogenetic level, grouping to two subclusters when aligned with other class III plant peroxidases. In situ hybridization analysis on expression of the three POXs in Norway spruce seedlings showed that mRNA coding for PX1 and PX2 accumulated in the cytoplasm of young, developing tracheids within the current growth ring where lignification is occurring. Function of the putative N-terminal secretion signal peptides for PX1, PX2 and PX3 was confirmed by constructing chimeric fusions with EGFP (enhanced green fluorescent protein) and expressing them in tobacco protoplasts. Full-length coding region of px1 was also heterologously expressed in Catharanthus roseus hairy root cultures. Thus, at least the spruce PX1 peroxidase is processed via the endoplasmic reticulum (ER) most likely for secretion to the cell wall. Thereby, PX1 displays correct spatiotemporal localization for participation in the maturation of the spruce tracheid secondary cell wall.     

Unique occurrence of pectin-like fibrillar cell wall deposits in xylem fibres of poplar

Using light and electron microscopic techniques, we studied the unique occurrence of fibrillar cell wall deposits in mature xylem fibres from poplar. These cell wall deposits lined the lumen-facing side of the wall, mainly in fibres next to vessel elements. Different lines of evidence point to the pectin-like nature of these fibrillar cell wall deposits. First, specific staining by Alcian Blue 8GX, a dye with high affinity for pectic substances. Second, the strongly reduced staining of the cell wall deposits in microscopic sections treated with pectolytic enzyme. Third, concomitant staining of pits, which are known to consist mainly of pectic substances. Given the pectin-like nature of the fibrillar cell wall deposits as well as their preferred occurrence in fibres neighbouring water-conducting vessel elements, a function for the fibrillar cell wall deposits in lateral water diffusion and stem water storage is hypothesised. The hypothesis is supported by the increased abundance of these cell wall deposits in wood tissue of a drought-sensitive poplar species.     

Presence of a basic secretory protein in xylem sap and shoots of poplar in winter and its physicochemical activities against winter environmental conditions

Journal of Plant Research - XSP25, previously shown to be the most abundant hydrophilic protein in xylem sap of Populus nigra in winter, belongs to a secretory protein family in which the...     

Drought‐induced xylem pit membrane damage in aspen and balsam poplar

Drought induces an increase in a tree's vulnerability to a loss of its hydraulic conductivity in many tree species, including two common in western Canada, trembling aspen (Populus tremuloides) and balsam poplar (Populus balsamifera). Termed ‘cavitation fatigue’ or ‘air‐seeding fatigue’, the mechanism of this phenomenon is not well understood, but hypothesized to be a result of damage to xylem pit membranes. To examine the validity of this hypothesis, the effect of drought on the porosity of pit membranes in aspen and balsam poplar was investigated. Controlled drought and bench dehydration treatments were used to induce fatigue and scanning electron microscopy (SEM) was used to image pit membranes for relative porosity evaluations from air‐dried samples after ethanol dehydration. A significant increase in the diameter of the largest pore was found in the drought and dehydration treatments of aspen, while an increase in the percentage of porous pit membranes was found in the dehydration treatments of both species. Additionally, the location of the largest pore per pit membrane was observed to tend toward the periphery of the membrane.     

Adaptation to high salinity in poplar involves changes in xylem anatomy and auxin physiology

To investigate the physiological basis of salt adaptation in poplar, we compared the effect of salt stress on wood anatomy and auxin physiology of the salt-resistant Populus euphratica and salt-sensitive Populus x canescens. Both poplar species showed decreases in vessel lumina associated with increases in wall strength in response to salt, however, in P. euphratica at three-fold higher salt concentrations than in P. x canescens. The predicted hydraulic conductivity of the wood formed under salt stress decreased in P. x canescens, while in P. euphratica, no significant effects of salt on conductivity and transpiration were observed. The concentration of free indole-3-acetic acid (IAA) decreased under salt stress in the xylem of both poplar species, but to a larger extent in P. x canescens than in P. euphratica. Only salt-treated P. euphratica exhibited an increase in IAA-conjugates in the xylem. Genes homologous to the auxin-amidohydrolase ILL3 were isolated from the xylems of P. euphratica and P. x canescens. For functional analysis, the auxin-amidohydrolase from P. x canescens was overexpressed in Arabidopsis. Transgenic Arabidopsis plants were more resistant to salt stress than the wild-type plants. Increased sensitivity of the transgenic Arabidopsis to IAA-Leu showed that the encoded hydrolase used IAA-Leu as a substrate. These results suggest that poplar can use IAA-amidoconjugates in the stem as a source of auxin to balance the effects of salt stress on auxin physiology.     

Distribution of glucomannans and xylans in poplar xylem and their changes under tension stress

Present work investigated glucomannan (GM) and xylan distribution in poplar xylem cells of normal- (NW), opposite- (OW) and tension wood (TW) with immunolocalization methods. GM labeling was mostly detected in the middle- and inner S(2) (+S(3)) layer of NW and OW fibers, while xylan labeling was observed in the whole secondary cell wall. GM labeling in vessels of NW and OW was much weaker than in fibers and mostly detected in the S(2) layer, whereas slightly stronger xylan labeling than fibers was detected in the whole secondary cell wall of vessels. Ray cells in NW and OW showed no GM labeling, but strong xylan labeling. These results indicate that GMs and xylans are spatially distributed in poplar xylem cells with different concentrations present in different cell types. Surprisingly, TW showed significant decrease of GM labeling in the normal secondary cell wall of gelatinous (G) fibers compared to NW and OW, while xylan labeling was almost identical indicating that the GM and xylan synthetic pathways in fibers have different reaction mechanisms against tension stress. Unlike fibers, no notable changes in GM labeling were detected in vessels of TW, suggesting that GM synthesis in vessels may not be affected by tension stress. GM and xylan was also detected in the G-layer with slightly stronger and much weaker labeling than the normal secondary cell wall of G-fibers. Differences in GM and xylan distribution are also discussed for the same functional cells found in hardwoods and softwoods.