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     

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.     

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.     

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.     

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     

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.     

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.     

5-hydroxyconiferyl aldehyde modulates enzymatic methylation for syringyl monolignol formation, a new view of monolignol biosynthesis in angiosperms

S-Adenosyl-L-methionine-dependent caffeate O-methyltransferase (COMT, EC 2.1.1.6) has traditionally been thought to catalyze the methylation of caffeate and 5- hydroxyferulate for the biosynthesis of syringyl monolignol, a lignin constituent of angiosperm wood that enables efficient lignin degradation for cellulose production. However, recent recognition that coniferyl aldehyde prevents 5-hydroxyferulate biosynthesis in lignifying tissue, and that the hydroxylated form of coniferyl aldehyde, 5-hydroxyconiferyl aldehyde, is an alternative COMT substrate, demands a re-evaluation of the role of COMT during monolignol biosynthesis. Based on recombinant aspen (Populus tremuloides) COMT enzyme kinetics coupled with mass spectrometry analysis, this study establishes for the first time that COMT is in fact a 5-hydroxyconiferyl aldehyde O-methyltransferase (AldOMT), and that 5-hydroxyconiferyl aldehyde is both the preferred AldOMT substrate and an inhibitor of caffeate and 5-hydroxyferulate methylation, as measured by K(m) and K(i) values. 5-Hydroxyconiferyl aldehyde also inhibited the caffeate and 5-hydroxyferulate methylation activities of xylem proteins from various angiosperm tree species. The evidence that syringyl monolignol biosynthesis is independent of caffeate and 5-hydroxyferulate methylation supports our previous discovery that coniferyl aldehyde prevents ferulate 5-hydroxylation and at the same time ensures a coniferyl aldehyde 5-hydroxylase (CAld5H)-mediated biosynthesis of 5-hydroxyconiferyl aldehyde. Together, our results provide conclusive evidence for the presence of a CAld5H/AldOMT-catalyzed coniferyl aldehyde 5-hydroxylation/methylation pathway that directs syringyl monolignol biosynthesis in angiosperms.     

Water stress-induced xylem hydraulic failure is a causal factor of tree mortality in beech and poplar

Background and Aims

Extreme water stress episodes induce tree mortality, but the physiological mechanisms causing tree death are still poorly understood. This study tests the hypothesis that a potted tree''s ability to survive extreme monotonic water stress is determined by the cavitation resistance of its xylem tissue.

Methods

Two species were selected with contrasting cavitation resistance (beech and poplar), and potted juvenile trees were exposed to a range of water stresses, causing up to 100 % plant death.

Key Results

The lethal dose of water stress, defined as the xylem pressure inducing 50 % mortality, differed sharply across species (1·75 and 4·5 MPa in poplar and beech, respectively). However, the relationships between tree mortality and the degree of cavitation in the stems were similar, with mortality occurring suddenly when >90 % cavitation had occurred.

Conclusions

Overall, the results suggest that cavitation resistance is a causal factor of tree mortality under extreme drought conditions.     

Profiling small RNA reveals multimodal substructural signals in a Boltzmann ensemble

As the biomedical impact of small RNAs grows, so does the need to understand competing structural alternatives for regions of functional interest. Suboptimal structure analysis provides significantly more RNA base pairing information than a single minimum free energy prediction. Yet computational enhancements like Boltzmann sampling have not been fully adopted by experimentalists since identifying meaningful patterns in this data can be challenging. Profiling is a novel approach to mining RNA suboptimal structure data which makes the power of ensemble-based analysis accessible in a stable and reliable way. Balancing abstraction and specificity, profiling identifies significant combinations of base pairs which dominate low-energy RNA secondary structures. By design, critical similarities and differences are highlighted, yielding crucial information for molecular biologists. The code is freely available via http://gtfold.sourceforge.net/profiling.html.     

Increase of xylan synthetase activity during xylem differentiation of the vascular cambium of sycamore and poplar trees

The activity of a -(1-4)-xylan synthetase, a membrane-bound enzymic system, was measured in particulate enzymic preparations (1,000 g and 1,000–100,000 g pellets) obtained from homogenates of cambial cells, differentiating xylem cells and differentiated xylem cells isolated from actively growing trees of sycamore (Acer pseudoplatamus) and poplar (Populus robusta). The specific activity (nmol of xylan formed min^–1 mg^–1 of protein) as well as the activity calculated on a per cell basis (nmol of xylan formed min^–1 cell^–1) of this enzymic system, markedly increased as cells differentiate from the vascular cambium to xylem. This increase is closely correlated with the enhanced deposition of xylan occurring during the formation of secondary thickening. The possible control of xylan synthesis during the biogenesis of plant cell wall is discussed.     

Diversity of endophytic bacterial communities in poplar grown under field conditions

Bacterial endophytes may be important for plant health and other ecologically relevant functions of poplar trees. The composition of endophytic bacteria colonizing the aerial parts of poplar was studied using a multiphasic approach. The terminal restriction fragment length polymorphism analysis of 16S rRNA genes demonstrated the impact of different hybrid poplar clones on the endophytic community structure. Detailed analysis of endophytic bacteria using cultivation methods in combination with cloning of 16S rRNA genes amplified from plant tissue revealed a high phylogenetic diversity of endophytic bacteria with a total of 53 taxa at the genus level that included Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes. The community structure displayed clear differences in terms of the presence and relative proportions of bacterial taxa between the four poplar clones studied. The results showed that the genetic background of the hybrid poplar clones corresponded well with the endophytic community structure. Out of the 513 isolates and 209 clones identified, Actinobacteria, in particular the family Microbacteriaceae, made up the largest fraction of the isolates, whereas the clone library was dominated by Alpha- and Betaproteobacteria. The most abundant genera among the isolates were Pseudomonas and Curtobacterium, while Sphingomonas prevailed among the clones.     

Characterization of peroxidases in lignifying peach fruit endocarp

Developing peach (Prunus persica L. Batsch `Redskin') fruit were used to characterize the role of peroxidases in lignification. During development, the endocarp of these drupes becomes lignified while the mesocarp remains parenchymatous. Acidic peroxidase from lignifying endocarp were similar to those of the fleshy mesocarp. The endocarp had a larger amount and number of basic peroxidases than the mesocarp. Cultured peach leaf cells are thought to be lignified because their walls give a positive reaction with phloroglucinol-HCI. These cells also secreted a basic peroxidase. Peroxidases were difficult to extract from endocarp tissue as they lignified. This was also demonstrated by tissue printing on nitrocellulose. Flesh, but not endocarp peroxidase was evident in tissue prints. This suggests that tissue printing may fail to reveal the presence of enzymes which are firmly attached to the cell.