Jean-Paul Joseleau

Molecular Phenotyping of the pal1 and pal2 Mutants of Arabidopsis thaliana Reveals Far-Reaching Consequences on Phenylpropanoid, Amino Acid, and Carbohydrate Metabolism

The first enzyme of the phenylpropanoid pathway, Phe ammonia-lyase (PAL), is encoded by four genes in Arabidopsis thaliana. Whereas PAL function is well established in various plants, an insight into the functional significance of individual gene family members is lacking. We show that in the absence of clear phenotypic alterations in the Arabidopsis pal1 and pal2 single mutants and with limited phenotypic alterations in the pal1 pal2 double mutant, significant modifications occur in the transcriptome and metabolome of the pal mutants. The disruption of PAL led to transcriptomic adaptation of components of the phenylpropanoid biosynthesis, carbohydrate metabolism, and amino acid metabolism, revealing complex interactions at the level of gene expression between these pathways. Corresponding biochemical changes included a decrease in the three major flavonol glycosides, glycosylated vanillic acid, scopolin, and two novel feruloyl malates coupled to coniferyl alcohol. Moreover, Phe overaccumulated in the double mutant, and the levels of many other amino acids were significantly imbalanced. The lignin content was significantly reduced, and the syringyl/guaiacyl ratio of lignin monomers had increased. Together, from the molecular phenotype, common and specific functions of PAL1 and PAL2 are delineated, and PAL1 is qualified as being more important for the generation of phenylpropanoids.

Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure.

Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula × Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.

Down-regulation of the AtCCR1 gene in Arabidopsis thaliana: effects on phenotype, lignins and cell wall degradability

Cinnamoyl CoA reductase (CCR; EC 1.2.1.44) is the first enzyme specific to the biosynthetic pathway leading to monolignols. Arabidopsis thaliana (L.) Heynh. plants transformed with a vector containing a full-length AtCCR1 cDNA in an antisense orientation were obtained and characterized. The most severely down-regulated homozygous plants showed drastic alterations to their phenotypical features. These plants had a 50% decrease in lignin content accompanied by changes in lignin composition and structure, with incorporation of ferulic acid into the cell wall. Microscopic analyses coupled with immunolabelling revealed a decrease in lignin deposition in normally lignified tissues and a dramatic loosening of the secondary cell wall of interfascicular fibers and vessels. Evaluation of in vitro digestibility demonstrated an increase in the enzymatic degradability of these transgenic lines. In addition, culture conditions were shown to play a substantial role in lignin level and structure in the wild type and in the effects of AtCCR1 repression efficiency.

Detection in situ and characterization of lignin in the G-layer of tension wood fibres of Populus deltoides

The occurrence of lignin in the additional gelatinous (G-) layer that differentiates in the secondary wall of hardwoods during tension wood formation has long been debated. In the present work, the ultrastructural distribution of lignin in the cell walls of normal and tension wood fibres from poplar (Populus deltoides Bartr. ex Marshall) was investigated by transmission electron microscopy using cryo-fixation–freeze-substitution in association with immunogold probes directed against typical structural motifs of lignin. The specificity of the immunological probes for condensed and non-condensed guaiacyl and syringyl interunit linkages of lignin, and their high sensitivity, allowed detection of lignin epitopes of definite chemical structures in the G-layer of tension wood fibres. Semi-quantitative distribution of the corresponding epitopes revealed the abundance of syringyl units in the G-layer. Predominating non-condensed lignin sub-structures appeared to be embedded in the crystalline cellulose matrix prevailing in the G-layer. The endwise mode of polymerization that is known to lead to these types of lignin structures appears consistent with such an organized cellulose environment. Immunochemical labelling provides the first visualization in planta of lignin structures within the G-layer of tension wood. The patterns of distribution of syringyl epitopes indicate that syringyl lignin is deposited more intensely in the later phase of fibre secondary wall assembly. The data also illustrate that syringyl lignin synthesis in tension wood fibres is under specific spatial and temporal regulation targeted differentially throughout cell wall layers.

Chemical and 13C N.M.R. studies on two arabinans from the inner bark of young stems of Rosa glauca

Abstract Two water-soluble arabinan fractions have been isolated from the inner bark of Rosa glauca stems. Methylation analysis and periodate oxidation revealed that the two polysaccharides have similar, highly branched structures consisting of α- L -arabinofuranose residues substituted (1→2), (1→3), and (1→5). The two arabinans differ in their degree of polymerization: 100 and 34, respectively. 13 C-N.M.R. spectroscopy has been used to distinguish between glycosidically substituted and free C-5 groups in the polymer, as well as to assign the methoxyl groups at positions 2, 3 and 5 of the methylated arabinans. The different types of linkages could also be identified. The results obtained by chemical N.M.R.-spectroscopic methods were in good agreement.

Structural studies of pectic substances from the pulp of grape berries

Abstract The structure of the neutral sugar side-chains of pectic substances extracted with water (WSP), dilute HCl (HP), and endopectinlyase (AIR/PEL) from an alcohol-insoluble residue (AIR) from the pulp of grape berries was investigated by using chemical methods (methylation, carboxyl-reduction, and periodate oxidation), enzymic degradation with an α- l -arabinofuranosidase, and 13 C-n.m.r. spectroscopy. The side chains of WSP are essentially a type II arabinogalactan, whereas those of HP and AIR/PEL are arabinan-like structures associated with minor proportions of type I and II arabinogalactans. The side chains are ∼80% degraded by the α- l -arabinofuranosidase, indicating that the α- l -arabinofuranosyl residues are mainly 3-linked to the (1→6)-galactan side-chains of the type II arabinogalactan. The three pectic fractions contain minor amounts of proteins rich in hydroxyproline and serine.

Investigation of the structure of a heteroxylan from the outer pericarp (beeswing bran) of wheat kernel

A heteroxylan isolated from the pericarp (beeswing bran) of wheat kernel and purified is shown to be a highly substituted glucuronoarabinoxylan in which 80% of the β-d-xylosyl residues carry one or two substituents. Single terminal α-l-arabinofuranosyl groups and short chains of 2-, 3-, 5-, and 2,3-linked arabinose residues are distributed along the β-(1→4)-linked backbone of the xylan core which also carries residues of glucuronic acid and its 4-O-methyl derivative. Graded acid hydrolysis and degradations with an endo-xylanase and an α-l-arabinofuranosidase indicate that both the arabinan and the xylan moieties are highly branched structures, and this is confirmed by 13C-n.m.r. spectroscopy of the heteroxyaln at various stages of degradation.

Altered lignin biosynthesis improves cellulosic bioethanol production in transgenic maize plants down-regulated for cinnamyl alcohol dehydrogenase.

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme involved in the last step of monolignol biosynthesis. The effect of CAD down-regulation on lignin production was investigated through a transgenic approach in maize. Transgenic CAD-RNAi plants show a different degree of enzymatic reduction depending on the analyzed tissue and show alterations in cell wall composition. Cell walls of CAD-RNAi stems contain a lignin polymer with a slight reduction in the S-to-G ratio without affecting the total lignin content. In addition, these cell walls accumulate higher levels of cellulose and arabinoxylans. In contrast, cell walls of CAD-RNAi midribs present a reduction in the total lignin content and of cell wall polysaccharides. In vitro degradability assays showed that, although to a different extent, the changes induced by the repression of CAD activity produced midribs and stems more degradable than wild-type plants. CAD-RNAi plants grown in the field presented a wild-type phenotype and produced higher amounts of dry biomass. Cellulosic bioethanol assays revealed that CAD-RNAi biomass produced higher levels of ethanol compared to wild-type, making CAD a good target to improve both the nutritional and energetic values of maize lignocellulosic biomass.

Characterisation of the oligosaccharides produced on hydrolysis of galactomannan with β-d-mannase

Abstract Treatment of hot-water-soluble carob galactomannan with β- d -mannanases from A. niger or lucerne seed affords an array of d -galactose-containing β- d -mannosaccharides as well as β- d -manno-biose, -triose, and -tetraose (lucerne-seed enzyme only). The d -galactose-containing β- d -mannosaccharides of d.p. 3–9 produced by A. niger β- d -mannanase have been characterised, using enzymic, n.m.r., and chemical techniques, as 6 1 -α- d -galactosyl-β- d -mannobiose, 6 1 -α- d -galactosyl-β- d -mannotriose, 6 3 ,6 4 -di-α- d -galactosyl-β- d -mannopentaose (the only heptasaccharide), and 6 3 ,6 4 -di-α- d -galactosyl-β- d -mannohexaose, 6 4 ,6 5 -di-α- d -galactosyl-β- d -mannohexaose, and 6 1 ,6 3 ,6 4 -tri-α- d -galactosyl-β- d -mannopentaose (the only octasaccharides). Four nonasaccharides have also been characterised. Penta- and hexa-saccharides were absent. Lucerne-seed β- d -mannanase produced the same branched tri-, tetra- and hepta-saccharides, and also penta- and hexa-saccharides that were characterised as 6 1 -α- d -galactosyl-β- d -mannotetraose, 6 3 -α- d -galactosyl-β- d -mannotetraose, 6 1 ,6 3 -di-α- d -galactosyl-β- d -mannotetraose, 6 3 -α- d -galactosyl-β- d -mannopentaose, and 6 4 -α- d -galactosyl-β- d -mannopentaose. None of the oligosaccharides contained a d -galactose stub on the terminal d -mannosyl group nor were they substituted on the second d -mannosyl residue from the reducing terminal.

An arabinogalactan from the culture medium of Rubus fruticosus cells in suspension

Abstract A water-soluble arabinogalactan, isolated from the extracellular medium of suspension-cultured cells of Rubus fruticosus , contained arabinose, rhamnose, galactose, and also protein (6.5%) and uronic acid (2.5%). Methylation analysis of the arabinogalactan and the arabinose-free product obtained by mild acid hydrolysis showed that the polysaccharide was a typical arabino-3,6-galactan in which rhamnose and glucuronic acid occupied non-reducing terminal positions. Successive Smith-degradations combined with methylation analysis and 13 C-n.m.r. spectroscopy revealed that the arabinogalactan contained a main chain of (→3)-linked β- d -galactopyranosyl residues with a high degree of branching at positions 6 by (1→6)-linked d -galactopyranosyl side-chains of various lengths, in which several contiguous residues were substituted at positions 3. The polymer is thus an arabinogalactan-protein belonging to the galactans of Type II.