Katia Ruel

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.

Structure and distribution of lignin in primary and secondary cell walls of maize coleoptiles analyzed by chemical and immunological probes.

Lignin is an integral constituent of the primary cell walls of the dark-grown maize (Zea mays L.) coleoptile, a juvenile organ that is still in the developmental state of rapid cell extension. Coleoptile lignin was characterized by (i) conversion to lignothiolglycolate derivative, (ii) isolation of polymeric fragments after alkaline hydrolysis, (iii) reactivity to antibodies against dehydrogenative polymers prepared from monolignols, and (iv) identification of thioacidolysis products typical of lignins. Substantial amounts of lignin could be solubilized from the coleoptile cell walls by mild alkali treatments. Thioacidolysis analyses of cell walls from coleoptiles and various mesocotyl tissues demonstrated the presence of guaiacyl-, syringyl- and (traces of)p-hydroxyphenyl units besidesp-coumaric and ferulic acids. There are tissue-specific differences in amount and composition of lignins from different parts of the maize seedling. Electron-microscopic immunogold labeling of epitopes recognized by a specific anti-guaiacyl/syringyl antibody demonstrated the presence of lignin in all cell walls of the 4-d-old coleoptile. The primary walls of parenchyma and epidermis were more weakly labeled than the secondary wall thickenings of tracheary elements. No label was found in middle lamellae and cell corners. Lignin epitopes appeared first in the tracheary elements on day 2 and in the parenchyma on day 3 after sowing. Incubation of coleoptile segments in H2O2 increased the amount of extractable lignin and the abundance of lignin epitopes in the parenchyma cell walls. Lignin deposition was temporally and spatially correlated with the appearance of epitopes for prolinerich proteins, but not for hydroxyproline-rich proteins, in the cell walls. The lignin content of coleoptiles was increased by irradiating the seedlings with white or farred light, correlated with the inhibition of elongation growth, while growth promotion by auxin had no effect. It is concluded that wall stiffness, and thus extension growth, of the coleoptile can be controlled by lignification of the primary cell walls. Primary-wall lignin may represent part of an extended polysaccharide-polyphenol network that limits the extensibility of the cell walls.

Redirection of the phenylpropanoid pathway to feruloyl malate in Arabidopsis mutants deficient for cinnamoyl-CoA reductase 1.

Cinnamoyl-CoA reductase 1 (CCR1, gene At1g15950) is the main CCR isoform implied in the constitutive lignification of Arabidopsis thaliana. In this work, we have identified and characterized two new knockout mutants for CCR1. Both have a dwarf phenotype and a delayed senescence. At complete maturity, their inflorescence stems display a 25–35% decreased lignin level, some alterations in lignin structure with a higher frequency of resistant interunit bonds and a higher content in cell wall-bound ferulic esters. Ferulic acid-coniferyl alcohol ether dimers were found for the first time in dicot cell walls and in similar levels in wild-type and mutant plants. The expression of CCR2, a CCR gene usually involved in plant defense, was increased in the mutants and could account for the biosynthesis of lignins in the CCR1-knockout plants. Mutant plantlets have three to four-times less sinapoyl malate (SM) than controls and accumulate some feruloyl malate. The same compositional changes occurred in the rosette leaves of greenhouse-grown plants. By contrast and relative to the control, their stems accumulated unusually high levels of both SM and feruloyl malate as well as more kaempferol glycosides. These findings suggest that, in their hypolignified stems, the mutant plants would avoid the feruloyl-CoA accumulation by its redirection to cell wall-bound ferulate esters, to feruloyl malate and to SM. The formation of feruloyl malate to an extent far exceeding the levels reported so far indicates that ferulic acid is a potential substrate for the enzymes involved in SM biosynthesis and emphasizes the remarkable plasticity of Arabidopsis phenylpropanoid metabolism.

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.

The maize ZmMYB42 represses the phenylpropanoid pathway and affects the cell wall structure, composition and degradability in Arabidopsis thaliana

The involvement of the maize ZmMYB42 R2R3-MYB factor in the phenylpropanoid pathway and cell wall structure and composition was investigated by overexpression in Arabidopsis thaliana. ZmMYB42 down-regulates several genes of the lignin pathway and this effect reduces the lignin content in all lignified tissues. In addition, ZmMYB42 plants generate a lignin polymer with a decreased S to G ratio through the enrichment in H and G subunits and depletion in S subunits. This transcription factor also regulates other genes involved in the synthesis of sinapate esters and flavonoids. Furthermore, ZmMYB42 affects the cell wall structure and degradability, and its polysaccharide composition. Together, these results suggest that ZmMYB42 may be part of the regulatory network controlling the phenylpropanoid biosynthetic pathway.

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.

Lignification in the flax stem: evidence for an unusual lignin in bast fibers

In the context of our research on cell wall formation and maturation in flax (Linum usitatissimum L) bast fibers, we (1) confirmed the presence of lignin in bast fibers and (2) quantified and characterized the chemical nature of this lignin at two developmental stages. Histochemical methods (Weisner and Maüle reagents and KMnO4-staining) indicating the presence of lignin in bast fibers at the light and electron microscope levels were confirmed by chemical analyses (acetyl bromide). In general, the lignin content in flax bast fibers varied between 1.5% and 4.2% of the dry cell wall residues (CWRs) as compared to values varying between 23.7% and 31.4% in flax xylem tissues. Immunological and chemical analyses (thioacidolysis and nitrobenzene oxidation) indicated that both flax xylem- and bast fiber-lignins were rich in guaiacyl (G) units with S/G values inferior to 0.5. In bast fibers, the highly sensitive immunological probes allowed the detection of condensed guaiacyl-type (G) lignins in the middle lamella, cell wall junctions, and in the S1 layer of the secondary wall. In addition, lower quantities of mixed guaiacyl–syringyl (GS) lignins could be detected throughout the secondary cell wall. Chemical analyses suggested that flax bast-fiber lignin is more condensed than the corresponding xylem lignin. In addition, H units represented up to 25% of the monomers released from bast-fiber lignin as opposed to a value of 1% for the corresponding xylem tissue. Such an observation indicates that the structure of flax bast-fiber lignin is significantly different from that of the more typical ‘woody plant lignin’, thereby suggesting that flax bast fibers represent an interesting system for studying an unusual lignification process.

Chemical analysis and immunolocalisation of lignin and suberin in endodermal and hypodermal/rhizodermal cell walls of developing maize (Zea mays L.) primary roots

Abstract. The composition of suberin and lignin in endodermal cell walls (ECWs) and in rhizodermal/hypodermal cell walls (RHCWs) of developing primary maize (Zea mays L.) roots was analysed after depolymerisation of enzymatically isolated cell wall material. Absolute suberin amounts related to root length significantly increased from primary ECWs (Casparian strips) to secondary ECWs (suberin lamella). During further maturation of the endodermis, reaching the final tertiary developmental state characterised by the deposition of lignified secondary cell walls (u-shaped cell wall deposits), suberin amounts remained constant. Absolute amounts of lignin related to root length constantly increased throughout the change from primary to tertiary ECWs. The suberin of Casparian strips contained high amounts of carboxylic and 2-hydroxy acids, and differed substantially from the suberin of secondary and tertiary ECWs, which was dominated by high contents of ω-hydroxycarboxylic and 1,ω-dicarboxylic acids. Furthermore, the chain-length distribution of suberin monomers in primary ECWs ranged from C16 to C24, whereas in secondary and tertiary ECWs a shift towards higher chain lengths (C16 to C28) was observed. The lignin composition of Casparian strips (primary ECWs) showed a high syringyl content and was similar to lignin in secondary cell walls of the tertiary ECWs, whereas lignin in secondary ECWs contained higher amounts of p-hydroxyphenyl units. The suberin and lignin compositions of RHCWs rarely changed with increasing root age. However, compared to the suberin in ECWs, where C16 and C18 were the most prominent chain lengths, the suberin of RHCWs was dominated by the higher chain lengths (C24 and C26). The composition of RHCW lignin was similar to that of secondary-ECW lignin. Using lignin-specific antibodies, lignin epitopes were indeed found to be located in the Casparian strip. Surprisingly, the mature suberin layers of tertiary ECWs contained comparable amounts of lignin-like epitopes.

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.

Lamellation in the S2 layer of softwood tracheids as demonstrated by scanning transmission electron microscopy

SummaryUltrathin cross sections of the tracheid wall in black spruce and silver fir have been examined at high resolution by scanning transmission electron microscopy (STEM) and by conventional transmission electron microscopy (TEM). For both softwoods, lamellation of the S2 layer was evident but the lamellae were seen more clearly in the STEM photomicrographs. The interlamellar distance was 7.1 nm in the case of spruce and 8.4 nm for silver fir.