Expression of a poplar cDNA encoding a ferulate-5-hydroxylase/coniferaldehyde 5-hydroxylase increases S lignin deposition in Arabidopsis thaliana (original) (raw)
Related papers
The Plant Journal, 2000
Ferulate 5-hydroxylase (F5H) is a cytochrome P450-dependent monooxygenase that catalyses the hydroxylation of ferulic acid, coniferaldehyde and coniferyl alcohol in the pathways leading to sinapic acid and syringyl lignin biosynthesis. Earlier studies in Arabidopsis have demonstrated that F5H overexpression increases lignin syringyl monomer content and abolishes the tissue-speci®city of its deposition. To determine whether this enzyme has a similar regulatory role in plants that undergo secondary growth, we over-expressed the F5H gene in tobacco and poplar. In tobacco, over-expression of F5H under the control of the cauli¯ower mosaic virus 35S promoter increased lignin syringyl monomer content in petioles, but had no detectable effect on ligni®cation in stems. By contrast, when the cinnamate 4-hydroxylase (C4H) promoter was used to drive F5H expression, there was a signi®cant increase in stem lignin syringyl monomer content. Yields of thioglycolic acid and Klason lignin in C4H± F5H lines were lower than in the wild-type, suggesting that F5H over-expression leads to a reduced deposition or an altered extractability of lignin in the transgenic plants. Histochemical analysis suggested that the novel lignin in C4H±F5H transgenic lines was altered in its content of hydroxycinnamyl aldehydes. Transgenic poplar trees carrying the C4H±F5H transgene also displayed enhanced lignin syringyl monomer content. Taken together, these data show that hydroxylation of guaiacyl-substituted lignin precursors controls lignin monomer composition in woody plants, and that F5H over-expression is a viable metabolic engineering strategy for modifying lignin biosynthesis in forest species.
The Effects on Lignin Structure of Overexpression of Ferulate 5-Hydroxylase in Hybrid Poplar1
PLANT PHYSIOLOGY, 2009
Poplar (Populus tremula 3 alba) lignins with exceedingly high syringyl monomer levels are produced by overexpression of the ferulate 5-hydroxylase (F5H) gene driven by a cinnamate 4-hydroxylase (C4H) promoter. Compositional data derived from both standard degradative methods and NMR analyses of the entire lignin component (as well as isolated lignin fraction) indicated that the C4H::F5H transgenic's lignin was comprised of as much as 97.5% syringyl units (derived from sinapyl alcohol), the remainder being guaiacyl units (derived from coniferyl alcohol); the syringyl level in the wild-type control was 68%. The resultant transgenic lignins are more linear and display a lower degree of polymerization. Although the crucial b-ether content is similar, the distribution of other interunit linkages in the lignin polymer is markedly different, with higher resinol (b-b) and spirodienone (b-1) contents, but with virtually no phenylcoumarans (b-5, which can only be formed from guaiacyl units). p-Hydroxybenzoates, acylating the g-positions of lignin side chains, were reduced by .50%, suggesting consequent impacts on related pathways. A model depicting the putative structure of the transgenic lignin resulting from the overexpression of F5H is presented. The altered structural features in the transgenic lignin polymer, as revealed here, support the contention that there are significant opportunities to improve biomass utilization by exploiting the malleability of plant lignification processes.
The Plant journal : for cell and molecular biology, 2012
The transcription factor MYB103 was previously identified as a member of the transcriptional network regulating secondary wall biosynthesis in xylem tissues of Arabidopsis, and was proposed to act on cellulose biosynthesis. It is a direct transcriptional target of the transcription factor SECONDARY WALL ASSOCIATED NAC DOMAIN PROTEIN 1 (SND1), and 35S-driven dominant repression or over-expression of MYB103 modifies secondary wall thickness. We identified two myb103 T-DNA insertion mutants and chemically characterized their lignocellulose by pyrolysis/GC/MS, 2D NMR, FT-IR microspectroscopy and wet chemistry. The mutants developed normally but exhibited a 70-75% decrease in syringyl (S) lignin. The level of guaiacyl (G) lignin was co-ordinately increased, so that total Klason lignin was not affected. The transcript abundance of FERULATE-5-HYDROXYLASE (F5H), the key gene in biosynthesis of S lignin, was strongly decreased in the myb103 mutants, and the metabolomes of the myb103 mutant and an F5H null mutant were very similar. Other than modification of the lignin S to G ratio, there were only very minor changes in the composition of secondary cell-wall polymers in the inflorescence stem. In conclusion, we demonstrate that F5H expression and hence biosynthesis of S lignin are dependent on MYB103.
Plant Cell, 2005
During lignin biosynthesis in angiosperms, coniferyl and sinapyl aldehydes are believed to be converted into their corresponding alcohols by cinnamyl alcohol dehydrogenase (CAD) and by sinapyl alcohol dehydrogenase (SAD), respectively. This work clearly shows that CAD-C and CAD-D act as the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis thaliana by supplying both coniferyl and sinapyl alcohols. An Arabidopsis CAD double mutant (cad-c cad-d) resulted in a phenotype with a limp floral stem at maturity as well as modifications in the pattern of lignin staining. Lignin content of the mutant stem was reduced by 40%, with a 94% reduction, relative to the wild type, in conventional b-O-4-linked guaiacyl and syringyl units and incorportion of coniferyl and sinapyl aldehydes. Fourier transform infrared spectroscopy demonstrated that both xylem vessels and fibers were affected. GeneChip data and real-time PCR analysis revealed that transcription of CAD homologs and other genes mainly involved in cell wall integrity were also altered in the double mutant. In addition, molecular complementation of the double mutant by tissue-specific expression of CAD derived from various species suggests different abilities of these genes/proteins to produce syringyl-lignin moieties but does not indicate a requirement for any specific SAD gene.
Plant and Cell Physiology, 2010
As a step toward a comprehensive description of lignin biosynthesis in Populus trichocarpa , we identifi ed from the genome sequence 95 phenylpropanoid gene models in 10 protein families encoding enzymes for monolignol biosynthesis. Transcript abundance was determined for all 95 genes in xylem, leaf, shoot and phloem using quantitative real-time PCR (qRT-PCR). We identifi ed 23 genes that most probably encode monolignol biosynthesis enzymes during wood formation. Transcripts for 18 of the 23 are abundant and specifi c to differentiating xylem. We found evidence suggesting functional redundancy at the transcript level for phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), 4-coumarate:CoA ligase (4CL), p -hydroxycinnamoyl-CoA:quinate shikimate p -hydroxycinnamoyltransferase (HCT), caffeoyl-CoA O -methyltransferase (CCoAOMT) and coniferyl aldehyde 5-hydroxylase (CAld5H). We carried out an enumeration-based motif identifi cation and discriminant analysis on the promoters of all 95 genes. Five core motifs correctly discriminate the 18 xylem-specifi c genes from the 77 non-xylem genes. These motifs are similar to promoter elements known to regulate phenylpropanoid gene expression. This work suggests that genes in monolignol biosynthesis are regulated by multiple motifs, often related in sequence.
Plant physiology, 1996
Cinnamyl alcohol dehydrogenase (CAD) catalyzes the last step in the biosynthesis of the lignin precursors, the monolignols. We have down-regulated CAD in transgenic poplar (Populus tremula X Populus alba) by both antisense and co-suppression strategies. Several antisense and sense CAD transgenic poplars had an approximately 70% reduced CAD activity that was associated with a red coloration of the xylem tissue. Neither the lignin amount nor the lignin monomeric composition (syringyl/guaiacyl) were significantly modified. However, phloroglucinol-HCl staining was different in the down-regulated CAD plants, suggesting changes in the number of aldehyde units in the lignin. Furthermore, the reactivity of the cell wall toward alkali treatment was altered: a lower amount of lignin was found in the insoluble, saponified residue and more lignin could be precipitated from the soluble alkali fraction. Moreover, large amounts of phenolic compounds, vanillin and especially syringaldehyde, were de...
Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins
Phytochemistry, 2004
A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8-O-4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and b-O-4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.
Coniferyl Aldehyde 5Hydroxylation and Methylation Direct Syringyl Lignin Biosynthesis in Angiosperms
Proceedings of The National Academy of Sciences, 1999
A central question in lignin biosynthesis is how guaiacyl intermediates are hydroxylated and methylated to the syringyl monolignol in angiosperms. To address this question, we cloned cDNAs encoding a cytochrome P450 monooxygenase (LsM88) and a caffeate O-methyltransferase (COMT) from sweetgum (Liquidambar styraciflua) xylem. Mass spectrometry-based functional analysis of LsM88 in yeast identified it as coniferyl aldehyde 5-hydroxylase (CAld5H). COMT expressed in Escherichia coli methylated 5-hydroxyconiferyl aldehyde to sinapyl aldehyde. Together, CAld5H and COMT converted coniferyl aldehyde to sinapyl aldehyde, suggesting a CAld5H͞COMT-mediated pathway from guaiacyl to syringyl monolignol biosynthesis via coniferyl aldehyde that contrasts with the generally accepted route to sinapate via ferulate. Although the CAld5H͞COMT enzyme system can mediate the biosynthesis of syringyl monolignol intermediates through either route, k cat ͞K m of CAld5H for coniferyl aldehyde was Ϸ140 times greater than that for ferulate. More significantly, when coniferyl aldehyde and ferulate were present together, coniferyl aldehyde was a noncompetitive inhibitor (K i ؍ 0.59 M) of ferulate 5-hydroxylation, thereby eliminating the entire reaction sequence from ferulate to sinapate. In contrast, ferulate had no effect on coniferyl aldehyde 5-hydroxylation. 5-Hydroxylation also could not be detected for feruloyl-CoA or coniferyl alcohol. Therefore, in the presence of coniferyl aldehyde, ferulate 5-hydroxylation does not occur, and the syringyl monolignol can be synthesized only from coniferyl aldehyde. Endogenous coniferyl, 5-hydroxyconiferyl, and sinapyl aldehydes were detected, consistent with in vivo operation of the CAld5H͞ COMT pathway from coniferyl to sinapyl aldehydes via 5-hydroxyconiferyl aldehyde for syringyl monolignol biosynthesis.
Forests
Plant UDP-glycosyltransferases (UGT) transfer sugars to small acceptor molecules and thereby play key roles in the biosynthesis of secondary metabolites, including phenylpropanoids. Some of those metabolites are involved in the xylem lignification of a broad range of terrestrial plants, particularly trees. Here, we focused on poplar UGT72B37, coding for an enzyme glycosylating monolignols by investigating CRISPR/Cas9 mutant lines. The cell wall characterization revealed a 10% lignin content increase in the xylem of three-month-old mutant lines compared to the wild type. No ectopic lignification was evidenced in the pith of the stems of the mutants, suggesting that the increased lignin deposition is restricted to lignified cell walls. The analysis of the expression level of lignin biosynthesis and polymerization genes did not show significant changes between the WT and the ugt72b37 mutants, except for CINNAMOYL-COA REDUCTASE 2 which was significantly upregulated by 1.2–1.5-fold. Noti...