Syringyl lignin biosynthesis is directly regulated by a secondary cell wall master switch.
ABSTRACT: Lignin is a major component of plant secondary cell walls and is derived from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monolignols. Among higher plants, S lignin is generally considered to be restricted to angiosperms, which contain the S lignin-specific cytochrome P450-dependent monooxygenase, ferulic acid/coniferaldehyde/coniferyl alcohol 5-hydoxylase (F5H). The transcription factor MYB58 directly regulates expression of monolignol pathway genes except for F5H. Here we show that F5H expression is directly regulated by the secondary cell wall master switch NST1/SND1, which is known to regulate expression of MYB58. Deletion of NST1 expression in Medicago truncatula leads to a loss of S lignin associated with a more than 25-fold reduction of F5H expression but only around a 2-fold reduction in expression of other lignin pathway genes. A detailed phylogenetic analysis showed that gymnosperms lack both F5H and orthologs of NST1/SND1. We propose that both F5H and NST1 appeared at a similar time after the divergence of angiosperms and gymnosperms, with F5H possibly originating as a component of a defense mechanism that was recruited to cell wall biosynthesis through the evolution of NST1-binding elements in its promoter.
Project description:Lycophytes arose in the early Silurian ( approximately 400 Mya) and represent a major lineage of vascular plants that has evolved in parallel with the ferns, gymnosperms, and angiosperms. A hallmark of vascular plants is the presence of the phenolic lignin heteropolymer in xylem and other sclerified cell types. Although syringyl lignin is often considered to be restricted in angiosperms, it has been detected in lycophytes as well. Here we report the characterization of a cytochrome P450-dependent monooxygenase from the lycophyte Selaginella moellendorffii. Gene expression data, cross-species complementation experiments, and in vitro enzyme assays indicate that this P450 is a ferulic acid/coniferaldehyde/coniferyl alcohol 5-hydroxylase (F5H), and is capable of diverting guaiacyl-substituted intermediates into syringyl lignin biosynthesis. Phylogenetic analysis indicates that the Selaginella F5H represents a new family of plant P450s and suggests that it has evolved independently of angiosperm F5Hs.
Project description:Ferulate 5-hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O-methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT-down-regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down-regulation of F5H in COMT-RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5-OH G units. Conversely, overexpression of F5H in COMT-RNAi transgenic plants reduced G units and increased 5-OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down-regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up-regulation and down-regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.
Project description:Conifers (softwoods) naturally lack syringyl units in their lignins, rendering lignocellulosic materials from such species more difficult to process than syringyl-rich hardwood species. Using a transformable Pinus radiata tracheary element (TE) system as an experimental platform, we investigated whether metabolic engineering can be used to create syringyl lignin in conifers. Pyrolysis-GC/MS and 2D-NMR analysis of P. radiata TE cultures transformed to express ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT) from Liquidambar styraciflua confirmed the production and incorporation of sinapyl alcohol into the lignin polymer. Transformation with F5H was sufficient for the production of syringyl lignin in TEs, but cotransformation with COMT improved its formation. In addition, lower levels of the pathway intermediate 5-hydroxyconiferyl alcohol were evidenced in cotransformation experiments, indicating that the introduction of the COMT overcame the inefficiency of the native pine methyltransferases for supporting sinapyl alcohol production.Our results provide the proof of concept that it is possible to generate a lignin polymer that contains syringyl units in softwood species such as P. radiata, suggesting that it might be possible to retain the outstanding fiber properties of softwoods while imbuing them with the lignin characteristics of hardwoods that are more favorable for industrial processing.
Project description:Certain plant cells synthesize secondary cell walls besides primary cell walls. This biosynthesis is strictly controlled by an array of transcription factors. Here, we show that SND1, a regulator of cell-wall biosynthesis, regulates abscisic acid (ABA) biosynthesis to ensure optimal plant growth. In Arabidopsis, the lack of SND1 and its homolog NST1 leads to the deficiency of secondary cell walls, preventing snd1nst1 double mutant seedlings from growing upright. Compared to wild type seedlings, the snd1 knockout mutant seedlings accumulated less anthocyanin and exhibited low tolerance to salt stress. Compared to wild type seedlings, the snd1 knockout seedlings were more sensitive to salt stress. Although SND1 can bind to the promoter of Myb46, we observed that SND1 binds directly to the promoter of the ABI4 gene, thereby reducing ABA levels under normal growth conditions. Thus, plants adjust secondary cell wall thickening and growth via SND1. SND1 has a dual function: it activates the Myb46 pathway, fostering lignin biosynthesis to produce sufficient cell wall components for growth, while maintaining a low ABA concentration, as it inhibits growth. This dual function of SND1 may help plants modulate their growth efficiently.
Project description:Background:During the chemical and biochemical decomposition of lignocellulosic biomasses, lignin is highly recalcitrant. Genetic transformation of plants to qualitatively and/or quantitatively modify lignin may reduce these recalcitrant properties. Efficient discovery of genes to achieve lignin manipulation is thus required. Results:To screen for new genes to reduce lignin recalcitrance, we heterologously expressed 50 enzymatic genes under the control of a cinnamate 4-hydroxylase (C4H) gene promoter, derived from a hybrid aspen, which is preferentially active in tissues with lignified cell walls in Arabidopsis plants. These genes encode enzymes that act on metabolites in shikimate, general phenylpropanoid, flavonoid, or monolignol biosynthetic pathways. Among these genes, 30, 18, and 2 originated from plants, bacteria, and fungi, respectively. In our first screening step, 296 independent transgenic plants (T1 generation) harboring single or multiple transgenes were generated from pools of seven Agrobacterium strains used for conventional floral-dip transformation. Wiesner and Mäule staining patterns in the stems of the resultant plants revealed seven and nine plants with apparent abnormalities in the two respective staining analyses. According to genomic PCR and subsequent direct sequencing, each of these 16 plants possessed a gene encoding either coniferaldehyde dehydrogenase (calB), feruloyl-CoA 6'-hydroxylase (F6H1), hydroxycinnamoyl-CoA hydratase/lyase (couA), or ferulate 5-hydroxylase (F5H), with one transgenic plant carrying both calB and F6H1. The effects of these genes on lignin manipulation were confirmed in individually re-created T1 transgenic Arabidopsis plants. While no difference in lignin content was detected in the transgenic lines compared with the wild type, lignin monomeric composition was changed in the transgenic lines. The observed compositional change in the transgenic plants carrying calB, couA, and F5H led to improved sugar release from cell walls after alkaline pretreatment. Conclusions:Simple colorimetric characterization of stem lignin is useful for simultaneous screening of many genes with the potential to reduce lignin recalcitrance. In addition to F5H, the positive control, we identified three enzyme-coding genes that can function as genetic tools for lignin manipulation. Two of these genes (calB and couA) accelerate sugar release from transgenic lignocelluloses.
Project description:The transcriptional regulation of phenylalanine metabolism is particularly important in conifers, long-lived species that use large amounts of carbon in wood. Here, we show that the Pinus pinaster transcription factor, PpNAC1, is a main regulator of phenylalanine biosynthesis and utilization. A phylogenetic analysis classified PpNAC1 in the NST proteins group and was selected for functional characterization. PpNAC1 is predominantly expressed in the secondary xylem and compression wood of adult trees. Silencing of PpNAC1 in P. pinaster results in the alteration of stem vascular radial patterning and the down-regulation of several genes associated with cell wall biogenesis and secondary metabolism. Furthermore, transactivation and EMSA analyses showed that PpNAC1 is able to activate its own expression and PpMyb4 promoter, while PpMyb4 is able to activate PpMyb8, a transcriptional regulator of phenylalanine and lignin biosynthesis in maritime pine. Together, these results suggest that PpNAC1 is a functional ortholog of the ArabidopsisSND1 and NST1 genes and support the idea that key regulators governing secondary cell wall formation could be conserved between gymnosperms and angiosperms. Understanding the molecular switches controlling wood formation is of paramount importance for fundamental tree biology and paves the way for applications in conifer biotechnology.
Project description:Cell wall recalcitrance is a major limitation for the sustainable exploitation of lignocellulosic biomass as a renewable resource. Species and hybrids of the genus Miscanthus have emerged as candidate crops for the production of lignocellulosic feedstock in temperate climates, and dedicated efforts are underway to improve biomass yield. However, nothing is known about the molecular players involved in Miscanthus cell wall biosynthesis to facilitate breeding efforts towards tailored biomass. Here, we identify a Miscanthus sinensis transcription factor related to SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1), which acts as a master switch for the regulation of secondary cell wall formation and lignin biosynthesis. MsSND1 is expressed in growth stages associated with secondary cell wall formation, together with its potential targets. Consistent with this observation, MsSND1 was able to complement the secondary cell wall defects of the Arabidopsis snd1 nst1 double mutant, and ectopic expression of MsSND1 in tobacco leaves was sufficient to trigger patterned deposition of cellulose, hemicellulose, and lignin reminiscent of xylem elements. Transgenic studies in Arabidopsis thaliana plants revealed that MsSND1 regulates, directly and indirectly, the expression of a broad range of genes involved in secondary cell wall formation, including MYB transcription factors which regulate only a subset of the SCW differentiation program. Together, our findings suggest that MsSND1 is a transcriptional master regulator orchestrating secondary cell wall biosynthesis in Miscanthus.
Project description:SECONDARY WALL-ASSOCIATED NAC DOMAIN1 (SND1) is a master regulator of fibre secondary wall deposition in Arabidopsis thaliana (Arabidopsis), with homologs in other angiosperms and gymnosperms. However, it is poorly understood to what extent the fibre-specific regulation of the SND1 promoter, and that of its orthologs, is conserved between diverged herbaceous and woody lineages. We performed a reciprocal reporter gene analysis of orthologous SND1 promoters from Arabidopsis (AthSND1), Eucalyptus grandis (EgrNAC61) and Populus alba × P. grandidentata (PagWND1A) relative to secondary cell wall-specific Cellulose Synthase4 (CesA4) and CesA7 promoters, in both a non-woody (Arabidopsis) and a woody (poplar) system. ?-glucuronidase (GUS) reporter analysis in Arabidopsis showed that the SND1 promoter was active in vascular tissues as previously reported and showed interfascicular and xylary fibre-specific expression in inflorescence stems, while reporter constructs of the woody plant-derived promoters were partial to the (pro)cambium-phloem and protoxylem. In transgenic P. tremula × P. alba plants, all three orthologous SND1 promoters expressed the GUS reporter similarly and preferentially in developing secondary xylem, ray parenchyma and cork cambium. Ours is the first study to reciprocally test orthologous SND1 promoter specificity in herbaceous and woody species, revealing diverged regulatory functions in the herbaceous system.
Project description:The fah1 mutant of Arabidopsis is defective in the accumulation of sinapic acid-derived metabolites, including the guaiacyl-syringyl lignin typical of angiosperms. Earlier results indicated that the FAH1 locus encodes ferulate-5-hydroxylase (F5H), a cytochrome P450-dependent monooxygenase (P450) of the general phenylpropanoid pathway. We have cloned the gene encoding this P450 by T-DNA tagging and have confirmed the identity of the cloned gene by complementation of the mutant phenotype. F5H shows 34% amino acid sequence identity with the avocado ripening-induced P450 CYP71A1 and 32% identity with the flavonoid-3',5'-hydroxylases of Petunia hybrida. In contrast, it shares much less homology with cinnamate-4-hydroxylase, a P450 that catalyzes the hydroxylation of cinnamic acid three steps earlier in the general phenylpropanoid pathway. Since the highest degree of identity between F5H and previously sequenced P450s is only 34%, F5H identifies a new P450 subfamily that has been designated CYP84.
Project description:Lignin is a key secondary cell wall chemical constituent, and is both a barrier to biomass utilization and a potential source of bioproducts. The Arabidopsis transcription factors MYB58 and MYB63 have been shown to upregulate gene expression of the general phenylpropanoid and monolignol biosynthetic pathways. The overexpression of these genes also results in dwarfism. The vascular integrity, soluble phenolic profiles, cell wall lignin, and transcriptomes associated with these MYB-overexpressing lines were characterized. Plants with high expression of MYB58 and MYB63 had increased ectopic lignin and the xylem vessels were regular and open, suggesting that the stunted growth is not associated with loss of vascular conductivity. MYB58 and MYB63 overexpression lines had characteristic soluble phenolic profiles with large amounts of monolignol glucosides and sinapoyl esters, but decreased flavonoids. Because loss of function lac4 lac17 mutants also accumulate monolignol glucosides, we hypothesized that LACCASE overexpression might decrease monolignol glucoside levels in the MYB-overexpressing plant lines. When laccases related to lignification (LAC4 or LAC17) were co-overexpressed with MYB63 or MYB58, the dwarf phenotype was rescued. Moreover, the overexpression of either LAC4 or LAC17 led to wild-type monolignol glucoside levels, as well as wild-type lignin levels in the rescued plants. Transcriptomes of the rescued double MYB63-OX/LAC17-OX overexpression lines showed elevated, but attenuated, expression of the MYB63 gene itself and the direct transcriptional targets of MYB63. Contrasting the dwarfism from overabundant monolignol production with dwarfism from lignin mutants provides insight into some of the proposed mechanisms of lignin modification-induced dwarfism.