Abstract
Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall1,2. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood3. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 51 print issues and online access
$199.00 per year
only $3.90 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Bonawitz, N. D. & Chapple, C. The genetics of lignin biosynthesis: connecting genotype to phenotype. Annu. Rev. Genet. 44, 337–363 (2010)
Vanholme, R. et al. Lignin biosynthesis and structure. Plant Physiol. 153, 895–905 (2010)
Bonawitz, N. D. & Chapple, C. Can genetic engineering of lignin biosynthesis be accomplished without an unacceptable yield penalty? Curr. Opin. Biotechnol. 24, 336–343 (2013)
Rogers, L. A. & Campbell, M. M. The genetic control of lignin deposition during plant growth and development. New Phytol. 164, 17–30 (2004)
Chen, F. & Dixon, R. A. Lignin modification improves fermentable sugar yields for biofuel production. Nature Biotechnol. 25, 759–761 (2007)
Hématy, K. et al. A receptor-like kinase mediates the response of Arabidopsis cells to the inhibition of cellulose synthesis. Curr. Biol. 17, 922–931 (2007)
Ralph, J. et al. Lignins: natural polymers from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochem. Rev. 3, 29–60 (2004)
Chapple, C. C., Vogt, T., Ellis, B. E. & Somerville, C. R. An Arabidopsis mutant defective in the general phenylpropanoid pathway. Plant Cell 4, 1413–1424 (1992)
Franke, R. et al. Changes in secondary metabolism and deposition of an unusual lignin in the ref8 mutant of Arabidopsis. Plant J. 30, 47–59 (2002)
Franke, R. et al. The Arabidopsis REF8 gene encodes the 3-hydroxylase of phenylpropanoid metabolism. Plant J. 30, 33–45 (2002)
Hoffmann, L. et al. Silencing of hydroxycinnamoyl-coenzyme A shikimate/quinate hydroxycinnamoyltransferase affects phenylpropanoid biosynthesis. Plant Cell 16, 1446–1465 (2004)
Schilmiller, A. L. et al. Mutations in the cinnamate 4-hydroxylase gene impact metabolism, growth and development in Arabidopsis. Plant J. 60, 771–782 (2009)
Reddy, M. S. et al. Targeted down-regulation of cytochrome P450 enzymes for forage quality improvement in alfalfa (Medicago sativa L.). Proc. Natl Acad. Sci. USA 102, 16573–16578 (2005)
Ruegger, M. & Chapple, C. Mutations that reduce sinapoylmalate accumulation in Arabidopsis thaliana define loci with diverse roles in phenylpropanoid metabolism. Genetics 159, 1741–1749 (2001)
Abdulrazzak, N. et al. A coumaroyl-ester-3-hydroxylase insertion mutant reveals the existence of nonredundant meta-hydroxylation pathways and essential roles for phenolic precursors in cell expansion and plant growth. Plant Physiol. 140, 30–48 (2006)
Weng, J. K. et al. Convergent evolution of syringyl lignin biosynthesis via distinct biosynthetic pathways in the lycophyte Selaginella and flowering plants. Plant Cell 22, 1033–1045 (2010)
Bonawitz, N. D. et al. REF4 and RFR1, subunits of the transcriptional coregulatory complex Mediator, are required for phenylpropanoid homeostasis in Arabidopsis. J. Biol. Chem. 287, 5434–5445 (2012)
Kornberg, R. D. The molecular basis of eukaryotic transcription. Proc. Natl Acad. Sci. USA 104, 12955–12961 (2007)
Kidd, B. N. et al. The Mediator complex subunit PFT1 is a key regulator of jasmonate-dependent defense in Arabidopsis. Plant Cell 21, 2237–2252 (2009)
Wathugala, D. L. et al. The Mediator subunit SFR6/MED16 controls defence gene expression mediated by salicylic acid and jasmonate responsive pathways. New Phytol. 195, 217–230 (2012)
Knight, H. et al. Identification of SFR6, a key component in cold acclimation acting post-translationally on CBF function. Plant J. 58, 97–108 (2009)
Imura, Y. et al. CRYPTIC PRECOCIOUS/MED12 is a novel flowering regulator with multiple target steps in Arabidopsis. Plant Cell Physiol. 53, 287–303 (2012)
Cerdán, P. D. & Chory, J. Regulation of flowering time by light quality. Nature 423, 881–885 (2003)
Besseau, S. et al. Flavonoid accumulation in Arabidopsis repressed in lignin synthesis affects auxin transport and plant growth. Plant Cell 19, 148–162 (2007)
Clifford, M. N. Specificity of acidic phloroglucinol reagents. J. Chromatogr. A 94, 321–324 (1974)
Lu, F. & Ralph, J. The DFRC method for lignin analysis. 2. Monomers from isolated lignins. J. Agric. Food Chem. 46, 547–552 (1998)
Ralph, J. et al. Effects of coumarate 3-hydroxylase down-regulation on lignin structure. J. Biol. Chem. 281, 8843–8853 (2006)
Vanholme, R. et al. Caffeoyl shikimate esterase is an enzyme in the lignin biosynthetic pathway in Arabidopsis. Science 341, 1103–1106 (2013)
Borevitz, J. O., Xia, Y., Blount, J., Dixon, R. A. & Lamb, C. Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis. Plant Cell 12, 2383–2394 (2000)
Gallego-Giraldo, L., Escamilla-Trevino, L., Jackson, L. A. & Dixon, R. A. Salicylic acid mediates the reduced growth of lignin down-regulated plants. Proc. Natl Acad. Sci. USA 108, 20814–20819 (2011)
Stout, J., Romero-Severson, E., Ruegger, M. O. & Chapple, C. Semidominant mutations in reduced epidermal fluorescence 4 reduce phenylpropanoid content in Arabidopsis. Genetics 178, 2237–2251 (2008)
Meyer, K. et al. Lignin monomer composition is determined by the expression of a cytochrome P450-dependent monooxygenase in Arabidopsis. Proc. Natl Acad. Sci. USA 95, 6619–6623 (1998)
Wagner, A. et al. CCoAOMT suppression modifies lignin composition in Pinus radiata. Plant J. 67, 119–129 (2011)
Weng, J. K., Akiyama, T., Ralph, J. & Chapple, C. Independent recruitment of an O-methyltransferase for syringyl lignin biosynthesis in Selaginella moellendorffii. Plant Cell 23, 2708–2724 (2011)
Mansfield, S. D., Kim, H., Lu, F. & Ralph, J. Whole plant cell wall characterization using solution-state 2D NMR. Nature Protoc. 7, 1579–1589 (2012)
Quideau, S. & Ralph, J. Facile large-scale synthesis of coniferyl, sinapyl, and p-coumaryl alcohol. J. Agric. Food Chem. 40, 1108–1110 (1992)
Tobimatsu, Y. et al. Hydroxycinnamate conjugates as potential monolignol replacements: in vitro lignification and cell wall studies with rosmarinic acid. ChemSusChem 5, 676–686 (2012)
Chen, F. et al. A polymer of caffeyl alcohol in plant seeds. Proc. Natl Acad. Sci. USA 109, 1772–1777 (2012)
Acknowledgements
This work was primarily funded by the Division of Chemical Sciences, Geosciences, and Biosciences, Office of Basic Energy Sciences of the US Department of Energy (DOE) through grant DE-FG02-07ER15905 to C.C. N.D.B. was supported in part by a fellowship from the Life Sciences Research Foundation. Y.T. and J.R. were funded by the US DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64944). J.I.K., P.N.C. and B.S.D. were supported as part of the Center for Direct Catalytic Conversion of Biomass to Biofuels (C3Bio), an Energy Frontier Research Center funded by the US DOE, Office of Science, Office of Basic Energy Sciences, award number DE-SC0000997. E.X. and M.L. were supported by the US DOE through grant DE-FG02-06ER64301 to M.L. and C.C. and by the Purdue University Office of Agricultural Research Programs. The authors acknowledge the support of the Bioinformatics Core at Purdue University.
Author information
Authors and Affiliations
Contributions
N.D.B. and C.C. were responsible for the conception, planning and organization of experiments. N.D.B. generated all Arabidopsis lines and all plant material used in the work, carried out metabolite analysis, interpretation of RNA-seq results and TGA lignin quantification, and was responsible for photography of whole plants. N.D.B. and J.I.K. harvested samples and prepared RNA for global transcript analysis. J.I.K. performed histochemical analyses and quantification of salicylic acid. N.A.A. assisted with cell-wall-bound phenylpropanoid quantification. J.M. carried out DFRC lignin analysis. P.N.C. and B.S.D. performed confocal microscopy, transmission electron microscopy, and cell wall thickness measurements. Y.T. and J.R. were responsible for NMR and gel-permeation chromatography analysis of lignin. E.X. and M.L. performed saccharification assays. The manuscript was primarily written by N.D.B. with critical input from other co-authors. Figures were prepared by N.D.B. with support from Y.T. and P.N.C.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Extended data figures and tables
Extended Data Figure 1 Disruption of MED5a/5b does not restore normal hydroxycinnamate ester biosynthesis to ref8 mutants.
Quantification of the hydroxycinnamate esters sinapoylglucose, sinapoylmalate, p-coumaroylglucose, p-coumaroylmalate and p-coumaroylshikimate in rosettes of 3-week-old plants of the indicated genotypes. Similar to ref8 mutants, med5a/5b ref8 plants fail to accumulate wild-type levels of the sinapate esters sinapoylglucose and sinapoylmalate, and instead hyperaccumulate the p-coumarate esters p-coumaroylglucose, p-coumaroylmalate and p-coumaroylshikimate. In each case, data are derived from five individual plants, except for ref8-2, where owing to their small size, three groups of five, seven and seven plants each were combined into independent pools. Error bars indicate s.d. in all cases.
Extended Data Figure 2 Disruption of MED5a/5b alleviates the flavonoid hyperaccumulation of ref8 mutants.
Quantification of the three major flavonol glucosides in rosettes of 3-week-old plants of the indicated genotypes. In each case, data are derived from five single plants, except for ref8-2, where three groups of five, seven and seven plants each were combined into independent pools. Error bars indicate s.d. K-(Rha-Glu)-Rha, kaempferol 3-O-[6′′-O-(rhamnosyl) glucoside] 7-O-rhamnoside; K-Glu-Rha, kaempferol 3-O-glucoside 7-O-rhamnoside; K-Rha-Rha, kaempferol 3-O-rhamnoside 7-O-rhamnoside.
Extended Data Figure 3 med5a/5b ref8-2 mutants show patterns of lignification similar to med5a/5b ref8-1 mutants.
Shown are thin sections of inflorescence stems of med5a/5b ref8-2 mutants stained with Mäule reagent (left) and phloroglucinol (right). Plants were grown and stained in parallel with those shown in Fig. 2a, b. Although med5a/5b ref8-2 mutant stems are somewhat thinner than those of med5a/5b ref8-1 mutants and show some morphological abnormalities, the overall staining patterns of med5a/5b ref8-1 and med5a/5b ref8-2 inflorescence stems are highly similar. The corresponding tissues of ref8-2 mutant plants could not be examined owing to their developmental arrest shortly after germination.
Extended Data Figure 4 ref8-1 mutants show thickening of the secondary cell wall that is rescued by disruption of MED5a/5b.
Left, distance map of cell wall thickness calculated from micrographs of representative samples of stem cross-sections of plants of the indicated genotypes. Right, quantification of cell wall thickness. N > 200 cells, with at least 100 measurements per cell for each sample. Error bars represent s.d. ***P < 0.001, difference from wild type (Student’s t-test).
Extended Data Figure 5 Lignin of med5a/5b ref8-1 mutant plants differs structurally from lignin of wild-type or med5a/5b mutant plants.
2D-NMR spectra of lignin from the indicated genotypes. The data shown are derived from a different region of the same spectra shown in Fig. 3. Colour-coded structures on right correspond to the major resonances in each spectrum.
Extended Data Figure 6 High-molecular-weight lignin polymers are underrepresented in med5a/5b ref8-1 mutants.
Shown are the results of gel-permeation chromatography of lignin from wild-type, med5a/5b and med5a/5b ref8-1 cell walls. The x-axis indicates the apparent molecular weight of individual lignin polymer fragments and is shown as a log scale. The y-axis shows the response of an ultraviolet-light detector normalized to the most abundant signal in each chromatogram. The most abundant signal in all samples corresponds to a molecular weight of ∼10,000 Da, whereas a secondary peak at ∼250,000 Da is significantly underrepresented in lignin derived from the med5a/5b ref8-1 mutant.
Extended Data Figure 7 Expression of lignin biosynthesis genes in wild-type, med5a/5b, ref8-1 and med5a/5b ref8-1 plants.
Shown is the expression of general phenylpropanoid and lignin biosynthesis genes in 3-week-old rosettes of plants of the indicated genotypes as determined using high-throughput sequencing of mRNA. The value shown on the y-axis refers to the number of reads unambiguously mapping to each gene, normalized for differences in the total number of reads between samples and for lane effects. *P < 0.05, difference from wild type, as determined by the DESeq algorithm using a Benjamini–Hochberg procedure to adjust for multiple testing.
Extended Data Figure 8 Expression of flavonoid biosynthesis genes in wild-type, med5a/5b, ref8-1 and med5a/5b ref8-1 plants.
Shown is the expression of flavonoid biosynthesis genes in 3-week-old rosettes of plants of the indicated genotypes as determined using high-throughput sequencing of mRNA. The value shown on the y-axis refers to the number of reads unambiguously mapping to each gene, normalized for differences in the total number of reads between samples and for lane effects. *P < 0.05, difference from wild type, as determined by the DESeq algorithm using a Benjamini–Hochberg procedure to adjust for multiple testing.
Extended Data Figure 9 A model for Mediator-dependent growth inhibition in Arabidopsis ref8 mutants.
Mutation or disruption of REF8 leads to direct alterations in the composition of the cell wall and other metabolic changes due to the loss of C3′H activity. Information on these changes is relayed to the nucleus by an at present unknown signalling pathway or sensor, resulting in massive changes in gene expression (represented by green and red transcripts in the model). Some of these transcriptional changes are directly dependent on MED5 (centre, illustrated as a direct MED5–transcription-factor interaction), whereas others are independent of MED5 (left) or are indirectly affected by MED5 (right), such as genes controlled by transcription factors that are themselves MED5-dependent targets. Ultimately, changes in the transcription of direct and/or indirect targets of MED5 result in inhibition of growth, sterility and indirect effects on cell wall architecture, all of which can be rescued by disruption of MED5.
Extended Data Figure 10 med5a/5b ref8-1 mutants show elevated levels of salicylic acid and disruption of SID2 does not rescue the stunted growth of the ref8-1 mutant.
a, Shown is the quantification of salicylic acid in 3-week-old rosettes of plants of the indicated genotypes. Data for each genotype are derived from three independently pooled samples representing 300 mg of whole rosette tissue each. Error bars represent s.d. **P < 0.01, difference from wild type (Student’s t-test). b, Shown are 3-week-old rosettes of representative plants of the indicated genotypes. The SID2 gene encodes the salicylic acid biosynthetic enzyme isochorismate synthase. The sid2-4 and ref8-1 sid2-4 plants shown are representative progeny of a single plant with the genotype sid2-4/sid2-4 REF8-1/ref8-1 that gave rise to both morphologically normal and dwarfed plants at a ratio of 3:1. N = 167 morphologically normal, 50 dwarfed; χ2 = 0.444, P = 0.502.
Supplementary information
Supplementary Information
This file contains Supplementary Tables 1-3 and additional references. (PDF 302 kb)
Source data
Rights and permissions
About this article
Cite this article
Bonawitz, N., Kim, J., Tobimatsu, Y. et al. Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant. Nature 509, 376–380 (2014). https://doi.org/10.1038/nature13084
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/nature13084
This article is cited by
-
The rapid-tome, a 3D-printed microtome, and an updated hand-sectioning method for high-quality plant sectioning
Plant Methods (2023)
-
Manipulating microRNA miR408 enhances both biomass yield and saccharification efficiency in poplar
Nature Communications (2023)
-
Research on adsorption mechanisms of levofloxacin over fungus chaff biochar modified by combination of alkali activation and copper-cobalt metallic oxides
Biomass Conversion and Biorefinery (2023)
-
Exposure of Eucalyptus to varied temperature and CO2 has a profound effect on the physiology and expression of genes related to cell wall formation and remodeling
Tree Genetics & Genomes (2022)
-
Overexpression of PnMYB2 from Panax notoginseng induces cellulose and lignin biosynthesis during cell wall formation
Planta (2022)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.