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Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant

Nature volume 509, pages 376–380 (2014)Cite this article

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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.

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Figure 1: Disruption of MED5a/5b rescues growth and fertility of Arabidopsis ref8 mutants.
Figure 2: Secondary cell wall composition and architecture of med5a/5b ref8-1 mutant plants is distinct from both wild-type and ref8-1 mutant plants.
Figure 3: Lignocellulosic material from med5a/5b ref8-1 plants contains lignin composed almost entirely of H subunits and shows substantially increased saccharification potential.
Figure 4: Disruption of MED5a/5b rescues the widespread transcriptional reprogramming of the ref8-1 mutant.

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Sequence Read Archive

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RNA-seq data have been deposited in the NCBI Sequence Read Archive under accession number SRP035272.

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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.

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Author notes

  1. Nicholas D. Bonawitz & Junko Maeda

    Present address: Present addresses: Dow AgroSciences LLC, 9330 Zionsville Road, Indianapolis, Indiana 46268, USA (N.D.B.); Department of Agronomy, University of Wisconsin-Madison, 1575 Linden Drive, Madison, Wisconsin 53706, USA (J.M.).,

Authors and Affiliations

  1. Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907, USA,

    Nicholas D. Bonawitz, Jeong Im Kim, Nickolas A. Anderson, Junko Maeda & Clint Chapple

  2. Department of Biochemistry, University of Wisconsin-Madison, Madison, 53706, Wisconsin, USA

    Yuki Tobimatsu & John Ralph

  3. Biosciences Center, National Renewable Energy Laboratory, Golden, 80401, Colorado, USA

    Peter N. Ciesielski & Bryon S. Donohoe

  4. Department of Agricultural and Biological Engineering and the Laboratory of Renewable Resources Engineering, Purdue University, West Lafayette, 47907, Indiana, USA

    Eduardo Ximenes & Michael Ladisch

  5. Department of Biological Systems Engineering, University of Wisconsin-Madison, Madison, 53706, Wisconsin, USA

    John Ralph

  6. DOE Great Lakes Bioenergy Research Center, and Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, 53726, Wisconsin, USA

    John Ralph

  7. Weldon School of Biomedical Engineering, Purdue University, West Lafayette, 47907, Indiana, USA

    Michael Ladisch

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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

Correspondence to Clint Chapple.

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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.

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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

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