Forward Genetic Screening for the Improved Production of Fermentable Sugars from Plant Biomass

<div><p>With their unique metabolism and the potential to produce large amounts of biomass, plants are an excellent bio-energy feedstock for a variety of industrial purposes. Here we developed a high-throughput strategy, using the model plant <em>Arabidopsis thaliana</em>, to identify mutants with improved sugar release from plant biomass. Molecular analysis indicates a variety of processes including starch degradation, cell wall composition and polar transport of the plant hormone auxin can contribute to this improved saccharification. To demonstrate translatability, polar auxin transport in maize was either genetically or chemical inhibited and this also resulted in increased sugar release from plant tissues. Our forward genetic approach using Arabidopsis not only uncovers new functions that contribute to cell wall integrity but also demonstrates that information gleaned from this genetic model can be directly translated to monocotyledonous crops such as maize to improve sugar extractability from biomass.</p> </div

Starch analysis of <i>rah</i> mutants <i>mur11</i>, <i>dpe2</i> and <i>sex4</i>.

<p>(A) Senesced stem tissue from starch mutants was treated with α-amylase and the amount of sugar from starch released was quantified using the anthrone method. Graph shows absorbance at 620 nm per 0.25 mg dry tissue; values are averages ± s.d. (<i>n</i> = 4). We repeated all experiments two times with similar results. (B) Post-amylase treatment, tissue from (A) was assayed by acid hydrolysis for residual sugar release using 1 M H₂SO₄. Graph shows absorbance at 620 nm per 0.25 mg dry tissue; values are averages ± s.d. (<i>n</i> = 3). (C) Acid hydrolysis of fresh leaf disc tissue from known starch mutants using 1 M H₂SO₄. Graph shows absorbance at 620 nm for ¼ of a leaf disc hydrolysate; values are averages ± s.d. (<i>n</i> = 4). We repeated all experiments at least three times with similar results. *, P<0.01 using Student's <i>t</i>-test.</p

Characterization of <i>rah</i> mutants.

<p>(A) Three-week old <i>Arabidopsis</i> plants grown in 96-well flats at 22°C under a 16 h/8 h light/dark cycle (<i>top panel</i>). Leaf 3 or 4 was excised from 21 day-old plants using a hole punch and subjected to acid hydrolysis using 1 M H₂SO₄ (<i>middle panel</i>). c, cotyledon; leaf numbers indicated. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0055616#s3" target="_blank">Results</a> of colorimetric anthrone assay illustrating that <i>rah</i> mutants release more sugars and turn a blue/green colour. Yellow indicates baseline levels of sugar release (<i>bottom panel</i>). (B) From the screen, 63 <i>rah</i> mutants and <i>mur11-1</i> were isolated and organized into 4 categories of low to high yielding sugar mutants. (C) Hierarchical cluster analysis of monosaccharide composition analysis by gas chromatography of <i>rah</i> mutants in 21 day-old seedlings. Values shown as a percentage relative to wild type (see Methods). Yellow indicates higher levels than wild type; blue, lower levels. (D) Clustered heatmap of sugar content as measured by the anthrone method from 63 <i>rah</i> mutants subjected to acid hydrolysis of fresh leaf tissue using 1 M H₂SO₄, acid hydrolysis of senesced stem tissue using 0.2 M H₂SO₄, or glucose content (using hexokinase method) of cellulase, cellulase+xylanase and cellulase+peroxidase digested senesced tissue and starch staining of 14 day-old seedlings. Values shown as a percentage relative to wild type (see Methods). Yellow indicates high levels of sugar; black, low levels of sugar. Mutants with similar characteristics have been classified into 3 subcategories (I, II and III) and outlined in red.</p

Screening for <i>wall hydrolysis sensitive (rah)</i> mutants.

<p>(A) Schematic of the production of ethanol from cellulosic biomass. For biomass pretreatment, dilute sulphuric acid is used to solubilize the hemicellulosic fraction and to disrupt the crystalline structure of cellulose so that hydrolyzing enzymes can easily access and convert cellulose to fermentable sugars. (B) Leaf discs of known cell wall mutants were subjected to acid hydrolysis using 1 M H₂SO₄ at 21 dag. Of the 30 cell wall mutants tested <i>mur11-1</i> showed a significant difference in saccharification relative to wild type. Graph shows absorbance at 620 nm for ¼ of a leaf disc hydrolysate; values are averages ± s.d. (<i>n</i> = 4). We repeated all experiments at least three times with similar results. Dotted red line denotes 2 standard deviations above wild type levels. (C) Fresh leaf disc tissue of <i>mur11-1</i> and a T-DNA allele of <i>MUR11</i>, <i>sac9-3</i> (SALK_058870), were assayed for increased saccharification using 1 M H₂SO₄ at 21 dag. Graph shows absorbance at 620 nm for ¼ of a leaf disc hydrolysate; values are averages ± s.d. (<i>n</i> = 8–10). *, P<0.01 using Student's <i>t</i>-test.</p

<i>pin</i> mutations and NPA treatment results in increased saccharification in <i>Arabidopsis</i> and maize.

<p>(A) Sugar release measured using anthrone method from ground, senesced stem tissue from <i>Arabidopsis</i> pin-shaped inflorescence mutants subjected to 0.2 M acid hydrolysis. Graph shows absorbance at 620 nm per 0.25 mg dry tissue; values are averages ± s.d. (<i>n</i> = 3). We repeated all experiments three times with similar results. Inset shows representative pin-shaped inflorescence in <i>Arabidopsis</i>. (B) Sugar release measured using anthrone method from ground, stems and leaves of 3 month-old maize inflorescence mutants, <i>bif2</i> and <i>ba1</i>, subjected to 0.2 M H₂SO₄ acid hydrolysis. Graph shows absorbance at 620 nm per 0.25 mg dry tissue; values are averages ± s.d. (<i>n</i> = 3–4). N, phenotypically normal siblings. Inset shows representative maize inflorescence mutant. (C) Wild type <i>Arabidopsis</i> 28 day-old seedlings grown on MS media supplemented with 0, 1 or 5 µM NPA and subjected to 0.2 M H₂SO₄ acid hydrolysis. Graph shows absorbance at 620 nm per 0.25 mg dry tissue; values are averages ± s.d. (<i>n</i> = 4). We repeated all experiments two times with similar results. (D) Two maize cultivars were grown for 4 weeks and then treated with 120 µM NPA for 2 weeks and the stems and leaves of the plant was subjected to 0.2 M H₂SO₄ acid hydrolysis and sugars released measured using the anthrone method. Graph shows absorbance at 620 nm per 0.25 mg dry tissue; values are averages ± s.d. (<i>n</i> = 3–6). *, P<0.01 using Student's <i>t</i>-test.</p

Phenotypic variation in <i>frb1</i> mutant seedlings.

<p>A. Wild-type <i>FRB1</i> seedling after 5 days growth on MS media. B. A 3-day old <i>frb1–1</i> seedling where the hypocotyl has spontaneously broken during germination. C. An example of a <i>frb1–1</i> seedling where the cotyledons have become fused as the seedling germinated. D. A <i>frb1–2</i> seedling showing severe cell dissociation. E. A two week old <i>FRB1</i> seedling grown on MS media. F. and G. At the same stage of growth as seedlings in E, <i>frb1–1</i> and <i>frb1–2</i> seedlings have little cell separation but many do have extensive fusions between leaves and other aerial organs. All scale bars equal 0.5. mm.</p

Sugar composition and sugar incorporation in membrane preparations from <i>FRB1</i> and <i>frb1</i> plants.

<p>A. Sugar composition derived from microsomal preparations of wild-type and <i>frb1</i>–<i>2</i>. Monosaccharides and uronic acids were identified based on retention time and comparison to pure standards and values are expressed in % of total area. Significant changes (two-tailed Student's t-test, n = 3, *: p<0.05; **: p<0.01) are marked with stars. Rha: rhamnose, Ara: arabinose, Gal: galacatose, Glc: glucose, Xyl-Man: unseparated peak containing xylose and mannose, GalA: galacturonic acid, GlcA: glucuronic acid. B. The total radioactivity in the chloroform insoluble material. C. The total radioactivity in the chloroform soluble material. See the experimental procedures for details.</p

Comparison of pectin methylesterase (PME) activity in <i>FRB1</i> and <i>frb1</i> alleles and analysis of esterified and non-esterified cell wall regions using FTIR.

<p>A. PME activity in <i>frb1</i> alleles is from 45–70% higher than in wild-type seedlings. Degree of methylesterification of the pectin used as substrate in PME assays is indicated as percentage. Each column is the average of three separate assays; error bars indicate ± standard deviation. All levels are significantly different with p<0.05 for all three <i>frb1</i> lines compared to wild-type B. Comparison of <i>FRB1</i> and <i>frb1</i> of FTIR scans. C. Close-up of region III from A. showing particular differences in the degree of methylesterification between <i>FRB1</i> and <i>frb1</i>.</p

GFP-FRB1 fusion protein accumulates in subcellular compartments.

<p>A. Hypocotyl cells of transgenic plants expressing GFP-FRB1 fusion protein under the control of a constitutive promoter. Note that the GFP accumulates in subcellular compartments (green). Red fluorescence is from chloroplasts. B. GFP-FRB1 fluorescent particles in the cortical cytoplasm. C. Higher magnification image showing the ring morphology of the GFP-tagged compartments. D. Hypocotyl cells of GFP-FRB1 plants treated with 100 µg/ml Brefeldin A for 15 min. Note the redistribution of the GFP-labeled compartments to aggregates, especially around nuclei. Some of the GFP signal has also become soluble. E. Transient over-expression of GFP-FRB1 fusion protein or F. mCherry (CD3–967) fusion protein in an epidermal tobacco cell. G. Micrograph showing overlap of E and F. Scale bars equal 10 µm in A–D and 20 µm in E–G</p

Xyloglucan oligosaccharide mass profile (OLIMP) of crude cell wall material from shoots (A, B, C) and its respective 4 M KOH fractions (D, E, F).

<p>A. and D. Representative spectra derived from <i>FRB1</i>. B. and E. Representative spectra derived from <i>frb1</i>. C. OLIMP analysis of shoots. D. OLIMP analysis of 4 M KOH fraction derived from shoots. Nomenclature of xyloglucan oligosaccharides are taken from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042914#pone.0042914-Neumetzler1" target="_blank">[38]</a> and references within. Stars indicate significant alterations with p<0.05.</p