Supplemental Material for Dolan and Chapple, 2018

File S1 contains the results of our differential expression analysis performed using EdgeR

MOESM1 of Genetic engineering of Arabidopsis to overproduce disinapoyl esters, potential lignin modification molecules

Additional file 1: Figure S1. UV spectra (a) and MS spectra under ESI (-) mode (b) of 1,2-DSG and compound 1

Phenotypic impacts of the <i>crw1-4</i> mutation in the B73 × Mo17 hybrid.

<p>Photograph of adjacent field plots planted with the B73×Mo17 hybrid (<b>A</b>) and the <i>crw1-4</i> near isogenic mutant hybrid (<b>B</b>) displaying extensive WCR beetle feeding and less rigid leaf architecture. <b>C</b>, Close up view of severe damage from foliar feeding by WCR beetles in <i>crw1-4</i>.</p

Epidermal lobing patterns in <i>crw1</i> and wild-type leaves.

<p>Representative cryoscanning electron micrographs comparing epidermal lobing pattern (indicated by arrowheads) in <i>crw1</i> leaf 7 (<b>A</b>), wild-type leaf 7 (<b>B</b>), <i>crw1</i> leaf 13 (<b>C</b>) and wild-type leaf 13 (<b>D</b>). Bar = 40 µm.</p

Representative TBO staining pattern of upper epidermis in the <i>crw1</i> mutant and wild-type leaves.

<p>Violet staining of upper epidermis from leaf 3 of wild-type (<b>A, E</b>), <i>crw1-4</i> mutant (<b>C</b>), and <i>crw1-1</i> mutant (<b>G</b>). <b>B and F</b>, aqua staining of wild type leaf 13. <b>D and H</b>, retention of violet staining in <i>crw1-4</i> (<b>D</b>) and <i>crw1-1</i> (<b>H</b>) leaf 13. <b>I-J</b>, cross section of <i>crw1-4</i> adult leaf 13 with all violet and no aqua staining (<b>I</b>) compared to aqua and violet staining (<b>J</b>) of wild-type leaf.</p

Vibrational Fingerprint Mapping Reveals Spatial Distribution of Functional Groups of Lignin in Plant Cell Wall

Highly lignified vascular plant cell walls represent the majority of cellulosic biomass. Complete release of the biomass to deliver renewable energy by physical, chemical, and biological pretreatments is challenging due to the “protection” provided by polymerized lignin, and as such, additional tools to monitor lignin deposition and removal during plant growth and biomass deconstruction would be of great value. We developed a hyperspectral stimulated Raman scattering microscope with 9 cm^–1 spectral resolution and submicrometer spatial resolution. Using this platform, we mapped the aromatic ring of lignin, aldehyde, and alcohol groups in lignified plant cell walls. By multivariate curve resolution of the hyperspectral images, we uncovered a spatially distinct distribution of aldehyde and alcohol groups in the thickened secondary cell wall. These results collectively contribute to a deeper understanding of lignin chemical composition in the plant cell wall

Comparison of cell wall bound hydroxycinnamic acids and lignin content isolated from Wild type and <i>crw1</i> (Mutant) foliage.

<p><b>A</b>, differences in foliar p-coumaric and ferulic acid levels from juvenile and adult stages of Wild type and Mutant. <b>B</b>, differences in lignin content of adult leaves of Wild type and Mutant. Each data point represents the mean ± standard deviation of 3 and 9 biological samples for hydroxycinnamic acids and lignin, respectively. Asterisks denote significant differences between Wild type and Mutant (unpaired <i>t</i> test: P<0.001).</p

Representative cryoscanning electron micrographs comparing leaf epidermis in <i>crw1</i> and wild-type leaves.

<p>Adaxial surface of leaf 3 of <i>crw1</i> (<b>A</b>) and wild-type (<b>E</b>), covered with crystalline epicuticular waxes. Adaxial surface of leaf number 7 of <i>crw1</i> (<b>B</b>) and wild-type (<b>F</b>) showing amorphous epicuticular waxes. All four different cell types of trichomes-prickle hair (Ph), macrohair (Mh), bicellular microhair (Bm) and bulliform cells (Bc) are present both in <i>crw1</i> (<b>C</b> and <b>D</b>) and wild-type (<b>G</b> and <b>H</b>) leaf 7. Bars A and E = 100 µm, B and F = 40 µm, C, D, G and H = 300 µm.</p

Phenotype of field grown <i>crw1</i>.

<p><b>A</b>, Initiation of WCR beetle feeding of <i>crw1</i> around week 5–6 after planting (left, area between arrowheads). <b>B</b>, Once initiated, the WCR beetle feeding results in complete loss of foliage during the adult stage (left, area between arrowheads). The wild type (W22 inbred) on the other hand does not show WCR beetle damage at the same stages (A and B right). <b>C and D</b>, stripping of epidermal tissue on underside of leaves in a basipetal fashion in <i>crw1</i> by WCR beetles. <b>E</b>, characteristic “window pane” pattern resulting due to WCR feeding of <i>crw1</i> leaves.</p

Representative uni-axial tensile stage scanning electron micrographs comparing fracture dynamics of wild-type and <i>crw1</i> adult leaves.

<p>The incipient crack propagation profile and breakage dynamics at the point of final failure of wild-type (<b>A</b>, area between arrowheads) and <i>crw1</i> (<b>B</b>, area between arrowheads). <b>C</b>, Additional fracture points along the axis of incipient crack propagation in <i>crw1</i> (area between arrowheads). Bar = 200 µm.</p