Determining the Syringyl/Guaiacyl Lignin Ratio in the Vessel and Fiber Cell Walls of Transgenic Populus Plants

Observation of the spatial lignin distribution throughout the plant cell wall provides insight into the physicochemical characteristics of lignocellulosic biomass. The distribution of syringyl (S) and guaiacyl (G) lignin in cell walls of a genetically modified Populus deltoides and its corresponding empty vector control were analyzed with time-of-flight secondary ion mass spectrometry (ToF-SIMS) and then mapped to determine the S/G lignin ratio of the sample surface and specific regions of interest (ROIs). The surface characterizations of transgenic cross-sections within 1 cm vertical distance of each other on the stem possess similar S/G lignin ratios. The analysis of the ROIs determined that there was a 50% decrease in the S/G lignin ratio of the transgenic xylem fiber cell walls

Highly Efficient Isolation of <em>Populus</em> Mesophyll Protoplasts and Its Application in Transient Expression Assays

<div><h3>Background</h3><p><em>Populus</em> is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function.</p> <h3>Methodology/Principal Findings</h3><p>We optimized a <em>Populus</em> leaf mesophyll protoplast isolation protocol and established a <em>Populus</em> protoplast transient expression system. We demonstrated that <em>Populus</em> protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the <em>Populus</em> protoplasts. Furthermore, we showed that the <em>Populus</em> protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events.</p> <h3>Conclusions/Significance</h3><p>This study established a method for efficient isolation of protoplasts from <em>Populus</em> leaf and demonstrated the efficacy of using <em>Populus</em> protoplast transient expression assays as an <em>in vivo</em> system to characterize genes and pathways.</p> </div

<i>Populus</i> leaf mesophyll protoplasts.

<p>(<b>A</b>) Optimal yield and quality of protoplasts can be isolated from one month-old <i>Populus</i> plants grown on MS medium in a magenta box. (<b>B</b>) High transfection efficiency is indicated with GFP signal.</p

Subcellular localization of various organelle markers in <i>Populus</i> protoplasts.

<p>(<b>A</b>) Plasma membrane; (<b>B</b>) Golgi apparatus; (<b>C</b>) Nucleus; (<b>G</b>) Peroxisome; (<b>H</b>) endoplasmic reticulum (ER); (<b>I</b>) An ubiquitously-localized protein (RACK1, Receptor for Activated C-protein Kinase 1). Shown in (<b>D</b>), (<b>E</b>), (<b>F</b>), (<b>J</b>), (<b>K</b>) and (<b>L</b>) are bright field images for fluorescent images of (<b>A</b>), (<b>B</b>), (<b>C</b>), (<b>G</b>), (<b>H</b>) and (<b>I</b>), respectively. The organelle markers were fused with mCherry fluorescent protein, and RACK1 was fused with YFP fluorescent protein. The mCherry signal was separated from chloroplast autofluorescence using spectral imaging and linear unmixing. The mCherry and YFP signals are false-colored green and the chloroplast autofluorescence is shown in red. Scale bar, 1 µm.</p

Energy sensing signaling in <i>Populus</i> protoplasts.

<p>(<b>A</b>) Semi-quantitative RT-PCR analysis of <i>PtrDIN6</i> transcripts in response to dark and hypoxia treatments. L-Light; D-Dark; H-Hypoxia. The <i>PtrUBQ10</i> gene was used as a control. (<b>B</b>) The change of <i>PtrDIN6</i> transcripts in response to overexpression of AthKIN10 protein. After transfection, protoplasts were incubated overnight to allow the expression of AthKIN10 before samples were harvested for qRT-PCR and western blot analysis. Western blot was used to detect the presence of the introduced HA-tagged AthKIN10 protein. The experiments were repeated three times with similar results. The averages of three technical replicates ± standard errors are presented in the graph. * indicates a significant difference (at P≤0.01, student’s t-test) between protoplasts expressing AthKIN10 and the control (ctrl). (<b>C</b>) The expression of three <i>Populus KIN10</i> homologues in transfected protoplasts examined by semi-quantitative RT-PCR. The expression of <i>PtrUBQ10</i> was used as an internal control. (<b>D</b>) The response of <i>PtrDIN6</i> transcript to the overexpression of three <i>Populus KIN10</i> homologues. The experiments were repeated three times with similar results. The averages of three technical replicates ± standard errors are shown. Protoplasts transfected with an empty vector was used as control (ctrl) for each comparison. (<b>E</b>) The activation of <i>PtrDIN6</i> by PtrKIN10 in a GUS reporter assay. For each co-transfection, a 35S::LUC (Luciferase) was included and the LUC activity was used to normalize GUS activity to account for the potential variations in the transfection efficiency. The averages of three technical replicates ± standard errors are shown. * indicates a significant difference (at P≤0.01, student’s t-test) between each treatment and the control (ctrl).</p

The response of <i>Populus</i> protoplasts to various plant hormone treatments.

<p>Shown are the change <i>POPTR_01s30560</i> transcript in response to different concentrations of NAA in protoplasts (A) and intact leaves (E), the change of <i>POPTR_14s08030</i> transcript in response to different concentrations of GA₃ in protoplasts (B) and intact leaves (F), the change of <i>POPTR_10s00320</i> transcript in response to different concentrations of BAP in protoplasts (C) and intact leaves (G), and the change of <i>POPTR_10s08300</i> transcript in response to different concentrations of ACC in protoplasts (D) and intact leaves (H). The protoplasts or intact leaves were incubated with various concentrations of plant hormones for 3h before being harvested for qRT-PCR analysis. The experiments were repeated three times with similar results. The averages of three technical replicates ± standard errors are shown. * indicates a significant difference (at P≤0.01, student’s t-test) between each treatment and the untreated control.</p

VACNFs penetrate <i>Populus</i> epidermis without damaging cells.

<p>(a) and (b) Micrographs of transverse sections through a <i>Populus</i> leaf showing individual nanofibers penetrating the cuticle and epidermis. The arrow in (a) shows a nanofiber reaching the base of an epidermal cell without penetrating the underlying palisade cell. Arrows in (b) indicate a nanofiber traversing the cytoplasm of the cell. (c) DIC micrograph of a leaf epidermis showing that carbon nanofiber impalement occurs in a grid pattern, similar to the original chip. (d) DIC micrograph of the same leaf depicted in (c) with the focal plane moved into the underlying palisade layer showing the absence of over-penetrant fibers. Results are representative of leaves from 2 separate plants. (e) and (f) Bright-field micrographs of <i>Populus</i> leaves after staining with DAB to detect H₂O₂ production. Leaves penetrated by carbon nanofibers (e) show no DAB staining and are similar to untreated areas of the leaf (f). The boxed inset in (e) shows a magnified image of the nanofiber-treated area, with black arrows indicating the location of carbon nanofibers in this image. (g) and (h) Leaves wounded with a cork borer (g) or abraded with carborundum (h) show areas stained dark brown by DAB deposition in reaction to H₂O₂ produced in the wound response. Black arrows in (h) indicate carborundum powder remaining on the leaf. Dashed arrows in (g) point to the cut edge of the leaf and solid arrows indicate staining with DAB. Similar results were obtained with leaves from 3 separate plants. (i) and (j) Transverse sections of a <i>Populus</i> leaf after carborundum abrasion, showing areas of (i) severe and (j) mild epidermal damage. The black arrow in (j) points to grit particles within the palisade layer. (k) DIC micrograph of a leaf epidermis after carborundum treatment showing abraded epidermal cells (denoted by white arrows). (l) DIC micrograph of the same leaf depicted in (k) with the focal plane moved into the underlying palisade layer, showing the presence of embedded grit particles (denoted by white arrows). Images (a), (b), (i) and (j) were obtained from thin sections of fixed (formalin), embedded (paraffin) and stained (toluidine blue) tissue; images (c), (d), (k) and (l) were obtained from fixed (ethanol-acetic acid) and cleared (chloral hydrate) tissue; images in (e)–(h) were obtained from stained (DAB) and decolorized (boiling ethanol) tissue. Details are provided in <i>Materials and Methods</i>.</p

Vertically-aligned carbon nanofiber arrays provide densely clustered, microscopic spikes that penetrate leaf tissue.

<p>(a) Scanning electron micrograph (SEM) of a small region of a VACNF array chip. Nanofibers are arrayed across this chip at a 10 μm pitch. Individual nanofiber heights were 20–25 μm. In the image, the chip is tilted 30° from perpendicular to the beam to show the aspect ratio of each nanofiber. (b) Application of a carbon nanofiber chip onto the adaxial surface of a <i>Populus</i> leaf. (c) SEM of a leaf impaled with a VACNF chip (20-μm pitch), showing fibers embedded in the epidermal cells of the adaxial surface as indicated by the arrows. The position of a minor vein adjacent to the site of impalement is marked by (*), and a region of unimpaled tissue is marked by (**). (d) A close-up view of the impalement site, showing individual carbon nanofibers that have broken off of the VACNF chip and remain embedded in the leaf epidermal cells, as indicated by arrows. The image was taken ~15 min after VACNF impalement. Excised leaf tissue was placed directly in the electron microscope, where it was dried <i>in vacuo</i> prior to imaging. Results shown are from single, typical experiments.</p

OG-CmPP16-1 moves through local <i>Populus</i> leaf vasculature following carbon nanofiber delivery.

<p>(a–d) Epifluorescence micrographs and (e) measured intensity at selected regions of interest (ROI). Fluorescence intensity was monitored at a main vein, a secondary vein, and a tertiary vein following introduction of the protein via a VACNF array. Veins are marked with numbers (1, 2, and 3, for the main, secondary and tertiary veins respectively) in (a) and with different styles of arrows in (b)–(d). Fluorescence micrographs were recorded at (a) 0 min, (b) 32 min, (c) 35 min, and (d) 42 min after VACNF delivery. In (e), fluorescence intensity is plotted for the main vein (circles), secondary vein (squares) and tertiary vein (triangles). The VACNF chip is located ~1 cm below and to the right of the imaged area. No correction for photobleaching was applied. Results shown are representative of 5 separate experiments (one plant per experiment).</p

VACNFs Deliver LYCH to symplast and apoplast in <i>Populus</i> leaf tissue.

<p>LYCH (1 mM) was applied to the adaxial surface of a <i>Populus</i> leaf, and a VACNF array was placed on top and pressed to penetrate the leaf surface. Uptake of the solution was allowed to proceed for 5 minutes, after which the leaf was removed from the plant, the chip area was excised with a scalpel and the chip was removed. The leaf tissue was gently washed to remove surface LYCH, sealed under a cover glass and imaged immediately using confocal laser scanning microscopy. Solid red circles indicate locations of observed nanofibers, whereas dashed red circles indicate locations where nanofibers would be expected but were not definitively observed. (left) LYCH fluorescence signal from the epidermal layer (false-colored white). (center) LYCH fluorescence signal from the palisade layer, 9 μm deeper in the leaf (false-colored green). Arrows indicate LYCH in the apoplast of the palisade layer. (right) Superposition of the left and center images.</p