Structure and Dynamics of <i>Brachypodium</i> Primary Cell Wall Polysaccharides from Two-Dimensional ¹³C Solid-State Nuclear Magnetic Resonance Spectroscopy

The polysaccharide structure and dynamics in the primary cell wall of the model grass <i>Brachypodium distachyon</i> are investigated for the first time using solid-state nuclear magnetic resonance (NMR). While both grass and non-grass cell walls contain cellulose as the main structural scaffold, the former contains xylan with arabinose and glucuronic acid substitutions as the main hemicellulose, with a small amount of xyloglucan (XyG) and pectins, while the latter contains XyG as the main hemicellulose and significant amounts of pectins. We labeled the <i>Brachypodium</i> cell wall with ¹³C to allow two-dimensional (2D) ¹³C correlation NMR experiments under magic-angle spinning. Well-resolved 2D spectra are obtained in which the ¹³C signals of cellulose, glucuronoarabinoxylan (GAX), and other matrix polysaccharides can be assigned. The assigned ¹³C chemical shifts indicate that there are a large number of arabinose and xylose linkages in the wall, and GAX is significantly branched at the developmental stage of 2 weeks. 2D ¹³C–¹³C correlation spectra measured with long spin diffusion mixing times indicate that the branched GAX approaches cellulose microfibrils on the nanometer scale, contrary to the conventional model in which only unbranched GAX can bind cellulose. The GAX chains are highly dynamic, with average order parameters of ∼0.4. Biexponential ¹³C <i>T</i>₁ and ¹H <i>T</i>_1ρ relaxation indicates that there are two dynamically distinct domains in GAX: the more rigid domain may be responsible for cross-linking cellulose microfibrils, while the more mobile domain may fill the interfibrillar space. This dynamic heterogeneity is more pronounced than that of the non-grass hemicellulose, XyG, suggesting that GAX adopts the mixed characteristics of XyG and pectins. Moderate differences in cellulose rigidity are observed between the <i>Brachypodium</i> and <i>Arabidopsis</i> cell walls, suggesting different effects of the matrix polysaccharides on cellulose. These data provide the first molecular-level structural information about the three-dimensional organization of the polysaccharides in the grass primary wall

Monosaccharide composition (mol%).

<p>(A) Monosaccharide composition of total cell walls extracted from the whole 4-week-old transgenic plants expressing AnF and wild type Col-0 plants. (B) Monosaccharide composition of cell wall fractions after pectin being removed. Analysis was done using stem, leaf and root tissues of 4-week-old transgenic Arabidopsis plants expressing AnF and wild type Col-0 plants. (C) Neutral monosaccharide composition (mol%) of AGP glycan. (D) Monosaccharide composition of cell wall fraction remaining after AGP removal. Analysis was done using stem, leaf, and root tissues of 4-week-old Arabidopsis plants. * Differences between transgenic lines and <i>Col</i>-0 are significant (n = 3, p<0.05).</p

Real-time qPCR analysis of <i>FUT</i> gene expression in transgenic lines AnF and wild type plants.

<p>Relative expression levels were calculated as comparison to the <i>ACTIN-2</i> reference gene, whose expression was not affected. 2^−ΔΔCt method was used for determining difference between transcripts copy numbers in wild-type and transgenic plants. * Differences between transgenic lines and <i>Col</i>-0 are significant. Analysis of <i>AtFUT</i> genes expression level were done separately for: (A) Stems. (B) Leaves. (C) Roots.</p

Post-Synthetic Defucosylation of AGP by <i>Aspergillus nidulans</i> α-1,2-Fucosidase Expressed in <i>Arabidopsis</i> Apoplast Induces Compensatory Upregulation of α-1,2-Fucosyltransferases - Fig 1

<p>(A) The expression cassette of the vector developed for Arabidopsis transformation. Abbreviations: CaMV 35S –Tetramer of Cauliflower Mosaic virus 35S RNA Promoter, YFP, yellow fluorescent protein coding sequence. (B) PCR analysis of genomic DNA from transgenic Arabidopsis lines transformed with microbial <i>A</i>.<i>nidulans</i> α-fucosidase expression cassette and wild type plants. Herbicide resistant lines were confirmed to harbor the full construct using four pairs of primers (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159757#pone.0159757.s003" target="_blank">S1 Table</a> for sequences). Lane 1—amplification of <i>AnF</i> genes from corresponding transgenic lines; Lane 5—amplification of the same genes from <i>Col</i>-0 wild type plant; Lane 2—amplification of hybrid fragment containing <i>AnF</i> gene linked to the <i>YFP</i> from mutant lines; Lane 6—amplification of the <i>AnF</i>-<i>YFP</i> fragment from <i>Col</i>-0 wild type plant; Lane 3—amplification of <i>YFP</i> gene from the AnF line; Lane 7 –amplification of <i>YFP</i> from <i>Col-0</i> wild type plant; Lane 4—amplification of <i>A</i>. <i>thaliana ACTIN</i>-2 gene fragment from AnF line, and Lane 8 –amplification of <i>ACTIN</i>-2 from <i>Col</i>-0 wild type plant. Analysis was done for three independent transgenic lines for each construction; picture shows results of PCR for single plant of each mutant line. (C) Western blot analysis of total proteins from apoplast of Arabidopsis AnF transgenic and wild type plants. The corresponding microbial fucosidase fused with YFP (116kDa) were found in transgenic lines and not in <i>Col</i>-0 control plants. Blots were produced using GFP monoclonal antibodies (1:5000 dilution).</p

Light and confocal microscopy images of different organs of 3-week old transgenic Arabidopsis plants expressing AnF.

<p>(A) Light microscopy image of AnF expressing plant root cells. (B) Localization of AnF protein fused with YFP in the root cells. (C) Light microscopy image of AnF expressing plant stem cells. (D) Localization of AnF protein fused with YFP in the stem cells. Bars = 0.2 mm.</p

Expression of pathogen response genes.

<p>Real-time qPCR evaluation of pathogen-related genes involved in immune response in (A) non-infected and (B) infected plants at 4 DPI. The samples were as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0150983#pone.0150983.g006" target="_blank">Fig 6B</a>, and the genes are described in the text.</p

Evans blue exclusion test.

<p>(A) Leaves of <i>im</i> and Col-0 were infiltrated with a <i>P</i>. <i>syringae</i> cell culture at a density of 10⁴ colony forming units (cfu), and bacterial growth was monitored daily for 4 days after infection (DPI). (B) Leaves were detached from 7 week-old Col-0 and <i>im</i> and stained with Evans blue. In some experiments, detached wild type leaves were infiltrated with a <i>P</i>. <i>syringae</i> cell culture (density of 10⁴ cfu) prior to staining. In the Evans blue exclusion test, living cells exclude the dye, while dead cells take it up.</p

Impaired Chloroplast Biogenesis in <i>Immutans</i>, an Arabidopsis Variegation Mutant, Modifies Developmental Programming, Cell Wall Composition and Resistance to <i>Pseudomonas syringae</i>

<div><p>The <i>immutans</i> (<i>im</i>) variegation mutation of Arabidopsis has green- and white- sectored leaves due to action of a nuclear recessive gene. <i>IM</i> codes for PTOX, a plastoquinol oxidase in plastid membranes. Previous studies have revealed that the green and white sectors develop into sources (green tissues) and sinks (white tissues) early in leaf development. In this report we focus on white sectors, and show that their transformation into effective sinks involves a sharp reduction in plastid number and size. Despite these reductions, cells in the white sectors have near-normal amounts of plastid RNA and protein, and surprisingly, a marked amplification of chloroplast DNA. The maintenance of protein synthesis capacity in the white sectors might poise plastids for their development into other plastid types. The green and white <i>im</i> sectors have different cell wall compositions: whereas cell walls in the green sectors resemble those in wild type, cell walls in the white sectors have reduced lignin and cellulose microfibrils, as well as alterations in galactomannans and the decoration of xyloglucan. These changes promote susceptibility to the pathogen <i>Pseudomonas syringae</i>. Enhanced susceptibility can also be explained by repressed expression of some, but not all, defense genes. We suggest that differences in morphology, physiology and biochemistry between the green and white sectors is caused by a reprogramming of leaf development that is coordinated, in part, by mechanisms of retrograde (plastid-to-nucleus) signaling, perhaps mediated by ROS. We conclude that variegation mutants offer a novel system to study leaf developmental programming, cell wall metabolism and host-pathogen interactions.</p></div

Sector identity is maintained during im leaf expansion.

<p>Images of the same <i>im</i> leaf were captured by light microscopy every two days from early expansion (day 0) to the attainment of full expansion (day 8). Bar = 5 mm.</p

Tissue anatomy and plastid numbers.

<p>Leaves from 4- and 8-week-old Col-0 and <i>im</i> (white and green sectors) were fixed, stained and examined by light microscopy. (A, D, G) <i>im</i> white; (B, E, H) <i>im</i> green; (C, F, I) Col-0; (A, B, C) 1 month-old; (D–I) 2 month-old. (A-F) Sections were stained with toluidine blue and plastids were counted using Image J software (NCBI website); (J) the sections were 500 nm thick and approximately 150 cells were analyzed for each tissue-type. Asterisks indicate significant difference (t-test, p < 0.01). (G-I) Sections from 2-month-old leaf tissues were stained with Schiff’s reagent for starch. TEM images of representative plastids from fully-expanded <i>im</i> white (K) and wild type leaves (L).</p