Heterogeneity and specificity of lignification in flax (linum usitatissimum L.) : by biochemical and microscopics tools

Le lin est une angiosperme annuelle dicotylédone connue pour ses fibres cellulosiques longues aux propriétés remarquables. Ces fibres se distinguent des cellules du bois par leur faible teneur en lignine et la distribution de ce polymère au sein des couches pariétales formant la fibre. Afin de mieux comprendre les facteurs contrôlant le taux de lignine dans les différents tissus de lin, nous avons entrepris une étude détaillée de la lignification au moyen d?approches microscopiques et biochimiques. Dans un premier temps, nos travaux ont souligné des similarités (lignine gaïacyle, liaisons condensées, détection du motif dibenzodioxocine et de la coniférine) entre la lignine de lin et celle des conifères. Une approche biochimique a montré que l?activité oxydasique majeure dépendrait des peroxydases. La partielle purification et des caractérisations initiales (électrophorèses natives, IEF, affinité de substrat) des peroxydases pariétales ont mis en évidence des différences importantes entre les isoformes du xylème, et celles des tissus externes (riches en fibres) de la tige. L?utilisation du chlorure de cérium a permis de préciser la distribution subcellulaire de l?H2O2 (catalyseur de polymérisation) dans la tige du lin au stade floraison. Dans les fibres longues, l?H2O2 n?a pu être détecté que dans la région médiane de la tige. En revanche l?H2O2 était présente dans toutes les cellules du xylème examinées. Des observations en ESEM et en microscopie confocale suggèrent que les fibres longues de lin forment un réseau dense qui limite la pénétration de protéines. L?ensemble de ces résultats souligne les différences entre le xylème et fibres longues pouvant expliquer en partie la faible lignification des fibres externes.The angiosperm flax is an annual dicotyledon grown for its cellulose-rich bast fibres that show interesting properties. These fibres are characterized by a low lignin level in the different layers of the fibre cell wall that distinguishes them from xylem cells. In order to obtain a better understanding of the factors controlling lignin levels in different flax stem tissues we have undertaken a detailed study of lignification using microscopic and biochemical approaches. Initial results underlined similarities (guaiacyl lignin, condensed bonds, presence of dibenzodioxocine and coniferin) between flax lignin and that of conifers. A biochemical approach showed that the major oxidase activity was provided by peroxidases. Partial purification and initial characterization (native electrophoresis, IEF, substrate affinities) of cell wall peroxidases indicated important differences between the isoforms present in xylem and outer (fibre-rich) stem tissues. The use of cesium chloride allowed us to determine the cellular locations of H2O2 (polymerisation catalyst) in flax stems at the flowering stage. In bast fibres, H2O2 was only detected in the median region of the stem. In contrast H2O2 was observed in all xylem cells examined. Observation by ESEM and confocal microscopy suggested that the cell walls of flax bast fibres form a dense network that limits protein penetration. Altogether, these results highlight a number of differences between flax xylem and bast fibres that could partially explain the low lignification of the external bast fibres

Stage-Specific Changes in the Water, Na+, Cl- and K+ Contents of Organelles during Apoptosis, Demonstrated by a Targeted Cryo Correlative Analytical Approach.

Many studies have demonstrated changes in the levels of several ions during apoptosis, but a few recent studies have reported conflicting results concerning the changes in water content in apoptotic cells. We used a correlative light and cryo-scanning transmission electron microscopy method to quantify water and ion/element contents simultaneously at a nanoscale resolution in the various compartments of cells, from the onset to the end of apoptosis. We used stably transfected HeLa cells producing H2B-GFP to identify the stages of apoptosis in cells and for a targeted elemental analysis within condensed chromatin, nucleoplasm, mitochondria and the cytosol. We found that the compartments of apoptotic cells contained, on average, 10% more water than control cells. During mitochondrial outer membrane permeabilization, we observed a strong increase in the Na+ and Cl- contents of the mitochondria and a strong decrease in mitochondrial K+ content. During the first step in apoptotic volume decrease (AVD), Na+ and Cl- levels decreased in all cell compartments, but remained higher than those in control cells. Conversely, during the second step of AVD, Na+ and Cl- levels increased considerably in the nucleus and mitochondria. During these two steps of AVD, K+ content decreased steadily in all cell compartments. We also determined in vivo ion status during caspase-3 activity and chromatin condensation. Finally, we found that actinomycin D-tolerant cells had water and K+ contents similar to those of cells entering apoptosis but lower Na+ and Cl- contents than both cells entering apoptosis and control cells

Nuclear shape and the condensation and disposition of chromatin were sufficient to identify cells i) entering apoptosis (ST 1), ii) in the course of apoptosis (ST 2 to ST 5) and iii) not engaged in apoptosis (ST 0).

<p>We confirmed this identification by TMRE staining, the localization of cytochrome-<i>c</i> (in the mitochondria, cytosol or nucleus) and the presence of activated CASP-3 and cleaved PARP.</p

Cryo-correlative fluorescence and scanning transmission electron microscopy (STEM) of cells in the different stages of apoptosis.

<p>For each stage, four images of the same cell from a single 80 nm-thick ultrathin cryo section are shown. Fluorescence imaging of H2B-GFP in the ultrathin cryo sections (first and second panels showing fluorescence intensity on a gray scale, and after application of a rainbow RGB look-up table, respectively) made it possible to analyze chromatin distribution and nucleus shape, to identify each stage of apoptosis. The cells characterized by fluorescence imaging were then imaged by STEM (third panel). Due to a strong natural electron contrast and good ultrastructural preservation, the compartments in the cytoplasm are easily identified. Merging of the fluorescence and STEM images recorded at the same magnification (last panel) is required for a clear identification of chromatin clumps before the targeted elemental analysis of condensed chromatin and nucleoplasm. The scale bar represents 1 μm.</p

“Elemental descriptors” gathering concentration of all the elements/ions considered, for each compartment (condensed chromatin, nucleoplasm, cytosol and mitochondria) in control cells, in cells during each stage of apoptosis and during stage 0.

<p>In the histograms, concentration of nitrogen was divided by 10 (N/10), whereas the concentration of sodium and chloride was multiplied by 3 (Na x 3 and Cl x 3).</p

Simultaneous 3D localization of cytochrome-<i>c</i> (Cc) and H2B-GFP showing Cc redistribution during specific stages of apoptosis.

<p>Anti-cytochrome-<i>c</i> antibody binding was imaged on fixed HeLa cells stably expressing H2B-GFP after the induction of apoptosis by 500 ng/mL AMD, for 7h and 15 minutes. Four images are shown for the same cell, at a given stage. On the first image, differential interference contrast (DIC) shows the shape of the cell, nucleus and nucleolus. The second image is an optical section passing through the middle of the nucleus showing the merge of images for Cc (red) and H2B-GFP (green). The third image is a 3D surface rendering of Cc alone (red). The last image is a simultaneous 3D transparent volume rendering of both Cc (red) and H2B-GFP (green). The scale bar represents 10 μm.</p

Targeted quantification of Na⁺, Cl^-, S and Mg²⁺ in the cytosol, mitochondria, condensed chromatin and nucleoplasm.

<p>Concentration of elements/ions (Na⁺, Cl^-, S and Mg²⁺) were determined by energy dispersive X-ray spectrometry in the cytosol, mitochondria, condensed chromatin and nucleoplasm of each of the following: i) control cells, ii) cells in the various stages of apoptosis (ST 1 to ST 5) and iii) cells in the ST 0 stage. Results, in mmol/L, are given as means ±SEM. (<i>n</i> = 3; 3 to 83 different cells per stage).</p

Simultaneous imaging of cell shape, mitochondrial potential and nuclear modifications at the onset and during the various stages of apoptosis.

<p>HeLa cells stably expressing H2B-GFP were stained with TMRE to study mitochondrial polarization. Simultaneous time-lapse confocal imaging of cell shape (DIC), TMRE and H2B-GFP was performed by two-photon excitation every five minutes for 7 hours and 15 minutes after the induction of apoptosis by the addition of 500 ng/mL AMD. (A) Traces for TMRE intensity (red line, relative to value reached at time 0.91 h) and nuclear volume (green line, relative to value at time 0 h) in a representative cell (cell #9 on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148727#pone.0148727.s004" target="_blank">S1</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0148727#pone.0148727.s006" target="_blank">S3</a> Movies). Mitochondrial depolarization began at 6 h 05 minutes and ended at 6 h 25 minutes, when nuclear volume began to decrease. (B) Cell shape, 3D structure of the TMRE signal, chromatin and nucleus. For each time point, one DIC image (left), one optical section for the red and green signals, a 3D view (surface rendering) of the TMRE signal and a 3D view (surface rendering) of both TMRE signal (red) and H2B-GFP (green) are shown. On DIC image, yellow dotted line indicates the limit of the cell. On 3D view of both TMRE signal (red) and H2B-GFP (green), the relative intensity of the red signal and the volume of the nucleus are indicated by the red and green labels, respectively. Typical chromatin and nucleus structures defined the main stages of apoptosis: stage 1 (ST 1) to stage 5 (ST 5). At the far right of the bottom row, one cell unaffected by AMD after 7 h and 15 minutes is defined as a stage 0 cell (ST 0). In this cell, TMRE staining appears as a 3D network of filaments and the angular nucleus contains a segregated nucleolus. The scale bar represents 10 μm.</p

Targeted quantification of water and of N, P and K⁺ in the cytosol, mitochondria, condensed chromatin and nucleoplasm.

<p>Water percentage was calculated by STEM imaging and the concentration of element/ions (N, P and K⁺) was calculated by energy dispersive X-ray spectrometry in the cytosol, mitochondria, condensed chromatin and nucleoplasm of each of the following: i) control cells, ii) cells in various stages of apoptosis (ST 1 to ST 5 stages) and iii) cells in the ST 0 stage. Results, in mmol/L, are given as means ±SEM (<i>n</i> = 3; 3 to 83 different cells per stage).</p