Phospholipid radioactive labeling and lipid extraction

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Phospholipid radioactive labeling and lipid extraction

Suspension cells were labelled with 37 MBq L-1 33Pi-orthophosphate per 7 mL suspension (circa 700 mg FW) during the specified time according to the procedure previously described by Krinke et al., 2007 (Krinke et al., 2007). Cells were dried from the cultivation media and dissolved in H50 buffer (175mM mannitol, 0.5mM CaCl2, 0.5mM K2SO4, 50mM Hepes, pH 8.9), 1g of cells to 10mL buffer. Cells were equilibrated for 3h at a rotary shaker before theatment. Lipids were extracted by adding into the flasks with 7 mL of equilibrated cells 2.14 volumes of ice-cold chloroform:methanol:37% (v/v) HCl (50:100:1.5, v/v). The mixture was transferred into tubes, and a two-phase system was induced by the addition of 0.7 volume of chloroform and 0.7 volume of 9% (w/v) NaCl in water. The tubes were vigorously shaken and left at 4°C overnight for two-phase formation. The upper phase was discarded and the lower organic phase was evaporated under nitrogen stream. Remaining lipids were dissolved in chloroform and stored at−20◦C until further use (Ruelland et al., 2002).
As for Arabidopsis 14-days-old seedlings (see 2.1 for growth conditions details), cultivation media was replaced by 100mM Tris-HCl buffer, pH 6.15, and plates with seedlings were kept for 2 hr at 22°C for pre-equilibration and then labelled with 33Pi for 1h. Flagellin was added to the wells and the reaction was stopped by adding 4mL of hot isopropanol to approximatively 400 mg of plant tissues followed by grinding in Potter‘s homogenizer with 3 volumes of chloroform:methanol:37% (v/v) HCl (50:100:1.5, v/v). The mixture was transferred into tubes, and a two-phase system was induced by the addition of 1.25 volumes of chloroform and 1.25 volumes of 9% (w/v) NaCl in water. The tubes were vigorously shaken and left at 4°C overnight for two-phase formation. The lower organic phase was evaporated under nitrogen stream, remaining lipids were dissolved in chloroform and stored at -20 °C until further use (modified from Folch et al. (Folch et al., 1957) and Drobak et al (Drøbak et al., 2000).
Adult leaf tissues (detached leaves from 4-5week old plants) were incubated for 2h in 100mM Tris-HCl buffer pH 5.7 to recover from wounding and labeled in the dark with 37 MBq [33P] orthophosphate per 200 mg FW in 100 mM Tris-HCl buffer (pH 6.15) for 14 h at 25◦C. After incubation, the tissues was washed from non-incorporated [33P]orthophosphate (Kalachova et al., 2013). To determine PLD activation in leaf tissues, leaf discs were incubated with 0.8% 1-butanol in Tris-HCl buffer (pH 6.15) for 1 h in the dark prior to treatment. All studied inhibitors were added 30 min prior to treatment. After reaction, the samples were fixed in liquid nitrogen. Control samples were incubated with the respective solvents (Munnik et al., 1996). Reaction was stopped by freezing in liquid nitrogen and lipids from approximately 300 mg of leaf tissues were extracted by adding 3.75 mL CHCl3/CH3OH/HCl (50:100:1, v/v) to each sample. A two-phase system was prepared by adding CHCl3 and 0.9% (w/v) NaCl. Samples were vortexed for 15 s and centrifuged for 2 min. The lower organic phase was washed with 3.75 mL CHCl3/CH3OH/1M HCl (3:48:47, v/v), dried under nitrogen gas stream and stored at−20◦C until further use (Munnik et al., 1996).

Thin-layer chromatography and scintillation counting

Lipids were dissolved in 100 μL CHCl3/CH3OH (2:1, v/v). Then samples (5 μl) were analyzed on thin-layer chromatography (TLC) plate (200 × 200 × 0.25 mm, Merck, Germany). To compare different samples, equal amounts of radioactivity were loaded on the same plate. Structural phospholipids and phosphatidic acid were separated in the acid solvent system composed of chloroform:acetone:acetic acid:methanol:water (10:4:2:2:1 [v/v/v/v]) (Lepage, 1967). Phosphoinositides were separated in the alkaline solvent system composed of chloroform:methanol:ammonia solution (5% [w/v]; 9:7:2 [v/v/v]). The TLC plates were soaked in potassium oxalate solution before heat activation (Munnik et al., 1994). Lipids were spotted by ATS4 automatic sampler (CAMAG). Radiolabeled spots were quantified by autoradiography using a Storm phosphorimager (Amersham Biosciences). Separated phospholipids were identified by co-migration with authentic non-labelled standards visualized by primuline staining (under UV light). For the evaluation of PLD activity, the chromatography was performed using the organic upper phase of ethyl acetate/iso-octane/acetic acid/H2O (12:2:3:10, v/v) mixture as developing solvent. [33P] phospholipids were visualized on TLC plates by autoradiography on X-ray film Retina XBM (Ukraine-Germany). Spots of equal areas were scrapped off and the radioactivity was quantified by liquid scintillation counting with SL-8 cocktail on Rack Beta 1219 counter (Wallack, Turku, Finland) (Kravets et al., 2010).

Methods concerning plant physiology

Superoxide formation was visualized in histochemical assay with Nitro Blue Tetrazolium (NBT, Sigma-Aldrich, N6876). All the reagents in the NBT staining procedure were prepared in a 100 mM Tris-HCl, pH 6.15. A 1-mL syringe with no needle was used to pressure infiltrate a spot area on one leaf half. For NBT staining, leaves were detached at 5, 60, and 120 min after infiltration and then were vacuum infiltrated with 10 mM NaN3 in 10 mM phosphate-potassium buffer pH 6.15 and immersed in 2 mL of the same buffer containing 0.1% (w/v) NBT at room temperature for 30 min. The blue precipitates of reduced NBT (formazan) were visualized after boiling the leaves in 96% (v/v) ethanol for 10 min (Sang et al., 2001b).

ROS visualization and measurement

For evaluation of ROS production, 7-day-old Arabidopsis thaliana suspension cells (Col-0) were washed and equilibrated for 3 h in agitation in Erlenmeyer flasks in assay buffer (10 mM Tris-HCl, 175 mM mannitol, pH 7.2). Then cells were transferred to 96-well plate (black, 175 μL of cells per well) and labeled with H2DCFDA (5.8 M) for 15 min. flg22 was added by dispenser in Tecan microplate reader and well fluorescence was measured in kinetics for 6h every 2 min (180 measure points). Measure parameters: sensibility: 35 (and 50: optional), wave length: emission =480 nm, reception = 528 nm Confocal fluorescence images were acquired using an IX81 inverted Olympus microscope equipped with a DSU spinning disk confocal system (Olympus France, RUNGIS, France), coupled to an Orca R2 CCD camera (Hamamatsu Corporation, Japon). Observations were performed with a 40 x objective (ph2 NA 0.6 LUCPlanFLN). Red fluorescence of chlorophyll was observed with an U-MNG2 filter offering band pass filtering of 530-550 nm for excitation and 590-800 nm for emission, while GFB green fluorescence was by a cube filter U-MWIBA3 with a band pass filtering of 460-495 nm for excitation and 510-550 for emission.
Cells images were observed by acquiring axial z stacks of pseudo-confocal images (30 μm from the base to the top, with a step of 2 μm). Image processing was executed using the ImageJ software (Schneider et al., 2012). Residual blurring were removed by spatial deconvolution: PSF were calculated using the ImageJ’s plugin PSF Generator (Richards and Wolf algorithm) (Griffa et al., 2010) and the deconvolution, properly speaking, was performed using the Richardson-Lucy algorithm implemented into the DeconvolutionLab ImageJ’s plugin (Vonesch and Unser, 2008) (15 iterations). These two last freeware programs are provided by the Biomedical Imaging Group of the EPFL (Ecole Polytechnique Fédérale de Lausanne) Switzerland.
For assay in seedlings, Arabidopsis plants were cultivated for 10 days in 14h/10h light, 22C in liquid MS media supplemented with 0.5% (w/v) sucrose and vitamins, pH 5.7, in a 96-well plate (1 seedling per well). Cultivation media was replaced by 200 μL of assay solution (50 mM TRIS-HCl, pH 8,5 buffer solution containing 70 μg.mL-1 luminol (09253 Sigma-Aldrich), 40 μg.mL-1 horseradish peroxidase and 0.1μM of flg22). Relative luminescence was measured during 1h with 2-min interval. Every well was measured separately and the data from 12 independent measurements were used in one set of experiments.

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Transcript abundance evaluation by qPCR

cDNA was synthetized by reverse transcription of 4 μg of total RNA using an oligo-dT primer and Superscript III kit (Invitrogen). Real-time PCR was performed in a Step One Plus system (Applied Biosystems) with the Power SYBR Green RT-PCR mastermix (Applied Biosystems). The amplification program was made of an initial denaturation at 95°C for 10 min, 35 cycles of amplification at 95°C for 20 sec, followed by 30 sec at Tm specific for each couple of primers and 45 sec at 72°C. The transcript abundance was standardized to that of At3g18780 and At5g11770 genes as references. A logarithm base 2 normalisation was applied to the average transcript abundance level to obtain the same data range as in transformed microarray data. The list of the primers used is given in the Table 2.1.

Statistical Analysis of Microarray Data

Experiments were designed with the statistics group of the Institute of Plant Sciences Paris-Saclay. For each array, the raw data comprised the logarithm of median feature pixel intensity at wavelengths 635 nm (red) and 532 nm (green) and no background was subtracted. An array-by-array normalization was performed to remove systematic biases. First, spots considered as badly formed features were excluded. Then a global intensity-dependent normalization using the loess procedure (Yang et al., 2002) was performed to correct the dye bias. Finally, for each block, the log-ratio median calculated over the values for the entire block was subtracted from each individual log-ratio value to correct print tip effects. Differential analysis was based on the log ratios averaged on the dye-swap. The technical replicates were averaged to get one log-ratio per biological replicate and these values were used to perform a paired t-test. A trimmed variance was calculated from spots which did not display extreme variance (Gagnot et al., 2008). The raw P-values were adjusted by the Benjamin-Hochberg method, which controls the Family Wise Error Rate in order to keep a strong control of the false positives in a multiple-comparison context. We considered as being differentially expressed the probes with a Benjamin-Hochberg P-value < 0.05.

In silico analysis of microarray data

Genes were classified using the Classification SuperViewer Tool developed by (Provart and Zhu, 2003). The classification source was set to Gene Ontology categories as defined by (Ashburner et al., 2000). The frequency of a category is normed that in the whole Arabidopsis set. The mean and standard deviation for 100 bootstraps of our input set were calculated to provide some idea as to over- or under-representation reliability. Similarity analysis were performed using tools developed by Genevestigator (Hruz et al., 2008). The ―Hierarchical clustering‖ tool works on the expression matrix defined by a microarray experiment selection and a gene selection. The similarities between expression profiles were determined using Pearson correlation for the measurement of distance. The “Biclustering” tool identifies groups of genes that are expressed above or under a set threshold ratio in a subset of conditions rather than in all conditions.
Motif analysis was carried using HOMER software (Heinz et al., 2010). Conserved motifs were searched in the promoter region 300 bp upstream of the Transcription Start Site (TSS) of genes up or down-regulated by ABA and/or SA. Basic options were used except for options: -S 5 –len 6. Top enriched de novo motif was then used as input to scan sequences for these motifs to determine their abundance among differentially ABA and/or SA regulated genes. MEME, AME (Bailey et al., 2009) and TAIR (https://www.arabidopsis.org/tools/bulk/motiffinder/) motif analysis tools were also used to strengthen the results.

Comparison of two sets of transcriptomic data

In the comparison of our ABA-regulated genes with previous SA or inhibitors transcriptome data, we first draw contingency tables. In response to either ABA, SA or to an inhibitor, genes can be up-regulated, down-regulated or unaffected by the treatment. When considering both the responses to ABA and to SA or to an inhibitor, this leads to 9 possibilities. The genes for each one of these 9 categories were counted. We can designate |A| as the number of genes in the gene set A (for instance ABA-induced genes), |B| is the number of genes in the gene set B (for instance SA-induced genes), and N is the total number of microarray probes that had a signal in the two experiments we cross. |A∩B| represents the observed number of genes common to gene set A and gene set B (i.e. genes induced by SA and ABA) while the theoretical number of genes predicted to be common for sets A and B can be calculated as (|A|/N) x (|B|/N) x N. The ratio between the observed number and the theoretical one is the representation factor (RF). To this RF can be associated P-value calculated by the hypergeometric distribution: ( ) ( )( )( )

Table of contents :

ABBREVIATIONS
INTRODUCTION
CHAPTER 1. Literature overview
CHAPTER 2 MATERIALS AND METHODS
2.1 Material used in the study
2.2. Pharmacological treatments
2.3. Methods concerning phospholipids analysis
2.3.1 Phospholipid radioactive labeling and lipid extraction
2.3.2. Thin-layer chromatography and scintillation counting
2.4. Methods concerning plant physiology
2.4.1 Histochemical assay
2.4.2. Stomatal assay
2.4.3 ROS visualization and measurement
2.4.4 Callose deposition evaluation
2.4.5 Pathogen assay
2.4.6 Root growth assay
2.5. Methods concerning gene expression analysis
2.5.1 RNA extraction
2.5.2 Transcript abundance evaluation by qPCR
2.5.3 Transcriptome Studies
2.5.4 Statistical Analysis of Microarray Data
2.5.5 In silico analysis of microarray data
2.5.6 Comparison of two sets of transcriptomic data
2.5.7 Data Deposition
2.6 Data analysis and statistics
CHAPTER 3 Involvement of phospholipase D and NADPH-oxidase in salicylic acid signaling cascade
CHAPTER 4 Basal phosphoinositide-dependent phospholipase C activity in Arabidopsis suspension cells as a signalling hub accounting for the overlap in transcriptome remodelling by abscisic or salicylic acid
CHAPTER 5 Phosphatidylinositol-dependent phospholipase C and diacylglycerol kinase are involved in primary responses to flagellin in plant cells
CHAPTER 6 The mutations of both type III phosphatidylinositol-4-phosphate kinases ß1 and ß2 isoforms lead to altered response to auxins and cytokinins in Arabidopsis roots
REFERENCES

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