This work was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences, Grant XDB27040106.

1. Sample preparation
<ol>
	<li>Harvest the control and stress treated seedlings (1 g) in an aluminum foil and flash freeze the samples in liquid nitrogen. 
	NOTE: Higher protein concentration is usually observed from two-week-old seedlings than that from mature plants. One gram of seedlings generates approximately 10 mg of protein lysate, which is enough for the MS analysis. All centrifugation steps take place at 16,000 x g in step 1.</li>
	<li>Grind frozen seedlings into a fine powder using a mortar and pestle filled with liquid nitrogen.</li>
	<li>Add 1 mL of lysis buffer ((6 M guanidine-HCl in 100 mM Tris-HCl, pH 8.5) with 10 mM Tris (2-carboxyethyl)phosphine hydrochloride (TCEP), 40 mM 2-chloroacetamide (CAA), protease inhibitor and phosphatase inhibitor cocktails) to the mortar and mix with the aid of pestle.</li>
	<li>Transfer the plant lysate to a 1.5 mL tube and heat at 95 &#176;C for 5 min.</li>
	<li>Place the tube on ice for 10 min. Sonicate the tube on ice for 10 s, pause for 10 s, and repeat thrice.</li>
	<li>Centrifuge the tube for 20 min and aliquot 150 &#181;L of plant lysate into a 1.5 mL tube.</li>
	<li>Add 600 &#181;L of 100% methanol into the tube. Vortex and spin down the tube.</li>
	<li>Add 150 &#181;L of 100% chloroform into the tube. Vortex and spin down the tube.</li>
	<li>Add 450 &#181;L of ddH2O into the tube. Vortex and centrifuge the tube for 3 min.</li>
	<li>Discard the upper aqueous layer. Add 600 &#181;L of 100% methanol into the tube. Centrifuge the tube for 3 min and then discard the solution.</li>
	<li>Wash protein pellets with 600 &#181;L of 100% methanol. Discard the solution and air dry the protein pellet.</li>
	<li>Resuspend protein pellets into 600 &#181;L of digestion buffer (12 mM sodium deoxycholate (SDC)/12 mM sodium lauroyl sarcosinate (SLS) in 100 mM Tris-HCl, pH 8.5). Sonicate the tube until the suspension is homogenized.</li>
	<li>Measure the protein concentration using a BCA kit and adjust the concentration to 4 &#181;g/&#181;L with the digestion buffer. Transfer 100 &#181;L of the lysate (400 &#181;g proteins) into a new tube.</li>
	<li>Add 292 &#181;L of 50 mM triethylammonium bicarbonate (TEAB) and 8 &#181;L of Lys-C (2.5 Unit/mL) to the tube. Incubate the tube at 37 &#176;C for 3 h.</li>
	<li>Add 100 &#181;L of 50 mM TEAB with 8 &#181;g trypsin to the tube. Incubate the tube at 37 &#176;C for 12 h.</li>
	<li>Add 25 &#181;L of 10% trifluoroacetic acid (TFA) to the tube. Vortex and centrifuge the tube for 20 min at 4 &#176;C.</li>
	<li>Transfer the supernatant into a conditioned desalting column for desalting. Dry the eluate using a vacuum centrifuge concentrator. 
	NOTE: Activate the resin with 1 mL of methanol followed by 1 mL of 0.1% TFA in 80% acetonitrile (ACN). Wash out the organic solvent with at least 3 mL of 0.1% TFA in 5% ACN. Load acidified peptide samples prepared in step 1.16 into the column and then wash the column with at least 3 mL of 0.1% TFA in 5% ACN. More washes may be needed for samples with high salt concentrations. Elute the phosphopeptides with 1 mL of 0.1% TFA in 80% ACN.</li>
</ol>
2. Tandem Mass Tag (TMT) labeling
<ol>
	<li>Resuspend the dried peptides into 100 &#181;L of 200 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), pH 8.5.</li>
	<li>Dissolve the TMT reagent of each channel (0.8 mg) in 40 &#181;L of anhydrous ACN. Vortex the TMT reagent tube for 5 min and spin it down.</li>
	<li>Transfer 40 &#181;L of TMT to the sample tube and incubate for 1 h at room temperature. 
	NOTE: In this experiment, the channel 126 to 128 were labeled with three biological replicates of plants without mannitol treatment, and the channel 129 to 131 were labeled with three biological replicates of plants with mannitol treatment.</li>
	<li>Add 8 &#181;L of 5% hydroxylamine and incubate for 15 min.</li>
	<li>Mix 6 samples in a 5 mL tube and add 14 &#181;L of 10% TFA.</li>
	<li>Add 3,876 &#181;L of 0.1% TFA to the tube. Vortex and spin down.</li>
	<li>Transfer the solution to a conditioned desalting column for desalting. Dry the eluate using an evaporator.</li>
</ol>
3. Preparation of IMAC stage tip
<ol>
	<li>Use a 16 G blunt-ended needle to penetrate a polypropylene frit disk. 
	NOTE: Hold the cutter perpendicular to the surface of the disk and roll the cutter a couple of times to make sure that the disk is completely excised. Place the disk in a Petri dish for storage. Place the needle into a 200 &#181;L pipette tip and push the frit into the tip using a plunger.</li>
	<li>Press the frit gently into the tip using a plunger to cap the end of tip.
	<ol>
		<li>Place the frit disk in tips with the same pressure, which provides better reproducibility. The reproducibility of stage tips production is important for constant back pressure, which affects the timing of the centrifuge steps and reproducibility between technical replicates.</li>
		<li>If the stage tips show high back pressure during conditioning step, discard the tips to avoid potential tip clogging when while loading samples. It could be that the disk might have been pressed too hard into position.</li>
	</ol>
	</li>
	<li>Invert Ni-NTA spin column and place on a 1.5 mL tube.</li>
	<li>Use a plunger to press the frit of Ni-NTA beads gently and push the beads into the tube. 
	NOTE: Ensure to push the frit gently to avoid the potential bead loss or adsorption on the spin column.</li>
</ol>
4. Preparation of Hp-RP stage tip
<ol>
	<li>Prepare Hp-RP stage tip as described for IMAC stage tip using a C8 disk. 
	NOTE: Do not apply large force to the disk because it may result in a densely packed frit and increase the back pressure of the stage tip. All centrifugation steps take place at 1,000 x g for 5 min in step 4.</li>
	<li>Suspend 1 mg of C18 beads in 100 &#181;L 100% methanol and pass the beads solution through the tip.</li>
	<li>Add 20 &#181;L of buffer 8 (80% ACN/20% 200 mM NH4HCO2, pH 10.0) to the stage tip and pass buffer 8 through the tip by centrifugation.</li>
	<li>Add 20 &#181;L of Buffer A (200 mM NH4HCO2) to the stage tip and pass buffer A through the tip by centrifugation. 
	NOTE: Balance the centrifuge during use and ensure that the Hp-RP stage tip is not completely dried.</li>
</ol>
5. Preparation of spin adaptor
<ol>
	<li>Use sharp end tweezers to puncture a hole at the center of the lid of a 1.5 mL tube.</li>
	<li>Insert the IMAC stage tip or Hp-RP stage tip into the hole of the spin adaptor. 
	NOTE: Ensure that the stage tip is not close to the bottom of spin adaptor.</li>
</ol>
6. Phosphopeptide enrichment using IMAC stage tip
<ol>
	<li>Suspend the 10 mg Ni-NTA beads with 400 &#181;L of loading buffer (6% acetic acid (AA), pH 3.0) and load all of the beads solution into per tip. Pass the solution through the tip by centrifugation. 
	NOTE: All centrifugation steps take place at 200 x g for 3 min in step 6 besides step 6.8.</li>
	<li>Load 100 &#181;L of 50 mM ethylenediaminetetraacetic acid (EDTA) to strip the nickel ions from IMAC stage tip and pass the solution through the tip by centrifugation.</li>
	<li>Load 100 &#181;L of loading buffer to the stage tip and pass the solution through the tip by centrifugation.</li>
	<li>Load 100 &#181;L of 50 mM FeCl3 in 6% AA to the stage tip and pass the solution through the tip by centrifugation. Fe3+ ions will chelate to IMAC stage tip.</li>
	<li>Load 100 &#181;L of loading buffer to condition the stage tip and pass the solution through the tip by centrifugation.</li>
	<li>Load 100 &#181;L of loading buffer with sample peptides prepared in step 2.7 to the stage tip and pass the solution through the tip by centrifugation. 
	NOTE: Take a new tube as a spin adaptor to collect the flow-through of sample and store the flow-through in the freezer for future analysis.</li>
	<li>Load 100 &#181;L of washing buffer (4.5% AA and 25% ACN) to the stage tip and pass the solution through the tip by centrifugation.</li>
	<li>Perform the second wash with loading buffer. Load 100 &#181;L of loading buffer to the stage tip and pass the solution through the tip using 1000 &#215; g centrifuge for 3 min. 
	NOTE: Ensure the washing buffer is replaced by loading buffer in the stage tip to eliminate the loss of phosphopeptides during elution step. Equilibrate the IMAC stage tip prior to phosphopeptide elution to ensure the concentration of ACN is below 5%.</li>
	<li>Trim the IMAC stage tip at the front with a scissors and place the trimmed IMAC stage tip inside the Hp-RP tip. Ensure that the two layers of stage tips do not touch the lid of the centrifuge.</li>
</ol>
7. Phosphopeptide fractionation using Hp-RP C18 stage tip
<ol>
	<li>Add 100 &#181;L of elution buffer (200 mM ammonium phosphate (NH4H2PO4)) to the trimmed IMAC stage tip and pass the solution through the two layers of stage tips by centrifugation. 
	NOTE: If the solution does not pass through the stage tip, increase the speed of spin down. All centrifugation steps take place at 1,000 x g for 5 min in step 7. All the Hp-RP buffers are listed in Table 1.</li>
	<li>Discard the IMAC stage tip using a tweezers and add 20 &#181;L of Buffer A to the Hp-RP stage tip and pass the buffer through the tip by centrifugation. 
	NOTE: Ensure all the elution buffer pass through the IMAC stage tip before discarding it.</li>
	<li>Add 20 &#181;L of Buffer 1 to elute fraction 1 and collect the eluate in a new tube by centrifugation. Repeat the process with Buffers 2, 3, 4, 5, 6, 7, and 8 to collect each fraction in a new tube. Dry the final eluate of each fraction using a vacuum concentrator. 
	NOTE: Prepare fresh fractionation buffers. Take 8 new tubes as the spin adaptor of each fraction. Collect the eluate of each fraction in a different spin adaptor. Phosphopeptides are not stable under basic conditions, so dry the eluates by a concentrator or acidified by 10% TFA immediately.</li>
</ol>
8. LC-MS/MS analysis and data analysis
<ol>
	<li>Add 5 &#181;L of 0.1% formic acid (FA) and analyze the sample by a mass spectrometer.</li>
	<li>Run a 90 min gradient with 6-30% buffer B (80% ACN and 0.1% FA) for each fraction.</li>
	<li>Fragment the top 10 labeled peptides using high collision energy dissociation (HCD). Complete a database search to identify phosphorylation sites.</li>
	<li>Load the raw files into the MaxQuant software, name the experiments, and set fractions and PTM. 
	NOTE: The tutorial of MaxQuant software can be found at https://www.maxquant.org/.</li>
	<li>Select reporter ion MS2 in the type of LC-MS/MS run and 6plex TMT as isobaric labels. Enable filter by PIF function and set the min PIF as 0.75.</li>
	<li>Select Acetyl (Protein N-term), Oxidation (M), and Phospho (STY) to the panel of variable modifications. Select Carbamidomethyl (C) as fixed modifications.</li>
	<li>Set digestion mode as specific, select Trypsin/P as digestion enzyme, and two missed cleavage.</li>
	<li>Set PSM false discovery rate (FDR) and protein FDR as 0.01. Set the minimum score for modified peptides as 40.</li>
	<li>Add Arabidopsis thaliana database to the panel of fasta files and run database searching.</li>
	<li>Load the txt file of Phospho (STY) sites to the Perseus software as the search is done. 
	NOTE: The detailed tutorials of Perseus software can be found at https://www.maxquant.org/perseus/.</li>
	<li>Filter out the reverse phosphorylation sites. Filter out the unlocalized phosphorylation sites using localization probability 75% as the cut-off.</li>
	<li>Use the intensities of phosphorylation sites for statistical analysis.</li>
</ol>

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</ol>

The authors declare no conflict of interest.

The dynamic range and complexity of plant proteome and phosphoproteome are still a limiting factor to depth of phosphoproteomics analyses. Despite the capability of single run LC-MS/MS analysis to identify 10,000 phosphorylation sites21,22, the coverage of the whole plant phosphoproteome is still limited. Therefore, a phosphoproteomic workflow that provides high sensitivity and superior separation efficiency is required in profiling the global view of plant signa...

High salinity, low temperature, and drought cause osmotic stresses, which is a major environmental factor that affects plant productivity1,2. Protein phosphorylation is one of the most significant post-translational modifications mediating signal perception and transduction in plant response to osmotic stress3,4,5. SNF1-related protein kinase 2s (SnRK2s) are involved in the osmotic stress signaling6. Nine of ten members of the SnRK2 family show significant activation in response to osmotic stress7,8. The snrk2.1/2/3/4/5/6/7/8/9/10 decuple (snrk2-dec) mutant having mutations in all ten SnRK2 displayed hypersensitivity to osmotic stress. In snrk2-dec mutant, the osmotic stress-induced accumulation of inositol 1,4,5-trisphosphate (IP3), abscisic acid (ABA) biosynthesis, and gene expressions are strongly reduced, highlighting the vital role of SnRK2s in osmotic stress responses6. However, it is still unclear how SnRK2s kinases regulate these biological processes. Profiling the phosphoproteomic changes in response to osmotic stress is an efficient way to bridge this gap and to delineate the osmotic stress-triggered defense mechanisms in plants.
Mass spectrometry (MS) is a powerful technique for mapping plant phosphoproteome9. Characterization of plant phosphoproteomics, however, remain a challenge due to the dynamic range of plant proteome and the complexity of plant lysate4. To overcome these challenges, we developed a universal plant phosphoproteomic workflow, which eliminates unwanted interferences such as from photosynthetic pigments and secondary metabolites, and enabling the deep coverage of plant phosphoproteome10. Several phosphopeptide enrichment methods such as immobilized metal ion affinity chromatography (IMAC) and metal oxide chromatography (MOC) have been developed for enriching phosphopeptides prior to MS analysis11,12,13,14,15,16. Acidic non-phosphopeptides co-purifying with phosphopeptides are the major interferences for phosphopeptide detection. Previously, we standardized the pH value and organic acid concentration of IMAC loading buffer to eliminate the binding of non-phosphopeptides, to obtain more than 90% enrichment specificity bypassing the pre-fractionation step11.
Sample loss in the multi-step process of phosphopeptide enrichment and fractionation hampers the sensitivity of phosphopeptide identification and the depth of phosphoproteomic coverage. Stop-and-go-extraction tips (stage tips) are pipette tips that contain small disks to cap the end of the tip, which can be incorporated with chromatography for peptide fractionation and cleaning17. Sample loss during the stage tip procedure can be minimized by avoiding sample transfer between the tubes. We have successfully implemented stage tip in Ga3+-IMAC and Fe3+-IMAC to separate low abundant multiple phosphorylated peptides from singly phosphorylated peptides, which improved the depth of human phosphoproteome15. In addition, the use of high pH reversed-phase (Hp-RP) stage tip has demonstrated the wider coverage of human membrane proteome compared to that of strong cation exchange (SCX) and strong anion exchange (SAX) chromatography18. Therefore, integrating IMAC and Hp-RP stage tip techniques can increase plant phosphoproteome coverage with simplicity, high specificity, and high throughput. We have demonstrated that this strategy identified more than 20,000 phosphorylation sites from Arabidopsis seedlings, representing an enhanced depth of plant phosphoproteome19.
Here, we report a stage tip-based phosphoproteomic protocol for phosphoproteomic profiling in Arabidopsis. This workflow was applied to study the phosphoproteomic perturbation of wild-type and snrk2-dec mutant seedlings in response to osmotic stress. The phosphoproteomic analysis revealed the phosphorylation sites implicated in kinase activation and early osmotic stress signaling. Comparative analysis of wild-type and snrk2-dec mutant phosphoproteome data leaded the discovery of a Raf-like kinase (RAF)-SnRK2 kinase cascade which plays a key role in osmore stress signaling in high plants.

<table><tbody><tr><td>1.5 mL tube</td><td>eppendorf</td><td>22431081</td><td>Protein LoBind, 1.5 mL, PCR clean, colorless, 100 tubes</td></tr><tr><td>200 &amp;micro;L pipet tip</td><td>Gilson</td><td>F1739311</td><td></td></tr><tr><td>2-chloroacetamide</td><td>Sigma-Aldrich</td><td>C0267</td><td></td></tr><tr><td>acetic acid</td><td>Sigma-Aldrich</td><td>5438080100</td><td></td></tr><tr><td>acetonitrile</td><td>Sigma-Aldrich</td><td>271004</td><td></td></tr><tr><td>ammonium hydroxide</td><td>Sigma-Aldrich</td><td>338818</td><td></td></tr><tr><td>ammonium phosphate monbasic</td><td>Sigma-Aldrich</td><td>216003</td><td></td></tr><tr><td>BCA Protein Assay Kit</td><td>Thermo Fisher Scientific</td><td>23227</td><td></td></tr><tr><td>blunt-ended needle</td><td>Hamilton</td><td>90516</td><td>Kel-F hub (KF), point style 3, gauge 16</td></tr><tr><td>C18-AQ beads</td><td>Dr. Maisch</td><td>ReproSil-Pur-C18-AQ 5 &amp;micro;m</td><td></td></tr><tr><td>C8 Empore disk</td><td>3 M</td><td>2214</td><td>47 mm</td></tr><tr><td>Centrifuge</td><td>eppendorf</td><td>22620444</td><td></td></tr><tr><td>chloroform</td><td>Sigma-Aldrich</td><td>CX1058</td><td></td></tr><tr><td>data analysis software</td><td></td><td>Perseus 1.6.2.1</td><td>https://maxquant.net/perseus/</td></tr><tr><td>ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma-Aldrich</td><td></td><td></td></tr><tr><td>formic acid</td><td>Sigma-Aldrich</td><td>5330020050</td><td></td></tr><tr><td>Frits</td><td>Agilent</td><td>12131024</td><td>Frits for SPE Cartridges</td></tr><tr><td>Guanidine hydrochloride</td><td>Sigma-Aldrich</td><td>50933</td><td></td></tr><tr><td>H2O</td><td>Sigma-Aldrich</td><td>1153334000</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Iron (III) chloride</td><td>Sigma-Aldrich</td><td>157740</td><td></td></tr><tr><td>LTQ-orbitrap</td><td>Thermo Fisher Scientific</td><td>Velos Pro</td><td></td></tr><tr><td>mass spectrometer</td><td>Thermo Fisher Scientific</td><td>LTQ-Orbitrap Velos Pro</td><td></td></tr><tr><td>methanol</td><td>Sigma-Aldrich</td><td>34860</td><td></td></tr><tr><td>nano LC</td><td>Thermo Fisher Scientific</td><td>Easy-nLC 1000</td><td></td></tr><tr><td>Ni-NTA spin column</td><td>Qiagen</td><td>31014</td><td></td></tr><tr><td>N-Lauroylsarcosine sodium salt</td><td>Sigma-Aldrich</td><td>L9150</td><td></td></tr><tr><td>plunger</td><td>Hamilton</td><td>1122-01</td><td>Plunger assembly N, RN, LT, LTN for model 1702 (25 &amp;mu;l)</td></tr><tr><td>search engine software</td><td></td><td>MaxQuant 1.5.4.1</td><td>&lt;u&gt;https://www.maxquant.org&lt;/u&gt;</td></tr><tr><td>SEP-PAK Cartridge 50 mg</td><td>Waters</td><td>WAT054960</td><td></td></tr><tr><td>sodium deoxycholate</td><td>Sigma-Aldrich</td><td>D6750</td><td></td></tr><tr><td>SpeedVac</td><td>Thermo Fisher Scientific</td><td>SPD121P</td><td></td></tr><tr><td>TMT 6-plex</td><td>Thermo Fisher Scientific</td><td>90061</td><td></td></tr><tr><td>Triethylammonium bicarbonate buffer</td><td>Sigma-Aldrich</td><td>T7408</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>91707</td><td></td></tr><tr><td>Tris(2-carboxyethyl)phosphine hydrochloride</td><td>Sigma-Aldrich</td><td>C4706</td><td></td></tr><tr><td>Trizma hydrochloride</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr></tbody></table>

phosphoproteomic strategy for profiling osmotic stress signaling in arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

To demonstrate the performance of this workflow, we exploited IMAC stage tip coupled with Hp-RP stage tip fractionation to measure the phosphoproteomic changes in wild-type and snrk2-dec mutant seedlings with or without mannitol treatment for 30 minutes. Each sample was performed in biological triplicates, and the experimental workflow is represented in Figure 1. The digested peptides (400 &#181;g) of each sample were labeled with one TMT-6plex channel, pooled and desalted. The phos...

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Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

phosphoproteomic-strategy-for-profiling-osmotic-stress-signaling

Research

JoVE Journal

Biochemistry

5.1K Views.  Carnegie Institute for Science. Hier wordt een fosfaatprotemische benadering gepresenteerd, namelijk stop and go extractietip op basis van phosfoproteom, die een hoge doorvoer en diepe dekking van Arabidopsis phosphoproteome biedt. Deze aanpak definieert het overzicht van osmotische stresssignalering in Arabidopsis.

Video: Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

A Simple Fractionated Extraction Method for the Comprehensive Analysis of Metabolites, Lipids, and Proteins from a Single Sample

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually determined by transcriptomic, proteomic, metabolomics and lipidomic measurements. In this protocol, we introduce a simple method for the reproducible extraction of metabolites, lipids and proteins from biological tissues using a single aliquot per sample. The extraction method is based on a methyl tert-butyl ether: methanol: water system for liquid: liquid partitioning of hydrophobic and polar metabolites into two immiscible phases along with the precipitation of proteins and other macromolecules as a solid pellet. This method, therefore, provides three different fractions of specific molecular composition, which are fully compatible with common high throughput 'omics' technologies such as liquid chromatography (LC) or gas chromatography (GC) coupled to mass spectrometers. Even though the method was initially developed for the analysis of different plant tissue samples, it has proved to be fully compatible for the extraction and analysis of biological samples from systems as diverse as algae, insects, and mammalian tissues and cell cultures.

A protocol for comprehensive extraction of lipids, metabolites and proteins from biological tissues using one sample is presented.

Understanding of complex biological systems requires the measurement, analysis and integration of multiple compound classes of the living cell, usually ...

A Flexible Low Cost Hydroponic System for Assessing Plant Responses to Small Molecules in Sterile Conditions

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for assessing plant responses to chemicals and other substances of interest is presented. This system is highly efficient in obtaining homogeneous and healthy seedlings of the C3 and C4 model species Arabidopsis thaliana and Setaria viridis, respectively. The sterile cultivation avoids algae and microorganism contamination, which are known limiting factors for plant normal growth and development in hydroponics. In addition, this system is scalable, enabling the harvest of plant material on a large scale with minor mechanical damage, as well as the harvest of individual parts of a plant if desired. A detailed protocol demonstrating that this system has an easy and low-cost assembly, as it uses pipette racks as the main platform for growing plants, is provided. The feasibility of this system was validated using Arabidopsis seedlings to assess the effect of the drug AZD-8055, a chemical inhibitor of the target of rapamycin (TOR) kinase. TOR inhibition was efficiently detected as early as 30 min after an AZD-8055 treatment in roots and shoots. Furthermore, AZD-8055-treated plants displayed the expected starch-excess phenotype. We proposed this hydroponic system as an ideal method for plant researchers aiming to monitor the action of plant inducers or inhibitors, as well as to assess metabolic fluxes using isotope-labeling compounds which, in general, requires the use of expensive reagents.

A simple, versatile, and low-cost in vitro hydroponic system was successfully optimized, enabling large-scale experiments under sterile conditions. This system facilitates the application of chemicals in a solution and their efficient absorption by roots for molecular, biochemical, and physiological studies.

A wide range of studies in plant biology are performed using hydroponic cultures. In this work, an in vitro hydroponic growth system designed for ...

Environment

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Developmental Biology

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure the strength of immune activation in Arabidopsis thaliana following treatment with elicitor molecules. Presented first is a method for capturing the rapidly-induced and dynamic oxidative burst, which can be monitored using a luminol-based assay. Presented second is a method describing how to measure immune-induced inhibition of seedling growth. These protocols are fast and reliable, do not require specialized training or equipment, and are widely used to understand the genetic basis of plant immunity.

Pattern-Triggered Oxidative Burst and Seedling Growth Inhibition Assays in Arabidopsis thaliana

This paper describes two methods for quantifying defense responses in Arabidopsis thaliana following exposure to immune elicitors: the transient oxidative burst, and the inhibition of seedling growth.

Plants have evolved a robust immune system to perceive pathogens and protect against disease. This paper describes two assays that can be used to measure ...

Immunology and Infection

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Biology

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to ~3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with 35S radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.

Here we describe a new method to study protein import into isolated chloroplasts under stress. The method is rapid and straightforward, and can be applied to study the consequences of different stress conditions for chloroplast protein import, and the corresponding regulatory mechanisms.

Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

Potato Resilience Analysis Under Climate-Related Stresses

High Throughput Image-Based Phenotyping for Determining Morphological and Physiological Responses to Single and Combined Stresses in Potato

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions over time. By using multiple imaging sensors, many traits of interest can be assessed, including plant biomass, photosynthetic efficiency, canopy temperature, and leaf reflectance indices. Plants are frequently exposed to multiple stresses under field conditions where severe heat waves, flooding, and drought events seriously threaten crop productivity. When stresses coincide, resulting effects on plants can be distinct due to synergistic or antagonistic interactions. To elucidate how potato plants respond to single and combined stresses that resemble naturally occurring stress scenarios, five different treatments were imposed on a selected potato cultivar (Solanum tuberosum L., cv. Lady Rosetta) at the onset of tuberization, i.e. control, drought, heat, waterlogging, and combinations of heat, drought, and waterlogging stresses. Our analysis shows that waterlogging stress had the most detrimental effect on plant performance, leading to fast and drastic physiological responses related to stomatal closure, including a reduction in the quantum yield and efficiency of photosystem II and an increase in canopy temperature and water index. Under heat and combined stress treatments, the relative growth rate was reduced in the early phase of stress. Under drought and combined stresses, plant volume and photosynthetic performance dropped with an increased temperature and stomata closure in the late phase of stress. The combination of optimized stress treatment under defined environmental conditions together with selected phenotyping protocols allowed to reveal&#160;the dynamics of morphological and physiological responses to single and combined stresses. Here, a useful tool is presented for plant researchers looking to identify plant traits indicative of resilience to several climate change-related stresses.

Author Spotlight: Unraveling Plant Responses to Abiotic Stresses Using the PlantScreen Robotic Platform

We designed an image-based phenotyping protocol to determine the morphological and physiological responses to single and combined heat, drought, and waterlogging treatments. This approach enabled the identification of early, late, and recovery responses at a whole plant level, particularly above-ground parts, and highlighted the necessity of using multiple imaging sensors.

High throughput image-based phenotyping is a powerful tool to non-invasively determine the development and performance of plants under specific conditions ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

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