We acknowledge the ultracentrifuge technical research services from Technology Commons in College of Life Science and the Instrumentation Center sponsored by Ministry of Science and Technology, National Taiwan University (Taiwan). We also thank Yu-Ling Liang for the technical support, and the Cheng lab members for critical reading of the manuscript. This work was supported by the Young Scholar Fellowship Einstein Program from the National Science and Technology Council in Taiwan under grant nos. NSTC 113-2636-B-002-007 to M.-C.C. M.-C.C. acknowledges the financial support from National Taiwan University.

1. Heat stress-treated Arabidopsis seedling sample preparation
<ol>
	<li>Plate 250 seeds for each replicate and each condition with a dropper on the filter paper placed on the Murashige and Skoog (MS) basal media agar plate without sucrose (pH 5.6) supplemented with 1.2% phytoagar. 
	NOTE: To plant seedlings on MS plates, sterile filter paper is placed on the plate to prevent seedling roots from penetrating deep into the medium, facilitating easier sampling. Seeds are then planted on top of the filter paper. To exclude the influence of sugars on plant growth and experimental results, it is recommended to use an MS medium that does not contain sucrose for cultivation.</li>
	<li>Wrap the MS plate containing planted seeds with aluminum foil to block light and incubate it at 4 &#176;C for 2 days to induce vernalization.</li>
	<li>Transfer the vernalized seeds to a growth chamber to be grown under a 16 h: 8 h, light-dark photoperiod at 22 &#176;C with a light intensity of 100 &#181;mol/m2/s.</li>
	<li>For heat stress samples, seal the MS medium plates containing 5-day-old seedlings with waterproof adhesive tape and place them in a 40 &#176;C water bath for 1 h.</li>
	<li>For samples recovering after heat treatment, cool the plates down immediately after heat treatment. Subsequently, place them in a growth chamber at 22 &#176;C for 2 h.</li>
	<li>Harvest 250 seedlings with treated or untreated into 1.5 mL microcentrifuge tubes for each replicate and freeze the samples in liquid nitrogen immediately. 
	NOTE: For each condition, two groups of samples were prepared. One is for the polysome profiling analyses, and the other one is for the non-polysomal and polysomal RNA extraction.</li>
</ol>
2. Sucrose gradient preparation
<ol>
	<li>Prepare the gradient solution containing the following concentrations of sucrose: 12.5%, 24.4%, 36.3%, 48.1%, and 60%. The solution composition includes 50 mM Tris-HCl, 25 mM KCl, 10 mM MgCl2, 100 &#181;g/mL Heparin, and 50 &#181;g/mL cycloheximide (CHX).</li>
	<li>Load the gradient solutions from high to low concentration, starting from the bottom and moving upwards. After adding each concentration of gradient solution, freeze the solution to a solid state before adding the next concentration. For each concentration of sucrose gradient, add 2.2 mL. This process will result in a discontinuous sucrose density gradient.</li>
	<li>Store the prepared sucrose gradient temporarily at -80 &#176;C and thaw overnight at 4 &#176;C before use.</li>
</ol>
3. Polysome profiling sample preparation
<ol>
	<li>Grind 250 5-day-old Col-0 seedlings, whether heat-stressed or untreated, that have been previously frozen into powder using a homogenizer at a frequency of 30 cycles/s for 1 min. 
	NOTE: When the number of plants with the same background is equal, they have the same RNA extraction yield. However, when comparing overexpressing transgenic or mutant plants with different backgrounds, the amount of plants used should be adjusted according to the plant conditions.</li>
	<li>To obtain an extraction solution containing non-polysomal and polysomal RNA, add 500 &#181;L of polysome extraction buffer (200 mM Tris-HCl, pH 8.0, 50 mM KCl, 25 mM MgCl2, 50 &#181;g/mL cycloheximide, 100 &#181;g/mL heparin, 2% PTE, 10% DOC, 400 U/mL Ribonuclease inhibitor) to each tube, and mix the samples by inverting and vortexing. Ensure that the samples are maintained at 4 &#176;C throughout the process.</li>
	<li>Centrifuge the extraction solution at 4 &#176;C, 12,000 x g for 10 min. Then, filter the supernatant through a 100 &#181;m cell strainer to obtain a clear and particle-free extraction solution. 
	NOTE: The particles present in the extraction solution may sediment during ultracentrifugation, potentially forming a pellet that could affect the accuracy of polysome profiling measurements.</li>
	<li>Load the filtered supernatant onto a pre-prepared sucrose gradient and ensure that it reaches equilibrium. 
	NOTE: Due to the high rotational speed of the ultracentrifuge, it is essential to ensure both safety and proper functioning of the ultracentrifuge. To achieve this, it is necessary to confirm that the sample weights are identical before rotation. Therefore, we utilize a precision balance to verify that the weights of the gradients are completely uniform.</li>
	<li>Centrifuge the sucrose gradient loaded with samples at 4 &#176;C, 210,000 x g for 3.5 h using an ultracentrifuge swinging-bucket rotor. Set the acceleration and deceleration rates to the maximum. After ultracentrifugation, non-polysomal and polysomal RNA are distributed according to their density in the corresponding sucrose gradient solution. 
	NOTE: In the sucrose gradient, non-polysomal RNA with lower density distributes in the upper layer, while polysomal RNA, characterized by higher density due to the presence of numerous ribosomes engaged in active translation on mRNA, distributes in the lower layer. Subsequently, polysome profiling can be measured using a density gradient fractionator.</li>
</ol>
4. Polysome profiling analysis
NOTE: A density gradient fractionator with a microvolume syringe pump is used to measure polysome profiling.
<ol>
	<li>Prepare the chase solution (70% sucrose, 10% glycerol, and 0.02% Bromophenol blue) for the microvolume syringe pump.</li>
	<li>Use software to control the density gradient fractionator. Calibrate the machine using deionized water. After calibration, use it to perform polysome profiling.
	<ol>
		<li>To perform baseline adjustment, turn on the UV/VISDetector until the light changes from red to green. Adjust the Recorder Offset knob to align the marker line, then turn the Baseline Adjust knob to the minimum open position. Set the Sensitivity knob on the UV/VISDETECTOR to level 2.0 and press the Auto Baseline button. Finally, use the Recorder Offset knob to adjust the desired baseline to 20. The calibrated process will vary depending on the brand of mechanism and different samples.</li>
	</ol>
	</li>
	<li>After ultracentrifugation, place the tubes with the samples onto the density gradient fractionator using the tube piercing system so the tube can be connected to the syringe pump with the chasing solution and the UV detector.</li>
	<li>Switch the density gradient fractionator to software control mode (REMOTE) and set the injection flow rate of the microvolume syringe pump to 3 mL/min.</li>
	<li>Initiate the software settings to begin and obtain the polysome profile graph using the fractionator by measuring OD254.</li>
</ol>
5. Isolation of non-polysomal and polysomal RNA
NOTE: For this part of the protocol, a different group of samples from the same batch was used after performing ultracentrifugation following the same steps as described previously.
<ol>
	<li>Based on the polysome profile measurement results, collect the non-polysomal and the polysomal RNA fractions separately. Calculate the volumes of solution for both non-polysomal and polysomal fractions and collect the non-polysomal fraction (upper part). 
	NOTE: According to the profiles, the fraction with more than two ribosomes is defined as a polysomal fraction, whereas the fraction with the 40S, 60S, and 80S rRNA peaks is defined as a non-polysomal fraction.</li>
	<li>Mix the target fractions of RNA thoroughly with pre-cold 2x high salt solution (400 mM Tris-HCl, pH 8.0, 100 mM KCl, 50 mM MgCl2).</li>
	<li>Add 8 &#181;L of diluted stock of Eukaryotic Poly-A RNA Control from the kit. 
	NOTE: For the spike-in normalization required for the seventh step. The Eukaryotic Poly-A RNA Control Kit includes B. subtilis genes, such as the DAP gene which are not present in plants, as reference genes. To determine the proportion of reference gene loss during the reaction after extraction, add an equal amount of the reagent containing RNA sample of DAP gene to different tubes with non-polysomal or polysomal RNA before extraction. This allows for the determination of the proportion of reference gene loss during the reaction after extraction and can be used for normalization purposes.</li>
	<li>Centrifuge the high-salt solution mixed RNA at 4 &#176;C, 450,000 x g, for 5 h using an ultracentrifuge swinging-bucket rotor.</li>
	<li>After ultracentrifugation, RNA will precipitate at the bottom of the centrifuge tube. Carefully pour off the supernatant from above to preserve the RNA pellet at the bottom.</li>
	<li>Quickly wash the RNA pellet with 500 &#181;L of pre-cold Diethylpyrocarbonate (DEPC)-treated water, repeating this step 3x. 
	NOTE: To prevent DEPC-treated water from re-dissolving the RNA during the washing process, it is necessary to pre-cool the DEPC-treated water before use to reduce its solubility. Additionally, the contact time between the DEPC-treated water and the RNA pellet during washing should be minimized.</li>
</ol>
6. Non-polysomal and polysomal RNA extraction
<ol>
	<li>Add 500 &#181;L of RNA extraction reagent and mix with the RNA sample to be extracted. Incubate for 10 min.</li>
	<li>Add 100 &#181;L of chloroform, mix thoroughly, and incubate for 10 min for phase separation.</li>
	<li>Centrifuge the reaction mixture at 4 &#176;C, 12,000 x g for 10 min. Transfer the upper clear aqueous layer containing RNA into a new tube, mix it gently with an equal volume of isopropanol, and incubate at -20 &#176;C for 2 h for RNA precipitation.</li>
	<li>After precipitation, centrifuge at 4 &#176;C, 12,000 x g for 10 min. Carefully discard the supernatant and retain the RNA pellet at the bottom.</li>
	<li>Wash the RNA pellet with 1 mL of pre-cold 75% ethanol. Centrifuge at 4 &#176;C, 12,000 x g for 10 min, discard the supernatant, and air dry the RNA pellet.</li>
	<li>Once the RNA pellet is dried, resuspend the RNA pellet in 30 &#181;L of DEPC-treated water and measure the RNA concentration (OD260) using a full spectrum (220 nm-750 nm) spectrophotometer.</li>
</ol>
7. Spike-in normalization
<ol>
	<li>Use 1000 ng of extracted RNA for reverse transcription done using a qPCR kit following the manufacturer&#39;s instructions. Both non-polysomal and polysomal RNA need to be reverse transcribed into cDNA.</li>
	<li>Mix 1 &#181;L of cDNA with DAP gene primers and use SYBR buffer to measure the expression level of the target gene as per the manufacturer&#39;s instruction. Next, use real-time PCR to detect DAP gene expression. 
	NOTE: Sequences of the primers of DAP gene are as follows: 
	Forward primer = 5&#39;-ATA AAA GAA TTC AGC TAA CGC TTC C-3&#39; 
	Reverse primer = 5&#39;-TTG TTT CTT TGC CTC TAT TGT ATC C-3&#39;</li>
	<li>Compare the expression levels of DAP genes in the measured non-polysomal and polysomal RNA groups and estimate the RNA content under conditions of proportional loss. Using the normalized RNA content, calculate the ratio of RNA in polysomes.</li>
</ol>

Analyzing Translational Regulation in Heat-Stressed Arabidopsis

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+phosphorylation+of+plant+translation+initiation+factors+by+Arabidopsis+thaliana+CK2+holoenzymes.">Differential phosphorylation of plant translation initiation factors by Arabidopsis thaliana CK2 holoenzymes.</a> J Biol Chem. 284 (31), 20602-20614 (2009).</li><li>Dennis, M. D., Person, M. D., Browning, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+plant+translation+initiation+factors+by+CK2+enhances+the+in+vitro+interaction+of+multifactor+complex+components.">Phosphorylation of plant translation initiation factors by CK2 enhances the in vitro interaction of multifactor complex components.</a> J Biol Chem. 284 (31), 20615-20628 (2009).</li><li>Chen, G. H., Liu, M. J., Xiong, Y., Sheen, J., Wu, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TOR+and+RPS6+transmit+light+signals+to+enhance+protein+translation+in+deetiolating+Arabidopsis+seedlings.">TOR and RPS6 transmit light signals to enhance protein translation in deetiolating Arabidopsis seedlings.</a> Proc Natl Acad Sci. 115 (50), 12823-12828 (2018).</li><li>Chang, H. H., Huang, L. C., Browning, K. S., Huq, E., Cheng, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+phosphorylation+of+carboxyl-terminal+eIF2&#945;+by+SPA+kinases+contributes+to+enhanced+translation+efficiency+during+photomorphogenesis.">The phosphorylation of carboxyl-terminal eIF2&#945; by SPA kinases contributes to enhanced translation efficiency during photomorphogenesis.</a> Nat Comm. 15 (1), 3467 (2024).</li><li>Liu, M. J., Wu, S. H., Chen, H. M., Wu, S. 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E., Chodasiewicz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+stress+granules:+trends+and+beyond.">Plant stress granules: trends and beyond.</a> Front Plant Sci. 12, 722643 (2021).</li><li>Hamada, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stress+granule+formation+is+induced+by+a+threshold+temperature+rather+than+a+temperature+difference+in+Arabidopsis.">Stress granule formation is induced by a threshold temperature rather than a temperature difference in Arabidopsis.</a> J Cell Sci. 131 (16), jcs216051 (2018).</li><li>Jang, G. J., Jang, J. C., Wu, S. 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The authors declare no conflict of interest.

This protocol outlines a straightforward and standardized method for measuring the translation efficiency of Arabidopsis seedlings. The critical steps of this protocol are ensuring RNA stability with secondary centrifugation and RNA extraction reagent extraction, as well as meticulous preparation of the sucrose gradient. Moreover, we provide critical steps for normalizing and quantifying the non-polysomal and polysomal RNA with the spike-in normalization method. It is very important that all procedures should be conducte...

Translation is crucial for organisms to synthesize functional proteins from mRNA, supporting essential cellular functions and biological processes like metabolism and signaling and enabling stress responses. Without translation, cells cannot produce vital proteins, impacting their structure, function, and regulation, thereby affecting sustaining life and fostering biological diversity1,2. Therefore, studying the translational efficiency of plants is crucial. Translation involves several essential steps. First, initiation occurs as mRNA binds to a ribosome, facilitated by initiation factors such as eIFs in eukaryotes, which identify the start codon, typically AUG. Next, elongation proceeds as transfer RNA (tRNA) molecules, each carrying specific amino acids, sequentially bind to the ribosome. Peptide bonds form between adjacent amino acids, elongating the polypeptide chain according to the mRNA sequence. Finally, termination is initiated upon encountering a stop codon (UAA, UAG, or UGA), recognized by release factors that prompt the ribosome to release the newly synthesized protein. Throughout translation, various eukaryotic initiation factors (eIFs), elongation factors, and ribosomal RNAs work together to ensure accuracy and efficiency3,4.
Previous studies have indicated that post-translational modifications play a critical role in regulating interactions among eIFs and thereby influence translation efficiency. In vitro research has revealed that CASEIN KINASE 2 (CK2) kinase phosphorylates eIF3c, eIF5, and eIF2&#946; to increase their interactions with each other and with eIF15,6. In dark, the E3 ligase CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) represses translation by inhibiting TOR-mediated phosphorylation of S6K-RPS6. The non-phosphorylated RPS6 is unable to form functional ribosomes, thereby halting translation7. Conversely, under light conditions, the SUPPRESSOR OF PHYA 105 (SPA1) kinase phosphorylates eIF2&#945; to facilitate eIF2 complex assembly and promote translation initiation8. These findings highlight the complex control mechanisms that regulate translation in response to environmental signals.
Moderate environmental stimuli can effectively promote translational processes to facilitate growth, such as photomorphogenesis8,9. However, when environmental factors are excessive, immobile plants need to evolve suitable regulatory mechanisms to mitigate damage caused by environmental stress10. In previous studies related to plant stress responses, the majority focused on regulation at the metabolic, hormonal, and transcriptional levels11,12,13,14. However, recent research has begun to highlight the influence of translational regulation on plant stress tolerance15,16,17. Plants can increase their stress tolerance by reducing translational efficiency, thereby minimizing unnecessary energy consumption. Due to the formation of non-membranous stress granules in plant cells, untranslated mRNA and associated proteins aggregate within them to reduce translational efficiency18. One of the common environmental stresses that plants often encounter is heat stress, which has been reported to induce the formation of stress granules within plant cells19,20. The global increase in average temperatures due to global warming severely affects crop yields21. Therefore, studying the physiological regulation of plants under heat stress is crucial. A previous study has shown that heat treatment of wheat resulted in a decrease in polysome-bound mRNA. However, mRNAs stored in stress granules were released and re-bound to ribosomes, facilitating translation after recovery22. Additionally, previous research has compared gene expression between total mRNA and polysome-bound mRNA in submerged plants16. The results indicated that the steady-state levels of mRNA associated with abscisic acid and abiotic stress responses slightly increased following submergence. Furthermore, the amount of polysome-bound mRNAs increased significantly. These results suggest that translation regulation might play a more critical role in controlling stress tolerance in plants. Therefore, an effective polysomal RNA isolation method is crucial for studying the translatome of stress-treated samples.
In this protocol, we modified the RNA isolation method from the high-risk and voluminous phenol/chloroform extraction with LiCl precipitation method to the small-scale phenol/guanidinium thiocyanate extraction method, which requires less volume. The former method involves direct mixing with polysomal fractions, resulting in a larger experimental waste9,15,23. In contrast, this modified approach utilizes differential density principles: polysomal RNA is first mixed with a high-salt, sugar-free solution and then precipitated by ultracentrifugation. Subsequently, RNA extraction is performed using a small volume of phenol/guanidinium thiocyanate reagent. This method effectively reduces the generation of organic waste, making our experiment more environmentally friendly. Additionally, the organic solvents used have lower toxicity. These reasons led us to adjust and improve the experimental procedures accordingly. Additionally, previous methods did not provide a comprehensive protocol for calculating translation efficiencies using spike-in normalization, which is essential for more in-depth translatomic analyses.
Here, we describe polysome profiling and polysomal RNA isolation protocol for investigating translation efficiency and translatomic analyses in Arabidopsis under heat shock stress. This protocol was employed to assess translation efficiency in the Col-0 wild type under normal, heat shock, and after-recovery conditions. Polysome profiling results and the percentage of polysomal RNA revealed alterations in translation efficiency following heat stress treatment in Arabidopsis seedlings.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1.5 mL eppendorf tube</td>
			<td>Labcon</td>
			<td>3012-870-000-9</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>13.2 mL centrifuge tube</td>
			<td>Beckman Coulter</td>
			<td>331372</td>
			<td>ultracentrifugation</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Honeywell</td>
			<td>32712</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Honeywell</td>
			<td>32211</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Cycloheximide (CHX)</td>
			<td>Sigma-Aldrich</td>
			<td>SI-C7698</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate (DEPC)</td>
			<td>Sigma-Aldrich</td>
			<td>D5758</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>32221</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>GeneChip Eukaryotic Poly-A RNA Control Kit</td>
			<td>Invitrogen</td>
			<td>900433</td>
			<td>Normalization</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Honeywell</td>
			<td>15523</td>
			<td>Normalization</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma-Aldrich</td>
			<td>SI-H3149</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>HiScript III RT SuperMix for qPCR kit</td>
			<td>Vazyme</td>
			<td>R323-01</td>
			<td>Normalization</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>J.T.Baker</td>
			<td>&#160;3040-01</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>SI-M8266</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>MS basal medium</td>
			<td>Phyto</td>
			<td>M524</td>
			<td>Plant culture</td>
		</tr>
		<tr>
			<td>Peak Chart Syringe Pump</td>
			<td>Brandel</td>
			<td>SYN4007LS</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Polyoxyethylene-10-Tridecyl-Ether (PTE)</td>
			<td>Sigma-Aldrich</td>
			<td>P2393</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>RNasin</td>
			<td>Promega</td>
			<td>N251B</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Sodium deoxycholate (DOC)&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>SI-D6750</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Sigma-Aldrich</td>
			<td>S5391</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>SYBR Green Supermix</td>
			<td>Bio-Rad</td>
			<td>BP170-8882</td>
			<td>Normalization</td>
		</tr>
		<tr>
			<td>TRI reagent</td>
			<td>MRC</td>
			<td>TR118</td>
			<td>RNA extraction</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>J.T.Baker</td>
			<td>&#160;4109-06</td>
			<td>Polysome profile</td>
		</tr>
		<tr>
			<td>Ultracentrifuge</td>
			<td>Beckman Coulter</td>
			<td>Optima L-100K</td>
			<td>ultracentrifugation</td>
		</tr>
		<tr>
			<td>UV/VISDETECTOR</td>
			<td>Brandel</td>
			<td>UA-6</td>
			<td>Polysome profile</td>
		</tr>
	</tbody>
</table>

translation efficiency test using polysome profiles under heat stress

Translational control of different genes under heat stress is a critical step for plant adaptation to the environment. Assessing the translational activities of various genes can help us understand the molecular mechanisms underlying plant resilience, contributing to the development of crops with enhanced stress tolerance in the face of global climate change. This paper presents a detailed methodology for assessing translation efficiency through polysome profiling in plants exposed to heat stress. The procedure is divided into three parts: heat stress treatment for Arabidopsis, translation efficiency test using polysome profiles, and calculation of translation efficiency by isolating non-polysomal and polysomal RNA based on the profile. In the first part, Arabidopsis plants are subjected to controlled heat stress conditions to mimic environmental challenges. The treatment involves exposing the plants to high temperatures for specified durations, ensuring consistent and reproducible stress induction. This step is crucial for studying the plant's physiological and molecular responses to heat stress. The second part involves the translation efficiency test using polysome profiling. Polysomes are extracted through sucrose gradient centrifugation, which separates mRNAs based on ribosomal loading. This allows for the examination of ribosome occupancy on mRNAs, providing insights into the translational control mechanisms under stress conditions. In the third part, RNA is isolated from both polysomal and non-polysomal fractions. Spike-in RNA is used to accurately measure the amount of RNA in each fraction. The calculation of translation efficiency is performed by comparing the distribution of mRNAs across these fractions under normal and heat stress conditions. The translation activities of specific genes are further assessed by performing quantitative real-time PCR (qRT-PCR) with ribosome-associated RNA and total RNA. This methodology focuses exclusively on the effects of heat stress, providing a detailed protocol for analyzing translational regulation in plants.

The wild type of Arabidopsis, Col-0, was grown on MS medium under a 16 h:8 h light photoperiod. For control, 5-day-old seedlings were used with no heat stress treatment. The heat stress group underwent 1 h of heat treatment at 40 &#176;C in a pre-heated water bath, while the recovery group was placed at 22 &#176;C for 2 h immediately after heat treatment. By employing different heat treatment conditions and recovery conditions, we can utilize subsequent steps to measure their translational efficiency.
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Author Spotlight: Polysome Profiling Protocol for Studying Translational Regulation in Arabidopsis Under Heat Stress

The protocol presented here involves polysomal profiling to isolate translatome, mRNAs associated with ribosomes, into non-polysomal and polysomal RNAs from Arabidopsis through sucrose density gradient centrifugation. This method demonstrates the translation efficiency of heat-stressed Arabidopsis.

Translation Efficiency Test Using Polysome Profiles Under Heat Stress

Translational control of different genes under heat stress is a critical step for plant adaptation to the environment. Assessing the translational ...

translation-efficiency-test-using-polysome-profiles-under-heat-stress

Research

JoVE Journal

Biology

1.2K Views.  National Taiwan University. The protocol presented here involves polysomal profiling to isolate translatome, mRNAs associated with ribosomes, into non-polysomal and polysomal RNAs from Arabidopsis through sucrose density gradient centrifugation. This method demonstrates the translation efficiency of heat-stressed Arabidopsis.

Video: Author Spotlight: Polysome Profiling Protocol for Studying Translational Regulation in Arabidopsis Under Heat Stress

Psychophysiological Stress Assessment Using Biofeedback 

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous system, previously thought to be outside of conscious control. By letting people observe signals from their own bodies, biofeedback enables them to develop greater awareness of their physiological and psychological reactions, such as stress, and to learn to modify these reactions. Biofeedback practitioners can facilitate this process by assessing people s reactions to mildly stressful events and formulating a biofeedback-based treatment plan. During stress assessment the practitioner first records a baseline for physiological readings, and then presents the client with several mild stressors, such as a cognitive, physical and emotional stressor. Variety of stressors is presented in order to determine a person's stimulus-response specificity, or differences in each person's reaction to qualitatively different stimuli. This video will demonstrate the process of psychophysiological stress assessment using biofeedback and present general guidelines for treatment planning.

Psychophysiological Stress Assessment Using Biofeedback

In this video we will describe one method of assessing person's psychophysiological reaction to stress using biofeedback. We will also present general guidelines for treatment planning.

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous ...

Platelet Adhesion and Aggregation Under Flow using Microfluidic Flow Cells

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions. Extracellular proteins, collagen I or von Willebrand factor are deposited within the microfluidic channel using active perfusion with a pneumatic pump. The matrix proteins are then washed with buffer and blocked to prepare the microfluidic channel for platelet interactions. Whole blood labeled with fluorescent dye is perfused through the channel at various flow rates in order to achieve platelet activation and aggregation. Inhibitors of platelet aggregation can be added prior to the flow cell experiment to generate IC50 dose response data.

The platelet adhesion cascade takes place in the presence of shear flow, a factor not accounted for in conventional (static) well-plate assays. This article reports on a platelet-aggregation assay utilizing a microfluidic well-plate format to emulate physiological shear flow conditions.

Platelet aggregation occurs in response to vascular injury where the extracellular matrix below the endothelium has been exposed. The platelet adhesion ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

An Easy Method for Plant Polysome Profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

Using a Thermal Camera to Measure Heat Loss Through Bird Feather Coats

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of thermoregulation in birds across a variety of ecological circumstances. However, few studies characterize the contribution of the feathers alone to thermoregulation. Several previous studies have established methods for measuring the insulation value of animal pelts, but they require destructive sampling methods that are problematic for birds, whose feathers are not distributed evenly across the skin. More information is needed about 1) how the contribution of feathers to thermoregulation varies both across and within species and 2) how feather coats may change over space and time. Reported here is a method for rapidly and directly measuring the thermal performance of feather coats and the skin using dried whole skin specimens, without the need to destroy the skin specimen. This method isolates and measures the thermal gradient across a feather coat in a way that measurements of heat loss and metabolic cost in live birds, which use behavioral and physiological strategies to thermoregulate, cannot. The method employs a thermal camera, which allows the rapid collection of quantitative thermal data to measure heat loss from a stable source through the skin. This protocol can easily be applied to various research questions, is applicable to any avian taxa, and does not require destruction of the skin specimen. Finally, it will further the understanding of the importance of passive thermoregulation in birds by simplifying and accelerating the collection of quantitative data.

This protocol describes the quantification of heat transmission through a flat skinned avian specimen using a thermal camera and hot water bath. The method allows obtaining of quantitative, comparative data about the thermal performance of feather coats across species using dried flat skin specimens.

Feathers are essential to insulation, and therefore to the cost of thermoregulation,&#160;in birds. There is robust literature on the energetic cost of ...

Analyzing the &alpha;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. Despite the development of multiple differentiation protocols, the generation of iPSC cardiomyocytes resembling an adult-like phenotype remains challenging. One major aspect of cardiomyocytes maturation involves the formation of a well-organized sarcomere network to ensure high contraction capacity. Here, we present a super resolution-based approach for semi-quantitative analysis of the &#945;-actinin network in cardiomyocytes. Using photoactivated localization microscopy a comparison of sarcomere length and z-disc thickness of iPSC-derived cardiomyocytes and cardiac cells isolated from neonatal tissue was performed. At the same time, we demonstrate the importance of proper imaging conditions to obtain reliable data. Our results show that this method is suitable to quantitatively monitor the structural maturity of cardiac cells with high spatial resolution, enabling the detection of even subtle changes of sarcomere organization.

Analyzing the &#945;-Actinin Network in Human iPSC-Derived Cardiomyocytes Using Single Molecule Localization Microscopy

The formation of a proper sarcomere network is important for the maturation of iPSC-derived cardiomyocytes. We present a super resolution-based approach that allows for the quantitative evaluation of the structural maturation of stem cell derived cardiomyocytes, to improve culture conditions promoting cardiac development.

The maturation of iPSC-derived cardiomyocytes is a critical issue for their application in regenerative therapy, drug testing and disease modeling. ...