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 &lt;i&gt;Arabidopsis&lt;/i&gt;

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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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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.

Author Spotlight: Polysome Profiling Protocol for Studying Translational Regulation in &lt;i&gt;Arabidopsis&lt;/i&gt; Under Heat Stress

Translation Efficiency Test Using Polysome Profiles Under Heat Stress

Our research investigates the translational regulation during heat stress in plants. By analyzing non-polysome-bound and polysome-bound RNA, we aim to study how plants regulate stress tolerance through translational control. So here we provide a polysome profiling and polysome RNA isolation protocol for investigating translation efficiencies in Arabidopsis under heat stress.In this protocol, we provide comprehensive and standardized methods for calculating translation efficiencies using spike-in normalization, which is essential for more in-depth translatomic analysis. Our protocol offers an easier and more efficient method for isolating polysome RNA by utilizing a smaller volume of organic buffer while achieving a higher RNA yield.

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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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.

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

Heat Stress Treatment of Arabidopsis Seedlings and Sucrose Gradient Preparation for Polysome Profiling and RNA Extraction

To begin, using a dropper, plate 250 Arabidopsis seeds for each group of conditions on filter paper placed on a Murashige and Skoog Basal Media agar plate without sucrose, supplemented with 1.2%phyto agar. Wrap the plate with aluminum foil to block light and place it in an incubator at four degrees Celsius for two days to induce vernalization. After two days, unwrap the foil and transfer the vernalized seeds to a growth chamber.Allow the plants to grow for five days. Next, seal the plate containing five-day-old seedlings with waterproof adhesive tape. Place the plate in a 40 degree Celsius water bath for one hour for heat stress treatment.Immediately after heat treatment, cool the plate down and transfer it to a growth chamber set at 22 degrees Celsius for two hours to allow recovery. Harvest 250 heat treated or untreated seedlings into 1.5 milliliter microcentrifuge tubes. Freeze the collected samples immediately in liquid nitrogen.Prepare the sucrose gradient solution with concentrations of 12.5%24.4%36.3%48.1%and 60%To prepare a discontinuous sucrose density gradient, first load 2.2 milliliters of 60%sucrose solution at the bottom of the tube. Then, place the tube in a 80 degrees Celsius freezer to freeze the sucrose solution to a solid state. After solidification, load 2.2 milliliters of each of the other sucrose concentrations in the series from the bottom to the top.Store the prepared sucrose gradient at 80 degrees Celsius until use.

Polysome Profiling and RNA Isolation for Translation Efficiency Analysis in Heat-Stressed Arabidopsis Seedlings

To begin, collect the frozen heat-stressed and untreated five-day-old Columbia-0 Arabidopsis seedlings into the liquid nitrogen container. Grind the seedlings using a homogenizer into powder for one minute. Then add 500 microliters of polysome extraction buffer to the tube.Invert and vortex the tube to mix the sample. Centrifuge the extraction solution at 12, 000G for 10 minutes at four degrees Celsius. Then, filter the supernatant through a 100-micrometer cell strainer to obtain a clear, particle-free extraction solution.Load the filtered supernatant onto the pre-prepared sucrose gradient and wait until it reaches equilibrium. Then, ultracentrifuge the gradient using a swinging bucket rotor at 210, 000G for 3.5 hours at four degrees Celsius with maximum acceleration and deceleration rates. The non-polysomal and polysomal RNA are separated based on their density in the corresponding sucrose gradient solution.Prepare the chase solution for the micro-volume syringe pump consisting of 70%sucrose, 10%glycerol, and 0.02%bromophenol blue. Mix the solution thoroughly for 30 to 40 minutes. After injecting the chasing solution into the gradient, using the software, control the density gradient fractionator.Then calibrate the machine with deionized water. After ultracentrifugation, place the tube onto the density gradient fractionator. Using the tube-piercing system, connect the tube to the syringe pump and the ultraviolet detector.Switch the density gradient fractionator to software control mode remote. Set the injection flow rate of the micro-volume syringe pump to three milliliters per minute. Initiate the process to obtain the polysome profile graph by measuring the optical density at 254 nanometers using the fractionator.Based on the polysome profile measurements, collect the non-polysomal and polysomal RNA fractions separately. Thoroughly mix the collected RNA fractions with pre-cold two-times high-salt solution. Then add eight microliters of a diluted stock of eukaryotic poly-A RNA control from the kit.Centrifuge the high-salt solution mixed RNA with a swinging bucket rotor at 450, 000G for five hours at four degrees Celsius. Carefully pour off the supernatant and wash the RNA pellet three times with 500 microliters of pre-cold DEPC-treated water. After the last wash, add 500 microliters of RNA extraction reagent to the RNA sample.Mix and incubate for 10 minutes. Add 100 microliters of chloroform and mix thoroughly. Incubate for 10 minutes to allow phase separation.Centrifuge the mixture at 12, 000G for 10 minutes at four degrees Celsius. Transfer the upper clear aqueous layer containing RNA into a new tube and mix it gently with an equal volume of isopropanol. Incubate the mixture at minus 20 degrees Celsius for two hours to precipitate the RNA.After precipitation, centrifuge again and discard the supernatant, leaving the RNA pellet at the bottom. Wash the RNA pellet with one milliliter of pre-cold 75%ethanol. Centrifuge at 12, 000G for 10 minutes at four degrees Celsius and air-dry the RNA pellet.Upon drying, resuspend the pellet in 30 microliters of DEPC-treated water. Measure the RNA concentration using a full-spectrum spectrophotometer at 220 to 750 nanometers, focusing on optical density at 260. The polysome profiling showed a distinct separation between the non-polysomal and polysomal fractions under normal conditions.Heat stress at 40 degrees Celsius significantly reduced the signal intensity of the polysomal fraction, and this effect was reversed after a two hour recovery at 22 degrees Celsius, bringing the signal back to control levels. RNA extraction results indicated that heat stress reduced the percentage of RNA in the polysomal fraction, which was restored after recovery at 22 degrees Celsius.

586 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.

Analyzing Translational Regulation in Heat-Stressed Arabidopsis

Summary