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 sampli...

<ol>
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	<li>Jackson, R. J., Hellen, C. U., Pestova, T. V. The mechanism of eukaryotic translation initiation and principles of its regulation. Nat Rev Mol Cell Biol. 11 (2), 113-127 (2010).</li>
	<li>Dennis, M. D., Browning, K. S. Differential phosphorylation of plant translation initiation factors by Arabidopsis thaliana CK2 holoenzymes. J Biol Chem. 284 (31), 20602-20614 (2009).</li>
	<li>Dennis, M. D., Person, M. D., Browning, K. S. Phosphorylation of plant translation initiation factors by CK2 enhances the in vitro interaction of multifactor complex components. J Biol Chem. 284 (31), 20615-20628 (2009).</li>
	<li>Chen, G. H., Liu, M. J., Xiong, Y., Sheen, J., Wu, S. H. TOR and RPS6 transmit light signals to enhance protein translation in deetiolating Arabidopsis seedlings. 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. The phosphorylation of carboxyl-terminal eIF2&#945; by SPA kinases contributes to enhanced translation efficiency during photomorphogenesis. Nat Comm. 15 (1), 3467 (2024).</li>
	<li>Liu, M. J., Wu, S. H., Chen, H. M., Wu, S. H. Widespread translational control contributes to the regulation of Arabidopsis photomorphogenesis. Mol Sys Biol. 8 (1), 566 (2012).</li>
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	<li>Hamada, T. et al. Stress granule formation is induced by a threshold temperature rather than a temperature difference in Arabidopsis. J Cell Sci. 131 (16), jcs216051 (2018).</li>
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</ol>

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.
<p cla...

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

Abstract