heat stress treatment of arabidopsis seedlings and sucrose gradient preparation for polysome profiling and rna extraction

Analyzing Translational Regulation in Heat-Stressed Arabidopsis

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.

Author Spotlight: Polysome Profiling Protocol for Studying Translational Regulation in Arabidopsis 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.

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.

heat-stress-treatment-arabidopsis-seedlings-sucrose-gradient

Research

JoVE Journal

Biology

156 Views. 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 pla...