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

228 visualizações. Para começar, usando um conta-gotas, coloque 250 sementes de Arabidopsis para cada grupo de condições em papel de filtro colocado em uma placa de ágar Murashige e Skoog Basal Media sem sacarose, suplementada com 1,2% de ágar fito. Enrole a placa com papel alumínio para bloquear a luz e coloque-a em uma incubadora a quatro graus Celsius por dois dias para induzir a vernalização. Após dois dias, desembrulhe o papel alumínio e transfira as sementes ver...

Vídeo: Tratamento por estresse térmico de mudas de Arabidopsis e preparação de gradiente de sacarose para perfil de polissomos e extração de RNA

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 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 ...

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.