Advanced Applications of Polysome Sequencing in Plant Research

Polysome sequencing (Polysome-seq) has emerged as a powerful tool for studying translational regulation in plants. By capturing mRNA fragments protected by ribosomes, this technology provides insights into gene expression at the translational level, surpassing the limitations of transcriptomic analyses alone. Below are key advanced applications of polysome sequencing in plant research, supported by specific studies and methodologies.

Unlocking Plant Translation: Overcoming Unique Challenges with Polysome Profiling

Polysome profiling in plant systems presents distinct hurdles that demand specialized methodological adjustments. The unique architecture of plant cells, including rigid cell walls, and their complex biochemistry, rich in secondary metabolites like polysaccharides and polyphenols, complicate the isolation of high-quality RNA for translation research. Despite these challenges, the core principle remains powerful: polysomes—multiple ribosomes on a single mRNA—provide a direct measure of translational activity. Sucrose density gradient centrifugation effectively separates this machinery into free mRNA, single ribosomes (often indicating suppressed translation), and polysomes (the hallmark of active protein synthesis).

Technical Adaptations for Plant-Specific Challenges

Success in plant studies hinges on customizing the protocol to account for plant-specific physiology and chemistry.

• Counteracting Interfering Metabolites: Plant tissues, primarily from higher plants, are rich in polysaccharides and polyphenols that can compromise an experiment.
  • When cells are lysed, polyphenols oxidize and form irreversible complexes with RNA, rendering it useless.
  • Polysaccharides co-purify with RNA due to similar physical properties, forming a gelatinous mess that inhibits essential enzymes.
  • To combat this, extraction buffers must be fortified with protective agents like polyvinylpyrrolidone (PVP) to bind polyphenols and 2-mercaptoethanol, thereby preventing oxidation.
• Decoding Organelle-Specific Translation: Plants possess semi-autonomous organelles—chloroplasts and mitochondria—that have their own translation systems. Researchers can exploit this by using a combination of translation inhibitors.
  • Adding cycloheximide inhibits cytoplasmic translation, while chloromycin blocks organellar translation.
  • This powerful strategy enables the parallel analysis of nuclear and organellar translation, which is crucial for understanding processes like photosynthesis.
• Broad Applicability Across Species: Through targeted optimizations, polysome profiling has been successfully applied to a wide range of plants, from the model organism Arabidopsis thaliana to crops like rice and tomato. For particularly challenging tissues, specialized extraction kits and optimized polysaccharide microarray chips have proven effective in linking cellular processes to important agricultural traits like drought resistance.

Experimental approaches to the investigation of plant organellar translation (Kwasniak-Owczarek M et al., 2024)

Advanced Application Scenarios

1. Analyzing Translation Regulation Under Abiotic Stress

Drought Response: In their study of rice drought stress, Kwasniak-Owczarek M et al., polysome analysis revealed that drought-tolerant varieties (such as Apo) maintain a higher polysome-to-monosome ratio, promoting the sustained synthesis of photosynthesis-related proteins, while sensitive varieties (IR64) exhibit significantly decreased translation efficiency.

Cold Adaptation: Rice mitochondrial translation requires pseudouridine-modified rRNA to adapt to low temperatures. Polysome sequencing revealed that deletion of the OsPUS1 gene leads to defects in ribosome biogenesis and reduced translation rate.

Heat Stress: Tian X et al., using multinuclear profiling, showed that TaMBF1c primarily affects the translation efficiency of a specific subset of genes, which are significantly enriched in "sequence-specific DNA binding" functions. This suggests that TaMBF1c may reprogram stress responses by regulating the synthesis of a class of transcription factors or other DNA-binding proteins. This study demonstrates that TaMBF1c, a key wheat heat stress protein, confers heat tolerance by regulating the translation efficiency of specific heat-responsive genes (particularly those involved in DNA binding and heat shock protein synthesis). This reveals a novel mechanism that goes beyond transcriptional regulation and operates at the level of protein synthesis, providing new targets for heat-tolerant wheat breeding.

2. Uncovering the Mechanism of Organelle Translation

Chloroplast Function Regulation: Kwasniak-Owczarek M et al. discovered that the RNA-binding protein SlRBP1 in tomatoes promotes polysome binding of photosynthesis-related mRNAs (such as psbA, encoding the D1 protein) by interacting with the translation initiation factor SleIF4A2. Silencing SlRBP1 leads to abnormal chloroplast development.

Mitochondrial Translation Initiation: Plant mitochondria lack the typical Shine-Dalgarno sequence found in bacterial mitochondria. Polysome profiling reveals that they initiate translation through protein-mRNA interactions, such as the specific binding of PPR proteins to the 5' UTR of mRNAs.

3. Identification of Translation Regulatory Elements (uORFs)

Seed Germination Regulation: Wang Z et al. discovered that the 5' UTR of the Arabidopsis ABA2 gene contains an upstream open reading frame (uORF) that represses translation of the main open reading frame (mORF), thereby regulating abscisic acid synthesis and seed dormancy. Polysome sequencing revealed that disrupting this uORF increased ABA2 translation and delayed germination.

Improving Crop Stress Tolerance: The rice OsABA2 uORF exists in two haplotypes (Hap1 and Hap2). Differences in their translation efficiency lead to varying panicle germination resistance, providing targets for breeding.

4. Integrated Epitranscriptomics

Polysome sequencing combined with m6A methylation sequencing revealed that under drought stress in rice, m6A modifications are enriched in polysome-bound mRNAs, promoting their translation efficiency and revealing a coupling mechanism between RNA modification and translation.

5. Discovering a New Function of Small RNAs in the Cellular "Translation Factory"

Using polysome sequencing technology, Yang X et al. broke through conventional wisdom. They discovered that small RNAs (such as miRNAs and some siRNAs) that exert gene silencing effects in maize and rice are not randomly distributed but are specifically enriched on membrane-bound polysomes attached to the endoplasmic reticulum. This suggests that the ER is not only a protein synthesis workshop, but also a key gene regulation center: miRNAs efficiently cleave target mRNAs here, and even some traditionally considered "non-coding" RNA precursors can be bound and processed by ribosomes here. This study reveals the existence of a sophisticated, spatially specific sRNA regulatory layer within the cell, thereby deepening our understanding of the gene silencing mechanism.

Enrichment of miRNAs on membrane-bound polysomes in maize and rice (Yang X et al., 2021)

6. Solving the Problem of Reduced Rice Yields Due to Water Conservation

Using multinucleosome sequencing and other technologies, Li W et al. discovered that water-saving treatment inhibits the activity of the rice target of rapamycin (TOR) signaling pathway, leading to a global decrease in protein translation efficiency, a key factor in the yield reduction. The study further revealed that TOR regulates translation through downstream molecular modules such as S6K and MAF1, and found that ammonium fertilizer can effectively activate TOR signaling, thereby enhancing nitrogen absorption and utilization efficiency and alleviating growth inhibition. This suggests that enhancing the TOR signaling pathway is a viable breeding strategy that could synergistically improve water and fertilizer use efficiency in rice, thereby reducing yield losses associated with water-saving cultivation.

7. Study of Intraspecific Diversity and Interspecific Divergence in Cotton

Using polysome analysis, Tian X et al. found that translational regulation exhibits greater variability and a faster evolutionary rate than transcriptional regulation. Within the same cotton species, the coefficient of variation at the translational level is greater than that at the transcriptional level, indicating that translational regulation can lead to more significant proteomic differences than transcriptional regulation. Among orthologous genes in different cotton species, the degree of divergence at the translational level (Δ value > 0) is significantly greater than that at the transcriptional level. This suggests that during cotton evolution, translational regulation has experienced more rapid divergence than transcriptional regulation, potentially contributing more to interspecies trait differences.

For the application of polysome sequencing in cancer research, please refer to "Applications of Polysome Sequencing in Cancer Research".

Navigating Challenges and Future Directions in Plant Translation Research

While powerful, plant polysome profiling faces specific technical hurdles that require careful consideration. A key limitation involves the selective analysis of organellar translation. Polysomes from chloroplasts can be contaminated by cytoplasmic complexes, necessitating optimized inhibitor cocktails for clean isolation. Furthermore, detecting translation events for low-abundance mRNAs remains challenging, even with advanced sequencing depth.

The future of the field, however, is bright and points toward greater resolution and dynamism. Two particularly promising avenues are emerging:

• The integration of single-cell polysome sequencing will finally allow researchers to dissect translational heterogeneity within complex plant tissues, revealing how individual cells contribute to overall function.
• The ongoing development of in vivo translation imaging technologies promises to move beyond static snapshots, enabling the real-time monitoring of protein synthesis in living plants.

These advancements will collectively provide a more holistic and dynamic understanding of gene regulation in plant biology.

For detailed information on polysome sequencing, please refer to Introduction to "Polysome Sequencing and Its Role in Translational Control".

For a comparison of polysome sequencing with other translation analysis techniques, please refer to "Comparing Polysome Sequencing with Other Translational Profiling Techniques".

References:

1. Kwasniak-Owczarek M, Janska H. Experimental approaches to studying translation in plant semi-autonomous organelles. J Exp Bot. 2024 Sep 11;75(17):5175-5187.
2. Wang Z, Zhang X, Zhou C, Cao X. Control of seed-to-seedling transition by an upstream open reading frame in ABSCISIC ACID DEFICIENT2. Proc Natl Acad Sci U S A. 2025 Jun 10;122(23):e2502155122.
3. Yang X, You C, Wang X, Gao L, Mo B, Liu L, Chen X. Widespread occurrence of microRNA-mediated target cleavage on membrane-bound polysomes. Genome Biol. 2021 Jan 5;22(1):15.
4. Gran P, Visscher TW, Bai B, Nijveen H, Mahboubi A, Bakermans LL, Willems LAJ, Bentsink L. Unravelling the dynamics of seed-stored mRNAs during seed priming. New Phytol. 2025 Sep;247(5):2196-2209.
5. Li W, Liu J, Li Z, Ye R, Chen W, Huang Y, Yuan Y, Zhang Y, Hu H, Zheng P, Fang Z, Tao Z, Song S, Pan R, Zhang J, Tu J, Sheen J, Du H. Mitigating growth-stress tradeoffs via elevated TOR signaling in rice. Mol Plant. 2024 Feb 5;17(2):240-257.
6. Tian X, Qin Z, Zhao Y, Wen J, Lan T, Zhang L, Wang F, Qin D, Yu K, Zhao A, Hu Z, Yao Y, Ni Z, Sun Q, De Smet I, Peng H, Xin M. Stress granule-associated TaMBF1c confers thermotolerance through regulating specific mRNA translation in wheat (Triticum aestivum). New Phytol. 2022 Feb;233(4):1719-1731.
7. Tian X, Wang R, Liu Z, Lu S, Chen X, Zhang Z, Liu F, Li H, Zhang X, Wang M. Widespread impact of transposable elements on the evolution of post-transcriptional regulation in the cotton genus Gossypium. Genome Biol. 2025 Mar 17;26(1):60.