RDM16 Alternative Splicing and Liquid-Liquid Phase Separation Contribute to Heat Tolerance in Plants

RDM16 Alternative Splicing and Liquid-Liquid Phase Separation Contribute to Heat Tolerance in Plants
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High-temperature heat stress constitutes a significant environmental challenge that profoundly affects plant growth, development, and reproductive success. Deciphering the mechanisms underlying heat stress responses and enhancing plant thermotolerance have emerged as focal areas in contemporary plant science research. As sessile organisms, plants have developed intricate signaling networks to perceive and adapt to temperature fluctuations in their surroundings. Among these adaptive strategies, mRNA alternative splicing (AS) serves as a crucial regulatory mechanism, facilitating the generation of diverse protein isoforms that bolster plant resilience under thermal stress.

In a recent study, we unveiled a novel mechanism underlying plant heat tolerance, identifying RNA-DIRECTED DNA METHYLATION 16 (RDM16) as a key player in thermo-resilience through phase separation. Intriguingly, two distinct splicing variants of RDM16, designated as RDL and RDS, were found to perform complementary roles in modulating plant responses to heat stress.

We conducted transcriptomic sequencing on wild-type Arabidopsis thaliana C24 plants subjected to heat stress at 37°C for varying durations. Comprehensive analysis of gene expression dynamics and KEGG enrichment pinpointed 19 genes encoding small nuclear ribonucleoproteins (snRNPs), integral spliceosome components, that were significantly upregulated in response to heat stress. Among these, RDM16 emerged as a central regulator of heat tolerance. Subcellular localization assays revealed that RDM16 undergoes heat-induced relocalization, forming distinct fluorescent puncta within the nuclear compartment. High-resolution confocal laser scanning microscopy of root-tip cells in transgenic plants further demonstrated that heat exposure elevated the abundance of RDM16 granules and induced morphological irregularities in nuclear puncta, strongly supporting its role in heat-induced phase separation and adaptive response mechanisms.

Further structural analysis of RDM16 revealed that its N-terminal region harbors a low-complexity domain (LCD), designated as CC1, which is indispensable for phase separation. Predictions from the PONDR (Predictor of Natural Disordered Regions) platform identified four intrinsically disordered regions (IDRs) within RDM16, highlighting their potential roles in liquid–liquid phase separation (LLPS). Functional dissection through truncation and site-directed mutagenesis pinpointed arginine residues within the IDR1 region as critical determinants of RDM16 condensation. Heat tolerance assays conducted on transgenic plants expressing mutated arginine residues (RDM16pro:RDM16mu-GFP) exhibited markedly reduced survival rates under heat stress compared to those expressing the wild-type RDM16 (RDM16pro:RDM16-GFP). These findings underscore the pivotal role of RDM16-mediated phase condensation in enhancing plant thermotolerance.

Interestingly, we identified two mRNA isoforms of RDM16: the longer isoform (RDL) and a shorter transcript (RDS), which lacks 79 base pairs in the second exon, resulting in premature translation termination and a truncated protein. RDL and RDS are heat-inducible but perform distinct roles in modulating heat tolerance. Protein interaction assays revealed that RDL and RDS physically interact, with RDS facilitating the condensation of RDL and thereby amplifying its function in heat tolerance. Heat tolerance assays in transgenic plants overexpressing RDS (RDS transgenic lines) demonstrated enhanced thermotolerance compared to wild-type plants. However, RDS overexpression failed to restore heat tolerance in plants with mutated RDL, indicating that RDS requires functional RDL to exert its heat-tolerant effects. Further hybridization experiments combining RDL and RDS confirmed that RDS synergistically enhances the heat tolerance conferred by RDL.

 

This study provides valuable insight into how plants regulate heat tolerance through coordinating mRNA alternative splicing and protein LLPS. The discovery of RDM16’s role in heat stress response through phase separation and the identification of the functional interplay between its splicing isoforms opens new avenues for the development of heat-tolerant crops. By leveraging these findings, it may be possible to enhance the resilience of crops against the growing threat of heat stress, ultimately contributing to global food security in the face of climate change.

Based on the above results, several questions remain that require further investigation. For instance, it remains unclear whether RDM16 directly regulates the splicing of HSFA3 pre-mRNA, which will necessitate evidence from genetic studies. Additionally, although phase separation is implicated in RDM16-mediated heat tolerance, the full scope of its contribution has yet to be determined. Furthermore, the specific characteristics of the condensates formed by RDM16 in response to temperature induction is still unknown, among other unresolved questions.

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Heat Stress
Life Sciences > Biological Sciences > Plant Science > Plant Stress Responses > Heat Stress
RNA splicing
Life Sciences > Biological Sciences > Genetics and Genomics > Molecular Genetics > Gene Expression > RNA splicing
Arabidopsis Thaliana
Life Sciences > Biological Sciences > Biological Techniques > Experimental Organisms > Model Plants > Arabidopsis Thaliana

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