Periodic Reporting for period 1 - MemoryRNA (MemoryRNA: the role of epitranscriptomics in thermomemory regulation)
Reporting period: 2024-09-01 to 2026-12-31
Plants possess remarkable molecular mechanisms that allow them to adapt to environmental fluctuations, including episodes of heat stress. When exposed to mild, non-lethal heat conditions, plants can “memorize” this experience and respond more effectively to future, more severe stress. This adaptive process, known as thermomemory, is crucial for survival under changing climates and contributes to long-term plant resilience and productivity. Understanding how plants establish, maintain, and recall thermomemory is therefore essential for developing crops capable of withstanding the increasing frequency of heatwaves associated with global climate change.
In recent years, chemical modifications of RNA molecules, collectively referred to as epitranscriptomic marks, have emerged as important regulators of gene expression. Among these modifications, N6-methyladenosine (m⁶A) and 5-methylcytosine (m⁵C) are particularly abundant and dynamic. They are installed by writer enzymes (methyltransferases), removed by erasers (demethylases), and recognized by reader proteins that interpret these marks to control RNA fate and function. These mechanisms influence RNA stability, translation, and localization, thereby fine-tuning the cellular response to developmental and environmental cues. Despite their recognized importance, the contribution of m⁶A and m⁵C pathways to heat stress adaptation and thermomemory has remained largely unexplored.
The overall goal of this project was to uncover the role of the plant epitranscriptome in regulating gene expression during thermomemory, using Arabidopsis thaliana as a model system. Specifically, the research aimed to:
i) Evaluate how writer, reader, and eraser proteins involved in m⁶A and m⁵C modifications influence the establishment and recovery of thermomemory through phenotypic assays of heat stress tolerance;
ii) Characterize the spatiotemporal expression patterns of selected m⁶A reader proteins in the shoot apical meristem (SAM) — a crucial region for growth regulation and developmental plasticity under stress;
iii) Combine molecular and imaging approaches, including RNA in situ hybridization and confocal laser scanning microscopy, to localize reader transcripts and protein accumulation in SAM tissues across thermomemory time points;
iv) Investigate the interaction between epitranscriptomic regulation and meristem maintenance genes to determine how RNA methylation pathways contribute to the maintenance of meristem activity under heat stress.
By integrating genetics, cell biology, and advanced microscopy, this project provides novel insights into how RNA methylation acts as a regulatory layer connecting environmental perception to gene expression plasticity. Understanding these mechanisms in Arabidopsis serves as a foundation for translating epitranscriptomic principles into crop systems, ultimately contributing to the development of heat-resilient plants and supporting global efforts toward sustainable agriculture under climate stress.
In recent years, chemical modifications of RNA molecules, collectively referred to as epitranscriptomic marks, have emerged as important regulators of gene expression. Among these modifications, N6-methyladenosine (m⁶A) and 5-methylcytosine (m⁵C) are particularly abundant and dynamic. They are installed by writer enzymes (methyltransferases), removed by erasers (demethylases), and recognized by reader proteins that interpret these marks to control RNA fate and function. These mechanisms influence RNA stability, translation, and localization, thereby fine-tuning the cellular response to developmental and environmental cues. Despite their recognized importance, the contribution of m⁶A and m⁵C pathways to heat stress adaptation and thermomemory has remained largely unexplored.
The overall goal of this project was to uncover the role of the plant epitranscriptome in regulating gene expression during thermomemory, using Arabidopsis thaliana as a model system. Specifically, the research aimed to:
i) Evaluate how writer, reader, and eraser proteins involved in m⁶A and m⁵C modifications influence the establishment and recovery of thermomemory through phenotypic assays of heat stress tolerance;
ii) Characterize the spatiotemporal expression patterns of selected m⁶A reader proteins in the shoot apical meristem (SAM) — a crucial region for growth regulation and developmental plasticity under stress;
iii) Combine molecular and imaging approaches, including RNA in situ hybridization and confocal laser scanning microscopy, to localize reader transcripts and protein accumulation in SAM tissues across thermomemory time points;
iv) Investigate the interaction between epitranscriptomic regulation and meristem maintenance genes to determine how RNA methylation pathways contribute to the maintenance of meristem activity under heat stress.
By integrating genetics, cell biology, and advanced microscopy, this project provides novel insights into how RNA methylation acts as a regulatory layer connecting environmental perception to gene expression plasticity. Understanding these mechanisms in Arabidopsis serves as a foundation for translating epitranscriptomic principles into crop systems, ultimately contributing to the development of heat-resilient plants and supporting global efforts toward sustainable agriculture under climate stress.
Experimental work during the reporting period focused on the phenotypic and molecular characterization of loss-of-function and hypomorphic mutants in genes encoding components of the m⁶A and m⁵C RNA methylation machinery, including writer, reader, and eraser proteins. These mutants were subjected to a well-established thermomemory assay, which evaluates plant survival and recovery after exposure to controlled heat priming and triggering treatments. The experimental setup enabled the distinction between plants capable of retaining thermomemory and those unable to recover after heat stress.
The phenotypic analyses revealed distinct thermo-responsive behaviors among the mutants compared with the wild-type (Col-0) plants. In particular, mutants plants exhibited variable thermomemory performance, indicating that these proteins contribute to differential regulation of recovery processes. Quantitative measurements of fresh weight and survival rates after stress supported these observations, providing a comprehensive overview of how epitranscriptomic regulators shape plant thermotolerance.
At the molecular and cellular levels, the project employed RNA in situ hybridization and confocal laser scanning microscopy to investigate the spatiotemporal expression dynamics of RNA-binding proteins and meristem maintenance genes within the shoot apical meristem (SAM). This approach allowed visualization of transcript and protein localization at different time points following heat priming and triggering. Transcripts of m⁶A reader proteins were detected in the SAM and emerging leaf primordia, showing a transient reduction shortly after priming, partial restoration within hours, and a return to control levels during recovery. These findings indicate that this expression is dynamically modulated by temperature changes and may play a role in re-establishing normal meristem function following heat exposure.
Parallel in situ analyses of the meristem marker genes revealed a temporary suppression of expression immediately after priming, followed by differential reactivation after several hours in wild-type and reader mutants. After the combined priming and triggering treatment, transcripts were undetectable in both genotypes, suggesting a general repression of meristem activity during severe stress. These results, together with the phenotypic assays, indicate that RNA methylation pathways act as regulatory layers influencing the transcriptional activity of meristem maintenance genes under stress conditions, possibly linking the epitranscriptomic control of mRNA fate with developmental resilience.
Overall, the project achieved the majority of its planned scientific deliverables and milestones for the reporting period. The phenotypic and transcript localization studies provided experimental evidence linking RNA methylation to thermomemory and heat stress adaptation, establishing a solid foundation for future mechanistic investigations. Ongoing analyses continue to identify specific molecular targets and signaling pathways affected by m⁶A and m⁵C modifications, paving the way for a deeper understanding of how epitranscriptomic mechanisms support plant thermotolerance.
The phenotypic analyses revealed distinct thermo-responsive behaviors among the mutants compared with the wild-type (Col-0) plants. In particular, mutants plants exhibited variable thermomemory performance, indicating that these proteins contribute to differential regulation of recovery processes. Quantitative measurements of fresh weight and survival rates after stress supported these observations, providing a comprehensive overview of how epitranscriptomic regulators shape plant thermotolerance.
At the molecular and cellular levels, the project employed RNA in situ hybridization and confocal laser scanning microscopy to investigate the spatiotemporal expression dynamics of RNA-binding proteins and meristem maintenance genes within the shoot apical meristem (SAM). This approach allowed visualization of transcript and protein localization at different time points following heat priming and triggering. Transcripts of m⁶A reader proteins were detected in the SAM and emerging leaf primordia, showing a transient reduction shortly after priming, partial restoration within hours, and a return to control levels during recovery. These findings indicate that this expression is dynamically modulated by temperature changes and may play a role in re-establishing normal meristem function following heat exposure.
Parallel in situ analyses of the meristem marker genes revealed a temporary suppression of expression immediately after priming, followed by differential reactivation after several hours in wild-type and reader mutants. After the combined priming and triggering treatment, transcripts were undetectable in both genotypes, suggesting a general repression of meristem activity during severe stress. These results, together with the phenotypic assays, indicate that RNA methylation pathways act as regulatory layers influencing the transcriptional activity of meristem maintenance genes under stress conditions, possibly linking the epitranscriptomic control of mRNA fate with developmental resilience.
Overall, the project achieved the majority of its planned scientific deliverables and milestones for the reporting period. The phenotypic and transcript localization studies provided experimental evidence linking RNA methylation to thermomemory and heat stress adaptation, establishing a solid foundation for future mechanistic investigations. Ongoing analyses continue to identify specific molecular targets and signaling pathways affected by m⁶A and m⁵C modifications, paving the way for a deeper understanding of how epitranscriptomic mechanisms support plant thermotolerance.
This project represents one of the first comprehensive and integrated studies combining genetic, molecular, and phenotypic analyses to investigate the role of RNA methylation in plant thermomemory. While earlier research has primarily focused on transcriptional and chromatin-based regulation of stress responses, this work highlights the importance of epitranscriptomic mechanisms—chemical modifications of RNA molecules—in fine-tuning the plant’s capacity to sense, memorize, and recover from heat stress. By focusing on the two major RNA methylation marks, N⁶-methyladenosine (m⁶A) and 5-methylcytosine (m⁵C), and their associated writer, reader, and eraser proteins, the project advances current understanding of how these regulatory systems contribute to both short-term acclimation and long-term memory of environmental stress.
The phenotypic characterization of mutants deficient in different components of the methylation machinery revealed that RNA methylation pathways have distinct and specific contributions to thermotolerance. Mutations in m⁶A writers, for instance, resulted in a pronounced reduction of heat recovery capacity, demonstrating that the deposition of methyl marks is essential for maintaining plant viability under recurrent heat exposure. Conversely, altered performance of reader mutants indicates that the interpretation of these marks is critical for executing the thermomemory program. These findings provide the first experimental evidence linking RNA modification dynamics with stress memory establishment in plants.
At the molecular and cellular levels, the project achieved progress beyond the state of the art by integrating RNA in situ hybridization and confocal laser scanning microscopy to visualize the spatiotemporal dynamics of RNA-binding proteins and meristem maintenance genes in the shoot apical meristem (SAM) during thermomemory. This approach allowed the detection of rapid, transient changes in gene expression following priming and triggering treatments, revealing that reader expression fluctuates in coordination with meristem recovery. Furthermore, the visualization of the meristem marker gene under the same conditions showed that RNA methylation pathways likely interact with developmental regulators to maintain meristem integrity under thermal stress. Together, these findings open new perspectives for understanding how post-transcriptional regulation contributes to the maintenance of developmental homeostasis in plants subjected to environmental fluctuations.
From a methodological perspective, this project also established new experimental resources that can be used by the broader scientific community. The generation and systematic phenotyping of RNA methylation mutants, combined with the design of gene-specific RNA in situ hybridization probes, constitute valuable tools for future research in plant molecular biology and epitranscriptomics. The integrated dataset produced during this project lays the groundwork for future epitranscriptomic and proteomic analyses aimed at identifying the direct mRNA targets of methylation-dependent regulation and assessing how these modifications influence mRNA stability, translation efficiency, and protein accumulation during stress recovery.
Looking forward, ongoing and planned work will extend these discoveries by applying high-throughput sequencing technologies and quantitative imaging approaches to identify specific thermomemory-associated transcripts that undergo dynamic methylation. This will provide mechanistic insights into how RNA methylation supports resilience to environmental stress. Ultimately, the project’s results and tools are expected to stimulate further research in plant epitranscriptomics, supporting both fundamental discoveries and potential applications in the development of stress-tolerant crop varieties capable of sustaining productivity in a warming climate.
The phenotypic characterization of mutants deficient in different components of the methylation machinery revealed that RNA methylation pathways have distinct and specific contributions to thermotolerance. Mutations in m⁶A writers, for instance, resulted in a pronounced reduction of heat recovery capacity, demonstrating that the deposition of methyl marks is essential for maintaining plant viability under recurrent heat exposure. Conversely, altered performance of reader mutants indicates that the interpretation of these marks is critical for executing the thermomemory program. These findings provide the first experimental evidence linking RNA modification dynamics with stress memory establishment in plants.
At the molecular and cellular levels, the project achieved progress beyond the state of the art by integrating RNA in situ hybridization and confocal laser scanning microscopy to visualize the spatiotemporal dynamics of RNA-binding proteins and meristem maintenance genes in the shoot apical meristem (SAM) during thermomemory. This approach allowed the detection of rapid, transient changes in gene expression following priming and triggering treatments, revealing that reader expression fluctuates in coordination with meristem recovery. Furthermore, the visualization of the meristem marker gene under the same conditions showed that RNA methylation pathways likely interact with developmental regulators to maintain meristem integrity under thermal stress. Together, these findings open new perspectives for understanding how post-transcriptional regulation contributes to the maintenance of developmental homeostasis in plants subjected to environmental fluctuations.
From a methodological perspective, this project also established new experimental resources that can be used by the broader scientific community. The generation and systematic phenotyping of RNA methylation mutants, combined with the design of gene-specific RNA in situ hybridization probes, constitute valuable tools for future research in plant molecular biology and epitranscriptomics. The integrated dataset produced during this project lays the groundwork for future epitranscriptomic and proteomic analyses aimed at identifying the direct mRNA targets of methylation-dependent regulation and assessing how these modifications influence mRNA stability, translation efficiency, and protein accumulation during stress recovery.
Looking forward, ongoing and planned work will extend these discoveries by applying high-throughput sequencing technologies and quantitative imaging approaches to identify specific thermomemory-associated transcripts that undergo dynamic methylation. This will provide mechanistic insights into how RNA methylation supports resilience to environmental stress. Ultimately, the project’s results and tools are expected to stimulate further research in plant epitranscriptomics, supporting both fundamental discoveries and potential applications in the development of stress-tolerant crop varieties capable of sustaining productivity in a warming climate.