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Temperature is an important signal that informs plants about their surroundings and daily and seasonal changes. In temperate climates, temperature variation throughout the year can reach up to 40°C, and usually, it is the cold that acts as a limiting factor for successful growth and development. The cold response is a multifaceted process that affects all levels of the organization, from molecular to organismal. There is an intertwined network of transcriptional changes, cold-triggered splicing events, and unspecific stress responses.

The aim of this thesis was to investigate the role of PORCUPINE (PCP/SmE1), a component of the core splicing complex called Sm-ring, in cold signaling and its connection to co-occurring events in the model plant Arabidopsis thaliana. Despite the functional redundancy of PCP and its homolog PORCUPINE-LIKE (PCPL/SmE2), their roles diverge due to the differential gene regulation in response to temperature. We showed a correlation between the level of the PCP transcript and plant phenotype and linked PCP expression to its introns. Then, we compared the transcriptome of the knockout PCP mutant, pcp-1, to other temperature-sensitive splicing mutants and showed a pool of differential splicing events that were PCP-specific. Some of these events were linked to the core components of the cold response. We hypothesized that at least part of the pleiotropic effects of the PCP loss in A. thaliana occur due to the misregulated splicing of these genes. We also identified a plausible connection between splicing and transcription through PCP as a component of the Sm-ring and an RNA Polymerase II regulator, CDKC;2. Here we found that the loss of CDKC;2 in the pcp-1 background rescued the cold-sensitive pcp-1 phenotype and restored transcriptional kinetics to the wild-type levels. Finally, we hypothesize that a broken Sm-ring requires an appropriate attenuation of the transcription rates to perform the splicing successfully.

Taken together, the work in this thesis demonstrates the complexity of the cold response mechanisms in A. thaliana and the central role of splicing components, such as PCP, for temperature acclimatization.

Plants are prone to genome duplications and tend to preserve multiple gene copies. This is also the case for the genes encoding the Sm proteins of Arabidopsis thaliana (L). The Sm proteins are best known for their roles in RNA processing such as pre-mRNA splicing and nonsense-mediated mRNA decay. In this study, we have taken a closer look at the phylogeny and differential regulation of the SmE-coding genes found in A. thaliana, PCP/SmE1, best known for its cold-sensitive phenotype, and its paralog, PCPL/SmE2. The phylogeny of the PCP homologs in the green lineage shows that SmE duplications happened multiple times independently in different plant clades and that the duplication that gave rise to PCP and PCPL occurred only in the Brassicaceae family. Our analysis revealed that A. thaliana PCP and PCPL proteins, which only differ in two amino acids, exhibit a very high level of functional conservation and can perform the same function in the cell. However, our results indicate that PCP is the prevailing copy of the two SmE genes in A. thaliana as it is more highly expressed and that the main difference between PCP and PCPL resides in their transcriptional regulation, which is strongly linked to intronic sequences. Our results provide insight into the complex mechanisms that underlie the differentiation of the paralogous gene expression as an adaptation to stress.

Alternative splicing occurs in all eukaryotic organisms. Since the first description of multiexon genes and the splicing machinery, the field has expanded rapidly, especially in animals and yeast. However, our knowledge about splicing in plants is still quite fragmented. Though eukaryotes show some similarity in the composition and dynamics of their splicing machinery, observations of unique plant traits are only starting to emerge. For instance, plant alternative splicing is closely linked to their ability to perceive various environmental stimuli. Due to their sessile lifestyle, temperature is a central source of information, allowing plants to adjust their development to match current growth conditions. Hence, seasonal temperature fluctuations and day-night cycles can strongly influence plant morphology across developmental stages. Here we discuss available data on temperature-dependent alternative splicing in plants. Given its fragmented state, it is not always possible to fit specific observations into a coherent picture, yet it is sufficient to estimate the complexity of this field and the need for further research. Better understanding of alternative splicing as a part of plant temperature response and adaptation may also prove to be a powerful tool for both fundamental and applied sciences.

Alternative splicing (AS) occurs mostly co-transcriptionally and is essential for plants’ transcriptomic adjustments to environmental stimuli. Transcriptional processes are regulated by the dynamic phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (RNAPII) via cyclin-dependent kinases (CDKs). Our understanding of AS and transcriptional regulations comes predominantly from fungal and animal studies. Plant-specific experimental data is limited even though they exhibit distinct mechanisms, which are not reflected in established models. We report that genetic loss and chemical inhibition of the Arabidopsis CDKC;2 reduces CTD phosphorylation and attenuates the low-temperature sensitivity of various splicing mutants. Our data show that low temperatures slow transcription rates, while the loss of CDKC;2 results in faster transcription rates under low-temperature conditions, which cannot be explained by currently available models on RNAPII regulation. This underscores the complexity of RNA processing regulation in plants and highlights the necessity for in-depth plant-specific investigations to establish more accurate models. 

Temperature plays a crucial role in plant growth and development, influencing numerous physiological processes throughout the plant life cycle. Ambient temperature fluctuations can significantly affect transcriptomic adjustments, which are essential for plants to adapt to ever-changing environmental conditions. Despite the known impacts of extreme temperatures on plant physiology, there remains a knowledge gap regarding the specific effects of moderate changes in ambient temperatures on transcriptomic responses. This study employs strand-specific mRNA sequencing (RNA-seq) to assess how different splicing-related mutants respond to varying ambient temperatures, providing a valuable resource to the research community. Analysis of our time-resolved temperature-regulated alternative RNA splicing data reveals that common and exclusive use of the splicing machinery plays pivotal roles in thermoresponsive growth. Furthermore, our analyses demonstrate that moderate temperature changes are translated into widespread transcriptomic responses, including adjustments of the circadian clock and significant splicing changes in light and temperature genes. These results highlight the importance of these particular signaling pathways in adapting to new temperature regimes and suggest future experiments to study the role of alternative RNA splicing in temperature adaptation. Taken together, our results provide insights regarding the role of RNA splicing in plant responses to ambient temperature changes, highlighting the biological relevance of transcriptomic adjustments in enhancing plant resilience and adaptation to climate variability.

SIGNIFICANCE STATEMENT:

• This is the first comprehensive study on how mutants involved in multiple steps of the splicing process modulate splicing activity in response to low and high ambient temperature changes.
• We assessed early and acclimated transcriptomic responses and created a valuable resource to investigate the biological outputs.