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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.

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.

• Appropriate abiotic stress response is pivotal for plant survival and makes use of multiple signaling molecules and phytohormones to achieve specific and fast molecular adjustments. A multitude of studies has highlighted the role of alternative splicing in response to abiotic stress, including temperature, emphasizing the role of transcriptional regulation for stress response. Here we investigated the role of the core-splicing factor PORCUPINE (PCP) on temperature-dependent root development.
• We used marker lines and transcriptomic analyses to study the expression profiles of meristematic regulators and mitotic markers, and chemical treatments, as well as root hormone profiling to assess the effect of auxin signaling.
• The loss of PCP significantly alters RAM architecture in a temperature-dependent manner. Our results indicate that PCP modulates the expression of central meristematic regulators and is required to maintain appropriate levels of auxin in the RAM.
• We conclude that alternative pre-mRNA splicing is sensitive to moderate temperature fluctuations and contributes to root meristem maintenance, possibly through the regulation of phytohormone homeostasis and meristematic activity.

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.