During early seedling development in darkness, plants undergo skotomorphogenesis (etiolation), a programme characterised by rapid hypocotyl elongation, the formation of an apical hook with closed cotyledons, and attenuated root growth. Exposure of etiolated seedlings to light triggers a swift switch to photomorphogenesis, enabling phototrophic growth through cotyledon opening, chlorophyll biosynthesis, and repression of hypocotyl elongation. This developmental transition is accompanied by extensive transcriptome reprogramming and widespread remodelling of alternative precursor mRNA splicing.

Work from our group and others has identified several splicing regulators that contribute to these light-dependent alternative splicing changes, uncovering a complex regulatory network with both auto- and cross-regulatory functions (Fig. 2A). Corresponding mutants exhibit defects in light-responsive alternative splicing and seedling development, highlighting the critical role of these proteins in this context.

Our current focus lies on the RS sub-family of serine/arginine-rich (SR) proteins, and we address two central questions: (i) How is specificity achieved among the four closely related RS proteins? (ii) What is the impact of phosphorylation and biomolecular condensation on splicing regulator function and light-dependent alternative splicing? (Fig. 2B). We tackle these questions through an integrated approach that combines in vitro assays to characterise the condensation behaviour of the RS proteins, in vivo experiments to resolve their subcellular localisation and alternative splicing functions, and physiological studies to assess the biological relevance of our findings for light-dependent development.

Further reading: Saile et al. (2025), Plant Physiology, 199(3), DOI: 10.1093/plphys/kiaf482

RNA can fold into highly dynamic and complex structures that encode functionality independent of the primary sequence. This inherent feature is also exploited by messenger RNAs, as exemplified by riboswitches – ligand-sensing domains within mRNAs that are employed for gene regulation. Early work on the thiamine pyrophosphate (TPP)-sensing riboswitch from plants revealed its function in alternative splicing control (Wachter et al., 2007). To investigate whether evolutionarily conserved mRNA structures play a more widespread role in plant alternative splicing, we established a bioinformatics search pipeline in collaboration with the group of Dr. Zasha Weinberg (University of Halle), followed by experimental testing of promising candidates such as the 45abc motif (Reinhardt et al., 2026) and the DEAD motif (Burgardt et al., 2026). Both motifs control alternative splicing in a structure-dependent manner and contribute to auto- and cross-regulation of the corresponding genes. Notably, overriding this regulatory mechanism by inducible expression of a DEAD-box helicase caused massive increases in cassette exon skipping and intron retention, suggesting an important role of RNA structures within the respective precursor mRNAs in alternative splicing regulation (Fig. 3). In our ongoing work, we examine the interplay between this helicase and splicing regulators in splice site choice and dissect the specific splicing-regulatory features of the DEAD-controlled helicases themselves.

In addition to intramolecular RNA structures, we investigate the potential role of RNA/DNA hybrids, so-called R-loops, in plant gene regulation as part of the 4R Research Training Group (https://4r-rtg.de/). Global profiling of R-loops by DRIP-seq (DNA:RNA immunoprecipitation followed by sequencing) alongside transcriptome-wide analysis of alternative splicing via RNA-seq will test for their co-occurrence in the context of light-triggered seedling responses. Furthermore, mutants with altered expression of R-loop-processing factors will be examined for potential changes in splicing and development.

Further reading: Reinhardt et al. (2026), The Plant Journal, DOI: 10.1111/tpj.71060

Association of trans-acting factors, such as splicing-regulatory proteins, with the precursor mRNA can alter the splicing outcome through various mechanisms – for example, by masking or promoting splice sites. The impact of splicing regulators on splice site choice can be highly dependent on their relative binding position. We previously demonstrated this for Polypyrimidine Tract Binding proteins (PTBs) from Arabidopsis thaliana: PTB binding upstream of a facultative cassette exon caused its skipping, whereas recruitment to intronic positions in close proximity but downstream of the cassette exon resulted in its inclusion (Fig. 4).

In our ongoing research, we aim to expand our understanding of the plant splicing code; specifically, how RNA/protein interactions contribute to defined alternative splicing outputs. To profile these interactions, we established an RNA editing-based approach in a transient assay (Loeser et al., 2024) and are currently extending it to stable mutant lines. Complementary transcriptome-wide data are generated by iCLIP (individual-nucleotide resolution cross-linking and immunoprecipitation).

Combining these datasets with global profiling of alternative splicing in mutants exhibiting altered levels of splicing regulators, together with splicing reporter studies to test the impact of individual cis-regulatory motifs, will reveal the functional impact of RNA/protein interactions on alternative splicing outcomes and provide deeper insight into the plant splicing code. This understanding is a prerequisite for deciphering the molecular principles underlying highly dynamic splicing outputs and for modulating splicing decisions to define their biological implications and potential impact on agriculturally relevant traits.

Further reading: Loeser et al. (2024), Plant Molecular Biology, 114(2), DOI: 10.1007/s11103-024-01414-3