Gross-Hardt, R. | Search bioRxiv

bioRxiv2024

The spliceosomal component GAMETOPHYTIC FACTOR 1 (GFA1) regulates a key photoperiodic switch

Plants have evolved sophisticated mechanisms to perceive and interpret daytime in order to flower at an optimal time point. Here we show that the spliceosomal component GAMETOPHYTIC FACTOR 1 (GFA1) constitutes a previously unrecognized key photoperiodic switch, that is essential for flowering in long days. We show that gfa1 hypomorphic (gfa1hyp) plants fail to initiate flowering in long-days (LD), which correlates with ectopic activation of the short-day (SD) flowering repressor ARABIDOPSIS THALIANA CENTRORADIALIS (ATC). Accordingly, flowering is restored upon inactivation of ATC in gfa1hyp mutants. A novel tissue-specific in-planta splice assay and comprehensive RNAseq profiling of gfa1hyp mutants indicate that GFA1 mediated pre-mRNA splicing is substrate specific, as previously suggested for GFA1 orthologs. Furthermore, we show that gfa1hyp mutants accumulate nonsense transcripts of the photoreceptor components PHYB and RRC1, suggesting inappropriate photoreceptor signaling as a potential cause for the ectopic activation of the SD characteristic profile in gfa1hyp. In fact, known downstream targets of the phytochrome system such as RS31, SR34a, SRp30 accumulate reduced amounts of light-dependent splice isoforms. Together, our data reveal a link between spliceosome composition and long-day flowering, based on complex transcriptional readouts in response to day length.

developmental biology↗

bioRxiv2021

A versatile mitochondria isolation- and analysis-pipeline generates 3D nano-topographies and mechano-physical surface maps of single organelles

Living eukaryotic cells typically contain large quantities of highly dynamic mitochondria, which sustain the cells energy and redox homeostasis. Growing evidence suggests that mitochondria can functionally differ among but also within cells. The extent and biological significance of mitochondrial diversity is still largely unexplored, due to technical limitations that hamper profiling of individual organelles. Previous measurements of the cells interior have shown that membrane-bound compartments respond to metabolic manipulation by changes in their surface stiffness, suggesting that mechano-physical properties are a valuable readout of mitochondrial function. We here present the establishment of a robust multi-step analysis pipeline that allows one to profile mechano-physical properties of single mitochondria at the nanoscale using Atomic Force Microscopy (AFM). Firstly, we developed a rapid cell-type specific isolation protocol (mRACE), which selectively functionalizes mitochondria with biotin, facilitating isolation by streptavidin decorated microbeads. We established the technique for human and rat cell cultures, the invertebrate Caenorhabditis elegans, and the model plant Arabidopsis thaliana. Based on this versatile tool, we detected diversity of mitochondrially associated proteins among different tissues, reflecting the trophic condition of the source material. Secondly, a rapid filtration-based mitochondria isolation protocol was established, which was combined with mRACE. Lastly, we established an AFM analysis platform, which generates 3D maps of the nano-topography and mechano-physical properties of individual mitochondria. The comparison of mitochondria with each other revealed an unprecedented diversity in their mechano-physical properties and suggests that shape is not the sole determining parameter for outer membrane stiffness. We expect our results to not only introduce a new dimension for basic mitochondrial research, but in addition to open the door for the exploitation of individual mitochondria for diagnostic characterization.

plant biology↗