Csicsely, E. | Search bioRxiv

bioRxiv2025

Expression levels of the Band-7 protein FLOTILLIN modulate salt tolerance, growth and development in the moss Physcomitrium patens

The Band-7 proteins, known as FLOTILLINs (FLOT), are present at the plasma membranes of most land plants. They function in clathrin-independent endocytosis and contribute to nodule formation following symbiotic infections. This study reveals that the single FLOT variant in Physcomitrium patens is located at the thylakoid membranes in chloroplasts, serving an unanticipated function. Phenotypic analysis of knockout and overexpression lines demonstrates that PpFLOT overexpression significantly impairs the high salinity tolerance of P. patens. Additionally, liquid protonema cultures of PpFLOT-OEX lines exhibited a distinct color change due to necrotic events and developed brachycyte-like cells. These changes correlate with the strength of PpFLOT expression and do not occur when these lines are cultivated on solid medium. Our study found that PpFLOT-OEX lines display increased chlorophyll and H2O2 production. We also discovered that PpFLOT is regulated by ABA and light, and its high expression can potentially affect retrograde signaling. Metabolomics and proteomics analyses revealed changes in the pigment and lipid composition as well as differentially accumulated proteins in PpFLOT mutant lines. We also observed changes in the expression of ion-transport related genes, accumulation of lipids crucial during pathogen defense, and differentially accumulated proteins taking part in multiple metabolomic pathways. Consequently, our study suggests a novel role for chloroplastic PpFLOT in plant terrestrialization, as it is putatively involved in Ca2+ and reactive oxygen species (ROS) signaling in response to abiotic and biotic stress, along with the light-dependent regulation of chlorophyll biosynthesis.

plant biology↗

bioRxiv2023

Identification and characterization of DICER-LIKE genes and their roles in Marchantia polymorpha development and stress adaptation

DICER-LIKE (DCL) proteins have a central role in plant small RNA (sRNA) biogenesis. The Marchantia polymorpha genome encodes four DCL proteins: two DCL1 homologs, MpDCL1a and MpDCL1b, MpDCL3 and MpDCL4. While MpDCL1a, MpDCL3 and MpDCL4 show high similarities to their orthologs in Physcomitrium patens and Arabidopsis thaliana, MpDCL1b shares only a limited homology with PpDCL1b, but it is very similar, in terms of functional domains, to orthologs in Anthoceros agrestis and Salvinia cucullata. We generated Mpdclge mutant lines via the CRISPR/Cas9 system and performed comprehensive phenotypic analyses of these mutant lines, under control and salt stress conditions as well as upon exogenous naphthaleneacetic acid (NAA) and abscisic acid (ABA) treatments to gain insights into the respective MpDCL functions. While Mpdcl1age mutants display severe developmental aberrations throughout their development, no adverse phenotypic changes are detectable in Mpdcl1bge and Mpdcl4ge mutants except the development of less and smaller male sexual organs (antheridiophores) when they are cultivated under photoperiod conditions supplemented with far-red light. Mpdcl3ge mutants display rosette-shaped thallus formation and overall faster development, but are not able to form antheridiophores. The rosette-shaped thallus development of Mpdcl3ge can be reverted to a wild-type-like thallus growth upon NAA treatment. Mpdcl1bge mutants can tolerate high levels of salt, whereas Mpdcl4ge mutants show higher salt sensitivity. Moreover, Mpdcl1age and Mpdcl3ge mutants show an ABA-hypersensitive phenotype. It can be concluded that the observed phenotypic alterations, under normal or treatment conditions, are linked to the mutations in the respective MpDCLs and hence to defective or altered sRNA biogenesis pathways in M. polymorpha. In conclusion, MpDCLs and their associated sRNAs regulate development, abiotic stress and phytohormonal response in M. polymorpha.

plant biology↗