Molecular and Phenotypic Characterization of Telomere Repeat Binding (TRBs) Proteins in Moss: Evolutionary and Functional Perspectives

Telomere repeat binding (TRB) proteins are plant-specific proteins with a unique domain structure distinct from telomere-binding proteins in animals and yeast. While extensively studied in seed plants, their role in early-diverging plant lineages remain largely unexplored. Here, we investigate TRB proteins in a model moss, Physcomitrium patens , to assess their evolutionary conservation and functional significance. Functional analysis using single knockout mutants revealed that individual PpTRB genes are essential for normal development, with mutants exhibiting defects in the two-dimensional (protonemal) stage and, more prominently, in the formation of three-dimensional (gametophore) structures. Some double mutants displayed telomere shortening, a phenotype also observed in TRB-deficient seed plants, indicating a conserved role for TRBs in telomere maintenance. Transcriptome profiling of TRB mutants revealed altered expression of genes associated with transcriptional regulation and stimulus response in protonema. Subcellular localization studies across various plant cell types confirmed that PpTRBs, like their seed plant counterparts, localize prevalently to the plant nucleus and mutually interact. In bryophytes, TRBs form a monophyletic group that mirrors the species phylogeny, whereas in seed plants, TRBs have diversified into two distinct monophyletic groups. Our findings provide the first comprehensive characterization of TRB proteins in non-vascular plants and demonstrate their conserved roles in telomere maintenance, with additional implications for plant development and gene regulation across land plant lineages.