AbstractPervasive transcription of eukaryotic genomes results in expression of long non-coding RNAs (lncRNAs) most of which are poorly conserved in evolution and appear to be non-functional. However, some lncRNAs have been shown to perform specific functions, in particular, transcription regulation. Thousands of small open reading frames (smORFs, <100 codons) located on lncRNAs potentially might be translated into peptides or microproteins. We report a comprehensive analysis of the conservation and evolutionary trajectories of lncRNAs-smORFs from the moss Physcomitrium patens across transcriptomes of 479 plant species. Although thousands of smORFs are subject to substantial purifying selection, the majority of the smORFs appear to be evolutionary young and could represent a major pool for functional innovation. Using nanopore RNA sequencing, we show that, on average, the transcriptional level of conserved smORFs is higher than that of non-conserved smORFs. Proteomic analysis confirmed translation of 82 novel species-specific smORFs. Numerous conserved smORFs containing low complexity regions (LCRs) or transmembrane domains were identified, the biological functions of a selected LCR-smORF were demonstrated experimentally. Thus, microproteins encoded by smORFs are a major, functionally diverse component of the plant proteome.