Mining of Small Open Reading Frame-Encoded Peptides from Chlamydomonas nivalis without Its Reference Genome

Small open reading frame (sORF)-encoded peptides (SEPs) participate in diverse biological processes, yet their identification in nonmodel organisms is limited by the lack of high-quality reference genomes. Here, we developed an integrative proteogenomic pipeline to discover SEPs in Chlamydomonas nivalis by combining sORFs predicted from its transcriptome with those derived from the genomes of the related species, Chloromonas eustigma and Volvox carteri. Using this strategy, we identified 978 endogenous SEPs supported by transcript evidence and 1 999 candidate SEPs supported by cross-species proteogenomic evidence. SEPs predicted from transcriptomic and related-species genomic data showed broadly similar physicochemical and evolutionary features. Importantly, incorporation of related-species genomic data substantially increased identification coverage, with over 95% of SEPs identified through these databases absent from the transcriptome-derived data set. Functional analysis identified several cold-responsive candidates, including an Lhca6-homologous SEP and several intrinsically disordered region-rich SEPs. Heterologous expression of selected candidates in C. reinhardtii further showed that two SEPs enhanced cold tolerance. These results demonstrate the value of integrating transcriptomic and related-species genomic resources for SEP discovery and provide functional evidence for SEP involvement in cold adaptation.