Functional Validation of Transcriptionally Active Microproteins/sORFs In Planta.

Advancements in biotechnology, bioinformatics tools, and high-throughput sequencing techniques have greatly enhanced our ability to identify small but significant molecules in plants that were previously thought to be junk. Small molecules, including microproteins (miPs) and micoRNA-encoded peptides (miPEPs), play a crucial role in various aspects of plant biology, such as growth, development, stress responses, hormonal signaling, and primary and secondary metabolism. Despite their importance, many of these molecules still need to be characterized due to the challenges associated with validating them in plants. Traditional validation methods, like protein sequencing and western blotting using specific antibodies, are often expensive and labor-intensive, which limits their use in large-scale studies. To address these challenges, alternative methods like promoter-reporter lines linked to a reporter gene such as β-glucuronidase (GUS) can offer a more efficient and cost-effective solution to validate transcriptionally active small peptides/ORFs in planta. The GUS assay allows for the direct visualization of gene activity in plant tissues, simplifying the validation of candidate molecules (small peptides/ORFs) and alternative transcripts. This approach speeds up the functional characterization of novel genes and molecules, ultimately advancing plant research and biotechnology applications focused on their functional characterization.