RUBY expression alters development and metabolism in Arabidopsis.

Genetically encoded reporters are indispensable tools for monitoring gene expression, transformation efficiency, and diverse cellular processes in plants. The betalain-based RUBY reporter has recently emerged as a convenient visual marker requiring no substrates or specialized equipment, and it has been widely used for a variety of applications. However, the developmental and metabolic consequences of its expression remain unexplored. Here, we show that independent transgenic lines of Arabidopsis thaliana expressing RUBY exhibit profound alterations in growth and reproductive development. RUBY-expressing plants were significantly smaller, produced fewer leaves, flowered later, and frequently developed malformed siliques with reduced seed yield. In addition, root architecture was severely impaired, with a marked reduction in the number of lateral roots. This phenotype was not rescued by indole-3-acetic acid (IAA). Targeted metabolomic profiling revealed widespread alterations in amino acid and hormone pools, including reduced indole-3-acetic acid and gibberellin, alongside accumulation of auxin precursors. Disruption of RUBY expression by suppressing the third enzyme (glucosyltransferase) restored the RUBY-associated phenotypes to wild-type. Together, our findings show that RUBY expression in Arabidopsis thaliana is not metabolically neutral; rather, it perturbs both development and metabolism by disrupting hormone and amino acid homeostasis. These results highlight reporter-associated trade-offs and underscore the necessity of regulated expression systems, such as inducible or tissue-specific expression, to expand the utility of RUBY in plant research and biotechnology.