GC3 codons enhance protein production in diverse GC- and AT-rich plant species.

Engineering translation holds great promise for maximizing protein yields in agriculture and biotechnology, but the diversity of plant genomes hinders predictable engineering. To identify mRNA features that broadly improve translation, we conducted a comparative translatome analysis across model plants. We found that codons with G or C at the third position (GC3) are consistently associated with higher translation efficiency. Experimental results confirmed that elevating GC3 increases both protein output and mRNA abundance, in both GC- and AT-rich species. Comparative analyses across 80 plant species, spanning a wide range of GC3 levels, show that GC3 content is positively correlated with translation efficiency. Additionally, high GC3-codon usage is conserved among endogenous high-abundance proteins, such as Rubisco small subunits and ribosomal proteins. Finally, tRNA availability likely explains why GC3 codons broadly enhance translation. Together, our results provide a simple guideline for codon optimization: increasing GC3 can enhance protein production across diverse plants.