QQS as a dual-mode regulator of carbon and nitrogen allocation in Arabidopsis

The Arabidopsis thaliana orphan gene QQS (Qua-Quine Starch) regulates carbon and nitrogen allocation, leading to increased protein accumulation, reduced starch content, and enhanced resistance to pests and pathogens. Although these physiological effects are well documented, the molecular basis of QQS function, specifically the relative contributions of its 59-amino-acid protein product and its transcript, remains unresolved. Here, we dissect QQS function using engineered Arabidopsis lines designed to favor either peptide-associated or transcript-associated activity. Synonymously recoded constructs were used to preserve the encoded peptide while altering RNA sequence features, whereas noATG constructs were used to disrupt canonical AUG-dependent translation initiation. Variants containing or lacking introns and untranslated regions were generated in both Columbia-0 (Col-0) and qqs knockout backgrounds and validated by RT-qPCR. Metabolic phenotyping, including iodine staining, quantitative starch assays, and total protein measurements, showed that constructs favoring either QQS peptide production or QQS transcript accumulation partially restored the high-starch, low-protein phenotype of the qqs mutant, although neither recapitulated the full metabolic effect of QQS overexpression. These results support separable contributions of the QQS transcript and its encoded microprotein to carbon and nitrogen allocation. Because construct-based approaches cannot fully exclude rare non-AUG or downstream translation initiation, especially in short ORFs, definitive confirmation of translation status will require ribosome profiling and targeted peptide detection.