Transcriptional activation of the orphan gene QQS by a MYB–NAC–GARP triad modulates starch and protein accumulation in Arabidopsis

The Arabidopsis-specific orphan gene Qua-Quine Starch (QQS) regulates starch and protein accumulation, yet the upstream mechanisms controlling its transcription remain poorly understood. Here, we investigated transcription regulators that modulate QQS expression and associated metabolic traits. Using yeast one-hybrid screening, luciferase reporter assays, chromatin immunoprecipitation-qPCR, electrophoretic mobility shift assays, and transient expression in Nicotiana benthamiana, we identified three transcription factors, AtMYR1, AtANAC87, and AtATS, that bind distinct cis-elements within the QQS promoter and activate its transcription. AtMYR1 physically interacted with both AtANAC87 and AtATS through domain-specific interactions, and combinatorial reporter assays indicated that AtANAC87 or AtATS enhances AtMYR1-mediated activation of the QQS promoter. Overexpression of each transcription factor increased QQS transcript levels and was associated with reduced starch accumulation and increased soluble protein content in leaves, whereas loss-of-function mutants showed the opposite pattern. Double-mutant and QQS rescue analyses further support a genetic relationship in which QQS acts downstream of these transcription factors in modulating leaf starch and soluble protein accumulation. Together, these findings define a MYB-NAC-GARP regulatory module upstream of QQS and provide insight into how transcriptional regulation of an orphan gene contributes to metabolic trait variation in Arabidopsis.