Two <i>AcMYB22</i> Alleles Differently Regulate Flavonoid Biosynthesis Resulting in Varied Flesh Color in Kiwifruit

Flavonoids are essential nutrient compounds in kiwifruit, yet the specific regulatory mechanism governing their biosynthesis remains poorly understood. In this study, we identified an R2R3-MYB transcription factor (TF), <i>AcMYB22</i>, associated with flavonoid biosynthesis in kiwifruit. Two alleles of <i>AcMYB22</i> were isolated: <i>AcMYB22-1</i> is exclusively present in the cultivar "Hongyang", while both <i>AcMYB22-1</i> and <i>AcMYB22-2</i> were identified in its mutant "H-16", with nine single nucleotide polymorphisms. Overexpression of <i>AcMYB22</i> in kiwifruit resulted in enhanced yellow flesh coloration with a <i>b</i>* hue value and a significant increase in flavonoid content. Moreover, transgenic kiwifruit plants of overexpressing <i>AcMYB22</i> exhibited more pronounced yellow leaves with red margins accompanied by significant increases in total flavonoid and anthocyanin levels. The expression levels of flavonoid biosynthesis genes were significantly upregulated in transgenic plants with notably higher increases in <i>AcMYB22-2</i> overexpressing plants compared to those overexpressing <i>AcMYB22-1</i>. Furthermore, yeast one-hybrid assays, electrophoretic mobility shift assays (EMSAs), and GUS activity assays confirmed that both AcMYB22-1 and AcMYB22-2 can physically bind to the promoters of <i>AcF3H</i> and <i>AcUFGT,</i> positively activating their transcription, with AcMYB22-2 exhibiting stronger activation activity than AcMYB22-1. These findings provide new insights into the regulatory mechanism of flavonoid biosynthesis in kiwifruit.