The shikimate pathway is critical for the biosynthesis of aromatic amino acids and a diverse array of secondary metabolites in plants, including anthocyanins. Erythrose-4-phosphate (E4P) serves as a crucial precursor in the shikimate pathway. Transaldolase (TA) and transketolase (TK) are two pivotal enzymes involved in E4P synthesis in plants through the oxidative pentose phosphate pathway (OPPP) and Calvin cycle pathways. During the coloring stage of flowers, a large number of anthocyanins accumulate. However, the source of E4P required for anthocyanin accumulation is still unknown. In this study, we characterized the TA and TK family members in petunia (Petunia hybrida), an important ornamental plant. Virus-induced gene silencing (VIGS) and RNAi techniques indicated that PhTA1 or PhTA2 silencing did not lead to visible phenotype change in petunia, while cosilencing of PhTK1-TK2 resulted in significantly lighter colors in flowers and leaves. The levels of anthocyanins, chlorophyll, E4P, flavonoids, and three aromatic amino acids all significantly decreased in PhTK1-TK2-silenced plants compared with the control. Additionally, cosilencing of PhTK1 and PhTK2 disrupted the flavonoid metabolome profile in petunia flowers. In summary, PhTK1 and PhTK2 provide the primary E4P source for anthocyanin biosynthesis.

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