The shikimate pathway is crucial for the production of aromatic amino acids and various secondary plant products, including anthocyanins. Phosphoenolpyruvate (PEP) is an important source for shikimate production. The pre-chorismate part of the shikimate pathway is confined to plastids. There are three sources of PEP in plastids. PEP can be imported into the plastids from cytoplasm via the PEP/phosphate translocator (PPT), and it can also be generated in plastids via enolase (ENO) and pyruvate orthophosphate dikinase (PPDK) catalysis. A large number of anthocyanins are synthesized in the flowers of most ornamental plants in the coloring stage. However, the source of PEP, the precursor of anthocyanin synthesis, is still unknown. Herein, Petunia hybrida PhENO1, PhPPT and PhPPDK genes were identified and their expression patterns and subcellular localization of encoded proteins were analyzed. Silencing of PhENO1, PhPPT, and PhPPDK alone and co-silencing of PhENO1 and PhPPDK or PhPPT and PhPPDK did not exhibit any visible phenotypic change compared with the control, while co-silencing of PhENO1 and PhPPT resulted in the flower color change from purple to light purple. The content of PEP, shikimate, flavonoids, anthocyanins, and aromatic amino acids were all significantly decreased in PhENO1 and PhPPT co-silenced plants. Co-silencing of PhENO1 and PhPPT did not affect the expression level of key genes in anthocyanin synthesis and shikimate pathways. Furthermore, co-silencing of PhENO1, PhPPT, and PhPPDK resulted in a phenotype similar to the co-silencing of PhENO1 and PhPPT. Altogether, our study suggested that PEP used for anthocyanin synthesis is mainly provided by PhENO1 and PhPPT, rather than PhPPDK.

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