Abstract
In plants, the shikimate pathway generally occurs in plastids and leads to the biosynthesis of aromatic amino acids. Chorismate synthase (CS) catalyses the last step of the conversion of 5-enolpyruvylshikimate 3-phosphate (EPSP) to chorismate, but the role of CS in the metabolism of higher plants has not been reported. In this study, we found that PhCS, which is encoded by a single-copy gene in petunia (Petunia hybrida), contains N-terminal plastidic transit peptides and peroxisomal targeting signals. Green fluorescent protein (GFP) fusion protein assays revealed that PhCS was localized in chloroplasts and, unexpectedly, in peroxisomes. Petunia plants with reduced PhCS activity were generated through virus-induced gene silencing and further characterized. PhCS silencing resulted in reduced CS activity, severe growth retardation, abnormal flower and leaf development and reduced levels of folate and pigments, including chlorophylls, carotenoids and anthocyanins. A widely targeted metabolomics analysis showed that most primary and secondary metabolites were significantly changed in pTRV2-PhCS-treated corollas. Overall, the results revealed a clear connection between primary and specialized metabolism related to the shikimate pathway in petunia.
Highlights
Phe biosynthesis as well as in the other route of Tyr biosynthesis via anthranilate, which is catalysed by A Sα and ASβ using chorismate as the substrate
The stomata of abaxial epidermal cells infected with pTRV2-PhCS were smaller than those of cells infected with pTRV2, and in particular, the results showed that the former exhibited a decreased width (Fig. 9G,H)
The enzymatic reaction catalysed by chorismate synthase (CS) is the last step in the shikimate p athway[28], and most petunia corollas synthesize large quantities of anthocyanins, which are derived from phenylalanine[55]
Summary
Phe biosynthesis as well as in the other route of Tyr biosynthesis via anthranilate, which is catalysed by A Sα and ASβ using chorismate as the substrate. A CS from a higher plant was first reported in 1986, and the activity of the enzyme was detected in tissue extracts and chloroplast preparations from pea (Pisum sativum)[6]. A Corydalis sempervirens CS was purified and characterized from a cell suspension culture, and a CS gene from a higher plant was first cloned[26]. Surveys of other species revealed that one to three CS genes were present in higher p lants[28]. The role of CS in metabolism in higher plants has not been reported. We characterized the single-copy PhCS gene in the petunia genome and analysed its localization. An analysis using GFP fusion proteins revealed that PhCS was localized in chloroplasts and in peroxisomes. Plants with reduced PhCS activity were generated using the virus-induced gene silencing (VIGS) approach. Our findings demonstrate a clear connection between primary and specialized metabolism in petunia
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