Petunias have trumpet-shaped flowers with fused petals that form a long corolla tube and an expanded corolla limb at the front. Flower buds are elongated before anthesis and harbour a similarly elongated immature pistil. Upon anthesis, the corolla limb opens and the stigma matures. Terpene synthase 1 (TPS1) is expressed in the corolla tube and synthesizes sesquiterpenes that are required for stigma maturation after anthesis. This suggests that a volatile signal emerges from the tubes, accumulates in flower buds and is perceived by developing stigmas upon anthesis. However, a receptor for such a signal had not yet been identified.
To identify such a receptor, these researchers performed RNA sequencing with tps1 mutant and wild-type stigmas before and after anthesis. Only expression of PhKAI2ia — one of the four KAI2 paralogues in petunia — was found to be increased in tps1 stigmas. Knockdown of PhKAI2ia by RNA interference mimics the small stigma and reduced seed number phenotypes of tps1 mutants, while — on the other hand — terpenoid emission is not affected. Gas phase complementation assays showed that (−)-germacrene D (but not its enantiomer, (+)-germacrene D) can complement the defects of stigmas in tps1-mutant tubes. Limited proteolysis mass spectrometry showed conformational changes of PhKAI2ia when exposed to (−)-germacrene D (but not to its enantiomer) and displacement assays showed that hydrolysis activity of PhKAI2ia is inhibited in a (−)-germacrene-D dose-dependent way. In the presence of (−)-germacrene D (but not its enantiomer), PhKAI2ia can interact with PhMAX2; this affects PhSMAX1 degradation, which suggests that signalling components that are downstream of PhKAI2ia are related to the canonical karrikin and strigolactone signalling pathways.
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