RESUMO
A barley diterpene synthase (HvKSL4) was found to produce (14S)-cleistantha-8,12-diene (1). Formation of the nearly planar cyclohexa-1,4-diene configuration leaves the ring poised for aromatization, but necessitates a deceptively complicated series of rearrangements steered through a complex energetic landscape, as elucidated here through quantum chemical calculations and labeling studies.
Assuntos
Diterpenos , Folhas de PlantaRESUMO
Terpenoids play important roles in plant defense. Although some terpene synthases have been characterized, terpenoids and their biosynthesis in wheat (Triticumaestivum L.) still remain largely unknown. Here, we describe the identification of a terpene synthase gene in wheat. It encodes a sesquiterpene synthase that catalyzes ß-patchoulene formation with E,E-farnesyl diphosphate (FPP) as the substrate, thus named as TaPS. TaPS exhibits inducible expression in wheat in response to various elicitations. Particularly, alamethicin treatment strongly induces TaPS gene expression and ß-patchoulene accumulation in wheat. Overexpression of TaPS in Arabidopsis successfully produces ß-patchoulene, verifying the biochemical function of TaPS in planta. Furthermore, these transgenic Arabidopsis plants exhibit resistance against herbivory by repelling beet armyworm larvae feeding, thereby indicating anti-herbivory activity of ß-patchoulene. The catalytic mechanism of TaPS is also explored by homology modeling and site-directed mutagenesis. Two key amino acids are identified to act in protonation and stability of intermediates and product formation. Taken together, one wheat sesquiterpene synthase is identified as ß-patchoulene synthase. TaPS exhibits inducible gene expression and the sesquiterpene ß-patchoulene is involved in repelling insect infestation.
Assuntos
Alquil e Aril Transferases/genética , Arabidopsis/genética , Plantas Geneticamente Modificadas/genética , Triticum/genética , Alquil e Aril Transferases/química , Alquil e Aril Transferases/farmacologia , Animais , Arabidopsis/química , Regulação da Expressão Gênica de Plantas/genética , Helmintos/efeitos dos fármacos , Helmintos/patogenicidade , Herbivoria/efeitos dos fármacos , Herbivoria/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Sesquiterpenos de Guaiano/metabolismoRESUMO
KEY MESSAGE: CYP71Z18 exhibited plastic substrate specificity to catalyze oxidation of multiple rice diterpenes and elevated chemical defense against the blast fungus in transgenic rice. Diversified plant specialized metabolism relies on corresponding biosynthetic enzymes with differential substrate specificity. CYP71Z18 catalyzed formation of maize phytoalexins including zealexin A1, the sesquiterpenoid phytoalexin, and diterpenoid phytoalexin dolabralexin, indicating catalytic promiscuity on different terpene substrates. Here substrate specificity of CYP71Z18 was further explored through microbial metabolic engineering and it was identified to accept multiple rice diterpenes as substrates for oxidation. One CYP71Z18 enzymatic product derived from syn-pimaradiene was identified as 15,16-epoxy-syn-pimaradiene by NMR analysis, which was further elaborated by CYP99A3 to generate C19 hydroxylated product. 15,16-epoxy-syn-pimaradien-19-ol exhibited inhibitory effect on spore germination and appressorium formation of the blast pathogen Magnaporthe oryzae. Overexpression of CYP71Z18 in rice resulted in accumulation of several new diterpenoids, indicating promiscuous activity in planta. Transgenic rice also showed stronger resistance against M. oryzae infection, suggesting elevated chemical defense through changed diterpenoid metabolism by CYP71Z18 overexpression. This investigation sheds light on plant metabolic engineering using plastic substrate specificity of P450s to strengthen disease resistance and potentially provide abundant lead compounds.
