RESUMEN
Production of a volatile phenylpropene; eugenol in sweet basil is mostly associated with peltate glandular trichomes (PGTs) found aerially. Currently only one eugenol synthase (EGS), ObEGS1 which belongs to PIP family is identified from sweet basil PGTs. Reports of the presence of eugenol in roots led us to analyse other EGSs in roots. We screened for all the PIP family reductase transcripts from the RNA-Seq data. In vivo functional characterization of all the genes in E. coli showed their ability to produce eugenol and were termed as ObEGS2-8. Among all, ObEGS1 displayed highest expression in PGTs and ObEGS4 in roots. Further, eugenol was produced only in the roots of soil-grown plants, but not in roots of aseptically-grown plants. Interestingly, eugenol production could be induced in roots of aseptically-grown plants under elicitation suggesting that eugenol production might occur as a result of environmental cues in roots. The presence of ObEGS4 transcript and protein in aseptically-grown plants indicated towards post-translational modifications (PTMs) of ObEGS4. Bioinformatics analysis showed possibility of phosphorylation in ObEGS4 which was further confirmed by in vitro experiment. Our study reveals the presence of multiple eugenol synthases in sweet basil and provides new insights into their diversity and tissue specific regulation.
Asunto(s)
Eugenol/metabolismo , Ocimum basilicum/fisiología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Raíces de Plantas/enzimología , Tricomas/enzimología , Secuencia de Aminoácidos , Eugenol/química , Cromatografía de Gases y Espectrometría de Masas , Regulación Enzimológica de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Inmunohistoquímica , Ocimum basilicum/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Hojas de la Planta/química , Hojas de la Planta/enzimología , Hojas de la Planta/metabolismo , Fenómenos Fisiológicos de las Plantas , Regiones Promotoras Genéticas , Secuencias Reguladoras de Ácidos Nucleicos , Nicotiana/fisiologíaRESUMEN
Spearmint produces and stores large amounts of monoterpenes, mainly limonene and carvone, in glandular trichomes and is the major natural source of these compounds. Towards producing heterologous monoterpenes in spearmint, we first reduced the flux into the native limonene pathway by knocking down the expression of limonene synthase (MsLS) by RNAi method. The MsLS RNAi lines exhibited a huge reduction in the synthesis of limonene and carvone. Detailed GC-MS and LC-MS analysis revealed that MsLS RNAi plants also showed an increase in sesquiterpene, phytosterols, fatty acids, flavonoids, and phenolic metabolites, suggesting an interaction between the MEP, MVA shikimate and fatty acid pathways in spearmint. Three different heterologous monoterpene synthases namely, linalool synthase and myrcene synthase from Picea abies and geraniol synthase from Cananga odorata were cloned and introduced independently into the MsLS RNAi mutant background. The expression of these heterologous terpene synthases resulted mainly in production of monoterpene derivatives. Of all the introduced monoterpenes geraniol showed the maximum number of derivatives. Our results provide new insights into MEP pathway interactions and regulation and reveals the existence of mechanisms for complex metabolism of monoterpenes in spearmint.
Asunto(s)
Liasas Intramoleculares/genética , Mentha spicata/enzimología , Ingeniería Metabólica/métodos , Monoterpenos/metabolismo , Cananga/enzimología , Cananga/genética , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Mentha spicata/química , Redes y Vías Metabólicas , Picea/enzimología , Picea/genética , Proteínas de Plantas/genética , Interferencia de ARNRESUMEN
Phytohormone auxin controls various aspects of plant growth and development. The typical auxin signalling involves the degradation of canonical Aux/IAA proteins upon auxin perception releasing the auxin response factors (ARF) to activate auxin-regulated gene expression. Extensive research has been pursued in deciphering the role of canonical Aux/IAAs, however, the function of non-canonical Aux/IAA genes remains elusive. Here we identified a non-canonical Aux/IAA gene, MsIAA32 from spearmint (Mentha spicata), which lacks the TIR1-binding domain and shows its involvement in the development of peltate glandular trichomes (PGT), which are the sites for production and storage of commercially important essential oils. Using yeast two-hybrid studies, two canonical Aux/IAAs, MsIAA3, MsIAA4 and an ARF, MsARF3 were identified as the preferred binding partners of MsIAA32. Expression of a R2R3-MYB gene MsMYB36 and a cyclin gene MsCycB2-4 was altered in MsIAA32 suppressed plants indicating that these genes are possible downstream targets of MsIAA32 mediated signalling. Ectopic expression of MsIAA32 in Arabidopsis affected non-glandular trichome formation along with other auxin related developmental traits. Our findings establish the role of non-canonical Aux/IAA mediated auxin signalling in PGT development and reveal species-specific functionalization of Aux/IAAs.