RESUMEN
Helianthus annuus (sunflower) displays non-glandular trichomes (NGT), capitate glandular trichomes (CGT), and linear glandular trichomes (LGT), which reveal different chemical compositions and locations in different plant tissues. With matrix-assisted laser desorption/ionization (MALDI) and laser desorption/ionization (LDI) mass spectrometry imaging (MSI) techniques, efficient methods were developed to analyze the tissue distribution of secondary metabolites (flavonoids and sesquiterpenes) and proteins inside of trichomes. Herein, we analyzed sesquiterpene lactones, present in CGT, from leaf transversal sections using the matrix 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) (mixture 1:1) with sodium ions added to increase the ionization in positive ion mode. The results observed for sesquiterpenes and polymethoxylated flavones from LGT were similar. However, upon desiccation, LGT changed their shape in the ionization source, complicating analyses by MSI mainly after matrix application. An alternative method could be applied to LGT regions by employing LDI (without matrix) in negative ion mode. The polymethoxylated flavones were easily ionized by LDI, producing images with higher resolution, but the sesquiterpenes were not observed in spectra. Thus, the application and viability of MALDI imaging for the analyses of protein and secondary metabolites inside trichomes were confirmed, highlighting the importance of optimization parameters.
Asunto(s)
Flavonoides/aislamiento & purificación , Helianthus/química , Lactonas/aislamiento & purificación , Hojas de la Planta/química , Sesquiterpenos/aislamiento & purificación , Tricomas/química , Ácidos Cumáricos/química , Gentisatos/química , Helianthus/metabolismo , Humanos , Hojas de la Planta/metabolismo , Metabolismo Secundario , Solventes/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tricomas/metabolismoRESUMEN
Sunflower is known to produce a variety of bisabolene-type sesquiterpenes and accumulates these substances in trichomes of leaves, stems and flowering parts. A bioinformatics approach was used to identify the enzyme responsible for the initial step in the biosynthesis of these compounds from its precursor farnesyl pyrophosphate. Based on sequence similarity with a known bisabolene synthases from Arabidopsis thaliana AtTPS12, candidate genes of Helianthus were searched in EST-database and used to design specific primers. PCR experiments identified two candidates in the RNA pool of linear glandular trichomes of sunflower. Their sequences contained the typical motifs of sesquiterpene synthases and their expression in yeast functionally characterized them as bisabolene synthases. Spectroscopic analysis identified the stereochemistry of the product of both enzymes as (Z)-γ-bisabolene. The origin of the two sunflower bisabolene synthase genes from the transcripts of linear trichomes indicates that they may be involved in the synthesis of sesquiterpenes produced in these trichomes. Comparison of the amino acid sequences of the sunflower bisabolene synthases showed high similarity with sesquiterpene synthases from other Asteracean species and indicated putative evolutionary origin from a ß-farnesene synthase.
Asunto(s)
Transferasas Alquil y Aril/aislamiento & purificación , Helianthus/enzimología , Tricomas/metabolismo , Transferasas Alquil y Aril/metabolismo , Helianthus/química , Resonancia Magnética Nuclear Biomolecular , EstereoisomerismoRESUMEN
Capitate glandular trichomes (CGT) of sunflower, Helianthus annuus, synthesize bioactive sesquiterpene lactones (STLs) within a short period of only a few days during trichome development. In the current project, the subcellular localization of H. annuus germacrene A monooxygenase (HaGAO), a key enzyme of the STL biosynthesis in sunflower CGT, was investigated. A polyclonal antibody raised against this enzyme was used for immunolabelling. HaGAO was found in secretory and stalk cells of CGT. This correlated with the appearance of smooth endoplasmic reticulum in both cell types. Stalk cells and secretory cells differed in form, size and types of plastids, but both had structures necessary for secretion. No HaGAO-specific immunoreaction was found in sunflower leaf tissue outside of CGT or in developing CGT before the secretory phase had started. Our results indicated that not only secretory cells but also nearly all cells of the CGT were involved in the biosynthesis of STL and that this process was not linked to the presence or absence of a specific type of plastid.
Asunto(s)
Helianthus/metabolismo , Lactonas/metabolismo , Sesquiterpenos/metabolismo , Tricomas/metabolismo , Vías Biosintéticas , Sistema Enzimático del Citocromo P-450/metabolismo , Helianthus/ultraestructura , Proteínas de Plantas/metabolismo , Tricomas/ultraestructuraRESUMEN
Uniseriate linear glandular trichomes occur on stems, leaves and flowering parts of Helianthus species and related taxa. Their metabolic activity and biological function are still poorly understood. A phytochemical study documented the accumulation of bisabolene type sesquiterpenes and flavonoids as the major constituents of the non-volatile metabolome of linear glandular trichomes in the common sunflower, Helianthus annuus. Besides known sesquiterpenes of the glandulone, helibisabonol and heliannuol type, four previously undescribed sesquiterpenes named glandulone D, E, F and helibisabonol C were identified by spectroscopic analysis. In addition, four known nevadensin type flavonoids varying in O-methoxy substitutions were found. None of them has previously been reported from Helianthus annuus.
Asunto(s)
Helianthus/metabolismo , Sesquiterpenos/aislamiento & purificación , Tricomas/metabolismo , Flavonoides/química , Lactonas/química , Metaboloma , Estructura Molecular , Hojas de la Planta/química , Sesquiterpenos/químicaRESUMEN
MAIN CONCLUSION: Sunflower trichomes fully develop on embryonic plumula within 3 days after start of germination. Toxic sesquiterpene lactones are produced immediately thereafter thus protecting the apical bud of the seedling against herbivory. Helianthus annuus harbors non-glandular and two different types of multicellular glandular trichomes, namely the biseriate capitate glandular trichomes and the uniseriate linear glandular trichomes. The development of capitate glandular trichomes is well known from anther tips on sunflower disk florets, but not from leaves and no information is yet available on the development of the linear glandular trichomes. Scanning electron microscopy of sunflower seedlings unravelled that within the first 40 h of seed germination all three types of trichomes started to emerge on primordia of the first true leaves. Within the following 20-30 h trichomes developed from trichoblasts to fully differentiated hairs. Gene expression studies showed that genes involved in the trichome-based sesquiterpene lactone formation were up-regulated between 72 and 96 h after start of germination. Metabolite profiling with HPLC confirmed the synthesis of sesquiterpene lactones which may contribute to protect the germinating seedlings from herbivory. The study has shown that sunflower leaf primordia can serve as a fast and easy to handle model system for the investigation of trichome development in Asteraceae.