Analytical Tools Developed for Nicotiana attenuata

Plants continuously adjust their metabolism to varying environmental conditions including the attack of herbivores and pathogens. Our group has substantial expertise in the identification and profiling of the volatile and non-volatile small molecules mediating some of these interactions. Figure 1 summarizes the techniques established in our group to routinely analyze a large array of chemically diverse biologically active metabolites. These analytical screens are developed in close collaboration with the molecular biological platform as they play an essential role in the early stages of characterization of transgenic lines.

Sample preparation: high throughput extraction procedures based on Fast-prep and Genogrinder instruments have been established for the extraction of large array of metabolites found in leaves, roots, flowers and other plant parts. Extractions, as well as sample clean-up, can be conducted in 96-well-plate formats to increase sample capacity and decrease costs for solid phase extraction. For automated purification of volatile and non-volatile substances we use an Agilent-HPLC connected to a fraction collector and an Agilent 7890A GC-FID connected to a Gerstel PFC.

 

Targeted HPLC-triple quadrupole-MS profiling: Temporal and spatial changes of phytohormone and oxylipin signatures, as well as of other small molecules, are routinely monitored by HPLC coupled to triple quadrupole mass spectrometer (Bruker EvoQ-Elite, 2 Vairan 1200 LC-qqq-MS instruments) using the multiple reaction monitoring mode (MRM). Quantification is performed in most cases using isotopically labeled internal standards. Here is a list of targets routinely screened in the group:

 

  • jasmonic acid (JA), JA derivatives (e.g. JA-amino acid conjugates, hydroxylated and carboxylated JA), and its precursors (e.g. 13-hydroperoxy-linolenic acid and 12-oxo-phytodienoic acid)
  • SA, abscisic acid (ABA) and ABA derivatives
  • auxins, cytokinins and gibberellins
  • dodecenoic acid derivatives originated from green leaf volatile (GLV) biosynthesis,
  • free fatty acids and its oxygenated forms,
  • 17-hydroxygeranyllinalool diterpene glycosides,
  • nicotine detoxification metabolites
  • amino acids
  • small peptides like glutathione.

 

 

Real time ethylene measurement: We are using a highly sensitive laser photo-acoustic detector with a detection limit of 0.3 ppb to measure ethylene emissions in real time from up to 6 plants in parallel.

 

Targeted and non-targeted analysis of volatile organic compounds: Headspace volatiles from leaves and flowers volatiles  (e.g. GLVs, mono- and sesquiterpenes and aromatic substances like benzyl acetone) collected in the glass house or field onto self-made. In the field (see Ecological platform) are conducted by passive adsorption using self-made pieces of silicone tubing (PDMS, polydimethylsiloxane), which are analyzed by GC-qMS (2 Shimadzu TDU-GC-MS instruments) after automated thermo-desorption. This technique facilitates the rapid screening of plant volatile bouquets involved in plant-plant and plant-animal communication. Real-time measurements in both, glasshouse and field conditions can be performed with a portable zNose. Volatile collections under constant flow and/or sample volume conditions are conducted using self-made super-Q traps with a constant flow-push-pull technique. After elution with dichloromethane, volatiles collected with super-Q traps are routinely analyzed by GC-ion trap-MS ( 1Varian 4000 GC-MS), GC-triple quad-MS (1 Bruker Scion GC-qqq-MS) and GC-FID (1 Varian 2000 GC-FID). Two-dimensional gas chromatography GCxGC-ToFMS (1 Leco Pegasus 4D GCxGC-ToF-MS) is used to obtain high chromatographic resolution. Raw data are processed using software from the vendors or a combination of open source software and classified using multivariate statistics.


Routine HPLC-UV/ELSD measurement of secondary metabolites
: Nicotine, phenolic derivatives, diterpene glycosides and O-acyl sugars are abundant Nicotiana attenuata secondary metabolites with established defensive functions against herbivores. Targeted screens based on HPLC-UV are routinely used in our group to quantify these metabolites. An evaporative light scattering detector (ELSD) is used for the in-line monitoring of Compounds with no UV chromophore, such as O-acyl sugars and diterpene glycosides.

Analytical and in silico resources for metabolomics:  Our group has developed a framework for the metabolomics analysis of plant extracts. Most of these analyses are conducted using an ultra-high pressure liquid chromatography (UPLC)-ESI/qTOF system (2 Bruker micro-ToF UPLC-ToF-MS instruments). The UHPLC system provides highly reproducible chromatographic conditions that allow for the analysis of large data sets targeting a wide range of secondary metabolites. Target primary metabolites are monitored after methoximation and silylation using GC-QQQ-MS and two-dimensional GCxGC-TOFMS and mass spectral information aligned with the Golm Metabolome DB.

For the structural interpretation of high resolution TOF data, we have adopted a versatile three-pronged approach integrating: 1) in vivo whole plant 15N and 13C isotope labeling; 2) a correlation-based grouping of the in-source fragmentation processes; and 3) targeted high resolution MS/MS and low resolution MSn measurements. Data analysis is done using vendor (collaboration with Bruker Daltonics) as well as open source softwares. Figure 2 provides an overview of the main equipment, software and public resources used by the metabolomics project group.

 

More information about the metabolomics research and its integration with genomics resources of  the molecular biology platform is available at the metabolomics project group webpage.