Application of PTR-TOF for unraveling metabolic steps in volatile isoprenoids biosynthesis

Our current progresses in understanding the biological and ecophysiological functions of plant volatiles are due to general advances in biochemical and molecular techniques and to the development of new instrumentation for the collection and analysis of plant volatile organic compounds. The contribution of PTR-MS and of new technologies improving the resolution, the frequency of acquisition, and the sensitivity of biogenic volatile organic compounds (BVOC) measurements are essential to further investigate these important aspects of the biosphere-atmosphere interaction in a changing world. Within MOMEVIP the University Innsbruck (UI) will provide instrumentation and expertise of ultra-sensitive and ultra-fast detection of volatile isoprenoid (VIP) emissions. Our instrumentation is based on proton transfer reaction mass spectrometry (PTR-MS) and its most recent evolution: proton transfer reaction time of flight (PTR-TOF) mass spectrometry. Within MOMEVIP joint experiments will be performed for detecting VIP emissions, and high molecular weight or semi volatile VIP, as well as VIP reaction products whose ecological functions and biochemical sources are still undisclosed. The recently developed PTR-TOF, will provide quantitative as well as exact mass information of almost all biogenic volatile organic compounds (BVOC) and reveal their atomic composition thus being able to differentiate e.g. between pure hydrocarbon, and oxygen containing BVOC. Nevertheless isomeric compounds such as different isomers of monoterpenes cannot be distinguished by PTR-TOF. Thus we will combine a GC-FID with a PTR-TOF. As emphasis will be on joint experiments among partners, mainly focusing on the mechanistic explanation of VIP-induced resistance to heat and oxidative stresses fast and quantitative information on VIP emissions and VIP reaction products will be very important. The PTR-TOF technique will be applied to elucidate metabolic pathways leading to the production and emission of oxygenated BVOC as well as VIP and other BVOC in leaves of plants by applying 13C-labeled precursor compounds. Plant enclosure measurements utilizing a standard PTR-MS instrument and 13C labeled compounds for the isoprene synthesis have been published (Schnitzler et al., 2004; Graus et al., 2004). Other metabolic pathways remain largely elusive such asthose generating a family of BVOC of large impact in the chemistry of the atmosphere (e.g. sesquiterpenes), and those generating methylsalycilate and methyljasmonate, two substances playing a role in plant interaction with abiotic and biotic stresses. The use of PTR-TOF and GC-PTR-TOF will substantially increase the capability to identify isotopically labeled BVOC, and the high time resolution is of essential importance to attribute BVOC labeling to specific biosynthetic pathways needed in the MOMEVIP consortium.

An incredibly large amount of VOC is steadily emitted into the shallow atmospheric layer of our planet. Volatile organic compounds (VOC) are carbon-containing compounds that will evaporate easily. VOC are released by the burning of fossil fuels, forest fires and of solvent use. Terrestrial vegetation is considered as the main emission source of VOC on a global scale. Oxidation of VOC in the presence of nitrogen oxides and sunlight produce ground-level or bad Ozone. Breathing ozone can trigger a variety of health problems for humans and can also have harmful effects on sensitive vegetation when sufficient ozone enters the plant leaves through the stomatal pores. The Collaborative Research Project MOMEVIP strategy was to put together different expertise, at the molecular and metabolic level, to unravel the mechanisms by which VOC protect plants from abiotic stresses such as heat, drought, and ozone. We as the Austrian (sub) project partner provided instrumentation and expertise for MOMEVIP, focused at testing and improving ultra-sensitive, fast and quantitative detection of plant VOC emissions. Detecting VOC in real-time, i.e. recognizing immediately, is possible by the PTR-TOF-MS method developed in Innsbruck. PTR-TOF-MS stands for proton transfer reaction time of flight mass spectrometry, and is called a soft ionization method. This method was further improved to determine even the isomeric structure of biogenic VOC emissions from stressed plants. One significant result of our sub project shows that the diterpenoid cis-abienol, a semi-volatile organic compound secreted by the leaf hairs (trichomes) of various tobacco varieties, protects the leaves from breathing" harmful ozone. Ozone is efficiently removed by chemical reactions with cis-abienol at the plant surface, forming oxygenated VOC (formaldehyde and methyl vinyl ketone) that are released into the air impacting atmospheric chemistry.