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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - VOCO (Biochemical link between plant volatile organic compound (VOC) emissions and CO2 metabolism - from sub-molecular to ecosystem scales)

Teaser

Summary

Plant metabolic processes exert a large influence on global climate and air quality through the emission of the greenhouse gas CO2 and volatile organic compounds (VOCs). Despite the enormous importance, processes controlling plant carbon allocation into primary and secondary metabolism, such as respiratory CO2 emission and VOC synthesis, remain unclear.

The overall goal of VOCO is the development of a novel technological and theoretical basis to couple investigation of CO2 fluxes and VOC emissions, establishing a mechanistic linkage between primary and secondary carbon metabolism. VOCO2 will evaluate carbon investment into VOCs, respiratory CO2 emission and the associated isotope effects among species with different plant functional traits, bridging scales from sub-molecular to the whole-plant and ecosystem processes in an interdisciplinary approach.

This radically new approach uses stable isotope fractionation of central metabolites (glucose, pyruvate) to trace carbon partitioning at metabolic branching points. A unique combination of cutting-edge technology (Î´13CO2 laser spectroscopy, high sensitivity PTR-TOF-MS and quantitative isotopic NMR spectroscopy) will allow a significant advancement through innovative leaf and whole-tree position-specific labelling experiments and an entire ecosystem assessment by merging complementary approaches.

Creation of a unique technological platform will allow an unprecedented assessment of carbon partitioning, bridging scales from sub-molecular to whole-plant and ecosystem processes in an interdisciplinary approach. Innovative positional 13C-labelling will break new ground quantifying real-time sub-molecular carbon investment into VOCs and CO2, enabling mechanistic descriptions of the underlying biochemical pathways coupling anabolic and catabolic processes, particularly the long overlooked link between secondary compound synthesis and CO2 emission in the light. This approach will permit the development of a novel mechanistic leaf model and its integration into a state-of-the-art ecosystem flux model.

VOCO2 will set a new dimension with a world-wide first ecosystem positional labelling experiment in the unique Biosphere 2 enclosure (Arizona, US). Jointly with the novel process-based ecosystem model, VOCO2 will open new frontiers for assessing biogenic emissions of greenhouse gases at the ecosystem scale. This will deliver important information for global change related aspects, as these greenhouse gases can impact atmospheric chemistry and enhance global warming.

Work performed

The goal of the first period was the development of the novel technological facility. This was achieved by merging state-of-the-art high sensitivity proton transfer ratio time of flight mass spectrometry (PTR-TOF-MS) with stable isotope CO2 laser spectroscopy (IRIS) coupled by an automated switching unit to self-build plant chamber systems (Fig. 1).
Additionally, VOCs of selected samples are identified by thermal desorption-gas chromatograph-mass spectrometry (TD-GC-MS, Markes International, Ltd. and Agilent Technologies, Inc.) with detection limits of < 5 pptv for most VOCs in air, collected on thermal desorption sample tubes. Leaf dark-respired Î´13CO2 are cross-referenced by my In-tube-Incubation technique (Werner et al. 2007) on a Âµgas GC autosampler interfaced to an ISOPRIME isotope ratio mass spectrometer (Elementar, Germany), allowing high precision without interference of background air.

The first tests showed the vast potential of combining these methods. However, they also indicated that due to the rapid dynamics in VOC and CO2 fluxes (ranging from seconds to minutes) precise, coordinated, and synchronised measurements are required, which are not attainable within a conventional setup. Currently, hard- and software of both instruments are coupled into one innovative device directly linked to an automated Teflon valve switching unit enabling synchronised sampling of 13CO2 and VOCs on several enclosures/atmospheric samples, calibration gases and background values. Moreover, a self-made hydrocarbon-free zero-air-generator had to be developed, as commercial available instruments turned out to be unable to produce the high amount of flow with sufficiently clean air. This unique infrastructure (PTR-TOF-IRIS) for automated real-time VOC and 13CO2 analysis (Fig. 1, Fasbender et al., manuscript in preparation) does now allow an unprecedented real-time detection of (13C)-carbon allocation into respiratory CO2 and VOC emission in intact plants and atmospheric trace gases and will be used throughout all subsequent experiments.

For work package 2, Different plant functional groups including high (e.g. Halimium halimifolium) and low (e.g. Oxalis triangularis) VOC-emitters, and tropical rainforest species of the dominant species of the Biosphere 2 are grown in the new walk-in growth chambers under optimal controlled conditions (e.g. 25ÂºC temperatures, 60% rel. humidity, 12 hour photoperiod). Measurements of net carbon assimilation, Î´13Cres and VOC emissions are made throughout the day and after darkening at the leaf and branch level simultaneously in custom-built plant cuvettes (Fig. 1). Leaf-respired Î´13CO2 is measured by in-tube incubation (Werner et al. 2007), given its higher sensitivity for natural abundance Î´13C, as interference with high background CO2 levels can be avoided. Additionally, leaf bulk Î´13C and thermal desorption tubes for TD-GC-MS are collected.
Position-specific 13C labelled metabolites enable tracing the fate of every single carbon atom of the molecule through the metabolic pathways, but in spite of its enormous potential, the application is still rare. Yet a huge step forward can be attained when combining position-specific 13C labelling with the PTR-TOF-IRIS (WP1), which allows now real-time recording of carbon partitioning into different pathways such as respiratory CO2 and VOCs in intact plants. Moreover, using central metabolites like pyruvate which are linking anabolic and catabolic metabolism, real-time information on carbon partitioning between these pathways can be retrieved. A plethora of difference VOC can be emitted by plants, which are synthesized in different metabolic pathways and cell compartments. Experiments will start exploring pathways with pyruvate (and acetyl-CoA) as precursor molecule and others metabolites will be added depending on progress and results. Many less-regarded volatile compounds have been detected in our first preliminary experiment.

Fasbender, L., YaÃ±ez-Ser

Final results

In the first work package we build a pioneering research facility (PTR-TOF-IRIS) where emergent technology from geosciences, such as the Proton-transfer-time-of-flight mass spectrometry and isotope laser spectroscopy, were coupled to resolve temporal dynamics of VOCs and CO2 at an unprecedented rate enabling research that goes significantly beyond the state-of-the-art.
The radically new approach of VOCO2 is based on molecular isotope analysis and will deliver a mechanistic description and model of carbon partitioning into different biochemical pathways. At the metabolic scale, the innovative setup of position-specific labelling breaks new ground in tracing real-time carbon allocation into VOCs and CO2 . First exciting results have been gain by detecting rare compounds such as volatile diterpens (kauren) emitted by plants (YaÃ±ez-Serrano et al., publication in preparation).

YaÃ±ez-Serrano, A. M., L. Fasbender, J. Kreuzwieser, C. Werner. Diterpene and other isoprenoid emissions by the Mediterranean shrub Halimnium halimifolium L. determined by the PTR-TOF-IRIS system. To be submitted to Scientific Reports by end of June