The ability of plants to fix light, carbon dioxide and nitrogen in a renewable manner, make them an attractive biological system for the production of bio-fuels, bio-polymers and even industrial chemicals. In order to use plants for such industrial applications, however, it is important to engineer their primary metabolism and to improve (a) carbon and inorganic nitrogen fixation (b) stress tolerance (c) nutritional content and composition (d) growth rate.. Metabolic Flux Analysis (MFA), an important tool for metabolic engineering of biological systems, can not be readily applied in a high-throughput manner to whole plants systems due to (i) difficulty in achieving a (pseudo) steady state, (ii) Incomplete information about plant's biochemical-network and compartmentalization of reactions, and (iii) High cost of labeled substrates for whole plant systems. Hence an alternate technique and experiment design strategy is needed to obtain the metabolic fingerprint of plants for identifying metabolic engineering targets in a high-throughput manner.

In this context, we carried out a time-series metabolomic analysis of a systematically perturbed Arabidopsis thaliana liquid culture system to study regulation of its primary metabolism and stress response. The biological system was studied under conditions of elevated CO₂ stress, salt (NaCl) stress, sugar (trehalose) signal, and hormone (ethylene) signal, applied individually; the latter three stresses also applied in combination with the CO₂ stress. Accurate polar metabolomic profiles were obtained using gas chromatography-mass spectrometry (GC-MS) and a novel data correction, validation and normalization strategy which significantly increased accuracy [1].

The metabolomic analysis of salt (NaCl) stress and the combined elevated CO₂ - salt (NaCl) stress identified important new regulatory information about salt stress response, resulting in new metabolic engineering targets for osmotic stress resistant plants. Trehalose (sugar) signal and Ethylene (plant hormone) helped similarly identification of better nitrogen fixation and faster growth targets respectively. The combined stress response showed different effect of elevated CO₂ in presence of salt and sugar signals.

In addition to identifying this important biological information, the results also demonstrate the advantages of dynamic, multiple-stress metabolomic analysis for obtaining metabolic fingerprint of complex eukaryotic systems. In this sense, it contributes in further advancing the computational and experimental, metabolic engineering and systems biology toolbox.

[1] Kanani H and Klapa MI, 2006. “Data Correction Strategy for Metabolomics Analysis using Gas Chromatography-Mass Spectrometry”, Metabolic Engineering, 2007, 9(1):35-51.

Author Current Address:

Dr. Harin H. Kanani: Pioneer Hi-Bred International - A DuPont Company, Johnston, Iowa – 50131, USA.

Dr. Maria I. Klapa: Institute of Chemical Engineering and High Temperature Chemical Processes, Foundation of Research and Technology, Hellas (FORTH/ICE-HT), Patras, Greece.