Tissue preparation using Arabidopsis.

The ability to track changes in the levels of many metabolites in plants has great utility in a number of biological contexts. A metabolomics experiment usually requires the comparison of different varieties in either a functional genomics context or in response to perturbation by an external treatment. Such treatments can result in subtle changes in the final chemical signature of the plant tissue, and therefore, any unwanted variance produced in the generation of that tissue must be minimised. Procedures for plant growth, harvesting, preparation of extracts, and the subsequent collection of data have been optimised to minimise experimental variation within the dataset. This chapter describes in detail how to generate reproducible Arabidopsis tissue suitable for a typical plant metabolomics experiment. Issues concerned with tissue sampling, harvesting, and storage are also discussed.

Metabolomic analysis of Arabidopsis reveals hemiterpenoid glycosides as products of a nitrate ion-regulated, carbon flux overflow.

An understanding of the balance between carbon and nitrogen assimilation in plants is key to future bioengineering for a range of applications. Metabolomic analysis of the model plant, Arabidopsis thaliana, using combined NMR-MS revealed the presence of two hemiterpenoid glycosides that accumulated in leaf tissue, to ~1% dry weight under repeated nitrate-deficient conditions. The formation of these isoprenoids was correlated with leaf nitrate concentrations that could also be assayed in the metabolomic data using a unique flavonoid-nitrate mass spectral adduct. Analysis of leaf and root tissue from plants grown in hydroponics with a variety of root stressors identified the conditions under which the isoprenoid pathway in leaves was diverted to the hemiterpenoids. These compounds were strongly induced by root wounding or oxidative stress and weakly induced by potassium deficiency. Other stresses such as cold, saline, and osmotic stress did not induce the compounds. Replacement of nitrate with ammonia failed to suppress the formation of the hemiterpenoids, indicating that nitrate sensing was a key factor. Feeding of intermediates was used to study aspects of 2-C-methyl-d-erythritol-4-phosphate pathway regulation leading to hemiterpenoid formation. The formation of the hemiterpenoids in leaves was strongly correlated with the induction of the phenylpropanoids scopolin and coniferin in roots of the same plants. These shunts of photosynthetic carbon flow are discussed in terms of overflow mechanisms that have some parallels with isoprene production in tree species.