Analytical Tools and Metabolomic Methods for Identifying Stilbenes and Other Defence Induced Compounds

As previously mentioned, stilbenes are phytoalexins produced naturally by several plants upon attack by pathogens such as bacteria or fungi. Because of their dynamic behaviour as responses to stress, their detection requires methods that can be used for monitoring their differential response in various phytopathologic situations. Various HPLC methods have been used for detection and quantification of stilbenes. Indeed, HPLC in gradient mode on reversed phase C18 columns provides a means to separate stilbenes directly in crude extracts without the need for derivatisation.

Many extraction methods have been tested in conjunction with the direct use of HPLC for stilbene profiling [103]. Most of the qualitative or quantitative analytical studies were performed with HPLC and ultraviolet (UV), fluorescence (FD) or electrochemical (ECD) detection [104]. We have demonstrated that fluorimetric detection is much more sensitive than UV detection and that its specificity allows simple pre-purification of grape berries juice and/or direct injection of wines [42]. The fluorescent properties of stilbenes have also been used for their in vivo detection and local assessment in grapevine leaves [105]. As discussed below, HPLC coupled to mass spectrometry (LC-MS) provides an even more sensitive and selective method for the detection of these polyphenols [106].

In general, all of these analytical approaches have been used in a targeted manner and require the use of well-defined stilbene standards for identification and quantification.

To obtain a deeper understanding of plant stress response phenomena at the level of production of phytoalexins, phytohormones and other stress-induced metabolites, non-targeted, holistic analytical approaches can be used. These studies are performed using 'metabolomics', which is defined as a universally applicable non-targeted analytical approach to identify and quantify the largest possible number of metabolites of a biological system. The complete set of primary and secondary metabolites of low molecular weight (MW < 1,000 Da) constitutes the 'plant metabolome'. The size of this metabolome is still unknown, but it is estimated to contain several thousand constituents [107].

Metabolomic profiling may actually provide the most 'functional' information of all of the 'omics' technologies [108]; it gives a broad view of the biochemical status of an organism that can be used to monitor significant metabolite variations. Indeed, because metabolites are the end products of the cellular regulatory processes, their levels can be regarded as the ultimate response of biological systems to genetic or environmental changes. Finally, this information can be used in conjunction with other systems biology approaches to assess gene function and provide a holistic view of a living system [109, 110].

Metabolomics, in contrast to 'hypothesis-driven' approaches for the study of plant stress responses, is a 'data-driven' approach that can generate new hypotheses in an unbiased manner. It has the potential to detect not only new stilbenes but also other related metabolites. To our knowledge, this type of approach has not yet been implemented to systematically search for stilbenes, but it has been successfully used to study other aspects of plant defence studies and in wine research.

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