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Identification of enzymes, regulators and transporters involved in different metabolic processes is the foundation to understand how organisms function. There are, however, many difficulties in identifying candidate genes as well as in proving their  in vivo  roles. In this review, we describe different approaches utilized in  Arabidopsis thaliana  to identify gene candidates and experiments required to prove the function of a given candidate. For example, we use the production of methionine-derived aliphatic glucosinolates that represent major defence compounds in  A. thaliana . Nearly all biosynthetic genes, as well as the first sets of regulators and transporters, have been identified. An array of approaches, i.e. classical mapping, quantitative trait loci (QTL) mapping, eQTL mapping, co-expression, genome wide association studies (GWAS), mutant screens and phylogenetic analyses, has been exploited to increase the number of identified genes. Here we summarize the lessons learned from the different approaches used over the years with the aim to help designing and combining new approaches in the future.

How to discover a metabolic pathway? An update on gene identification in aliphatic glucosinolate biosynthesis, regulation and transport