# An Integrated Metabolomics Study of Glucosinolate Metabolism in Different Brassicaceae Genera
## 3 Discussion  
In this study, we systematically examined the variations in glucosinolates, their precursors, and their breakdown products among 12 commonly consumed vegetables of the Brassicaceae family using an integrated metabolomics approach. The results from our principal component analysis (PCA) analysis of the 12 vegetables studied (Figure 2) agreed well with the results from our previous studies [30; 48]. In the present study, we are able to distinguish the same 12 vegetables using PCA, based on the profiles of volatile compounds [48] and glucosinolates [30]. For volatile organic compounds, 1490 features were subjected to PCA after cleaning the data of features present in blanks and with too low detection frequencies, with the vegetable samples separated into 12 clusters that correspond to each genus. Further study on glucosinolate profile using PCA of 36 samples also resulted in clear separation into 12 clusters, which can be further clustered into four groups that show close similarity to morphological taxonomical classification. In order to gain a better understanding of the glucosinolate and relevant synthetic pathways, we constructed a comprehensive map by integrating our data with data from previous studies (Figure 3). The levels of precursor glucosinolates correlate well with levels of their downstream breakdown isothiyanate products. Moreover, the types and abundances of glucosinolates differ significantly among different genera. This is in reminiscence to previous reports that the glucosinolate profiles of Brassicaceae vegetables could be useful for their taxonomical classification, sometimes even at the level of different cultivars [30; 49; 50]. Figure 4: Representative pathway behavior in three vegetables. A heat map of abundances of metabolites detected involved in the pathways from amino acids to glucosinolates and isothiyanates in three different vegetables which grow in different environments (aboveground broccoli, damp soil or submerged watercress and belowground radish). The data was collected using an untargeted approach and the presented heat maps depict the discovery set of three samples and validation data consisting of 5 samples. The isothiyanates could not all be included in the validation dataset. The intensities of metabolites in the heat map were expressed as relative levels in broccoli, radish and watercress. Asterisks denote statistical significance by Student’s \(t\)-test (\(p<0.05\)). ITC: isothiyanate. Our findings on the three vegetables from different growing environments (Figure 4) reveal that the synthesis of different secondary metabolites in the plant could be either dictated by the growing environment or vice versa, i.e., the plant need to have secondary metabolites to adapt to the growing environment for defense against insects, microorganisms, and nematodes. Further studies are needed to examine how these secondary metabolites react to their potential pests or in different growing environments. Although amino acids are usually considered as glucosinolates' precursors, we did not observe association between glucosinolates and amino acids (Figure 1). The correlations in metabolite abundance have shed light on certain functions of a biosynthetic pathway or relationship between precursors and their breakdown products. For example, one of the few amino acids that correlate with alanine is leucine (Figure 1), which can function as alanine's precursor. However, detecting correlations is by no means necessary for such relationships to exist. At any given time, metabolites may be converted to others, or their synthesis may be regulated by different pathways. Understanding the activity of pathways therefore often requires comprehensive information. For example, [PERSON] and colleagues described the preferential formation of isothiyanates over nitriles and epithionitriles as species specific, with isothiyanates being the preferential product in radish (_R. sativus_), but not in _B. rapa_ and _B. olercea_.