## 3 Discussion  
The development and optimization of an untargeted metabolomics method aiming to investigate the volatiles of a biological system represents a crucial step for a successful analysis. HS-SPME followed by GC-HRMS has been shown to be a promising approach in metabolomics studies, and previous studies showed that the SPME fibre with DVB/CAR/PDMS coating phase provided high recovery and reproducibility for analysis of plant VOCs with a wide range of polarity [31]. As plants produce most of the volatiles only after their cells are ruptured, for this study, we first modified a laboratory blender to disintegrate and homogenize the vegetable samples for the production and release of VOCs in an enclosed environment, as shown in Figure S1. After blending, a direct HS-SPME method was employed by exposing a SPME fibre to the headspace of the crushed vegetative tissues in the sealed  
Figure 5: Spearman correlations of identified VOCs in 12 Brassicaceae vegetables. Correlation coefficients (\(r\)) were in the range of \(-1\) to 1. Only significant correlations (\(p<0.05\)) are shown in the heatmap. Blue (\(r=-1\)) and red (\(r=1\)) represent the most significantly negative and positive correlations, respectively. White (0) represents no significant correlation. vessel. There are several advantages of using the present approach. First, the procedure enables capture of the rapid-releasing VOCs soon after the vegetative tissues are being disrupted, reducing VOCs losses during sample pre-treatment in an open environment. Secondly, this approach avoids or minimizes further reactions such as oxidation or reduction of the unstable VOCs. Thirdly, the VOCs captured through the direct HS-SPME method would reflect the metabolic pathways in fresh vegetables more accurately, since it may avoid unexpected breakdown byproducts or metabolites under conditions that disrupt normal metabolic activities of fresh vegetables. In addition, extraction of the VOCs at 37 \({}^{\circ}\)C mimics the possible volatile components released and VOCs profiles obtained for the chewing of raw vegetables, such as during the consumption of salads. This newly developed sampling approach represents an alternative simplified means of VOCs sampling from plant tissues. It has been observed that GLVs can be released from plants within seconds when leaves are injured or suffer from biotic/abiotic stresses, and reach maximum intensity in 30 min [32; 33]. Trace amounts of GLVs are usually released by plants under normal physiological conditions, but large amounts can be released under stressed conditions [34]. In recent years, GLVs have been extensively studied and emerged as key players in plant defence as well as plant communication with insects and neighbour plant [35]. For instance, previous studies showed that 3-(Z)-hexenol and its related aldehydes have demonstrated fungicidal and bactericidal activities [36]. In addition to their biological functions, GLVs usually provide fruits and vegetables with their characteristic 'fresh green' aroma [35], which are appreciated by consumers and therefore have the potential to be exploited in industrial biotechnology. GLVs are produced by the autolytic oxidative breakdown of membrane lipids including linoleic acid and linoleic acid through the LOX pathway (Figure 3), and their levels varied among plant species and stage of development. In our study, various GLVs were released from green leafy vegetables such as watercress, rocket, kai lan, choy sum, pak choi, broccoli and Brussels sprouts, while much lower levels of these VOCs were emitted from cabbage, Chinese cabbage cauliflower, radish and cherry radish (Figure 2). As the vegetables samples used in this study were mature vegetables, the variations mainly reflected the species-specific differences in VOCs emissions upon vegetative tissue damage. Volatile terpenoids originate from the cytosolic mevalonic acid (MVA) pathway (sesquiterpenes, terpenes and geranyllinal inalool) and the plastitial methylerythnitol phosphate (MEP) pathway (monoterpenes) (Figure 3) [1].