Important Odorants of Four Brassicaceae Species, and Discrepancies between Glucosinolate Profiles and Observed Hydrolysis Products

It is widely accepted that the distinctive aroma and flavour traits of Brassicaceae crops are produced by glucosinolate (GSL) hydrolysis products (GHPs) with other non-GSL derived compounds also reported to contribute significantly to their aromas. This study investigated the flavour profile and glucosinolate content of four Brassicaceae species (salad rocket, horseradish, wasabi, and watercress). Solid-phase microextraction followed by gas chromatography-mass spectrometry and gas chromatography-olfactometry were used to determine the volatile compounds and odorants present in the four species. Liquid chromatography-mass spectrometry was used to determine the glucosinolate composition, respectively. A total of 113 compounds and 107 odour-active components were identified in the headspace of the four species. Of the compounds identified, 19 are newly reported for ‘salad’ rocket, 26 for watercress, 30 for wasabi, and 38 for horseradish, marking a significant step forward in understanding and characterising aroma generation in these species. There were several non-glucosinolate derived compounds contributing to the ‘pungent’ aroma profile of the species, indicating that the glucosinolate-derived compounds are not the only source of these sensations in Brassicaceae species. Several discrepancies between observed glucosinolates and hydrolysis products were observed, and we discuss the implications of this for future studies.


Introduction
Crops of the Brassicaceae family are grown all over the world, and they form an important part of many different cuisines and cultures [1]. Some species are noted for their distinctive, and often very strong, tastes and flavours. Armoracia rusticana (horseradish), Eruca sativa ('salad' rocket), Eutrema japonicum (wasabi), and Nasturtium officinale (watercress) are four such examples, which are noted for their pungent, peppery, and aromatic organoleptic properties [2][3][4].
Horseradish and wasabi produce large roots that are grated and used as a condiment in many cultures across the world, most notably in Eastern Europe and the United Kingdom (horseradish) and Japan (wasabi; [5]). Horseradish is a vegetative perennial that grows widely in temperate regions [6], whereas wasabi can only be grown in a very few locations, owing to its sensitivity to temperature change and root oxygen availability [7]. Wasabi is traditionally cultivated in damp river valleys of Japan, although commercial operations have been established elsewhere, such as in the UK.
Salad rocket originates from the Middle East and has spread throughout the Mediterranean basin [8]. It has become naturalised on every inhabited continent and is considered cial operations have been established elsewhere, such as in the UK.
Salad rocket originates from the Middle East and has spread throughout the Mediterranean basin [8]. It has become naturalised on every inhabited continent and is considered an invasive weed in some regions. Watercress has similarly become naturalised (for example in North America) and grows in shallow rivers and streams. It can be cultivated commercially on large scales using artificial growing 'pools' flooded with stream water. Its leaves and shoots are a popular salad and sandwich garnish, and they can also be made into soups [9].
It is widely accepted that the distinctive aroma and flavour traits are produced by glucosinolate (GSL) hydrolysis products (GHPs [10]). GSLs are sulphur-containing secondary metabolites produced by Brassicales plants in response to biotic and abiotic stress [11]. Myrosinase enzymes are responsible for the hydrolysis of GSLs in water to form a plethora of GHPs, the conformation of which can be determined by the presence of enzyme cofactors, pH level, metallic ion concentration, and the precursor GSLs side-chain structure [8]. These products include: isothiocyanates (ITCs), nitriles, epithionitriles, indoles, oxazolidine-2-thiones, and other diverse products that result from tautomeric rearrangements ( Figure 1). Previous work has reported olfactometry data for each of these crops; however, data are generally very scarce. Only five studies of E. sativa volatile compounds have been published in the last twenty years [3,[12][13][14][15]. Very little information regarding wasabi root volatile composition and aroma is available outside of Japanese language journals [7,16]. Similarly, very little information is available for watercress, with only four papers published in the last 40 years [17][18][19][20]. Horseradish is the most well characterised of these four species, but still, only six studies of note have been published in the last 50 years [6,[21][22][23][24][25].
There is also an 'elephant in the room' regarding previous reports of GHPs present in aroma profiles of these Brassicaceae vegetables. Many of the reported GHP compounds are derived from GSL precursors that are not regularly reported as part of the profile for each respective species. In E. sativa, for example, GHPs such as methyl ITC, Previous work has reported olfactometry data for each of these crops; however, data are generally very scarce. Only five studies of E. sativa volatile compounds have been published in the last twenty years [3,[12][13][14][15]. Very little information regarding wasabi root volatile composition and aroma is available outside of Japanese language journals [7,16]. Similarly, very little information is available for watercress, with only four papers published in the last 40 years [17][18][19][20]. Horseradish is the most well characterised of these four species, but still, only six studies of note have been published in the last 50 years [6,[21][22][23][24][25].
There is also an 'elephant in the room' regarding previous reports of GHPs present in aroma profiles of these Brassicaceae vegetables. Many of the reported GHP compounds are derived from GSL precursors that are not regularly reported as part of the profile for each respective species. In E. sativa, for example, GHPs such as methyl ITC, 3-butenyl ITC, 1-isothiocyanato-4-methylpentane, and 5-(methylsulfanyl)pentanenitrile have all been previously reported [13]. The GSL precursors to these compounds (glucocapparin, GCP; gluconapin, GNP; 4-methylpentyl GSL, 4MP; and glucoberteroin, GBT; respectively) have never been reliably or consistently reported as being part of the GSL profile in this species [26,27]. This begs the question whether these identifications are correct or if the reports of GSL components in these species are incomplete. This may be due to a lack of sensitivity in reported mass spectrometry methods or because there is a lack of analytical standards to confirm compound identities. Another possibility is that there is a post-hydrolysis modification of GHPs, either by enzymatic means or through reactions with other phytochemical components, or as part of thermolytic reactions during gas chromatography. Very few examples of such modifications have been reported within the literature [28], but this may explain the presence of some GHPs within profiles of species where the GSL precursor is absent.
Other non-GSL derived compounds have also been reported to contribute significantly to the aromas of Brassicaceae crops. 2-Isopropyl-3-methoxypyrazine has been found to produce a strong pea, or green, pepper-like aroma in horseradish, for example ([6] Figure 2). In rocket, 'green-leaf' volatiles such as 3-hexenal and 1-penten-3-one have also been highlighted as having high odour potency [12]. The role of these compounds in aroma generation in GSL-containing crops is often not fully appreciated, and there are many diverse compounds with equally high odour intensities to GHPs present within the volatile bouquet.  Table 2); * two separate peaks present for this compound; $ tentatively identified. Figure 2. Chemical structures of volatile compounds found in horseradish, rocket, wasabi, and watercress samples (numbers in parentheses refer to compound codes in Table 2); * two separate peaks present for this compound; $ tentatively identified. The aims of this study were to (i) identify and describe in detail the key odorants of four Brassicaceae species (salad rocket, horseradish, wasabi, and watercress) by gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS), and (ii) associate the observed GHPs with their respective GSL profiles by liquid chromatography-mass spectrometry (UPLC-MS/MS). Our goal was to improve upon existing compound characterisation and odour descriptors for compounds in salad rocket, horseradish, wasabi, and watercress. The contribution of pungency by GHPs to aroma profiles is well studied; however, other aroma traits generated by non-GHPs are not well described for these species, and they likely create distinctive and subtle sensory characteristics. Additionally, detailed GSL compositions and MS/MS spectra for rocket, wasabi, and watercress is presented, highlighting discrepancies with observed hydrolysis products.

Samples
Individual horseradish and wasabi roots were purchased from Morrison's supermarket (Reading, UK) and The Wasabi Company (Dorchester, UK) respectively. Watercress was purchased as whole bags of leaves from ASDA supermarket (Reading, UK). Salad rocket was grown in controlled environment conditions at the University of Reading using seeds donated by Elsoms Seeds Ltd. (Spalding, UK) and designated RS4 and RS8. Seeds of each cultivar were sown into module trays containing peat-based seedling compost and germinated at 30 • C (daytime; 25 • C night). Lighting conditions were set to a long-day cycle (16 h light, 8 h dark). Light intensity was set to 380 µmol m −2 s −1 . Humidity was ambient. Plants were considered mature upon the development of 10 to 15 leaves. Harvested leaves were taken intact and placed inside a Zip-loc bag.
Roots and leaves were placed in a fridge upon either purchase or harvest (4 • C) until further analysis was performed.

Headspace Solid Phase Microextraction (SPME)
Samples of respective leaf and root tissues were homogenised by means of a commercial blender for 30 s, and 2 g of each was weighed into a SPME vial of 15 mL fitted with a screw cap. Samples were left aside for 10 min for the enzymatic hydrolysis of GSLs to take place. After exactly 10 min, 2 mL of saturated CaCl 2 was added in order to cease the enzymatic reactions. After equilibration at 40 • C for 10 min, a 50/30 µm DVB/CAR/PDMS fibre was exposed to the headspace above the sample for 20 min. Three biological replicates were prepared for GC-MS analysis, and two replicates for each of the three assessors were prepared for the GC-O analysis.

GC-MS Analysis of SPME Extracts
After extraction, the SPME device was inserted into the injection port of an Agilent 7890A gas chromatography system coupled to an Agilent 5975C detection system equipped with an automated injection system (CTC-CombiPAL). A capillary column HP-5MS (30 m × 0.25 mm × 0.25 µm film thickness) (Agilent, Santa Clara, CA, USA) coated with (5% Phenyl Methyl Silox) was used for the chromatographic separation of volatile compounds. The oven temperature program used was 2 min at 40 • C isothermal and an increase of 4 • C/min to 250 • C. Helium was used at 3 mL/min as carrier gas. The sample injection mode was splitless. Mass spectra were measured in electron ionisation mode with an ionisation energy of 70 eV, the scan range from 20 to 280 m/z and the scan rate of 5.3 scans/s. The data were controlled and stored by the HP G1034 Chemstation system. Identities were confirmed by running the samples on a Stabilwax-DA (30 m × 0.25 mm × 0.25 µm film thickness) polar column from Restek (Bellefonte, PA, USA). Volatile compounds were identified or tentatively identified by comparison of each mass spectrum with spectra from authentic compounds analysed in our laboratory, or from the NIST mass spectral database (NIST/EPA/NIH Mass Spectral database, 2014), or spectra published elsewhere (see Supplementary Data S1 for GC-MS chromatograms and compound fragmentation spectra). A spectral quality value >80 was used alongside linear retention index to support the identification of compounds where no authentic standards were available. LRI was calculated for each volatile compound using the retention times of a homologous series of C 6 -C 25 n-alkanes and by comparing the LRI with those of authentic compounds analysed under similar conditions. The compound peak areas were normalised and converted to the relevant abundance of each component as a percentage of the total peak area.

GC-O Analysis of SPME Extracts
After extraction, the SPME device was inserted into the injection port of an Agilent 7890B Series ODO 2 (SGE) GC-O system equipped with a non-polar HP-5MS column (30 m × 0.25 mm × 0.25 µm film thickness). The outlet was split between a flame ionisation detector and a sniffing port. The contents of the SPME fibre were desorbed for 3 min in a split/splitless injection port, in splitless mode, onto five small loops of the column in a coil, which were cooled in solid carbon dioxide and contained within a 250 mL beaker. The injector and detector temperatures were maintained at 280 • C and 250 • C, respectively. During desorption, the oven was held at 40 • C. After desorption, the solid carbon dioxide was removed from the oven. The oven was maintained at 40 • C for a further 2 min and then, the temperature was raised at 4 • C/min to 200 • C and at 8 • C/min to 300 • C. Helium was the carrier gas, and the flow rate was 2.0 mL/min. Three assessors were used for the detection and verbal description of the odour active components of the SPME extracts. Each assessor participated in three training sessions for each sample species prior to scoring sessions. Each assessor evaluated by sniffing each sample in duplicate and documented the odour description, retention time, and odour intensity (OI) on a seven-point scale (2)(3)(4)(5)(6)(7)(8), where <3 = weak, 5 = medium, and 7 = strong. Only those odours that were detected by all three assessors were recorded in the results. n-Alkanes C 6 -C 25 were analysed under the same conditions to obtain LRI values for comparison with the GC-MS data.

Glucosinolate (GSL) Extraction
GSL extraction was performed as per the protocol presented by [29] with modifications. Briefly, 40 mg of dried leaf powder was placed into Eppendorf tubes and put into a heat block (80 • C for ten minutes). Afterwards, 1 mL of preheated methanol water (70% v/v) was added to dried powder, vortexed vigorously, and placed in a water bath (75 • C) for 20 min. Samples were cooled and centrifuged at full speed for five minutes at room temperature (≈22 • C); the supernatant was collected and filtered (0.22 µm PVDF Acrodisc syringe filters; VWR, Lutterworth, UK). Crude extracts were dried using a centrifugal evaporator and resuspended in 1 mL of LC-MS-grade H 2 O and stored at −80 • C until analysis. Immediately before analysis by UPLC-MS, samples were diluted five-fold with LC-MS-grade H 2 O.  Table 1). Then, MS/MS spectra were compared to authentic standards and available literature sources [30][31][32][33][34][35][36][37][38]. Pentyl GSL (PEN), isobutyl GSL (ISO), glucoputranjivin (GPJ), and butyl GSL (BUT) were tentatively identified due to the possible presence of isomers [38], and/or no reliable reference MS spectra could be found in the literature. Total ion chromatograms of glucosinolates identified were included in the Supplementary Data (S2).
MS/MS settings for the PIS method were as follows: samples were analysed in the negative ion mode with a scan range of 70-820 m/z. A collision energy of 25 eV and a scan speed of 30,000 u per s −1 . For the quantification of GSLs, a Multiple Reaction Monitoring (MRM) method was established. Based on the fragmentation observed in the PIS method, confirmation and quantification transitions were established (Table 1). Dwell times for each precursor and product ion were set to 5 s.

Volatile Compounds
The volatile compounds identified in the headspace of the four Brassicales species are listed in Table 2, detailing their PubChem compound identification (PubChem CID) as well as their linear retention indices (LRI) in a polar and non-polar column. Semiquantitative characterisation results are also shown in Table 2 as relative area. ITCs and alcohols were the chemical classes of compounds dominating the volatile profile of the samples with other compounds such as aldehydes, esters, and terpenes also present.
GC-olfactometry analysis of the samples yielded a total of 107 odorants across the four species, which are presented in Table 3. Qualitative differences were observed between the samples with horseradish and watercress yielding a total of 52 and 51 odorants, respectively. Green/grassy, radish, sulphury, and horseradish were some of the terms that were mostly used by the assessors to describe the odours. Additionally, a total of 46 odorants of unknown identity were detected within the headspace of the four Brassicales analysed that may contribute to the odour profiles of these crops. These compounds matched no corresponding peaks and LRI values within the GC-MS data. This suggests that the compounds responsible for generating the perceived aromas were present at levels below the detection threshold of the instrumentation used. The number and diversity of unidentified compounds and aromas is indicative of the fact that characterisation of the species' volatile profiles is far from complete, and it is likely that many more will be discovered in future studies.
Despite its high relative abundance, (Z)-3-hexen-1-ol produced only a weak, green, radishy aroma (Table 3) in rocket leaves. (E)-2-Hexenal by comparison was 2.9-fold less abundant in relative terms but produced a slightly stronger aroma, described by assessors as green, and apple-like. Hexanal (43) produced a pungent, green, grassy aroma of relatively high intensity, which has not been previously described in rocket to our knowledge.
2-Isopropyl-3-methoxypyrazine (102) by comparison was of low relative abundance in the rocket headspace (0.3%; Table 2) but was found to have one of the strongest aromas in rocket (rotten, potato-like, vegetative; Table 3). 1-Isothiocyanato-4-methylpentane had a weak aroma and was given a tentative new description of 'musty', as no previous studies have reported an odour for this compound.
The ITC erucin (23) is known for its anticarcinogenic properties, but its aroma was only recently described [12]. In agreement with a previous report, this compound produced a radishy aroma of weak intensity. Interestingly, the compound previously associated with characteristic "rocket-like" aroma (sativin, 20) [12] was relatively weak-smelling. In this study, the compound was found to have a burnt, rubbery, and soil-like aroma. This suggests that sativin is not the main driver of pungency or aroma in 'salad' rocket. Other compounds such as ethyl vinyl ketone (65; [41]), hexanal, 3-butenyl ITC (11) [48,54], and several unknown compounds (see final paragraph in this section) all had descriptions of pungency at higher intensities than sativin (Table 3). It may also be likely that no single compound is responsible for this attribute of rocket aroma but rather several.
Pentyl ITC (15, 0.6%) not previously identified in 'salad' rocket produced a strong odour, which was characterised as cabbage-like, green, and rotten (Table 3, Figure 2). As will be discussed in Section 3.2, we have tentatively identified pentyl GSL (Table 1) as a significant and previously unreported component of the GSL profile of 'salad' rocket, which gives rise to this ITC compound.
Other compounds not previously identified in 'salad' rocket included 2-phenylethanol, 4-heptenal and 2-sec-butyl-3-methoxypyrazine. 2-Phenylethanol (37, 0.2%) was noted to impart a floral aroma at a medium-weak intensity (Table 3). This compound is derived from phenylalanine and has been found to contribute to aroma and flavour in many foods, such as tomatoes [66]. 4-Heptenal (45) occurred in rocket leaves with a relative abundance of 0.1% (Table 2). Despite this low amount in terms of the overall volatile profile, the compound was perceived at a medium intensity by the assessors (Table 3) and described as grassy and green. This compound has been variously described as mushroom-like [50], fatty and fishy [67], and potatoey [68]. The variation in these descriptions may be associated with the isomerisation of the compound, which could not be resolved in this study. 2-sec-Butyl-3-methoxypyrazine (106, 0.2%) has been reported in several Brassicales species, such as white mustard, rapeseed [60], and horseradish [25], but not in E. sativa (Figure 2). It has been variously described as having a pea-like, musty, green, and bell pepper-like aroma. Assessors described the compound as earthy, similar to rotten potatoes, and vegetablelike. Despite its relatively low abundance, it was perceived as a medium-weak smelling compound in the sample headspace.

Wasabi
A total of 43 compounds were identified or tentatively identified in the headspace of wasabi roots (Table 2) with 30 compounds newly described for the species, making this a significant step forward in the understanding of aroma composition in wasabi roots. Two peaks of near-identical spectra were observed and identified as allyl ITC (5/6, 8.6%/52.1%) and were the most abundant compounds, which agrees with previous observations [7]. It is unknown why two distinct peaks were formed in this manner, and further investigation may be required to determine the isomeric differences responsible for the separation. 4-Pentenyl ITC (14, 12.4%) was also found to be high in terms of overall relative abundance.
Despite having near identical spectra, compounds 5 and 6 presented distinct differences in aroma and intensity. Peak 6 was characterised as being very pungent (Table 3) and having garlic, mustard, and horseradish-like qualities. Allyl ITC is one of the most well characterised ITCs and is well known for these properties [1,48,63] (Figure 2). Peak 5 by contrast had no discernible aroma in wasabi but was apparent in horseradish (see Section 3.1.3). 4-Pentenyl ITC likewise exhibited a pungent aroma and strong odour intensity but also had peppery, sulphurous, and musty notes. This compound is commonly reported in Brassicaceae crops [69]. 3-Butenyl ITC (11,4.2%) was scored as a high odour intensity compound, despite its much lower relative abundance and was described as having a pungent, green, and aromatic odour.
Several other GHP odorants are newly reported in wasabi including cyclopropane ITC, isoamyl ITC, pentyl ITC, 1-isothiocyanato-4-methylpentane, and benzyl ITC. Cyclopropane ITC (7, 0.1%) is likely to be a cyclic reaction product of allyl ITC and has been previously reported in brown mustard [49] (Figure 2). To our knowledge, no odour description of this compound has been previously made, but assessors described it as sulphurous, horseradish, garlic, and onion-like ( Table 3). The high odour intensity score indicates that it is a significant component of wasabi aroma. Isoamyl ITC (13, 0.4%) was described as having a pungent grassy aroma and being of high odour intensity. This compound is not commonly reported in Brassicales species, but it is used as a food additive [70]. Most ITC compounds are noted for their sulphurous and mustard-like potency in Brassicales; however, the contribution of grassy aroma ITCs to volatile compound bouquets has not been previously appreciated or fully understood. Pentyl ITC (15, 0.1%) and 1-isothiocyanato-4methylpentane (16, <0.1%) were observed and shared the same odour characteristics as in 'salad' rocket (see Section 3.1.1.). Benzyl ITC (22, <0.1%) was reported to have a rotten grass and cooked aroma of medium-weak intensity. Similar to isoamyl ITC, this compound is not regularly reported as a constituent of Brassicales headspace, but these data indicate that even in very low relative abundance, it is odour active.
Another interesting compound was also found in wasabi headspace: cyclohexyl isocyanate (101, <0.1%; Figure 2). This has been previously reported in black mustard [53], though it is unclear if it is related to or derived from GHPs. Assessors perceived this odour having a medium-strong intensity and described it as peppery, cooked, and potato-like (Table 3). This is a tentative new odour description for this compound, and our data suggest it to be an important constituent of wasabi aroma.
Two additional compounds not previously identified in wasabi were 1-octen-3-ol and eucalyptol. 1-Octen-3-ol (34, <0.1%), despite its very low relative intensity in root tissue headspace (Table 2, Figure 2), exhibited a high odour intensity imparting a mushroom-like odour in agreement with previous descriptions [50]. Eucalyptol (97, <0.1%), previously observed in Brassicales crops [18] but not in wasabi (Figure 2), was found to have a mediumstrong, characteristic eucalyptus, and mint aroma, and it is likely an important component of the overall volatile bouquet.
As stated in Section 3.1.2, allyl ITC was identified as two distinct peaks (5 and 6). As in wasabi, 6 was of the greatest abundance and odour intensity, producing a very strong, pungent, garlic, mustard, and horseradish-like aroma (Table 3), whereas 5 produced a medium intensity, pungent horseradish smell. Thus far, the presence of two peaks has not been addressed or explained satisfactorily within the literature, with only one previous paper reporting the same phenomenon of separate and distinct allyl ITC peaks [71]. Secbutyl ITC (9) produced a medium-weak intensity aroma that was vegetative and radish-like ( Table 3). The compound was also present in wasabi at a medium intensity. The compound has been previously reported in horseradish as having green, chemical, and mustard like aromas [21], and it is known to activate the human Transient Receptor Potential Ankyrin 1 (TRPA1). This receptor is known to act in response to environmental irritants, and several ITCs identified in this study are known to activate it to varying degrees (isopropyl ITC, 3; isobutyl ITC, 10; allyl ITC, 5/6; 3-butenyl ITC, 11; 4-pentenyl ITC, 14; benzyl ITC, 22; phenylethyl ITC, 24; [72]). Phenylethyl ITC (24) is known to be a key constituent of horseradish aroma, and our data are in agreement with previous reports [73]. Assessors described the compound as radish and gooseberry-like, with a sweet note. It had a high odour intensity and contributed significantly to the odour profile of roots. Likewise, 4pentenyl ITC (14) was observed to have the same odour attributes as previous reports [1] and those found for wasabi in this study, but at a lower intensity. By contrast, pentyl ITC (15) was present at much lower relative intensities to other GHPs (0.2%, Table 2) but produced a strong, green, rotten, and cabbage-like aroma.
Thiiraneacetonitrile (77) has been previously reported in horseradish [22] and is an epithionitrile hydrolysis product of sinigrin. To our knowledge, no previous studies have described the odour of this compound. We found it to have a sweaty, gas-like aroma of medium intensity (Table 3).
2-Isopropyl-3-methoxypyrazine (102, 0.1%) and 2-sec-butyl-3-methoxypyrazine (106, 0.4%) have been previously described and characterised in horseradish roots [25] as having green and pepper-like aromas. Our data agree with previous reports but found the compounds to be of very high aroma intensity, despite relatively low abundances within the headspace (Table 3). Assessors described the compounds as rotten, earthy, potato-like, and vegetative.
We report several compounds previously unidentified in horseradish including GHPs, isocyanates, alcohols, aldehydes, and a ketone and ester. As in wasabi root, cyclopropane ITC (7, 0.1%) produced an intense aroma containing horseradish, garlic, onion, and sulphur notes (Table 3). Therefore, it is likely to be a significant contributor to root odour and the volatile profile, despite its very low abundance, which may be a reason why it has not been previously detected and/or reported.
As discussed in Section 3.1.2, it is unknown if the presence of isocyanates is linked with GSLs and their hydrolysis products. Allyl isocyanate (99, <0.1%) and phenethyl isocyanate (107, 0.4%) were both observed for the first time in horseradish roots. Given that high abundances of allyl ITCs (5/6) and phenethyl ITC (24) were observed, it seems likely that isocyanates may be derived from them and/or directly from parent GSLs. Isocyanates are not commonly reported in the literature, and their formation may be because of asyet-unstudied enzymatic or post-hydrolysis modification processes. Allyl isocyanate was described as having a weak musty and burnt plastic aroma; and phenethyl isocyanate was described as being pungent, with ground pepper and horseradish-like quality at a medium intensity. We are not aware of any previous odour descriptions for these compounds, so these are tentative new characterisations.
In the esters group, methyl salicylate (60, <0.1%), a common compound throughout the plant kingdom, has previously been identified in Brassicales as part of systemic acquired resistance response to herbivory [75], and it was identified for the first time as a volatile constituent of horseradish headspace ( Table 2). Its aroma is characteristic of, and present in, plants such as wintergreen. Assessors described its odour as medicinal and camphorous at a medium intensity (Table 3). Ethyl decanoate (61, <0.1%), known to be present in B. oleracea [59], was described by the assessors as green and waxy (Table 3; [65]). Benzyl tiglate (113, <0.1%) was reported at low relative abundance, but a perceptible musty aroma was apparent for this compound.
The presence of D-limonene (96, 0.1%) is reported for the first time in horseradish root. It is known to be a constituent of B. oleracea headspace [76], but its sensory contribution to Brassicales is not well defined. Assessors found this compound to have a medium-weak intensity aroma of lemon and being vegetable-like (Table 3 [ 57]). This agrees with previous descriptions of the odour properties of the compound.
Finally, 6-methylquinoline (111, <0.1%), an aromatic compound, produced an unpleasant hydrogen sulphide and egg-like aroma of medium-weak intensity, and it has not been previously reported.
1-Penten-3-ol is a compound present widely in Brassicales species [77]. It exhibited a high intensity in watercress leaves, producing a sulphurous and cabbage-like aroma. These attributes are often attributed to ITCs and other sulphur-containing compounds; however, our data suggest that some of these characteristics in watercress could be attributed to this alcohol. (Z)-3-Hexen-1-ol by comparison was much higher in relative abundance but produced a medium intensity aroma that was green and radishy (Table 3).
Phenethyl ITC (aroma attributes described in Section 3.1.2.) had one of the highest intensity aromas in watercress, along with phenylacetaldehyde (52, 0.2%). The latter, similar to phenethyl ITC, is derived from phenylalanine, but occurs in much lower abundance (Table 2, Figure 2). Its aroma was described as honey-sweet (Table 3) and is likely a significant contributor to watercress odour that has previously gone unrecognised.
Several sulphur-containing compounds, aldehydes, alcohols, ketones, not previously identified in watercress were identified. Methyl thiocyanate (2, 0.5%) imparted a sulphury and oniony note, with a medium intensity. This compound is known to be a GSL hydrolysis product of GCP (methyl GSL), but as will be discussed in Section 3.2, this compound was not detected in the UPLC-MS/MS analysis. Therefore, we suggest that it is not directly derived from this GSL and may be a degradation product of other GSL hydrolysis products within the tissues and headspace of the tested Brassicaceae.
Cyclopentyl-1-thiaethane (8, 0.5%) was detected, and uniquely present in watercress compared with the other three species tested ( Table 2). Little is known about this compound in a biological context. It produced a sweaty, sulphury, medium-weak intensity aroma that is tentatively described in this species for the first time (Table 3).
One ester, (Z)-pent-2-en-1-yl acetate (57, 0.1%), was only detected in watercress, and it produced a medium-weak aroma. It was described as sulphury and rotten, and we are not aware of any previous odour attributes associated with this compound. As such, this is a tentative first description.
Octyl ITC (21, 0.2%) has been previously identified in horseradish [64] but not watercress to our knowledge. Its exact derivation and parent GSL are unclear in the literature, though watercress has been reported to contain glucohirsutin (GHS, (RS)-8methylsulfinyl)octyl GSL; [38]). This will be discussed further in Sections 3.2 and 3.3. Assessors found the compound to be of medium aroma strength having a green and vegetative character. Again, we are unaware of previous odour descriptions for this compound.
Of note is the high abundance of PEN (m/z 388). It seems unlikely that a GSL of such relatively high concentration has gone undetected in previous analyses. Therefore, we postulate that previous studies may have attributed the negative ion mass incorrectly to that of PRO, which is also m/z 388 ( Table 1). The authentic standard of PRO did not match the retention time or MS/MS spectra of PEN, and it was found in only very low concentrations by comparison. While PEN is only a tentative identification (due to the possibility of other isomeric GSLs such as glucojiaputin and 3-methylbutyl GSL), the presence of pentyl ITC (17) within the headspace of rocket makes this the most likely identification. See Section 3.3 for further discussion.

Discrepancies between Identified Glucosinolate Hydrolysis Products and Glucosinolate Profile Precursors
There is often an 'elephant in the room' regarding volatile GSL hydrolysis products and reported GSL profiles in Brassicales crops: there are often GSLs found with no corresponding hydrolysis products, or more troublingly, hydrolysis products observed but no GSL precursor. Table 5 presents a list of the GSL-derived compounds identified within the headspace of 'salad' rocket, wasabi, and watercress, alongside their expected GSL precursors. It is apparent from our data that the present study is no exception when it comes to discrepancies of this nature, and there is a need to find a robust solution to prevent the inaccurate reporting of both GSLs and their volatile hydrolysis products.
As discussed in previous sections, compounds such as methyl thiocyanate (2) may be produced from degradation of other hydrolysis products. Others such as the presence of sec-butyl ITC (9) in rocket, butyl ITC (12) in wasabi, and octyl ITC (21) in watercress cannot be so easily explained. There are several explanations with varying levels of likelihood: firstly, the most likely is that the ITCs and other GSL hydrolysis products have been identified incorrectly, and that they belong to other parent GSLs present within the analysed tissues. Despite researchers' best efforts to obtain authentic standards to test and match MS profiles, this is not always possible or affordable, and so there is a heavy reliance upon reference libraries. These are often incomplete and not always accurate. It is also possible that the high temperatures utilised in GC-MS cause thermolytic reactions to occur in GHPs, thus yielding compounds not 'naturally' produced by tissues. The next possibility is that the GSLs responsible for producing hydrolysis products are below the LOD of the MS/MS method. In this case, this is unlikely, as the LOD for each standard β-ionone (98). Several non-GSL derived compounds also share similar pungent odour characteristics with GHPs, indicating that the latter may not be the only source of these sensations in Brassicales crops. There is also a clear need for the improvement of mass spectral libraries and the availability of GSL and GHP standards in order to overcome discrepancies between GSL profiles and the reported volatiles derived therefrom.