S‐Methyl‐l‐Cysteine Sulfoxide: A Hidden Layer of Defences Against Herbivorous Insects in Brassicaceae

ABSTRACT

Plants defend themselves against herbivorous insects through diverse morphological and biochemical traits. Non‐protein amino acids (NPAAs) are an important component of the plant metabolome, although their defensive function remains largely unexplored. Here, we investigated the role of S‐methyl‐l‐cysteine sulfoxide (SMCSO), a sulphur‐containing NPAA accumulated in Brassicaceae, in mediating plant defence against herbivorous insects. SMCSO was quantified in inflorescences, leaves (young and old) and roots (primary and secondary) of 14 Brassicaceae species. Additionally, feeding tests on artificial substrates supplemented with physiological SMCSO levels were conducted with both generalist and specialist herbivorous insects feeding on one of the different plant parts studied. In line with the optimal defence theory, we found higher SMCSO levels in reproductive than vegetative tissues, and in young leaves and primary roots compared to old leaves and secondary roots, respectively. SMCSO also exerted a consistent phagodeterrent effect on generalist herbivores, while specialists showed variable responses from deterrence to stimulation. This study provides the first evidence of the influence of this compound on herbivore feeding behaviour, with effects depending on the diet breadth. These findings broaden our understanding of plant chemical defence complexity and highlight the ecological role of NPAAs in plant–insect interactions.

Summary Statement

We show that S‐methyl‐l‐cysteine sulfoxide (SMCSO), an abundant sulphur‐containing non‐protein amino acid found in Brassicaceae, accumulates in fitness‐relevant tissues and influences the feeding behaviour of herbivorous insects in a diet breadth‐dependent manner, revealing its previously overlooked role in plant chemical defence.

1. Introduction

Plants rely on multiple defence strategies to overcome the challenges posed by herbivores, among which chemical defences are central (Mithöfer and Boland 2012). Non‐volatile specialised metabolites such as cyanogenic glycosides, alkaloids, polyphenols or terpenoids are known to protect plants against herbivores by acting as deterrents or toxins (Schoonhoven et al. 2005; Mithöfer and Boland 2012). Next to these metabolites, non‐protein amino acids (NPAAs), which are structural analogues of proteinogenic amino acids not encoded for protein synthesis, may also contribute to plant defence (Huang et al. 2011). NPAAs can cause adverse biological reactions in insects through misincorporation into proteins and disruption of metabolic processes (Rosenthal 2001; Bown et al. 2006; Samardzic et al. 2021). However, although numerous NPAAs have been described, few studies have investigated their direct defensive function against herbivores through behavioural effects such as deterrence (Huang et al. 2011; Jander et al. 2020).

Glucosinolates have historically been the prime focus of chemical ecologists studying brassicaceous plants. Indeed, these specialised metabolites have a wealth of biological effects, including defensive ones (Kumar et al. 2017; Eugui et al. 2022; Malhotra et al. 2023). Besides glucosinolates, Brassicaceae accumulate the sulphur‐rich NPAA S‐methyl‐l‐cysteine sulfoxide (SMCSO; a.k.a methiin), which is often found in greater levels than glucosinolates (i.e., 1%–4% vs. 0.1%–0.6% dry weight, respectively) (Mae et al. 1971; Marks et al. 1992). Also found within the plant tissues of many members of the Fabaceae (Leguminosae) and Alliaceae (∼Liliaceae) families (Rose et al. 2005; Sors 2008), SMCSO has primarily been studied for its role in the organoleptic qualities of plant products and its potential health benefits for humans (Coode‐Bate et al. 2019; Hill et al. 2023; 2025). Few investigations however have sought for an ecological role of SMCSO (Lee et al. 2024) and its function, if any, remains largely unexplored.

Three facts suggest that SMCSO may play a defensive role in plants. First is its spatial distribution across plant tissues. Given the metabolic cost of defence production, the optimal defence theory predicts that constitutive defences should be allocated to tissue of high value and vulnerability (e.g., reproductive organs, young leaves and primary roots) (McKey 1974). Consistently, it has been repeatedly reported that SMCSO is present in greater levels in reproductive organs than in vegetative ones, in young than in old leaves, and in primary than in secondary roots, although systematic investigations are lacking (Morris and Thompson 1956; Whittle et al. 1976; Dan et al. 1999; Kubec and Dadáková 2009).

Second is the spatial separation between SMCSO and its catabolic enzyme. Indeed, plant defences are often stored in a non‐active form that is spatially separated from its bioactivating enzymes unless tissues are damaged, which limits the production of autotoxic compounds (Bones and Rossiter 2006; Morant et al. 2008; Shimada et al. 2018). In the case of SMCSO, contact with its vacuole‐residing catabolic enzyme (i.e., a cysteine sulfoxide lyase) occurs only after tissue disruption. The following reactions lead to the formation of volatile compounds characteristic of the cabbage flavour, including dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS) (Figure 1; Friedrich et al. 2022; Andernach et al. 2024), also known for their neurotoxic properties (Dugravot et al. 2003; Danner et al. 2015).

Figure 1.

Enzymatic hydrolysis of S‐methyl‐l‐cysteine sulfoxide (SMCSO) leading to the formation of volatile organic compounds, i.e., dimethyldisulfide (DMDS) and dimethyltrisulfide (DMTS). Highlighted are the compounds discussed in this study. Adapted from Friedrich et al. (2022).

Finally, the ingestion of high levels of SMCSO has been associated with severe health issues in ruminants, largely due to dimethyl disulphide formation. This phenomenon was historically referred to as “kale anaemia factor” (Grant et al. 1968; Whittle et al. 1976; Smith 1980) leading to the development of low‐content cultivars for animal food (Bradshaw and Wilson 2012; Bradshaw 2021). However, in humans, where SMCSO intake is substantially lower, emerging evidence even points to possible health benefits, including antioxidant, antihyperglycemic, and anti‐inflammatory effects (Castro et al. 2021; Lemos et al. 2021; Hill et al. 2023; 2025). In non‐ruminants, potential toxicity thresholds remain unclear, and although a defensive role of this compound against herbivorous insects has been suggested, no study has tested this hypothesis so far.

Here, we investigated whether SMCSO acts as a defence against herbivorous insects. We hypothesised that if SMCSO is indeed defensive, it should be allocated to tissues of high value and vulnerability, as expected by the optimal defence theory. To test this hypothesis, we assessed its distribution within reproductive parts (inflorescences), leaves (young and old) and roots (primary and secondary) of 14 wild and cultivated species along the Brassicaceae phylogeny. From this screening, contrasted physiological levels of SMCSO were identified for each tissue across the plant panel studied. Using a pure SMCSO standard, feeding tests with artificial substrates supplemented with these levels were conducted to assess their effect on a panel of generalist and specialist herbivorous insects that feed on different plant parts (i.e., inflorescences vs. leaves vs. roots). As expected from the generalist‐specialist paradigm, generalists (i.e., feeding on different plant families) should be clearly affected by specific defence traits produced by plants while specialists (i.e., feeding on closely related plant taxa belonging to the same family) could show negative, but also neutral or even positive effects due to targeted adaptations (Ali and Agrawal 2012). Our results shed light on the ecological role of this major component of the Brassicaceae metabolome and pave the way for future research on the evolutionary history of SMCSO as a defence in this plant family and counteradaptations in specialist herbivores.

2. Materials and Methods

2.1. Plants

To investigate the evolution of SMCSO as a defence, its spatial distribution across plant tissues was assessed in 14 wild and cultivated species along the Brassicaceae phylogeny. This panel included 11 species from the Brassicodae supertribe (i.e., Brassica carinata, B. juncea, B. macrocarpa, B. montana, B. napus, B. nigra, B. oleracea B. rapa and Raphanus sativus, Sinapis alba and S. arvensis), one specie from the Camelinodae supertribe (i.e., Camelina sativa), one species from the Hesperodae supertribe (i.e., Hesperis matronalis) and one species from the Heliophilodae supertribe (i.e., Iberis amara) (Hendriks et al. (2023) (Figure 2A and Supporting Information: Table S1). Seedlings were grown in 3.5 × 3.5 × 5 cm cells filled with fertilised substrate (Premier Tech Horticulture) in a climatic chamber (20 ± 1°C, 70 ± 10% RH, 8:16 L:D photoperiod) and watered twice a week with a fertiliser solution (N‐P‐K: 2.5‐5‐2.5). Plants were sampled at the ‘green‐yellow bud stage’, that is, BBCH 55–57 (Lancashire et al. 1991), except for B. macrocarpa, B. montana, B. oleracea and H. matronalis which were harvested earlier at full vegetative development, that is, BBCH 39, as these species do not produce inflorescences without vernalisation.

Figure 2.

The level of SMCSO varies depending on the species and tissue. (A) Phylogenetic tree depicting relationships between species selected for this study (supertribes according to Hendriks et al. (2023)). Accessions and origins of selected species are indicated in Supporting Information: Table S1. (B) Estimated marginal mean (EMMean) level (± SE, µmol/g DW) of SMCSO in whole plants according to the species. Letters indicate significant differences. n = 32–40 per species. (C) Heatmap of mean normalised levels of SMCSO (levels autoscaled by species from zero (white) to high (dark green)) across tissues of the selected plant species. Mean ± SE levels (µmol/g DW) of SMCSO are indicated for each species and tissue. Tissues sampled from 4‐ to 12‐week‐old plants of the selected 14 species. n = 8 per treatment with two plants per replicate. SE, standard error; SMCSO, S‐methyl‐l‐cysteine sulfoxide. [Color figure can be viewed at wileyonlinelibrary.com]

2.2. Insects

To assess the effect of SMCSO on herbivorous insects, six insect species feeding on different plant parts were used (Table 1). Generalist insects included adults of the pollen beetle Brassicogethes aeneus F. (Coleoptera: Nitidulidae) as flower bud feeders, third‐instar caterpillars of the cotton leafworm Spodoptera littoralis Boisduval (Lepidoptera: Noctuidae) as leaf feeders, and third‐instar larvae of the rose chafer Pachnoda marginata Drury (Coleoptera: Scarabaeidae) as root feeders. Specialist insects included adults of the cabbage seedpod weevil Ceutorhynchus assimilis Paykull (Coleoptera: Curculionidae) as flower bud feeders, adults of the cabbage stem flea beetle Psylliodes chrysocephala L. (Coleoptera: Chrysomelidae) as leaf feeders, and first‐instar larvae of the cabbage root fly Delia radicum L. (Diptera: Anthomyiidae) as root feeders.

Table 1.

SMCSO levels tested for feeding tests with each insect species.

Tissue consumed	Diet breadth	Species	SMCSO levels tested (µmol/g DW)
Flower buds	Generalist	Brassicogethes aeneus	SMCSO levels found in flower buds: Lowest: 52.3 (found in B. napus) Highest: 125.4 (found in S. alba)
	Specialist	Ceutorhynchus assimilis
Leaves	Generalist	Spodoptera littoralis	SMCSO levels found in leaves: Lowest: 5.45 (found in C. sativa) Highest: 176.87 (found in B. oleracea)
	Specialist	Psylliodes chrysocephala
Roots	Generalist	Pachnoda marginata	SMCSO levels found in roots: Lowest: 2.02 (found in S. alba) Highest: 22.77 (found in R. sativus)
	Specialist	Delia radicum

Abbreviation: S‐methyl‐l‐cysteine sulfoxide.

Delia radicum and P. chrysocephala originated from laboratory colonies and were maintained as described in Menacer et al. (2021) and Giguère et al. (2025), respectively. Spodoptera littoralis eggs were kindly provided by Emmanuelle Jacquin‐Jolly (iEES, Paris, France) and larvae were reared as described in Rharrabe et al. (2014). Pachnoda marginata was purchased from a commercial supplier (Grillonshop, Flaviac, France) and maintained at 22 ± 1°C, 60 ± 10% RH, 8:16 L:D. Larvae were used immediately upon receipt without adding any food other than the food provided by the supplier (i.e., decomposed wood). Brassicogethes aeneus and C. assimilis were collected from unsprayed winter oilseed rape crops (Brittany, France), maintained at 18 ± 1°C, 70 ± 10% RH, 8:16 L:D and fed with field‐collected oilseed rape inflorescences.

2.3. Chemicals

Methanol (UHPLC‐MS grade, ≥ 99.9%), chloroform (UHPLC‐MS grade, ≥ 99.5%) and ultrapure water (UHPLC‐MS grade) were purchased from Carlo Erba Reagents (Val de Reuil, France); BABA (10 mM) and adonitol (20 mM) used for the internal standard mixture, as well as a commercial mixture of 18 amino acids (100 µM) used for the external amino acid standards, were purchased from Sigma‐Aldrich (St. Louis, MO, USA); S‐methyl‐l‐cysteine sulfoxide (SMCSO) standard (≥ 98%) was obtained from Cayman Chemical Company (Ann Arbor, MI, USA); AccQ‐Tag Ultra Derivatization Kit was obtained from Waters (Milford, MA, USA); acetonitrile (UHPLC‐MS grade, ≥ 99.9%) was purchased from Carlo Erba Reagents (Val de Reuil, France).

2.4. Sample Preparation and SMCSO Quantification

To assess the distribution of SMCSO across plant tissues, the inflorescence, the two youngest leaves, the two oldest leaves, the primary root and the secondary roots were harvested from the same plants, flash‐frozen in liquid nitrogen and freeze‐dried for 72 h. Frozen samples were then ground using a bead‐based homogeniser (BeadBugTM, stainless steel balls 2.8 mm) for 1 min at 3500 rpm. For each tissue, eight batches of two plants each were assembled by mixing equal levels of dry powders. Plants were paired identically for all tissues.

SMCSO was extracted from 1 to 5 mg DW, by addition of 1 ml of acidified methanol (1% formic acid). Samples were then vacuum‐dried, resuspended in 50 µL of ultrapure water, vortexed, placed in an ultrasonic bath for 5 min, and centrifuged for 5 s at room temperature. SMCSO was finally derivatized as in Ourry et al. (2018) and quantified on an Acquity UPLC‐DAD system (Waters Corporation) according to Jubault et al. (2008). A SMCSO standard was used to generate calibration curves. Confirmation of the identity of the compound was achieved using an Acquity UPLC‐DAD‐MS (TQD) system. As part of the method, all other free amino acids were quantified alongside SMCSO.

2.5. Feeding Tests

To assess the potential deterrent effect of SMCSO on insects, dual‐choice tests using artificial substrates were conducted. Artificial substrates consisted of 3% agar disks (Ø = 5 mm, h = 2 mm) supplemented with sucrose as a natural feeding stimulant (Chapman 2003), to which ‘physiological’ levels of SMCSO were added depending on the experiment. SMCSO levels were the absolute lowest and highest ones found for each tissue across the plant panel studied, that is, for flower buds, leaves (old and young leaves combined) and roots (primary root and secondary roots combined), excluding species for which no SMCSO was detected (see Section 3 and Table 1). Starvation durations, sucrose levels and the number of individuals per test were defined for each insect species based on preliminary experiments (Supporting Information: Table S2).

For all insect species, individuals were placed in Petri dishes (Ø = 35 mm, except for D. radicum larvae Ø = 26 mm and P. marginata larvae Ø = 55 mm) and the number of individuals feeding on each disk was counted every 5 min for 45 min. Each individual feeding on a disk was counted as one feeding observation. Observations were made using a numerical binocular microscope (Adonstar™ ADSM302) for D. radicum larvae. Based on preliminary observations, individuals were actually feeding when on or under a disk. Three experiments were conducted per species: control versus lowest SMCSO level, control versus highest level, lowest versus highest level. Thirty replicates were performed per experiment.

2.6. Statistical Analyses

All statistical analyses were performed in the R software version 4.5.0 (R Core Team 2025). SMCSO levels in whole plants were calculated as the sum of the levels in each plant tissue. These whole‐plant levels were then compared among species using a Wald test applied to a Linear Mixed Model (LMM) with the tissue as random factor (R packages ‘car’ (Fox et al. 2001) and ‘lme4’ (Bates et al. 2015). SMCSO levels within plants were analysed using a Wald test applied to an LMM, with tissue as independent variable and species as random factor. Response variables were systematically square‐root transformed to ensure a better model fit. For feeding tests, the number of feeding observations per disk over the 45‐min observation period was summed. This total number of feeding observations was then compared between the two treatments, separately for each experiment and insect species, using a Wald test applied to a Generalised Linear Mixed Model (GLMM) (distribution: negative binomial, link function: log). The treatment was used as fixed factor and the replicate as random factor. When needed, pairwise comparisons of estimated marginal means (EMMeans) were performed using the R package ‘emmeans’ (Lenth et al. 2025) and p‐values adjusted using the false discovery rate correction (Benjamini and Hochberg 1995).

3. Results

3.1. SMCSO Allocation in Brassicaceae Is Species and Tissue‐Dependent

Among all free amino acids analysed, SMCSO was one of the most abundant (see Supporting Information: Figure S1 for the mean level of all free amino acids, including SMCSO, in whole plants and in each tissue). Significant differences in the total level of SMCSO were observed according to the species (χ² = 2020.4, df = 13, p < 0.001, Figure 2B), with two species not producing SMCSO regardless of the tissue, namely I. amara and H. matronalis, and variations up to two folds were observed among other species (Figure 2B). In all species producing SMCSO, a tissue‐dependent allocation was observed (Figure 2C).

3.2. SMCSO Is Preferentially Allocated to Tissues of High Value and Vulnerability

The total level of SMCSO in whole plants was not significantly different whether the plants were sampled at the flower bud stage or at the full vegetative stage (χ² = 0.12, df = 1, p = 0.728). At both developmental stages, significant differences in SMCSO levels were observed between tissues (flower bud stage: χ² = 936.41, df = 4, p < 0.001; full vegetative stage: χ² = 385.57, df = 4, p < 0.001) (Figure 3, see Supporting Information: Figure S2 for analyses per plant species). In species sampled at the flower bud stage, SMCSO was more concentrated in reproductive tissues than in vegetative tissues, with 1.7–14.6 times higher levels in the former. In all species, SMCSO was more concentrated in leaves than in roots. Finally, higher levels were found in young leaves compared to old leaves, and in the primary root compared to secondary roots (Figure 3 and Supporting Information: Figure S2). Since the distribution of SMCSO in the plant could be the result of a more general allocation pattern of free amino acids, correlations between the level of SMCSO and the level of each free amino acid were assessed, both in whole plants and in each tissue (Supporting Information: Figure S3). Although significant correlations were observed between SMCSO and some specific amino acids (e.g., α‐alanine, asparagine and methionine), these were not consistent across all tissues. The consistent allocation pattern of SMCSO was then unique to this amino acid.

Figure 3.

SMCSO is preferentially allocated to reproductive tissues, to young leaves and to the primary root. Estimated marginal mean (EMMean) level (± SE, µmol/g DW) of SMCSO according to the tissue (inflorescences [yellow]), young and old leaves (green), tap and fine roots (brown) all species confounded, for species sampled at the flower bud stage (left), and species sampled at the full vegetative stage (right). Different letters indicate statistically significant differences between inflorescences, leaves and roots, while asterisks indicate significant differences between two treatments (*p < 0.05, **p < 0.01 and ***p < 0.001). n = 64 per tissue with two plants per replicate for species sampled at the flower bud stage, n = 32 per tissue with two plants per replicate for species sampled at the full vegetative stage. SE, standard error; SMCSO, S‐methyl‐l‐cysteine sulfoxide. [Color figure can be viewed at wileyonlinelibrary.com]

3.3. SMCSO Is Consistently Deterrent for Generalist Herbivores, but Ranges From Deterrent to Stimulant for Specialist Species

Among the generalist insects tested, the flower bud feeder B. aeneus, fed significantly less on substrates supplemented with SMCSO compared to the control, and showed a preference for the lowest SMCSO level compared to the highest (Figure 4, see Supporting Information: Table S3 for detailed results). Similarly, the leaf feeder S. littoralis and the root feeder P. maginata fed significantly less on substrates supplemented with the highest level of SMCSO compared to the control or to the lowest level, while no difference was observed when given a choice between the lowest level and the control.

Figure 4.

SMCSO is mostly deterrent for generalist insects, while it ranges from deterrent to stimulant for specialist species. Estimated marginal mean (EMMean) total number (± SE) of feeding observations on each disk per replicate over the 45‐min feeding test. Disks were artificial substrates supplemented with sucrose only (control [white]) or with physiological levels of SMCSO (i.e., the lowest [light colour] and highest levels [dark colour] found in inflorescences [yellow], young and old leaves [green], tap and fine roots [brown]) in the plant panel studied; Table 1. Results are shown for generalist (left) and specialist (right) insects feeding on the different plant parts (i.e., inflorescences, leaves and roots). Detailed statistics of Wald tests applied to GLMM are indicated in Supporting Information: Table S3. Asterisks indicate significant differences (*p < 0.05, **p < 0.01 and ***p < 0.001). n = 30 per experiment. GLMM, Generalised Linear Mixed Model; SE, standard error; SMCSO, S‐methyl‐l‐cysteine sulfoxide. [Color figure can be viewed at wileyonlinelibrary.com]

Among the specialist insects tested, the leaf feeder P. chrysocephala fed significantly less on the substrate supplemented with the highest level of SMCSO when compared to the lowest one, while no significant difference was observed when SMCSO‐supplemented substrates, at either level, were compared to the control. In contrast, the flower bud feeder C. assimilis fed significantly more on disks supplemented with the highest level of SMCSO than on the control (Figure 4, see Supporting Information: Table S3 for detailed results), whereas no significant difference was observed when given a choice between the lowest level and the control or between the two SMCSO levels. Strikingly, the root feeder D. radicum fed significantly more on SMCSO‐supplemented disks regardless of their level compared to the control, and preferred the highest level than the lowest one.

4. Discussion

NPAAs are an important but often overlooked component of the plant specialised metabolome. Although their biochemical diversity is well described, their ecological functions, especially the mediation of interactions with herbivores through defence mechanisms, remain underexplored. This study reports a non‐uniform distribution of the sulphur‐containing NPAA SMCSO across Brassicaceae, with a specific allocation to tissues of high value and vulnerability. It provides the first evidence of the influence of this compound on the feeding behaviour of herbivorous insects, with distinct effects depending on their diet breadth. The ecological and evolutionary relevance of our findings are discussed below.

Plant chemical defences require a substantial investment of energy and resources. For instance, glucosinolate synthesis by Brassicaceae is estimated to consume 15% of the total photosynthetic energy of the plant (Bekaert et al. 2012). Therefore, rather than maximising defence production in all tissues, which would divert resources from other processes (i.e., growth and reproduction), plants should allocate them to tissues that are crucial for their fitness, that is, reproductive tissues, young leaves and primary root (McKey 1974; Rhoades and Cates 1976; Tsunoda et al. 2017). Here, we found that the distribution of SMCSO within Brassicaceae was consistent with these predictions and with previous observations (Morris and Thompson 1956; Mae et al. 1971; Whittle et al. 1976; Dan et al. 1999). This intra‐plant allocation pattern, which was not only the result of a general pattern of amino acids allocation, is similar to that observed for well‐known defence compounds such as alkaloids, proteinase inhibitors, glucosinolates or DIMBOA (Bravo and Copaja 2002; Damle et al. 2005; Alves et al. 2007; Tsunoda et al. 2017, 2018; Gershenzon and Ullah 2022). This pattern was also consistent in all species sampled, regardless of whether they were sampled at the flower bud stage or at the full vegetative stage. Total levels of SMCSO were found similar between the two developmental stages, although previous studies have reported an overall increase in SMCSO with plant maturity, after flowering or during regrowth (Arnold and Lehmkuhler 2014). The accumulation of SMCSO in young leaves at the vegetative stage may reflect a potential storage in these tissues before flowering, and a subsequent reallocation towards inflorescences. This would be consistent with strategies observed in monocarpic plants, where both nutrients and chemical defences such as glucosinolates are known to be redirected to flowers and seeds where they enhance and protect the reproductive output (Brown et al. 2003; van Leur et al. 2006). The pattern observed here is therefore not only consistent with tissue value and vulnerability, but may also align with an adaptive defence strategy prioritising reproductive success. Further investigation into the largely unresolved biosynthetic SMCSO pathway, including its production sites, transport mechanisms and regulatory processes, is however essential for a better understanding of its production and allocation dynamics.

When consumed at high doses, SMCSO has toxic effects on ruminants, rats and fowls (Smith 1980; Steven et al. 1981; Maxwell 1981; Stoewsand 1995; Hill and Roberts 2020). It also has antimicrobial properties against plant pathogens (Kyung and Fleming 1994; Pedras and Yaya 2015). Strikingly, very few studies have suggested an influence on plant interactions with herbivorous insects. Only can be mentioned Ourry et al. (2018) who reported an increase in SMCSO content in response to herbivory by D. radicum larvae, and Hervé et al. (2014) who found a negative correlation between SMCSO level and egg production in B. aeneus. More recently, Lee et al. (2024) showed that while SMCSO had no lethal effects on bees, chronic exposures led to significant reductions in body and head weights. The present screening facilitated the identification of contrasted levels of SMCSO in each tissue studied, which were then used to assess their effect on the feeding behaviour of a panel of herbivorous insects. Consistent with predictions based on their diet breadth (Ali and Agrawal 2012), we observed a shift between generalist and specialist species. In generalists (i.e., B. aeneus, S. littoralis, and P. marginata), SMCSO consistently acted as a phagodeterrent. This clearly suggests that it can serve as an overlooked defensive compound in Brassicaceae, adding a layer of complexity to the well‐characterised glucosinolate‐based defences. It also questions the post‐ingestive effects of this compound, e.g. toxicity or digestibility reduction, which were not evaluated here. In specialist herbivores, SMCSO deterred the leaf feeder P. chrysocephala although less markedly than generalists, while it was neutral or even phagostimulant for the flower bud feeder C. assimilis, and phagostimulant in all cases for the root feeder D. radicum. The reduced feeding observed in the leaf feeder P. chrysocephala at high SMCSO levels may reflect an exposure to levels exceeding those naturally encountered by this species, as it typically feeds on plants younger than those tested here (i.e., 2–3 leaves stage; Ekbom et al. 1995) and containing lower SMCSO levels (Mae et al. 1971; Griffiths and Smith 1989; Edmands et al. 2013), but it still points toward a defensive role of SMSCO. Neutrality and stimulation observed in the flower bud feeder C. assimilis and the root feeder D. radicum are closer to what is expected for specialists. Indeed, specialised metabolites have often been diverted to host‐recognition cues in specialist herbivores (Nishida 2014). In the case of the root feeder D. radicum, feeding stimulation is consistent with the fact that SMCSO volatile breakdown products (i.e., DMDS and DMTS) act as host‐recognition cues at levels emitted by healthy plants (Lamy et al. 2018). As for many other defence compounds, a new question arises from the present results as how do specialists of Brassicaceae deal with SMCSO and its catabolites. From what is known from other defence compounds, several adaptations are possible including rapid excretion, enzymatic detoxification, and even sequestration for their own defence (reviewed in Heidel‐Fischer and Vogel 2015). Our findings highlight the need for studies investigating the effects of SMCSO on insect physiology and performance, to assess potential detrimental or beneficial (e.g., antioxidant) effects, as observed in other study systems (i.e., rodents, ruminants and humans; Lemos et al. 2021; Smith 1978; Traka et al. 2019).

Plant metabolic profiles often reflect evolutionary adaptations to selective pressures exerted by herbivorous insects (Edger et al. 2015). In this study, we observed an optimised allocation of SMCSO within plant tissues, as well as contrasting effects on generalist and specialist insects, consistent with the presence of a lineage‐specific defence mechanism in response to selective pressures from herbivores. Although SMCSO is produced by plant species from multiple families, it was found absent in H. matronalis and I. amara, suggesting possible independent losses during evolution. This suggests variable selective pressures that led to lineage‐specific changes in the SMCSO biosynthetic pathway, as observed in some Brassicaceae supertribes for glucosinolates (Bird et al. 2025). Combining data on SMCSO production across plant taxa with patterns of herbivore specialisation could offer valuable insights into the role of this compound in shaping coevolutionary interactions between plants and their animal antagonists.

In conclusion, the present study demonstrates an underappreciated defensive role of the NPAA SMCSO in Brassicaceae. To fully elucidate the ecological role of this compound in plant–insect interactions, future studies should include a broader diversity of herbivorous insects, particularly piercing‐sucking feeders which were not assessed here, as well as mutualistic insects such as pollinators (as recently initiated by Lee et al. 2024). Importantly, it appears also necessary to validate the SMCSO defensive function in planta. This will require manipulation of SMCSO levels (i.e., overexpression or suppression) independently of other metabolites to precisely determine its contribution to the plant defence. The present results broaden our understanding of the complexity and diversity of plant chemical defences and highlights the potential ecological role of NPAAs in shaping plant–insect interactions.

Conflicts of Interest

The authors declare no conflicts of interests.

Supporting information

Supporting Material Revised.

Bellec, L. , Maire C. L., Marnet N., Paty C., Cortesero A.‐M., and Hervé M. R.. 2025. “ S‐Methyl‐l‐Cysteine Sulfoxide: A Hidden Layer of Defenses Against Herbivorous Insects in Brassicaceae.” Plant, Cell & Environment 48: 8960–8969. 10.1111/pce.70179.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.