Effects of Magnesium Sulphate Fertilization on Glucosinolate Accumulation in Watercress (Nasturtium officinale)

Abstract

Watercress is a nutrient-dense, aquatic leafy vegetable with significant public health and economic potential. Hydroponically cultivated watercress can offer greater nutritional benefits due to the controlled delivery of specific nutrients. From an agronomist’s perspective, watercress has the advantage of optimized environmental resource efficiency, achieved through reduced energy, chemical, and water consumption, as well as its short cultivation cycle. Glucosinolates (GSLs) in watercress enhance sustainable agriculture by naturally protecting crops from pests and diseases, reducing the need for chemical inputs. They also increase market value and shelf-life, supporting resource-efficient and profitable farming. Within the pharmaceutical space, GSLs are well-known for their chemo preventive and anti-inflammatory properties. This review aims to summarize research findings, critically evaluate existing studies to highlight current knowledge, and identify research gaps, and to guide future investigations. The synthesis of the reviewed literature demonstrates that increased sulphate generally improves GSL content. However, not many studies have looked specifically at how magnesium sulphate (MgSO₄) affects watercress. This review highlights the specific impact of MgSO₄ on GSL production in watercress, which could provide valuable insights for optimizing nutrient management in hydroponic systems and enhancing the health benefits of this nutrient-dense crop.

1. Introduction

Different cultivation approaches, including hydroponics, have been employed to evaluate the accumulation of key metabolites and plant performance metrics such as fresh and dry biomass, leaf area, and root hair development [1]. Glucosinolates (GSLs), the principal sulphur (S) containing secondary metabolites in the Brassicaceae family, exhibit variable accumulation depending on environmental S availability and S fertilization levels [2]. Studies in model and crop species like Arabidopsis and Brassica have demonstrated that S-containing metabolites, including GSLs, are sensitive to variations in nutrient composition, especially S availability [3]. Magnesium sulphate (MgSO₄) is a widely used nutrient in hydroponic systems. Magnesium (Mg²⁺) acts as a cofactor in numerous enzymatic reactions within secondary metabolism, while sulphate (SO₄²⁻) contributes directly to GSL biosynthetic precursors [4]. Although hydroponic systems provide the advantage of precise nutrient manipulation and can be used to evaluate the biochemical sensitivity of watercress to cultivation conditions, few studies have specifically investigated how MgSO₄ influences GSL biosynthesis in watercress. Preliminary findings suggest that hydroponic conditions, including the use of microbubbles and tailored nutrient regimes, can modulate GSL accumulation in watercress [5].

Watercress’s long-standing use in traditional medicine is supported by numerous pharmacological studies highlighting its therapeutic potential [6,7]. This nutrient-dense crop exhibits a range of therapeutic effects, such as antioxidants, anti-inflammatory, heart-protective, skin-healing, antibacterial, and anticancer activities [8]. Additionally, watercress leaves can serve as natural remedies, acting as diuretic, cleansing, cough-relieving, toothache-soothing, and blood sugar-lowering agents [9]. Experimental and human trials suggest that watercress intake may enhance lipid metabolism and influence inflammatory responses associated with cardiovascular and respiratory diseases [10]. Within cancer models, watercress extracts demonstrate growth-inhibiting, cell death-promoting, and cancer-preventive activities, thus exhibiting targeted toxicity against malignant cells while safeguarding the healthy ones [11,12]. The hydrolysis product formed from GSL breakdown by myrosinases (2-Phenethyl isothiocyanate) has demonstrated anticancer properties by inhibiting phase I metabolic enzymes and enhancing the activity of phase II detoxification enzymes [13]. These medicinal properties are primarily attributed to watercress’s high content of GSLs, flavonoids, vitamin C, and carotenoids, which collectively help to promote human and animal health [14,15]. Beyond their medicinal value, these bioactive constituents are closely linked to the crop’s characteristic flavor profile. Brassica family crops, including watercress, are abundant sources of (GSLs).

Watercress is characterized by its distinctive peppery flavor and abundant phytonutrients, particularly GSLs [16]. Garden cress is incorporated into soups, sandwiches, and salads for its sharp, zesty taste. It is also consumed as sprouts, and both the fresh and dried seed pods can serve as a spicy condiment [17]. In a culinary assessment by Renna et al. [18], watercress that grows devoid of soil was regarded as the initial step in the culinary process for preparing the dishes that were part of the assessment process. Vegetables like watercress produce a wide range of specialized compounds, such as phenolics, terpenoids, alkaloids, and S-containing substances, which enhance their nutritional benefits, taste, and fragrance. Watercress particularly, is a remarkable provider of essential minerals such as Mg, iodine, copper, phosphorus, calcium, and iron, along with flavonoids, particularly kaempferol, quercetin, and rutin which are well known for their antioxidant, anti-inflammatory, and heart-protective properties [19,20]. The characteristic odor and pungency of Brassica crops are caused by isothiocyanates, which are degradation products of GSLs [21]. Importantly, these biochemical pathways are central to the health-promoting and ecological functions of GSLs.

The biosynthesis of GSLs occurs through three distinct stages as shown in Figure 1. The first stage begins with the corresponding amino acid. The carbon backbone of amino acids other than tryptophan may be extended via a series of iterative reactions that can repeat as many as six cycles. Next, the fundamental GSL scaffold is assembled. Finally, the resulting GSLs may experience numerous downstream tailoring reactions, including hydroxylation, methylation, oxidation, desaturation, or benzoyl substitution, most of which modify the side chain [22,23]. These GSLs are then subject to hydrolysis by the enzyme myrosinase, which is a critical step in the breakdown process (Figure 2a). The biosynthesis of GSLs culminates in a crucial sulfation step which provides the key sulfated functional group required for GSL biological activity (Figure 2b). Please see Section 4 “The Science Behind GSLs” for details.

2. Review Methodology

This study employs a review methodology characterized by a rigorous, transparent, and methodologically sound process for GSL biosynthesis in hydroponically grown watercress in response to. The approach encompasses the formulation of explicit inclusion and exclusion criteria, the execution of an exhaustive search across multiple peer-reviewed academic databases, critical evaluation of the methodological quality of selected studies, and the synthesis of findings to derive evidence-based conclusions or to elucidate unresolved gaps within the current body of knowledge. In preparing this review, the authors conducted a literature search using these databases: Google Scholar, Web of Science, ScienceDirect, Scopus and PubMed. The keywords used were “Brassica”, “watercress”, “glucosinolates”, “hydroponics”, magnesium sulphate and sulphur assimilation. Inclusion criteria were limited to English-language studies published between 1 January 2000 and 1 October 2025. Data extraction focused on the effects of MgSO₄ fertilization on GSL accumulation in Brassica vegetables. Additional data were extracted on genetic factors and environmental determinants of GSL levels in Brassica. The GSL biosynthesis, composition, and their implications for agriculture and human health were also explored.

The biochemical modulation of GSL biosynthesis in hydroponically grown watercress in response to sulphate fertilization remains an underexplored area, despite its significant implications for functional food production and plant secondary metabolism. While GSL pathways have been partially characterized in watercress through transcriptomic and metabolic profiling, previous studies have largely overlooked the effects of mineral nutrient inputs, particularly sulphate, within controlled hydroponic systems. Previous studies have predominantly focused on genetic manipulation, model species such as Arabidopsis thaliana, or broad comparisons between cultivation systems (e.g., aquaponics vs. hydroponics), without dissecting the role of targeted sulphate enrichment, but suggesting the potential of sulphate modulation as a strategic lever to enhance the nutritional and health-promoting properties of watercress.

In this review, we highlight recent advancements regarding the role of GSLs in watercress, drawing primarily from studies on S fertilization effects, the biosynthetic pathways of GSLs within the Brassicaceae family, and the influence of environmental, epigenetic, and mineral nutrient factors, particularly S and Mg, on GSL biosynthesis and metabolism. Our objective is to critically assess how sulphate fertilization influences GSLs profiles in hydroponically grown watercress, thereby linking plant physiological responses with targeted biochemical regulation.

3. History of Watercress

Based on data from the Food and Agriculture Organization of the United Nations, cruciferous vegetable cultivation spanned over 0.48 million hectares in 2020 with significant year-on-year growth, particularly for cabbage, Chinese cabbage, and cauliflower, although production remains geographically limited [25]. According to the USDA [26], a total of 638 acres of watercress were harvested for the fresh market across the United States. Of this, 22 acres were harvested in California, while Kentucky accounted for 7 acres of freshly harvested watercress. The origins of watercress trace back to ancient Greece and Rome, where it was utilized as both a nutritional staple and a remedy for various ailments, such as digestive problems and scurvy [27]. Its continued popularity throughout the Middle Ages and Victorian era for its nutritional and therapeutic benefits laid the foundation for modern studies [28]. Watercress is identified by the US Centers for Disease Control and Prevention (CDC) as the crop offering the greatest nutrient density per calorie [22]. Interest in watercress is driven in part by its unique phytochemical profile, particularly its content of GSLs, which are chemically stable and biologically inert, comprising a fundamental framework containing a sulphated oxime moiety linked to thioglucose.

4. The Science Behind Glucosinolates

The GSLs are S and nitrogen (N) containing secondary metabolites found mainly in cruciferous vegetables like watercress, broccoli, and mustard. The GSLs are typically water-soluble and remain stable within undamaged plant tissues. However, high intake of raw cruciferous vegetables can interfere with thyroid function, due to goitrogenic compounds. The GSLs are composed of a fundamental framework containing a sulphated oxime moiety linked to thioglucose, with side chains originating from amino acids (Figure 2) [23,29]. Amino acid chain elongation (Figure 3) only happens for certain GSLs (mainly methionine-derived). At this chain elongation stage, GSL biosynthesis pathway begins in the cytosol, where methionine or phenylalanine undergo transamination via branched-chain amino acid aminotransferase 4 (BCAT4), resulting in the formation of 2-oxo acids [30]. These intermediate compounds are subsequently transported into the chloroplast by Bile Acid Transporter 5 (BAT5), where the enzymes involved in side-chain extensions are situated [31]. Once inside the chloroplast, the enzyme methylthioalkylmalate synthase (MAM) facilitates the condensation of 2-oxo acids with acetyl-CoA, yielding a 2-malate derivative [32]. In the elongation of aromatic GSLs, MAM3 was identified as a substrate for phenylalanine, indicating a potential involvement in the biosynthesis of aromatic GSLs [33]. As illustrated in Figure 3, the resulting 2-malate derivative is then converted to a 3-malate derivative by the isopropylmalate isomerase 1 (IPM1) complex [34]. During the concluding phase of the elongation process, the extended 2-oxo-acid either proceeds through additional rounds of elongation or is converted via transamination by branched-chain amino acid aminotransferase 3 (BCAT3) into the respective amino acid, which subsequently participates in the formation of the core structure [35].

After chain elongation, Met is activated to aldoxime and nitrile oxide by cytochrome P450 (CYP83) and conjugated with glutathione (GSH) by GSH-S-transferase. Following these initial activation and conjugation steps, the pathway advances into the metabolic rearrangement phase. The process of metabolic rearrangement initiates with the oxidation of the elongated amino acid into aldoximes which are converted to thiohydroximic acids. The activated aldoxime is subsequently linked to glutathione (GSH), which serves as a S source, resulting in the formation of the corresponding thiohydroximate intermediate (Figure 4). The GSL core structure is then formed and a third stage of biosynthesis proceeds with various R-group modifications, such as hydroxylation, methoxylation, oxidation, desaturation, and benzoylation [30].

The biosynthesis of GSLs in Brassicaceae vegetables involves a sequence of five enzymatic reactions: oxidation, sequential oxidation, carbon-S bond cleavage, glycosylation, and sulfation. The key enzymes involved in Brassicaceae GSL biosynthesis include CYP79, CYP83, UGTs, and sulfotransferases as shown in Figure 4. Firstly, the oxidation of the precursor amino acid (e.g., tryptophan, phenylalanine, methionine) into the corresponding aldoxime is catalyzed by cytochrome P450 monooxygenases of the CYP79 family, in a NADPH- and O₂-dependent mechanism, yielding the aldoxime intermediate [36]. Second, a sequential oxidation of the aldoxime by CYP83 enzymes generates reactive aci-nitro or nitrile oxide intermediates, which are conjugated to glutathione via glutathione-S-transferases to produce S-alkyl-thiohydroximates (Figure 1) [37]. Third, carbon–S bond cleavage is carried out by the lyase SUR1 which processes the glutathione-conjugated intermediate into thiohydroximic acid (Figure 1), effecting release of glutamate residues and forging the C–S bond essential to the thioglucose attachment [38]. Fourth, a glycosylation step is performed by UDP-glucosyltransferases of the UGT74 family (e.g., UGT74B1, UGT74C1), which transfers UDP-glucose to the thiohydroximate to yield desulfoglucosinolate, forming the S-glycosidic linkage [39,40]. Finally, a sulfation reaction, mediated by desulfoglucosinolate sulfotransferase enzymes, transfers a sulphate moiety from 3′-phosphoadenylyl sulphate (PAPS) onto the desulfoglucosinolate substrate to yield the mature GSL, releasing adenosine 3′,5′-bisphosphate (PAP) as a by-product [41]. This is a rate-limiting step since the availability of PAPS and sulfotransferase activity can constrain GSL accumulation.

5. Glucosinolates in Agriculture

GSLs are of immediate practical importance to farmers because they underpin multiple key benefits, primarily through their roles in plant defense, soil health, and breeding strategies. They act as inducible chemical defenses that reduce herbivore damage and pathogen incidence, providing biofumigant activity when Brassicaceae residues are incorporated into soil, and offering clear targets for breeding programs aimed at enhancing host resistance or tailoring seed/forage quality. Together these effects can improve yield stability, reduce reliance on synthetic pesticides, and contribute to integrated pest-management strategies. Growth of cruciferous crops is affected by a variety of fungi, bacteria, aphids, and other harmful insects. GSL-mediated defense mechanisms against bacterial infections have been demonstrated, whereby exposure to Burkholderia cepacia, Pseudomonas syringae, and Xanthomonas campestris pv. campestris (Xcc) triggered an increase in the expression of GSL biosynthetic pathways [42]. Likewise, GSL-driven defense mechanisms against fungal pathogens have been proven, whereby water-based plant extracts rich in isothiocyanates are can reduce the in vitro growth of Alternaria brassicicola, a fungal pathogen that infects cruciferous crops, by approximately 50% [43]. Additionally, a comparison of GSL-deficient biosynthetic (gsm1-1) mutants with wild-type Arabidopsis plants showed that isothiocyanates are key contributors to Arabidopsis defense against infections by Plectosphaerella cucumerina, Botrytis cinerea, Fusarium oxysporum, and Peronospora parasitic [44].

Despite long-lived knowledge that plants rich in GSL can suppress soil-dwelling plant pests [45], scientists were eager to investigate chemical dynamics within and around roots during pest suppression. They found that B. nigra, B. juncea, and B. napus plants immediately release m/z 60, a compound which indicates formation of an isothiocyanate from sinigrin, when their roots are injured or attacked by cabbage root fly larvae [46]. Both farming practices and breeding research have shown that GSL levels can be adjusted to balance pest resistance with food and feed safety for example, low-GSL varieties for seed meal and feed, or higher levels for pest control and biofumigation. However, some herbivores may take advantage of certain GSL profiles, so the choice of cultivar should consider the local pest issues. Further, GSLs aide farmers to enhance crop protection and breeding strategies are optimized to local soils, climates and production goals [47]. Beyond their agronomic relevance, the significance of GSLs extends to human nutrition, where their unique biochemical properties underpin the health-promoting qualities of cruciferous vegetables.

6. Glucosinolates in the Human Diet

The GSLs contribute to the health benefits of cruciferous vegetables and distinguish them from other plant types as shown in

Table 1. Glucosinolate content in Brassicaceae plants.

Glucosinolate	1 Chemical Structure	Source	Breakdown Product	Health Relevance	References
Glucotropaeolin		Red cabbage, mustard	Benzyl isothiocyanate	Neuroprotection and diabetes control agent	[51,52]
		Papaya seeds
Glucoerucin		Rocket, broccoli	4-methylthiobutyl isothiocyanate (erucin)	Anticancer	[53]
Neoglucobrassicin		Red cabbage, Chinese cabbage	N-methoxyindole-3-carbinol	Anticancer	[54]
Glucobrassicanapin		Turnip green and swede	4-methylsulfinylbutyl isothiocyanate	Antimicrobial activity	[55]
Glucoraphanin		Cauliflower, broccoli, kale	Sulforaphane	Anticancer	[56,57]
Glucobrassicin		Cabbage, brussels sprout	Indole-3-carbinol	Anticancer	[58]
Gluconasturtiin		Watercress, broccoli, turnips	2-phenylethyl isothiocyanate.	Antioxidant, anti-inflammatory	[59]
Glucoraphasatin		Radish sprouts	4-methylsulfanyl-3-butenyl isothiocyanate	Antioxidant and anticancer	[60]
Glucoiberin		White cauliflower, savoy cabbage	3-Methylsulfinyl propyl isothiocyanate	Chemo preventive	[61]
Progoitrin		Mustard greens, turnip, rapeseed	5-Ethenyl-1,3-oxazolidine-2-thione	Inhibition of thyroid hormone synthesis	[62]
Sinigrin		Mustard, horseradish	Allyl isothiocyanate	Antimicrobial and pungency	[63]

¹ Chemical structures were obtained from the online database of NLM (National Library of Medicine).

. The watercress plant harbors a diverse range of bioactive compounds, such as quercetin, lutein, β-carotene, phenolic acids, and GSLs [48,49]. GSLs may increase or decrease in vegetables during storage. They can be broken down or leached out during processing, or retained through heat-induced inactivation of myrosinase. These compounds are metabolized by plant-derived myrosinase in the small intestine or by microbial myrosinase in the colon. Isothiocyanates are taken up from both the small intestine and the colon, and their byproducts can be detected in human urine within two to three hours following the consumption of Brassica vegetables. Further, 4-methylsulfinylbutyl (glucoraphanin) GSL, present in broccoli, breaks down into the isothiocyanate sulforaphane, a compound that inhibits the cell cycle and induces programmed cell death, helping to suppress tumor development [50]. Given the structural and functional diversity of GSLs across plant species, it is important to consider the distinct physiological and pharmacological roles of individual compounds.

7. Pharmacological Potential of Watercress

Among the five individual GSLs, gluconasturtiin (phenethyl GSL) is the major GSL in leaves and stems of watercress. Gluconasturtiin has historically been employed in managing diabetes [64]. Watercress extracts enriched with gluconasturtiin or polyphenolic compounds have been reported to exert cytotoxic effects on a wide range of human cancer cell lines, including those of the breast, colon, lung, liver, prostate, cervix, melanoma, leukemia, and myeloma [65]. The medicinal leaf is also well known for its mucus-clearing, toothache-relieving, and estrogen-inhibiting properties [66]. Consumption of raw watercress has also been shown to elevate plasma antioxidant concentrations, particularly lutein and β-carotene [67]. Watercress can also be used in phytotherapy, rhinitis, and homeopathic medicines [16]. As a natural remedy, leaves of watercress can serve as a diuretic, detoxifier, expectorant, pain reliever for toothaches, and blood sugar-lowering agent [9]. In hypercholesterolemic rats, watercress intake was associated with increased activities of superoxide dismutase and catalase, alongside reduced hepatic levels of glutathione (GSH), glutathione reductase (GR), glutathione peroxidase (GPx), and malondialdehyde (MDA) [68]. Watercress has also been documented to possess antimicrobial activity [69] and anti-tubercular activity [70]. Furthermore, watercress juice has been found to regulate hyperglycemia by inhibiting α-glucosidase activity while enhancing the functions of lipase and α-amylase [71]. The use of oil in treating heat and chemical-induced burn wounds in rabbits has been demonstrated [72]. The antihypertensive activity and the anti-ulcer capability of watercress have also been reported [73,74], documenting the effectiveness of the ethanol-based extract of watercress as a supportive agent in the recovery of injury-induced ulcers on the dorsal surface of rat tongues. An antihyperlipidemic activity study demonstrated that flavonoids, phenolic constituents, and glycosides from the plant contribute to cholesterol-lowering, lipid-reducing effects [75] and improved urinary system health [76]. Building on these pharmacological insights, it is critical to examine the biosynthetic pathways and accumulation patterns of GSLs, which underpin many of the health-related properties of watercress.

8. Literature Gaps Relevant to Glucosinolates in Watercress

8.1. Glucosinolate Accumulation Under Soilless Conditions

Hydroponic systems offer controlled nutrient management that shapes plant metabolic profiles, making the understanding of flow rate effects essential for maximizing both crop performance and system efficiency. A study by Hameed et al. [77] substantiates this, since altering ammonium/nitrate (NH₄⁺/NO₃⁻) ratios generates distinct metabolomic profiles in lettuce across soil, substrate, and hydroponic systems, offering candidate markers for enhancing N efficiency. Moreover, studies have probed watercress growth, nutrient uptake efficiency, and antioxidative enzyme activity within hydroponic environments [78,79,80]. In Brassica vegetables, results from an analysis identified 23 principal metabolites, chiefly GSLs and flavonoids, which played a pivotal role in differentiating the metabolic profiles of watercress leaves cultivated in hydroponic systems [81]. Despite increasing interest in biofortification and controlled-environment agriculture, the biochemical response of watercress to mineral nutrient modulation in hydroponic systems, particularly sulphate fertilization, remains poorly understood. Existing studies have explored GSL biosynthesis through genetic manipulation and environmental perturbation in model species or under soil-based cultivation [82], yet there is a lack of consolidated evidence addressing how sulphate availability influences GSL accumulation in watercress under soilless conditions. This gap limits the development of nutrient management strategies aimed at optimizing both yield and phytochemical richness in controlled environments. By synthesizing cross-disciplinary literature on sulphate-driven modulation of GLSs in hydroponically grown watercress, this review provides a critical foundation for targeted nutrient interventions, offering new insights for functional food production, sustainable horticulture, and public health nutrition. Thus, insights into nutrient modulation in watercress directly intersect with the growing emphasis on functional foods in public health and agriculture.

8.2. Impact of Magnesium Sulphate on Glucosinolate Accumulation

Mg is essential for numerous enzymatic functions, including energy metabolism, while sulphate is employed as a major pathway for producing various S-containing metabolites, including amino acids (cysteine and methionine), glutathione (GSH) and GSLs as shown in Figure 5. A study by Kopsell et al. [83] shows that MgSO₄ fertilization promotes GSL synthesis in watercress. One key finding across multiple studies was that moderate concentrations of MgSO₄ can increase the total GSL content in plants [84]. This effect is likely due to the activation of S assimilation pathways and gene regulation mechanisms involving v-myb avian myeloblastosis viral oncogene homolog (MYB) transcription factors [85]. Analogous work in broccoli sprouts showed that 50 mM MgSO₄ treatment increased total GSLs by approximately 42% within 24 h, though with some yield penalty [86]. This reduced biomass result aligns with previous findings in Brassica species, where increased S availability enhances secondary metabolite accumulation while concurrently impairing growth through resource reallocation or stress signaling [87]. Together, these examples illustrate a classic biofortification trade-off: enhanced secondary metabolism achieved at the expense of primary growth (both fresh and dry biomass). Conversely, a study by Lu et al. [88] showed that Mg fertilization significantly improved the yield but reduced nutritional quality. From an agronomic perspective, enhancing GSLs while minimizing biomass loss requires careful management to balance nutritional quality with yield and production efficiency [89]. Further, success depends on crop-specific optimization of dosage, timing, and genotype to achieve a favorable balance between enhanced nutraceutical value and agronomic productivity [90]. Thus, understanding how MgSO₄ modulates this pathway could offer novel strategies to enhance the nutritional and functional value of watercress cultivated in controlled environments. To effectively investigate these nutrient–metabolite interactions, cultivation platforms with controlled growing conditions are essential.

8.3. Impact of Magnesium Sulphate on Glucosinate Accumulation in Hydroponically Grown Watercress

Hydroponic systems offer an ideal platform for investigating biochemical interactions due to their accuracy in nutrient delivery and environmental control [92]. MgSO₄ contributes about 13% S by weight in the commonly used hydroponic solutions such as Hoagland solution [93]. It is a low-cost hydroponic fertilizer containing 10% Mg and 16% kieserite, which are both highly water-soluble and quick-acting sources of Mg [94]. It is chosen for its high solubility, nutrient supply duality, and compatibility with other salts [95,96], and its ability to trigger secondary metabolites including GSLs [97]. Other sources of hydroponic S fertilization such as sodium sulphate (Na₂SO₄), ammonium sulphate ((NH₄)₂SO₄), potassium sulphate (K₂SO₄) and calcium sulphate (CaSO₄) are less soluble, may introduce cations that can unbalance the nutrient solution and may be toxic to plant stress if supplied in high doses [98,99]. Further, a study by Ali et al. [100] investigated the relationship between S availability and GSL biosynthesis. In their experiment, (Lepidium latifolium, which hails from the mustard family and first formally described by Carl Linnaeus) was supplied with varying levels (0.5 and 1.0 mM) of sulphate sources. Further, MgSO₄, zinc sulphate (ZnSO₄), and their mixtures were also added. Application of these salts led to marked increases in GSL concentrations, reaching up to 29% and 38% with MgSO₄ and ZnSO₄, respectively.

9. Factors Influencing Glucosinolate Biosynthesis and Accumulation

9.1. Effect of Sulphur Fertilization on the Glucosinolate Content

S and N have been demonstrated to impact GSL concentrations across a range of vegetable crops. A review by Falk et al. [2] concluded that sulphur fertilization commonly results in marked increases in GSL content across Brassica species, sometimes up to a 50% increase, depending on species and treatment. A study by Barickman et al. [61] confirms this since both N and S applications showed a positive association with gluconasturtiin levels, which varied between 1.5 and 19.6 µmol per gram of dry weight. Sulphur, an essential nutrient for plant growth and development, fulfills various functions in metabolic processes and adaptation to stress within plants. The elements’ contribution to improving the yield and quality of cruciferous crops has attracted growing interest in agricultural studies [101]. Given that GSLs can constitute as much as 30% of the total S pool within plant tissues, their synthesis and accumulation are likely to be closely linked to the plant’s overall S nutritional status [2]. Further, S is found in protein-related amino acids like methionine and cysteine, as well as in vitamins such as biotin and thiamine, phytochelatins, chlorophyll, coenzyme A, and S-adenosylmethionine [102]. The S nutrient is frequently used as a soil amendment and is particularly crucial for the growth of crops such as onions, garlic, and members of the cruciferous family like broccoli and cauliflower [101]. This nutrient is an integral part of the GSL’s core structure, and it is directly incorporated into the thioglucoside bond. Haneklaus et al. [103], along with Zenda et al. [3], provide an overview of how S supplementation influences the GSL levels in various agricultural crops. Further studies by Krumbein et al. [104] show that higher S availability enhances the levels of certain GSLs, such as glucobrassicanapin, progoitrin, gluconapin, and sinigrin, in Brassica juncea L., as well as the overall GSL content in Brassica rapa. Sulphur fertilization can satisfy the nutrient demand of the crop and increase the GSL content. This is shown by a study by Traka et al. [105] which shows that broccoli hybrids with elevated glucoraphanin levels had 2.5–3 times more glucoraphanin content, due to improved S uptake. A study by Kopsell et al. [83] showed that, in watercress tissues, the levels of aromatic, indole, and overall GSLs were influenced by variations in N supply, while aliphatic, aromatic, and total GSL contents were affected by adjustments in S nutrition. A study by Kim et al. [106] found that when MgSO₄ was applied through the roots of broccoli, the levels of glucoraphanin rose significantly within 24 h. Additionally, a study by Szulc et al. [107] agrees with previous findings, in that their study found the overall GSLs content, along with the combined amount of alkenyl GSL, is significantly greater in oilseed rape seeds treated with 60 kg·ha⁻¹ of elemental S applied to the soil. Further, another study showed that S application led to an increase in indole GSL levels in cabbage [108]. In germinating broccoli sprouts, S fertilization enhances the GSL glucoraphanin [109]. Treatments which use amino acids alone or with S and boron, culminate in the highest GSL concentration [86]. Conversely, when S fertilizer is not used, the total amount of aliphatic GSLs in the plants is notably reduced compared to treatments with sulphur fertilization [110]. These findings highlight the broader influence of environmental conditions on GSL accumulation, extending beyond nutrient supplementation alone.

9.2. Effect of Magnesium on Glucosinate Content

Generally, Mg enhances carbon assimilation, which in turn leads to production of carbon-based secondary metabolites such as GSLs. Mg’s role as a central atom in chlorophyll and as a cofactor in GSL biosynthesis may explain its dual influence on yield and nutritional quality [111]. Research by Gui-Xiao et al. [112] showed that an appropriate combination of nitrogen and Mg significantly enhanced photosynthetic performance, stabilized antioxidant activity and internal hormone balance, thereby promoting plant development and boosting the fresh biomass of Chinese kale. Notwithstanding, in Brassica species, excessive application of N fertilizer lowers the relative content of GSLs, thereby diminishing the nutritional and therapeutic significance of these vegetables [113]. Specifically, Mg acts as a key cofactor for enzymes needed for side-chain elongation of methionine-derived GSLs biosynthesis such as MAM [114]. Further, it acts as a divalent ion necessary to stimulate sulfotransferase enzymes in the process of GSL biosynthesis [115]. Conversely, elevated Mg levels suppress ATP-sulphurylase activity, the enzyme responsible for catalyzing the initial step of sulphate assimilation [116]. Therefore, Mg is crucial for playing a dual role by controlling S assimilation and GSL production in plants.

9.3. Environmental Factors Affecting Glucosinolate Biosynthesis

The level of GSLs is influenced by the surrounding environmental factors [117] which affect the conversion of GSLs into isothiocyanates, including low pH, iron (II) ions, and proteins involved in thiocyanate formation. Temperature, light, physical damage, and insect herbivory also affect GSL production and composition [118]. A study by Antonious et al. [119] showed that environmental pressure on developing plants can elevate the levels of GSLs, vitamin C, and overall phenolic compounds in Brassica shoots, but it does not enhance the total output of these phytochemicals per unit of land area. In Arabidopsis thaliana there is regulatory influence of photoperiod and S availability, where diurnal variation, HY5-mediated light signaling, and nutrient stress distinctly modulate GSL biosynthesis [120]. Additionally, GSL synthesis is regulated by phytohormones, plant growth regulators, agrochemicals, pesticides, metals, atmospheric gases, and other stimuli [121]. Further, agricultural conditions such as soil composition, fertilizers, cultivation methods, and habitat changes, along with environmental variables like season, light, temperature, water availability, humidity, CO₂ levels, and nutrient supply, further impact the levels of GSLs in Brassicaceae crops [122] and gluconasturtiin in watercress [64]. Beyond these external and agronomic factors, intrinsic regulatory mechanisms, such as epigenetic modifications, also play a critical role in determining GSL content by modulating gene expression.

9.4. Transcriptomic, Epigenetic and Metabolomic Factors Affecting Glucosinolate Biosynthesis

De novo transcriptome sequencing and organ-specific GSL profiling in watercress identified approximately 33 candidate biosynthetic genes and quantified eight GSLs, establishing a foundational molecular framework, though lacking integration with hydroponic or nutrient-focused systems [49]. Transgenic approaches employing Brassica oleracea-derived transcription factors (e.g., IQD1, MYB29) in watercress hairy root cultures achieved significant GSLs enhancement of 2.6–4.7-fold yet were restricted to non-soil systems without nutrient modulation [123]. The amount of GSLs is influenced by epigenetic changes, which have been shown to affect metabolic composition by regulating the expression of metabolism-related genes [39]. At the epigenetic level, factors such as DNA methylation, histone modification patterns, chromatin structure, and non-coding RNAs influence the transcriptional accessibility of gene clusters involved in biosynthetic pathways [124]. As shown in Figure 6, epigenetic regulation modulates GSL biosynthetic pathways, influencing both the quantity and profile of GSLs produced in Brassica species [125]. Like Heterochromatin Protein (LHP1), a component of the polycomb repressive complex 1 that modifies histones, plays a role in both development and stress adaptation. In the LHP1 mutant, levels of indolic GSLs are moderately decreased, while aliphatic GSLs are significantly increased [106]. Comparative metabolomics of watercress cultivated under hydroponic, aquaponic, and soil-based systems revealed higher GSL diversity and abundance, particularly gluconasturtiin, in aquaponics [81]. Understanding how the transcriptome, epigenetic regulation, and the metabolome influence GSL profiles provides a foundation for exploring how targeted nutrient interventions, such as MgSO₄ supplementation, can optimize phytochemical accumulation in watercress.

9.5. Intra-Genic Variation in Glucosinolate Biosynthesis

In 12 commonly eaten Brassicaceae vegetables, precursor GSL levels strongly correlate with their resulting metabolites, and GSL types and amounts vary significantly among plant genera [47]. These variations align with morphological classifications and suggest genus-specific biosynthetic pathways. Understanding these genus-specific biosynthetic variations underscores the importance of key nutrients, particularly S, in modulating GSL profiles and overall plant development. A study by Kittipol et al. [126] further exemplifies intragenic variation in regulatory genes such as MYB28/HAG1 and its specific homologues Bna.HAG1.A9 and Bna.HAG1.C2 modulates aliphatic GSL levels across tissues, reinforcing the link between genotype-specific biosynthesis and resulting metabolite accumulation. Robin and co-workers [127] also researched how, intragenic differences, in this case differences in expression of specific biosynthetic genes like ST5b and GSL-OH, translate into distinct GSL profiles among cabbage genotypes. Building on these genotype-specific insights in model Brassicaceae crops, focus on watercress, integrating broader physiological and agronomic perspectives.

10. Conclusions

This narrative review synthesizes current research on GSL biosynthesis and accumulation in watercress and related cruciferous species, highlighting genetic, environmental, and nutritional influences while addressing a critical gap: the role of targeted sulphate fertilization in hydroponically grown watercress. Further, the review revealed the impact of organic and inorganic amendments on the GSL concentrations in various vegetable crops; however, very few studies investigated the effect of MgSO₄. Additionally, this review highlights the increasing recognition of watercress as a nutrient-dense, environmentally efficient crop with substantial economic and public health potential, particularly through hydroponic cultivation. The relevance of this review lies in the fact that watercress contains bioactive compounds such as GSLs, which is why watercress has anti-inflammatory, antimicrobial, hepatoprotective and cardioprotective effects. Supplementation with Mg sulphate can boost GSL accumulation by supporting both S metabolism and the activity of key biosynthetic enzymes. Given the established influence of sulphate supply on GSL profiles in multiple Brassica crops, the sulphate levels employed in analogous experiments, such as 0.5–1.0 mM sulphate or 50 mM MgSO₄ in broccoli, offer a biologically meaningful starting point for designing future hydroponic studies in watercress. Concentrations of at least 20 mM can impair growth and alter S homeostasis. Future research should define the dose–response thresholds for GSL accumulation. Further, the respective roles of Mg and sulphate should be elucidated through multivariable nutrient interaction designs, coupled with isotope-based metabolic tracking to determine how S assimilation responds to environmental factors such as light quality and temperature. Elucidating the role of MgSO₄ in the GSL biochemical pathway can enable the development of targeted strategies to enhance phytochemical accumulation under controlled hydroponic cultivation of watercress, leading to improved agricultural productivity and public health benefits.