Epigenetic Regulation of Glucosinolate Biosynthesis: Mechanistic Insights and Breeding Prospects in Brassicaceae

Abstract

Glucosinolates (GSLs) are nitrogen- and sulfur-containing secondary metabolites central to the defense, development, and environmental responsiveness of Brassicaceae species. While the enzymatic steps and transcriptional networks underlying GSL biosynthesis have been extensively characterized, mounting evidence reveals that chromatin-based processes add a critical, yet underexplored, layer of regulatory complexity. Recent studies highlight the roles of DNA methylation, histone modifications, and non-coding RNAs in modulating the spatial and temporal expression of GSL biosynthetic genes and their transcriptional regulators in response to developmental cues and environmental signals. This review provides a comprehensive overview of GSL classification, biosynthetic pathway architecture, transcriptional regulation, and metabolite transport, with a focus on emerging epigenetic mechanisms that shape pathway plasticity. We also discuss how these insights may be leveraged in precision breeding and epigenome engineering, including the use of CRISPR/dCas9-based chromatin editing and epigenomic selection, to optimize GSL content, composition, and stress resilience in cruciferous crops. Integrating transcriptional and epigenetic regulation thus offers a novel framework for the dynamic control of specialized metabolism in plants.

1. Introduction

Glucosinolates (GSLs) are a structurally diverse family of sulfur- and nitrogen-containing secondary metabolites predominantly found in the Brassicaceae. They function as key chemical defenses against a broad range of herbivores and pathogens and also contribute to abiotic stress adaptation, including osmotic adjustment under drought or nutrient limitation [1,2,3,4]. GSL structural diversity arises from variation in side-chain chemistry, determined by the amino acid precursors from which they are derived: aliphatic GSLs from methionine, alanine, leucine, isoleucine, or valine; indolic GSLs from tryptophan; and aromatic GSLs from phenylalanine or tyrosine [5,6,7,8]. Each class exhibits unique physicochemical properties such as solubility, reactivity and volatility that influence their ecological functions and metabolic fate [9,10,11,12].

The composition and abundance of GSLs in Brassicaceae species are shaped by both genetic factors and environmental inputs. While genotype determines the baseline capacity for GSL biosynthesis, the final GSL profile is modulated by external cues such as temperature, light, soil fertility, plant developmental stage, and canopy position [13,14,15,16]. For instance, nutrient deficiencies (e.g., sulfur or nitrogen) and harvest timing can significantly shift GSL content and composition, often with trade-offs between yield and defense chemistry [17]. Experimental drought stress in Brassica oleracea have been shown to increase total leaf GSL levels by up to 85%, particularly enriching indolic compounds [18]. These observations underscore the plasticity of the GSL metabolic network in response to both endogenous and exogenous stimuli [19].

Upon tissue disruption, GSLs are hydrolyzed by myrosinases to yield bioactive products such as isothiocyanates, nitriles, and thiocyanates that act as deterrents to herbivores and pathogens while also contributing to osmoprotection [20,21,22,23]. In addition to their ecological roles, these hydrolysis products have garnered attention for their potential health benefits, particularly in cancer chemoprevention and anti-inflammatory responses [24,25,26,27]. At the transcriptional level, decades of genetic and biochemical studies have established R2R3-MYB and bHLH transcription factors as core regulators of GSL biosynthesis [28,29,30,31,32]. However, these regulators alone cannot account for the rapid, tissue-specific, and reversible shifts in GSL production observed during development or under stress. Emerging evidence now implicates chromatin-based regulatory processes including DNA methylation, histone modifications and non-coding RNAs as dynamic and reversible mechanisms that fine-tune GSL gene expression without altering the underlying DNA sequence [33,34,35]. For example, light-induced recruitment of histone deacetylases to aliphatic GSL loci leads to deacetylation of H3K9, chromatin condensation, and transcriptional repression. Moreover, genes involved in methionine chain elongation exhibit selective depletion of the activating H3K4me3 mark, suggesting an epigenetic mechanism that restricts constitutive expression and enhances environmental responsiveness [36,37,38].

Transcriptional control is further integrated into broader environmental signaling networks. R2R3-MYB transcription factors such as MYB28, MYB29, and MYB76 regulate aliphatic GSL biosynthesis, while MYB34, MYB51, and MYB122 govern indolic GSL production. These MYBs are themselves regulated by light signaling via phytochrome B and the COP1/SPA ubiquitin ligase complex, which modulates MYB stability and activity to balance growth and defense [39,40,41,42]. Disruption of this module (e.g., in cop1-4, cop1-6, or spa1/3/4 mutants) leads to a ~50% reduction in both aliphatic and indolic GSLs and alters the expression of several biosynthetic genes [43]. Notably, downregulation of MYB34 is accompanied by upregulation of MYB51 and MYB122, indicating a feedback mechanism that stabilizes indolic GSL output under altered signaling [44]. This photomorphogenic regulatory axis provides a mechanistic entry point into chromatin-level regulation of specialized metabolism. The presence of bivalent histone marks such as H3K4me3 and H3K27me3 at MYB loci further supports the hypothesis that environmental cues interface with the GSL biosynthetic machinery through epigenetic plasticity.

Despite these advances, key gaps remain in our understanding of how chromatin dynamics regulate the GSL pathway [45,46]. First, there is no genome-wide epigenomic map detailing DNA methylation and histone modification landscapes across aliphatic, indolic, and aromatic GSL loci. Second, we lack systematic studies comparing how environmental and developmental signals (e.g., light, jasmonate, drought and flowering) reshape these epigenetic signatures. Third, the interplay between distinct epigenetic layers such as DNA methylation, histone post-translational modifications and small/long non-coding RNAs has yet to be fully elucidated at the level of individual genes. Fourth, translational applications in Brassica crops remain underdeveloped, leaving significant potential untapped in breeding or agronomic modulation of GSL profiles.

This review synthesizes current understanding of the biosynthetic architecture and regulatory landscape of glucosinolate metabolism, with particular emphasis on emerging epigenetic mechanisms DNA methylation, histone modifications and non-coding RNAs that dynamically influence GSL gene expression. We critically assess how these regulatory layers interface with environmental and hormonal cues, and explore their potential for targeted manipulation in Brassicaceae crop improvement. In the following sections, we first provide an overview of glucosinolate biosynthesis in Brassicaceae, highlighting the enzymatic steps and classical transcriptional regulators. We then detail the current evidence for epigenetic regulation of GSL metabolism, focusing on DNA methylation, histone modifications, chromatin remodeling, and non-coding RNAs, and how these mechanisms integrate with environmental and hormonal signaling. Finally, we discuss translational perspectives, including epigenome-based breeding, CRISPR/dCas9-mediated chromatin editing, and stress-priming strategies, and identify key research priorities for bridging mechanistic insights to applied crop improvement.

2. Overview of the GLS Biosynthesis

2.1. Classification and Structures: Aliphatic, Indolic, Aromatic GSLs

GLSs are a class of sulfur and nitrogen containing secondary metabolites structurally characterized by a common thioglucoside-linked core and a variable side chain derived from amino acids [19]. Based on the nature of their amino acid precursors, GSLs are broadly classified into three major groups: aliphatic, indolic and aromatic (Table 1). This classification not only reflects biosynthetic origin but also underpins functional and ecological diversity across species [46,47].

Aliphatic GSLs are the most abundant class in Arbidopsis thaliana and many Brassica crops, are derived primarily from methionine and, less frequently, from branched-chain amino acids such as valine, leucine and isoleucine [48,49,50]. They exhibit substantial side-chain variability due to chain elongation and secondary modifications, and are associated with strong deterrent and toxic activities against herbivores and pathogens [51,52,53,54,55]. Indolic GSLs are synthesized from tryptophan and tend to be more responsive to environmental and hormonal stimuli. The hydrolysis products of indolic GSLs serve as reactive molecular signals with antimicrobial and auxin-like functions, underpinning their importance in stress-induced metabolic reprogramming and hormonal signaling crosstalk [56,57,58,59]. Aromatic GSLs derived from phenylalanine or tyrosine, are less structurally diverse and less prevalent in most species, but remain significant in certain Brassicaceae (e.g., Sinapis alba), where they contribute to specific defense and flavor profiles [60,61,62,63,64].

While all three classes share a conserved thioglucoside-sulfate core structure, the chemical diversity imparted by side-chain variation and post-synthetic modifications governs their biological specificity, storage stability and bio activation potential [65]. Importantly, the biosynthetic genes involved in these classes show differential transcriptional and spatial expression patterns, often under control of distinct transcription factors and signaling pathways [66]. These differences lay the groundwork for investigating class-specific epigenetic regulation, particularly in response to stress, developmental reprogramming and ecological adaptation.

Table 1. Classification of glucosinolates based on amino acid precursors.

GSL Class	Precursor Amino Acid(s)	Example GSLs	Characteristic Functions	References
Aliphatic	Methionine, Valine, Leucine, Isoleucine	Glucoraphanin, Gluconapin, Singrin	- Major defense against herbivores (e.g., caterpillars, aphids) - Sulfur storage and redistribution - Hydrolysis products exhibit strong antimicrobial and antifungal activity - Allelopathic suppression of competitors	[48,49,50,51,52,53,54,55]
Indolic	Tryptophan	Glucobrassicin, Neoglucobrassicin	- Highly inducible under biotic stress and hormonal (JA, SA) signaling - Antimicrobial defense and pathogen signaling - Precursors for auxin-like compounds, linking defense to development - Key role in wound signaling and systemic acquired resistance	[56,57,58,59]
Aromatic	Phenylalanine, Tyrosine	Gluconasturtiin, Sinalbin	- Species-specific defense - Antifungal activity - Flavor and aroma contribution in mustard and condiments	[60,61,62,63,64]

Table 1. Classification of glucosinolates based on amino acid precursors.

GSL Class	Precursor Amino Acid(s)	Example GSLs	Characteristic Functions	References
Aliphatic	Methionine, Valine, Leucine, Isoleucine	Glucoraphanin, Gluconapin, Singrin	- Major defense against herbivores (e.g., caterpillars, aphids) - Sulfur storage and redistribution - Hydrolysis products exhibit strong antimicrobial and antifungal activity - Allelopathic suppression of competitors	[48,49,50,51,52,53,54,55]
Indolic	Tryptophan	Glucobrassicin, Neoglucobrassicin	- Highly inducible under biotic stress and hormonal (JA, SA) signaling - Antimicrobial defense and pathogen signaling - Precursors for auxin-like compounds, linking defense to development - Key role in wound signaling and systemic acquired resistance	[56,57,58,59]
Aromatic	Phenylalanine, Tyrosine	Gluconasturtiin, Sinalbin	- Species-specific defense - Antifungal activity - Flavor and aroma contribution in mustard and condiments	[60,61,62,63,64]

2.2. Biosynthetic Pathway Overview

Glucosinolate biosynthesis is a highly modular and evolutionarily fine-tuned metabolic system. While it occurs in multiple tissues, its activity is most prominent in young photosynthetically active leaves, correlating with developmental cues, herbivore pressure, and physiological status [67,68]. GSLs are then transported to reproductive, storage and defensive organs via specialized membrane transporters [69,70]. The spatial separation between synthesis and accumulation underlies the biological economy of GSL deployment in the plant’s defense arsenal [71]. The biosynthetic pathway comprises three core stages [72]

(i)

Side-chain elongation of amino acid precursors (Specific to aliphatic GSL)

(ii)

Formation of the core

(iii)

Post-synthetic modifications that diversify biological function [46].

Beyond mere biochemical linearity, these steps represent regulatory nodes sensitive to environmental and hormonal signals, offering multiple levels of transcriptional control, post-transcriptional, and potentially epigenetic, making GSL biosynthesis a paradigm of adaptive, specialized metabolism.

Stage I—Amino acid chain elongation (Aliphatic GSLs)

The first stage of aliphatic glucosinolate biosynthesis expands methionine-derived precursors through a pathway similar to leucine biosynthesis, thereby establishing the structural and functional diversity that defines this GSL class. This process is initiated by branched-chain aminotransferases (BCAT3/4), which transaminate methionine to yield 2-oxoacids. These intermediates condense with acetyl-CoA via methylthioalkylmalate synthases (MAM1/3), forming methylthioalkylmalate derivatives (Table 2). Isopropylmalate isomerase (IPMI) then rearranges these molecules, followed by oxidative decarboxylation via isopropylmalate dehydrogenase (IPMDH). The resulting elongated 2-oxoacid may re-enter the elongation cycle for further extension or be transaminated back to an elongated methionine derivative [73,74].

The GSL-ELONG (ELONG) gene family encodes enzymes central to the methionine side-chain elongation process and is a major determinant of natural variation in aliphatic GSL profiles [75,76,77]. Allelic variation at these loci contributes to chemotypic diversity within Arabidopsis and Brassica species, and homoeologous gene replacement has been successfully exploited in B. rapa to modulate GSL content via marker-assisted selection [77,78]. Likewise, GSL-ALK genes, which function downstream in determining the degree of side-chain desaturation, play a pivotal role in shifting the balance between specific aliphatic GSLs, such as reducing progoitrin while enriching glucoraphanin in B. napus through targeted silencing [79].

The MAM and ELONG loci represent key evolutionary innovations within the Brassicales, underpinning adaptation to ecological niches by enabling species- and tissue-specific metabolic diversification. Their copy number variation, promoter polymorphisms, and tissue-specific expression patterns make them compelling candidates for epigenetic modulation in response to environmental cues. Indeed, such upstream control points could facilitate rapid reconfiguration of aliphatic GSL output under biotic stress without requiring de novo activation of the full biosynthetic pathway [80].

Stage II—Core structure formation

The second biosynthetic stage represents the conserved central module in glucosinolate biosynthesis, wherein structurally diverse amino acid precursors converge into a unified pathway leading to the formation of the characteristic thioglucoside core [51]. This process is shared across aliphatic, indolic and aromatic GSL classes. The first committed step is catalyzed by cytochrome P450 monooxygenases of the CYP79 family, which convert amino acids into their corresponding aldoximes with CYP79F1 and CYP79F2 acting in aliphatic precursors and CYP79B2 and CYP79B3 on indolic ones [81,82,83]. These aldoximes are subsequently oxidized into aci-nitro compounds by CYP83A1 (For Aliphatic GSLs) or CYP83B1 (for indolic GSLs). The resulting electrophilic intermediates undergo conjugation with reduced gluthathion (GSH) via glutathione S-transferases (GTSs), forming S-alkyl-thiohydroximates [84,85,86,87]. This conjugate is then processed through three sequential enzymatic steps:

(i)

C-S bond cleavage catalyzed by the C-S lyase SUR1;

(ii)

Glucosylation by UDP-glucosyltransferase UGT74B1;

(iii)

Sulfation by sulfotransferases SOT16, SOT17 and SOT18.

Resulting in the production of the mature glucosinolates core structure with a side chain defined by the initial precursor. Although often viewed as a linear and constitutive biochemical process, recent findings reveal that the regulation of this core module is far more dynamic [6,45,88,89]. Transcriptional plasticity of key genes-particularly CYPs, GSTs and UGTs- in response to developmental cues and abiotic or biotic stress underscores its integration within broader physiological networks. Notably, several of these loci exhibit bivalent chromatin signatures, including the coexistence of activating (H3K4me3) and repressive (H3K27me3) histone marks [90,91]. This epigenetic configuration suggests a poised regulation state, enabling rapid transcriptional activation or silencing and highlights the potential for epigenetic switch mechanisms to modulate glucosinolates biosynthesis in a context-dependent manner.

Stage III—Secondary modification of side chains

The final stage of glucosinolates biosynthesis involves a series of side chain modifications that impart remarkable chemical diversity and functional specificity to the core structure of the glucosinolates (Table 2). These post-synthetic transformations are catalyzed by a suite of specialized enzymes, including AOP2 and AOP3, which introduce alkenyl or hydroxyl groups through 2-oxoglutarate-dependent deoxygenation [92], GS-OH for hydroxylation and FMOGS-OX enzymes that mediate sulfur oxidation [93,94,95,96,97,98,99]. Additional modifications, such as methylthiolation, catalyzed by thiomethyltransferases (TMTs), and desaturation or epoxidation, further alter the physicochemical properties of the molecules. These structural changes profoundly influence the biological activity of the GSL-derived hydrolysis products, particularly isothiocyanates and nitriles, which are central to plant defense, microbial resistance, and nutritional effects in humans [100]. Importantly, this chemical diversification enables context-dependent modulation of bioactivity, allowing plants to fine-tune defense responses based on developmental stage, environmental stimuli or abiotic stresses.

From a regulatory standpoint, the genes controlling side-chain modification are highly dynamic and represent evolutionary hotspots under diversifying selection [100,101,102,103]. Their transcription is known to be responsive to biotic and abiotic cues, including herbivory, pathogen attack, sulfur availability, and hormonal signaling via jasmonates and salicylates. However, while transcriptional regulation of these loci—often mediated by MYB or WORKY transcription factors—has been relatively well-characterized, the role of epigenetic regulation remains poorly understood [103]. Notably, the conditional and tissue-specific expression patterns of modification genes suggest a potential role for chromatin-based regulatory mechanisms, such as histone modifications, DNA methylation, or micro RNA-mediated silencing, in fine-tuning GSL output. As such, this late stage of GSL biosynthesis may represent an underexplored but strategically positioned regulatory interface, offering flexibility for rapid adaptation and stress-induced metabolic reprogramming.

Table 2. Enzymes involved in GSL biosynthesis and their regulation.

Biosynthetic Stage	Key Enzymes	Gene Family	Regulation Type	Evidence for Epigenetic Regulation	References
Chain Elongation	MAM1/2/3, BCATs	MAM, BCAT	Transcriptional (MYB28/29)	Limited (H3K4me3 depletion)	[37,50,72,73,74,75,93]
Core Structure Formation	CYP79s, CYP83s, GSTs, UGT74B1, SUR1, SOT16/17/18	CYP, GST, UGT, SOT	MYB28/34, stress-responsive	Partial (bivalent chromatin marks)	[81,82,83,84,85,86,87,104,105,106]
Secondary Modifications	AOP2/3, GS-OH, FMOGS-OX	2OGD, FMO	MYB, WRKY	Not yet studied	[92,93,94,95,96,97,98,99]
Transport	GTR1, GTR2	NPF	Sulfur-, JA-responsive	Likely (heterochromatic localization)	[100,101,102]

Table 2. Enzymes involved in GSL biosynthesis and their regulation.

Biosynthetic Stage	Key Enzymes	Gene Family	Regulation Type	Evidence for Epigenetic Regulation	References
Chain Elongation	MAM1/2/3, BCATs	MAM, BCAT	Transcriptional (MYB28/29)	Limited (H3K4me3 depletion)	[37,50,72,73,74,75,93]
Core Structure Formation	CYP79s, CYP83s, GSTs, UGT74B1, SUR1, SOT16/17/18	CYP, GST, UGT, SOT	MYB28/34, stress-responsive	Partial (bivalent chromatin marks)	[81,82,83,84,85,86,87,104,105,106]
Secondary Modifications	AOP2/3, GS-OH, FMOGS-OX	2OGD, FMO	MYB, WRKY	Not yet studied	[92,93,94,95,96,97,98,99]
Transport	GTR1, GTR2	NPF	Sulfur-, JA-responsive	Likely (heterochromatic localization)	[100,101,102]

These biosynthetic modules, their transport systems and the layered transcriptional controls provide multiple regulatory entry points. The presence of poised chromatin states, stress-induced transcriptional plasticity and tissue-specific expression patterns suggest that epigenetic regulation is an underappreciated but potentially central mechanism shaping GSL diversity and adaptability in Brassicaceae.

3. Compartmentalization and Transport

Following biosynthesis, GSLs are actively sequestered in vacuoles, spatially isolated from myrosinases, the enzymes responsible for their hydrolytic activation. This compartmentalization prevents premature activation and autotoxicity, preserving the chemical integrity of GSLs until triggered by tissue disruption or specific environmental stimuli. Beyond intracellular storage, GSLs are distributed across organs and tissues via specialized transporters, enabling not only distribution but also fine-tuned spatial and temporal modulation of defense capacity. Key actors include GTR1 and GTR2, members of the Nitrate/Peptide Transporter Family (NPF), which mediate the proton-coupled symport of GSLs across cellular membranes [102,103]. These transporters facilitate both intercellular allocation and long-distance translocation through vascular tissues. Their expression is developmentally regulated and environmentally responsive, allowing dynamic redistribution of GSLs in response to ontogeny, nutrient status, and biotic stress.

Rather than acting as passive conduits, GSL transporters represent a strategic regulatory checkpoint within the metabolic network [70,107]. Arabidopsis chromatin profiling has revealed that GTR1 and GTR2 loci are embedded within AT-rich, heterochromatic regions, suggesting that their transcription may be modulated by chromatin state [71,107,108]. Histone modifications and changes in chromatin accessibility could thus enable rapid shifts in GSL distribution under fluctuating environmental conditions. In this way, GSL transport functions not merely as a distribution mechanism, but as a downstream regulatory layer potentially governed by epigenetic and chromatin-based signals.

4. Integrative Control of Glucosinolates Pathway Commitment: Hormonal Crosstalk, Metabolic Feedback, and Chromatin Dynamics

At the transcriptional apex, subgroup 12 R2R3-MYB transcription factors (TFs) MYB28, MYB29 and MYB76, govern the expression of aliphatic GSL biosynthetic genes, whereas subgroup 13 MYBs, MYB34, MYB51and MYB122, regulate indolic GSL loci [31,40,41,106]. These MYBs operate in concert with bHLH partners and JAZ–MYC signaling modules, which provide hormone-dependent amplification, particularly under jasmonate (JA) induction [109,110,111]. Cross-regulatory interactions, notably the mutual repression between aliphatic and indolic MYB subgroups, are thought to sharpen branch specificity, yet their mechanistic basis remains unresolved. Evidence from other plant regulatory systems provides instructive parallels: in the phenylpropanoid pathway, Arabidopsis MYB4 represses gene expression by occupying promoter elements and excluding activator MYBs; in anthocyanin regulation, R3-MYB repressors such as CPC in Brassica napus attenuate transcription by competing with PAP1 for bHLH partners, thereby disrupting MBW complex formation; and in developmental contexts, the Arabidopsis co-repressor TOPLESS (TPL) exemplifies how EAR-motif transcription factors can recruit repressive complexes, including Mediator components, to block transcriptional activation. These precedents collectively suggest that competitive promoter occupancy and co-repressor recruitment represent plausible mechanistic frameworks for MYB cross-regulation in glucosinolate biosynthesis, warranting future experimental validation [40,41,112].

The chromatin landscape constitutes a pivotal regulatory layer for both inducibility and homeostasis in glucosinolate biosynthesis. In Arabidopsis thaliana, HY5 recruits the histone deacetylase HDA9 to aliphatic GSL promoters, reducing H3K9ac and thereby constraining basal expression while preserving the capacity for rapid JA-induced activation [36,37,88,113,114,115,116]. Concurrently, methionine chain-elongation genes exhibit selective loss of the activating H3K4me3 mark while retaining H3K4me2 and basal transcript levels, a chromatin signature indicative of epigenetic poising. Such poising is mechanistically distinct from generic active or repressive states and often reflects the juxtaposition of restrictive modifiers (e.g., HDA9, PRC2-mediated H3K27me3) with permissive features such as residual H3K4me2 or SWI/SNF-mediated chromatin accessibility. Similar poised configurations have been documented in other plant contexts, including immune priming, where low H3K4me3 facilitates rapid re-induction, and vernalization, where Polycomb-mediated H3K27me3 repression remains environmentally reversible. By analogy, the GSL chromatin architecture likely represents a tailored poising strategy that balances energetic economy with inducibility, although the specific histone writers, erasers, and potential lncRNA scaffolds establishing these locus-specific signatures remain to be elucidated [38].

Beyond TF–hormone crosstalk, pathway commitment is reinforced by mutual antagonism among MYB subgroups: elevated MYB28 suppresses MYB34/MYB51 expression, and vice versa [44]. Whether such repression reflects promoter competition, co-repressor recruitment, or chromatin blockade is not yet resolved [117]. Metabolic feedback adds further control, as accumulation of indole-3-acetaldoxime attenuates flux through the indolic branch, though the molecular sensors mediating this response are still unknown [118].

Transcriptional control of GSL biosynthesis emerges as a multilayered network integrating MYB paralogs, hormone-responsive modules, metabolic feedback, and chromatin-based regulation [119,120]. Dissecting the quantitative contribution of each layer through native-locus epitope tagging, single-cell chromatin profiling, and inducible degron systems will be essential to understand how Brassicaceae plants achieve rapid yet precise switching between aliphatic and indolic defense programs. Glucosinolate biosynthesis is thus not a static, linear pathway but a dynamic, modular system, in which each enzymatic phase functions as a potential control node responsive to internal and external cues (Figure 1) [4,5,120,121,122,123,124,125]. While transcriptional regulation has been extensively characterized [16,106,126,127,128,129], the epigenetic dimension remains comparatively underexplored. Elucidating how histone modifications, DNA methylation, lncRNAs, and microRNAs intersect with canonical transcriptional networks may open new avenues for rationally fine-tuning GSL content and composition to enhance defense and nutritional quality in Brassicaceae crops.

Environmental cues such as biotic and abiotic stresses activate hormonal signaling pathways (e.g., jasmonic acid, ethylene, salicylic acid, abscisic acid), which interact with epigenetic regulators and diverse transcription factor families (MYB, bHLH) to control the expression of biosynthetic genes. These genes encode enzymes involved in amino acid chain elongation, core structure formation, and side-chain modification, ultimately leading to the accumulation of distinct glucosinolate profiles.

5. Epigenetic Control of Glucosinolates Biosynthesis

5.1. DNA Methylation and Its Functional Implications

In plants, DNA methylation occurs in three sequence contexts CG, CHG, and CHH (where H = A, T, or C) and is maintained or established through the coordinated action of DNA methyltransferases and demethylases [130]. While traditionally associated with transcriptional silencing, methylation at gene bodies or promoters can fine-tune expression, influencing the responsiveness of metabolic genes to environmental or developmental cues [131,132,133]. Genome-wide methylome analyses in Arabidopsis thaliana reveal that several GSL biosynthetic and regulatory loci, including MYB28, CYP79F1, and AOP2, harbor context-specific DNA methylation patterns [40]. These patterns often overlap with transposable elements or repeat-rich promoter regions, suggesting potential involvement of the RNA-directed DNA methylation (RdDM) pathway in establishing and maintaining these states [134,135,136,137,138].

Although functional studies remain limited, current evidence suggests that DNA methylation contributes to distinguishing basal from inducible transcriptional states. For example, stress-induced demethylation at promoters of defense-related cytochrome P450 genes has been reported in A. thaliana, raising the possibility that similar mechanisms operate at GSL loci [139,140,141,142]. Conversely, hypermethylation could act as a gatekeeper, restricting unnecessary metabolic investment in GSL production under non-inductive conditions [91,143]. The proximity of key MAM and AOP genes to methylated transposons raises the intriguing hypothesis that RdDM-mediated heterochromatinization may serve as a rapid, reversible switch for branch-specific regulation [118].

Despite these insights, direct causal links between methylation changes and GSL output remain largely untested. Most available data are correlative, lacking targeted manipulation of methylation states at native loci [45,89]. Moreover, methylation dynamics in Brassica crops remain underexplored, with few studies integrating methylome profiling with metabolite quantification [144,145]. Addressing this gap will require locus-specific perturbation approaches, such as CRISPR/dCas9-TET or -DNMT fusions, combined with high-resolution methylome mapping under contrasting environmental regimes [145,146,147,148,149,150]. Such experiments could clarify whether DNA methylation acts primarily as a long-term stability mechanism, a short-term environmental sensor, or both, in the context of GSL biosynthesis.

5.2. Histone Modifications and Chromatin State

Histone post-translational modifications (PTMs) play a central role in regulating chromatin accessibility and transcriptional competence of GSL biosynthetic genes [151]. Activating marks such as histone H3 lysine 4 trimethylation (H3K4me3), H3 lysine 36 trimethylation (H3K36me3), and histone acetylation (e.g., H3K9ac, H3K27ac) are generally associated with transcriptionally active chromatin, whereas repressive marks such as H3 lysine 27 trimethylation (H3K27me3) and H3 lysine 9 dimethylation (H3K9me2) are linked to Polycomb-mediated silencing or heterochromatin formation [152,153,154,155].

In Arabidopsis thaliana, chromatin immunoprecipitation sequencing (ChIP-seq) studies have revealed that several core GSL biosynthetic genes, including CYP79F1, UGT74B1, and SOT16, display bivalent chromatin signatures characterized by the co-occurrence of H3K4me3 and H3K27me3 [36,90,155,156,157]. Such a “poised” configuration is thought to allow rapid transcriptional activation upon environmental induction while preventing constitutive expression that would be metabolically costly [157,158,159].

Functional examples illustrate how histone marks interface with environmental signaling. Light–JA cross-talk in aliphatic GSL regulation involves HY5-mediated recruitment of the histone deacetylase HDA9 to MYB28 and MYB29 promoters, leading to H3K9ac removal and reduced basal transcription [36,40]. This repression, however, enhances the dynamic range of induction upon jasmonate treatment, effectively “priming” the loci for strong, signal-dependent activation [109]. In another example, methionine chain-elongation genes (MAM1, MAM3) exhibit selective depletion of the activating H3K4me3 mark while maintaining H3K4me2 and basal expression, suggesting a fine-tuned chromatin configuration that balances readiness for induction with suppression of unnecessary flux [153,155,158,160].

Despite these insights, our understanding of the histone-modifying enzymes acting at GSL loci remains unclear. The identity of specific histone methyltransferases (“writers”), demethylases (“erasers”), and chromatin readers that establish and interpret these marks is unknown [154]. Likewise, the potential involvement of tissue-specific or stress-responsive histone acetyltransferases (HATs) in GSL regulation has not been systematically investigated in Arabidopsis or Brassica crops [161]. A comprehensive chromatin profiling strategy that integrates histone mark mapping with transcriptome and metabolome data across developmental stages and environmental conditions could reveal how histone PTMs orchestrate branch-specific regulation [154,161]. Targeted epigenetic editing could provide causal evidence, transforming correlative observations into mechanistic understanding.

5.3. Chromatin Remodeling and 3D Genome Architecture

Beyond chemical histone modifications, nucleosome positioning and higher-order genome architecture may also influence the inducibility of GSL biosynthetic genes. At present, no direct evidence exists for chromatin remodeling or 3D spatial clustering of GSL loci. However, insights from other specialized metabolic pathways suggest these mechanisms could provide rapid and reversible regulation. For example, SWI/SNF and CHD chromatin remodelers regulate defense-related gene activation in Arabidopsis, and chromatin looping has been implicated in the coordinated expression of clustered terpenoid and alkaloid biosynthetic genes in other plant systems [162]. Such precedents point to the possibility that similar remodelers or higher-order chromatin contacts could facilitate co-regulation of GSL biosynthetic modules, particularly under stress conditions [163,164]. High-throughput chromatin conformation capture (Hi-C) provides genome-wide maps of 3D genome folding, whereas Capture-C offers high-resolution, locus-focused interaction profiles; neither approach has yet been applied to GSL loci in Arabidopsis or Brassica, representing a key opportunity for future research. Future research integrating chromatin accessibility assays (ATAC-seq), 3D genome mapping, and targeted functional interrogation will be essential to determine whether spatial genome organization contributes to dynamic regulation of GSL metabolism.

5.4. Non-Coding RNAs as Epigenetic Modulators

Non-coding RNAs (ncRNAs) are emerging as important regulators of plant specialized metabolism, with potential but largely unexplored roles in GSL biosynthesis. They can influence gene expression both post-transcriptionally and through chromatin-based mechanisms. Among them, microRNAs (miRNAs), small interfering RNAs (siRNAs), and long non-coding RNAs (lncRNAs) are the most plausible modulators of GSL pathway activity. In Arabidopsis thaliana, miR828 and miR858 regulate R2R3-MYB transcription factors involved in secondary metabolism, suggesting that similar regulation could extend to GSL-related MYBs such as MYB28, MYB29, MYB34, and MYB51. Although direct evidence for these GSL-specific MYBs is lacking, miRNA-mediated repression is likely to fine-tune pathway flux in response to developmental or environmental cues. In Brassica napus, miRNAs responsive to drought stress and secondary metabolism have been identified, but their functional link to GSL regulation remains untested [165,166,167,168,169].

Long non-coding RNAs (lncRNAs) add a further, largely unexplored dimension. In Arabidopsis, the COLDAIR and COOLAIR lncRNAs recruit Polycomb repressive complex 2 (PRC2) to the FLC locus during vernalization, establishing stable yet reversible epigenetic silencing [153,155,165]. By analogy, lncRNAs could act as scaffolds for chromatin modifiers at GSL loci, particularly those under Polycomb control, or function as competing endogenous RNAs (ceRNAs) that sequester miRNAs, thereby modulating MYB transcript stability. In Brassica rapa, stress-responsive lncRNAs have been identified under cold treatment, but their role in secondary metabolism remains undefined.

The lack of integrated ncRNA–GSL studies thus represents a major gap, especially in Brassica crops where ncRNA resources are still limited and functional analyses are scarce. Future progress will require combined strategies such as small RNA sequencing, lncRNA annotation, chromatin profiling, and functional validation through degradome sequencing and CRISPR/dCas9-based ncRNA dissection. These approaches could establish whether ncRNAs act upstream, downstream, or in parallel with epigenetic regulators, offering new opportunities to fine-tune GSL metabolism in crops.

5.5. Integration with Environmental and Developmental Cues

Epigenetic mechanisms in GSL biosynthesis, such as histone marks, DNA methylation and non-coding RNAs, are inherently dynamic and closely tuned to both development stage and environmental challenge [170,171,172]. Increasing evidence indicates that epigenetic priming enables stress-responsive genes to react more rapidly and robustly to recurring stimuli. In Arabidopsis and other plant models, transient exposures to drought, cold or salinity induce chromatin modification, including H3K4me3 enrichment at stress-responsive promoters and altered H3K27me3 deposition that poise specific loci for accelerated reactivation upon re-exposure [173,174,175,176,177,178,179].

In plants, stress-induced chromatin states can persist beyond the initial stimulus, providing a basis for somatic memory and, in some cases, even transgenerational inheritance [180,181,182,183]. For example, drought-induced “memory genes” retain sustained H3K4me3/H3K4me2 enrichment, thereby maintaining a transcriptionally competent yet inactive configuration that allows more rapid re-induction upon subsequent stress [179]. Likewise, pathogen priming leaves defense-related loci marked by residual H3K4me3 and accessible chromatin, a configuration that underlies immune memory in Arabidopsis. Stress-triggered changes in DNA methylation can also be mitotically and meiotically stable, modulating secondary metabolism and defense responses across developmental stages and into the next generation [120]. Although direct evidence at GSL biosynthetic or regulatory loci is still lacking, the strong inducibility of MYB28/29 (aliphatic) and MYB34/51/122 (indolic) transcription factors suggests that they may be embedded within such memory-associated chromatin states [110,125,126]. This perspective highlights stress-induced epigenetic memory as a likely contributor to enhanced GSL accumulation upon recurrent stress exposure, representing an important yet largely unexplored frontier.

Advancing this field will require temporally resolved chromatin mapping during both stress exposure and recovery, integrated with multigenerational epigenomic profiling to assess stability and inheritance of stress-induced marks. Coupling these approaches with single-cell transcriptomics and chromatin accessibility assays (e.g., ATAC-seq) will provide high-resolution insights into the regulatory dynamics of individual cell types. Crucially, causal interrogation using CRISPR/dcas9-based epigenetic editors can move beyond correlation to establish whether GSL biosynthetic and regulatory genes exhibit true epigenetic metabolic memory. Such integrative strategies promise not only to clarify chromatin-level control of GSL metabolism, but also to lay the groundwork for exploiting stress memory as a non-genetic avenue for enhancing resilience and nutritional value in Brassica crops.

Glucosinolate regulation also displays several distinctive features compared with other specialized metabolic pathways [68]. First, the pathway is partitioned into two major branches, aliphatic and indolic, each controlled by distinct MYB transcription factor subgroups that exhibit mutual repression [117]. This cross-antagonism is a rare example of transcription factor crosstalk sharpening pathway specificity, contrasting with the largely linear hierarchies observed in phenylpropanoid or alkaloid biosynthesis. Second, chromatin-based mechanisms add another layer of regulation, exemplified by the selective loss of H3K4me3 at methionine chain-elongation genes, a chromatin signature consistent with epigenetic “poising” that enables rapid inducibility. Such poising has not yet been reported in other major classes of plant defense metabolites. Third, environmental integration is uniquely illustrated by HY5-mediated recruitment of the histone deacetylase HDA9 to GSL promoters, which directly couples light signaling with chromatin remodeling. Finally, metabolic feedback via indole-3-acetaldoxime provides an additional control layer, suggesting a rare integration of metabolic, transcriptional, and epigenetic mechanisms that sets GSL regulation apart from other specialized metabolic networks.

While most mechanistic evidence for epigenetic regulation of stress memory comes from Arabidopsis thaliana, the extent to which similar chromatin states operate in Brassica crops remains largely unexplored (Figure 2). Recent methylome surveys in Brassica rapa and B. napus under drought and salt stress have reported stress-responsive changes in DNA methylation, yet these studies remain descriptive and have not been directly linked to GSL metabolism [125,126,158,164]. Moreover, functional analyses of histone modifiers or ncRNAs in Brassica are limited, underscoring a major translational gap. Bridging this divide will require applying high-resolution epigenomic tools and targeted validation in crop species to assess whether the principles uncovered in Arabidopsis translate into conserved regulatory logic for GSL biosynthesis and stress resilience in Brassicaceae crops.

6. Future Research Priorities

Despite major advances in deciphering the biosynthetic steps of GSL metabolism, many aspects of its regulatory complexity, evolutionary diversification and biotechnological application remain unresolved. Future research should prioritize a more detailed functional characterization of GSL metabolism in cruciferous crops, not only to enhance biological understanding but also to unlock economic value through optimized chemotypes. Investigating the haplotype variation and allelic effects of key regulatory genes, such as BnaC2. MYB could guide precision breeding efforts aimed at tailoring seed GSL content for food, feed or industrial uses. In parallel, dissecting the upstream regulatory networks-including the spatial, developmental, and environmental control of MYB, bHLH, and WRKY transcription factors-will be essential for unraveling the layered control of GSL biosynthesis, particularly in the aliphatic pathway.

A critical and underexplored dimension of GSL regulation involves the role of epigenetic mechanisms. The dynamic responsiveness of GSL biosynthesis to external stimuli strongly suggests the involvement of chromatin-based regulatory systems, such as histone modifications (e.g., H3K27me3, H3K4me3), DNA methylation, and small RNAs. These epigenetic marks could enable developmental priming or stress-induced reprogramming of GSL biosynthetic and transport genes, providing a heritable and reversible layer of metabolic control. Targeted studies employing epigenomic profiling, ChIP-seq, and CRISPR-dCas9 epigenetic editors may uncover novel regulatory hubs, especially in non-model species [147,156,159,182]. Such insights could not only deepen fundamental understanding but also pave the way for epigenome-informed breeding strategies in Brassica oleracea and related crops.

In this context, functional validation via CRISPR–dCas9 epigenome editors offers a powerful and precise approach to interrogate the causal role of chromatin modifications in regulating GSL biosynthesis. Unlike traditional gene knockouts, this system employs catalytically inactive Cas9 (dCas9) fused to chromatin-modifying enzymes such as histone acetyltransferases, demethylases, or DNA methyltransferases to selectively alter the epigenetic landscape at targeted GSL biosynthetic or regulatory loci without changing the underlying DNA sequence. This enables reversible and spatially controlled modulation of gene expression, offering a unique tool to dissect the contribution of specific histone marks or DNA methylation states to GSL pathway flux. In Arabidopsis and emerging Brassica models, such precision epigenetic editing can validate the functional relevance of bivalent chromatin states observed at key MYB or CYP loci and explore their responsiveness to environmental or hormonal stimuli.

Although epigenome editing and epigenome-informed breeding hold considerable promise for the targeted modulation of GSL biosynthesis, several practical and conceptual constraints must be acknowledged. A central limitation lies in the stability and heritability of epigenetic marks: while certain DNA methylation patterns can persist through mitotic and, in some cases, meiotic divisions, many histone modifications and stress-induced chromatin states are subject to reprogramming, thereby constraining their long-term utility in breeding. Moreover, the polyploid architecture of Brassica crops introduces additional complexity, as duplicated loci may display divergent regulatory dynamics and complicate precise epigenetic targeting. Beyond biological challenges, regulatory frameworks also shape translational potential. In the European Union and other regions, epigenome-editing tools such as CRISPR/dCas9 remain subject to GMO-like legislation, raising questions about their acceptance and deployment in commercial breeding pipelines. Collectively, these factors underscore that the integration of epigenetic approaches into crop improvement will require not only fundamental advances in understanding epigenetic stability and plasticity, but also innovative translational strategies and regulatory clarity to fully realize their potential in sustainable agriculture.

As summarized in

Table 3. Future research priorities in the epigenetic regulation of GSL biosynthesis.

Priority Area	Key Research Questions	Recommended Approaches	Expected Outcomes/Applications
Epigenomic landscape mapping	Which GSL biosynthetic and regulatory loci are associated with dynamic chromatin modifications?	ChIP-seq (H3K4me3, H3K27me3, H3K9ac), ATAC-seq, whole-genome bisulfite sequencing (WGBS) in Arabidopsis and Brassica spp.	Identification of epigenetically regulated genes controlling GSL biosynthesis and transport under developmental or stress conditions
Functional validation of epigenetic marks	Do specific chromatin modifications causally influence GSL gene expression and metabolite accumulation?	CRISPR/dCas9-based epigenome editing (targeting histone marks or DNA methylation), mutant analysis of writers/erasers (e.g., SDG8, REF6)	Mechanistic understanding of how chromatin states control GSL pathway activation and repression
Non-coding RNA regulation	What roles do small RNAs (miRNAs, siRNAs) and long non-coding RNAs play in GSL pathway regulation?	sRNA-seq, degradome sequencing, lncRNA annotation, target prediction and validation	Discovery of RNA-based regulatory modules influencing GSL biosynthesis, potentially modulating tissue- or stress-specific expression
Epigenotype × environment interactions	How do environmental stimuli reshape the epigenetic regulation of GSL pathways?	Controlled environment experiments (light, drought, JA/SA treatments) + time-resolved epigenomic profiling	Insights into chromatin-mediated plasticity of GSL responses under abiotic and biotic stress
Epigenetic variation in crop germplasm	Are there natural or induced epialleles linked to GSL content or composition in Brassica species?	EpiGWAS, methylation-sensitive markers, bisulfite epiQTL mapping across accessions	Identification of stable epigenetic variants for breeding high-GSL or stress-resilient cultivars
Translational applications	Is it possible to exploit epigenetic regulation as a tool to optimize GSL composition for both crop improvement and dietary benefits?	Epigenetic priming, seed treatments, CRISPR/dCas9 epi-engineering, chromatin-targeted agrochemicals	Development of epigenome-informed breeding or treatment strategies to enhance crop value and resilience

, the epigenetic regulation of GSL biosynthesis remains an emerging field with substantial knowledge gaps across both model and crop systems. Future investigations should prioritize integrative approaches that combine chromatin profiling, non-coding RNA discovery, and functional validation via CRISPR-based epigenome editing. Such efforts are essential not only to decode the chromatin-level control of GSL metabolic genes, but also to exploit epigenetic variation for breeding stress-resilient and nutritionally enriched Brassicaceae crops.

Moreover, future research should examine how quantitative trait loci (QTL) for seed and leaf GSL content interact with environmental variables, particularly under abiotic stress or low sulfur conditions. In this regard, integrating epigenetic plasticity with QTL mapping may help explain genotype × environment interactions. The regulation of GSL transporters, such as GTR1 and GTR2, also warrants further attention, due to their central function in regulating metabolite partitioning and tissue-specific distribution across developmental and environmental contexts [100,101]. Finally, comparative evolutionary analysis across the Brassicales could shed light on the origin and diversification of GSL pathway genes, including epigenetic remodeling events associated with domestication. A more holistic understanding of the epigenetic, hormonal, and transcriptional interplay in GSL metabolism holds significant potential for developing climate-resilient, biofortified, and ecologically tailored cruciferous crops.

7. Conclusions

Over the past two decades, GSL research in Brassicaceae has evolved from pathway elucidation to an increasingly nuanced understanding of its regulatory complexity. This review highlights that epigenetic regulation, through DNA methylation, histone modifications, chromatin remodeling and non-coding RNA, forms a dynamic and multi-layered control system that enables plants to fine-tune GSL biosynthesis in response to developmental cues and environmental challenges. These chromatin-based mechanisms integrate with transcription factor networks, hormonal signaling, and metabolic feedback to achieve precise, branch-specific modulation of defense chemistry, balancing ecological fitness with the metabolic economy.

Despite these advances, the epigenetic dimension of GSL regulation remains an underexplored frontier. Much of the current evidence is correlative, derived largely from Arabidopsis thaliana, and rarely linked directly to quantitative changes in GSL composition in agronomically relevant Brassica crops. Bridging this gap will require integrative, multi-omics approaches that combine high-resolution epigenomic profiling with transcriptomic and metabolomic datasets under both controlled and field conditions. Functional validation through locus-specific epigenome editing, using CRISPR/dCas9-based DNA methylation or histone modification tools, will be essential to move from association to causation.

Looking ahead, harnessing epigenetic regulation offers a promising route to design crops with tailored GSL profiles for improved pest resistance, enhanced nutritional value, and optimized flavor. Epigenetic-assisted breeding, the identification of stable and selectable epialleles, and the strategic deployment of stress-priming interventions could deliver these traits without permanent genomic alteration, offering potential advantages in regulatory acceptance and public perception. Furthermore, expanding epigenetic studies into diverse Brassicaceae species, including landraces and wild relatives, may uncover naturally occurring chromatin states that can be leveraged for crop improvement.

In this context, the integration of mechanistic epigenetics with plant breeding and agronomy heralds a new era of chromatin-informed crop design. By combining detailed molecular insight with translational innovation, the next phase of GSL research has the potential to not only advance fundamental plant biology but also deliver tangible benefits for sustainable agriculture, food security, and human health.