Transcriptomic and metabolomic profiling to unravel sex-dependent mechanisms and the role of exogenous glutathione in Marchantia polymorpha under cadmium stress

Abstract

Cadmium (Cd) contamination poses a persistent threat to plant fitness and ecosystem stability, yet the molecular basis of sex-dependent Cd tolerance in dioecious plants remains unclear. In this study, the dioecious early land plant Marchantia polymorpha was employed to investigate the transcriptomic and metabolomic responses, physiological and morphological variations, and the remedial effects of exogenous glutathione (GSH). Physiological analyses revealed pronounced sexual dimorphism: females accumulated significantly more Cd (82.97 ± 0.75 mg·kg⁻¹) than males (61.80 ± 0.10 mg·kg⁻¹) and suffered more severe chlorosis and oxidative damage, whereas males maintained lower Cd burdens and exhibited stronger antioxidant enzyme activities, including superoxide dismutase (SOD) and catalase (CAT). Transcriptomic analysis identified 4,547 differentially expressed genes (DEGs) in females and 4,102 in males under Cd stress, with GO and KEGG enrichment revealing that females preferentially reprogrammed photosynthesis and defense-related pathways, while males activated carbohydrate metabolism and cell wall remodeling. Metabolomic profiling further uncovered sex-divergent metabolic flux, with females accumulating naringenin chalcone (log₂FC = 3.88) and males specifically upregulating quercetin biosynthesis via F3’H. Integrated transcriptomic and metabolomic analysis highlighted three core pathways—flavonoid biosynthesis, tyrosine metabolism, and GSH metabolism—as central hubs of sex-specific Cd responses. Exogenous GSH application alleviated Cd-induced toxicity in a sex-dependent manner: males showed greater reduction in Cd accumulation (32.58%) compared to females (8.36%) and more efficient restoration of redox homeostasis, while females exhibited stronger recovery of photosynthetic pigments and flavonoid accumulation. This study provides a molecular-level perspective for understanding sex-dependent heavy metal adaptation in early land plants and offers a rationale for sex-aware phytoremediation strategies.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08772-8.

Introduction

Heavy metal (HM) pollution stands as one of the most formidable environmental challenges of the modern era. Cadmium (Cd) is a highly toxic metal that reduces plant growth and development [1]. Among HMs, Cd is particularly hazardous due to its potent toxicity, high mobility, and propensity for bioaccumulation, posing a serious threat to both ecosystem stability and human health [2–4]. In plants, Cd exposure initiates a cascade of toxic events: it disrupts the uptake and homeostasis of essential nutrients, impairs the function of key enzymes involved in photosynthesis and respiration, and potently induces the generation of reactive oxygen species (ROS) [5–8]. The resulting oxidative burst inflicts widespread damage, including protein oxidation, lipid peroxidation, and DNA lesions, ultimately suppressing growth and viability [4, 9, 10].

To counteract Cd toxicity, plants have evolved a multi-tiered defense system. The cell wall serves as the first barrier, sequestering Cd ions extracellularly. Intracellularly, free Cd²⁺ is chelated by thiol-rich ligands such as glutathione (GSH) and phytochelatins (PCs). These complexes are subsequently trafficked and compartmentalized into the vacuole, primarily via tonoplast-localized ATP-binding cassette (ABC) transporters, achieving effective detoxification [8–11]. Concurrently, a sophisticated antioxidant network—encompasses enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), as well as non-enzymatic antioxidants including GSH and flavonoids—is activated to maintain redox homeostasis [12–14]. While these core mechanisms are well established, how they are differentially regulated between sexes in dioecious plants under Cd stress remains poorly understood.

In terrestrial ecosystems, approximately 5–6% of angiosperms and about 60% of liverworts are dioecious [15, 16]. Sexual dimorphism in these species extends beyond reproductive structures to fundamental differences in physiology, resource allocation, and stress adaptation, largely governed by evolutionary trade-offs between reproduction and defense [11]. A prevailing hypothesis posits that because females typically allocate more resources to reproduction for seed and fruit development, males may invest more in stress defense, thereby exhibiting greater tolerance [17–19]. Supporting this view, male Populus deltoides has shown superior antioxidant capacity and growth under lead stress [20]. However, this pattern is not universal; for instance, female Silene latifolia exhibits greater adaptive fitness under copper stress despite higher accumulation [21]. These discrepancies indicate that sex-specific stress responses arise from interactions between species-specific traits and stressor identity, the underlying molecular mechanisms remain largely unresolved, calling for suitable model systems.

The dioecious early land plant Marchantia polymorpha offers a compelling model to address this gap. Unlike angiosperms with large, complex genomes and limited genetic tools, M. polymorpha possesses a small, well-annotated genome, simple thalloid morphology, short life cycle, and established transformation protocols [22–24]. Additionally, its high surface-area-to-volume ratio and predominantly passive uptake mechanism make it highly effective at accumulating Cd, combining model organism tractability with bioindicator properties [25, 26]. Our previous single-omics analysis revealed distinct sex-specific responses under Cd stress: male transcriptional and metabolic profiles were enriched in flavonoid biosynthesis, whereas female responses were more associated with signal transduction pathways [27, 28].

To move beyond single-omics approaches, we employed an integrated transcriptomic and metabolomic strategy to obtain a comprehensive landscape of sex-specific Cd responses [29, 30]. However, multi-omics profiling alone can only reveal correlative associations. To functionally validate the role of GSH metabolism, we adopted an exogenous GSH intervention strategy for three reasons: (i) the priming of plant endogenous defense systems via exogenous compounds represents a potent strategy for enhancing stress resilience [1, 5, 31–34]; (ii) GSH serving as a major antioxidant, a phytochelatin precursor, and a key node in the ascorbate–glutathione (AsA-GSH) cycle [35, 36]; (iii) exogenous GSH has been shown to mitigate Cd toxicity in various crops, providing a well-established experimental paradigm [37–39].

This study was designed to address three pivotal questions: (1) How is sex-specific transcriptional and metabolic reprogramming achieved in M. polymorpha under Cd stress, and what are the core divergent pathways? (2) How do key metabolic pathways differ in their response patterns between sexes? (3) Does exogenous GSH confer sex-specific alleviation of Cd-induced damage? By integrating multi-omics profiling with targeted pharmacological validation, this study aims to elucidate the molecular basis of sex-dependent heavy metal adaptation in a dioecious early land plant and to provide a conceptual framework for developing sex-aware phytoremediation strategies.

Results

Overview of experimental strategy

To unravel gender-dependent Cd response mechanisms in M. polymorpha, we used a time-resolved multi-omics strategy combined with physiological validation. Male and female gametophytes were exposed to 100 mg L⁻¹ CdCl₂ (renewed every 48 h). Transcriptomic profiling was performed at 96 h (acute phase), and metabolomic profiling at 20 d (chronic phase). This design captured both early transcriptional reprogramming and long-term metabolic adaptation. The datasets were integrated to identify sex-specific pathways. To functionally validate glutathione metabolism, exogenous GSH (100 and 200 mg L⁻¹) was applied under Cd stress, and physiological parameters were measured.

Transcriptome sequencing and data quality assessment

Transcriptome analysis was performed on 12 samples (CK and Cd-treated, both sexes). A total of 609,348,400 high-quality clean reads were generated, with Q20 > 98.00%, Q30 > 94.00%, and a mapping rate exceeding 88.29% (Supplementary Table S1). Biological replicates showed high consistency (r ≥ 0.91, Fig. 1A). PCA clearly separated samples by treatment (PC1: 65.52%) and sex (PC2: 9.97%), indicating distinct transcriptional responses to Cd stress and inherent sexual dimorphism in gene expression profiles (Fig. 1B).

Fig. 1.

Quality assessment of transcriptome sequencing data. A Pearson Correlation Coefficients Analysis between biological replicates. B Principal Component Analysis (PCA) of all samples

Identification and functional enrichment of DEGs

Having established the reliability of the transcriptomic data, we next identified DEGs in response to Cd stress. Differential expression analysis identified 4,547 DEGs in females (2,086 upregulated, 2,461 downregulated) and 4,102 DEGs in males (2,019 upregulated, 2,083 downregulated) under Cd stress (Fig. 2A). The Venn diagram further revealed 1,612 upregulated and 1,573 downregulated DEGs shared by both sexes, alongside sex-specific DEGs—474 upregulated and 888 downregulated in females, and 407 upregulated and 510 downregulated in males (Fig. 2B), indicating prominent sexual dimorphism in transcriptional responses. RT-qPCR validation of six randomly selected DEGs showed expression patterns highly consistent with RNA-Seq fold changes (Supplementary Fig. S1), confirming the reliability of the transcriptomic data.

Fig. 2.

Analysis of DEGs in male and female M. polymorpha under Cd stress. A Histogram of upregulated and downregulated DEGs; B Venn diagram of DEGs between groups

Gene Ontology (GO) enrichment analysis of Cd-responsive DEGs revealed distinct sex-specific functional biases (Fig. 3A, B; Supplementary Fig. S2; Supplementary Tables S2–S5). In females, DEGs were significantly enriched in biological processes related to photosynthesis (including light reactions and light harvesting), secondary metabolism (phenylpropanoid biosynthesis), defense response, and response to abiotic stimuli. Consistently, cellular component terms were highly enriched in photosynthetic structures, such as thylakoids and photosynthetic membranes. In contrast, male DEGs were predominantly enriched in biological processes associated with small molecule metabolism, hexose biosynthesis, lignin metabolism, and phenylpropanoid metabolism, with cellular component terms enriched in the cell wall and external encapsulating structures.

Fig. 3.

GO and KEGG enrichment analysis of DEGs. A, B GO enrichment of DEGs in (A) female and (B) male M. polymorpha under Cd stress. C, D KEGG pathway enrichment analysis of DEGs in (C) female and (D) male M. polymorpha under Cd stress. GeneRatio represents the proportion of DEGs associated with a given term relative to the total number of DEGs. Bubble size corresponds to the number of DEGs assigned to each term, and color indicates the adjusted P‑value (FDR < 0.05)

Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (q-value < 0.05) further refined these functional trends (Fig. 3C, D). Phenylpropanoid biosynthesis was significantly enriched in both sexes, suggesting a core conserved pathway in Cd stress response. However, female DEGs were significantly enriched in phenylpropanoid biosynthesis, photosynthetic carbon fixation, and plant-pathogen interaction pathways, consistent with the GO results. Male DEGs, in contrast, exhibited robust enrichment in carbohydrate metabolism and microbial metabolism in diverse environments pathways.

Combined GO and KEGG analyses indicated that female M. polymorpha transcriptional responses to Cd stress are more focused on photosynthesis, secondary metabolism, and defense-related processes, whereas male responses are geared towards carbohydrate metabolism and cell wall restructuring.

Enrichment analysis of DAMs

KEGG enrichment analysis identified both shared and sex-specific metabolic signatures (Fig. 4A, B). Both sexes showed significant enrichment in metabolic pathways, phenylpropanoid biosynthesis, tyrosine metabolism, and ABC transporters, albeit with distinct enrichment strengths. Females exhibited a stronger bias toward secondary metabolite biosynthesis and glycine, serine and threonine metabolism, whereas males showed prominent enrichment in linoleic acid metabolism and branched-chain amino acid biosynthesis (valine, leucine, and isoleucine).

Fig. 4.

KEGG pathway enrichment analysis of DAMs of female (A) and male (B) M. polymorpha under Cd stress. MetaRatio represents the enrichment factor of each pathway. Bubble size corresponds to the number of DAMs mapped to the pathway, and color indicates the P‑value

Three core metabolic pathways involved in Cd stress response

To identify potential regulatory couplings between gene expression and metabolite accumulation, we performed Pearson correlation analysis between DEGs and DAMs (Supplementary Table S6) co-enriched in the same KEGG pathways. A heatmap revealed distinct correlation patterns between sexes (Fig. 5), and the complete correlation coefficients are provided in Supplementary Table S7.

Fig. 5.

Gene-metabolite correlation heatmaps for core stress-response pathways in M. polymorpha under Cd stress. A Flavonoid biosynthesis, B Glutathione metabolism, and C Tyrosine metabolism pathways are shown, with female (left) and male (right) M. polymorpha heatmaps displaying Pearson correlation coefficients between key DEGs and DAMs. Genes are grouped by enzyme function (top color bar), and asterisks represent statistical significance (*P < 0.05, **P < 0.01)

Divergent flux in the flavonoid biosynthesis pathway

The flavonoid biosynthesis pathway (map00941) exhibited a high degree of transcriptional mobilization, with 32 DEGs in females and 31 in males (Supplementary Table S8, S9), accounting for over 55% of the pathway’s total genetic architecture. Under Cd stress, both sexes upregulated early-stage genes, including C4H, HCT, C3’H, CHS, and F3’H, while suppressing downstream CHI and F3H (Fig. 6B). However, the resulting metabolic flux revealed distinct sexual dimorphism. In females, high CHS expression coupled with limited downstream conversion led to a significant accumulation of naringenin chalcone (log₂FC = 3.88, P = 1.81 × 10⁻²), while apigenin (log₂FC = −1.59, P = 2.16 × 10⁻⁴) and luteolin (log₂FC = −1.19, P = 8.45 × 10⁻³) were significantly reduced. Consistently, CHS expression was strongly correlated with naringenin chalcone accumulation in females (Fig. 5A). In contrast, males exhibited specific upregulation of F3’H (MARPO_0022s0191), which drove the synthesis of quercetin (log₂FC = 2.40, P = 3.15 × 10⁻⁴), and F3’H expression correlated specifically with quercetin in males (r = 0.92, P < 0.01).

Fig. 6.

Integrated transcriptomic and metabolomic profiling of three core pathways in M. polymorpha under Cd stress. A Tyrosine metabolism, B Flavonoid biosynthesis, C Glutathione metabolism. Heatmaps show log₂FC of key genes and metabolites in females and males under Cd stress

Suppression of the tyrosine metabolism pathway

Cd toxicity significantly impacted the tyrosine metabolism pathway (map00350), with 41.76% of pathway genes identified as DEGs in females and 31.87% in males (Supplementary Table S10, S11). Transcriptional analysis revealed a coordinated upregulation of TAT and HPD, which correlated with a massive accumulation of 4-hydroxyphenylpyruvate (Female: log₂FC = 12.298, P = 2.03 × 10⁻³; Male: log₂FC = 10.629, P = 1.29 × 10⁻⁵). Despite this upstream activation, the terminal branch of the pathway was severely inhibited: downregulation of DDC and PPO led to a marked reduction in dopamine content in both sexes (Fig. 6A), which is consistent with the correlation analysis (Fig. 5C). Notably, the dopamine reduction was more severe in males (log₂FC = −1.3704, P = 2.13 × 10⁻²) than in females (log₂FC = −1.0991, P = 3.45 × 10⁻²).

Orchestration of the glutathione metabolism pathway

Females and males had 51.79% and 46.43% DEGs enriched in the glutathione metabolism pathway (map00480), respectively (Supplementary Table S12, S13). Both sexes mounted a robust response by upregulating genes involved in GSH synthesis (OPLAH, GCL, GSS) and maintenance (GSR, IDH, 6PGD, G6PD), while simultaneously downregulating the gene associated with GSH degradation (GGT) (Fig. 6C). Notably, the expression of genes involved in glutathione degradation exhibited sexual dimorphism: the DHAR gene was significantly downregulated only in males (log₂FC = −2.27, P = 3.11 × 10⁻⁷), whereas GPX was significantly downregulated only in females (log₂FC = −1.03, P = 3.80 × 10⁻³). The upregulation of IDH and 6PGD was significantly more pronounced in females than in males. This robust transcriptional orchestration suggests that the glutathione system may serve as a metabolic cornerstone for Cd tolerance in female M. polymorpha.

Alleviation of Cd toxicity by exogenous GSH

Phenotypic and photosynthetic pigment

Exposure to 100 mg L⁻¹ CdCl₂ for 20 d induced severe chlorosis and growth inhibition in both male and female M. polymorpha, with females exhibiting more severe damage (Fig. 7). The co-application of exogenous GSH effectively alleviated these symptoms.

Fig. 7.

Effects of exogenous GSH on phenotype in male and female M. polymorpha under Cd stress

Consistent with the phenotypic observations, Cd stress significantly reduced chlorophyll (Chl) content in both sexes (P < 0.05; Table 1). Total Chl content decreased by 77.94% in males and 60.23% in females relative to their respective controls. Exogenous GSH significantly mitigated this chlorophyll degradation. In females, total Chl content increased by 121.67% and 98.33% under Cd + GSH100 and Cd + GSH200 treatments, respectively, compared to the Cd-only group. The corresponding increases in males were 64.08% and 84.47%. Despite this restoration, Chl levels in males remained higher than in females under all Cd-exposed conditions. The Chl a/b ratio was significantly elevated by Cd stress. Exogenous GSH further increased this ratio in females but had no significant effect in males.

Table 1.

Chlorophyll content in M. polymorpha under Cd stress with exogenous GSH

	Chl a (mg·g−1)	Chl b (mg·g−1)	Total Chl(mg·g−1)	Chl a/Chl b
F_CK	0.146 ± 0.0026a	0.126 ± 0.0004a	0.272 ± 0.0027a	1.161 ± 0.0207de
F_Cd	0.036 ± 0.0023f	0.024 ± 0.0012 g	0.060 ± 0.0035 g	1.490 ± 0.0217b
F_Cd + GSH100	0.086 ± 0.0058d	0.048 ± 0.0033e	0.133 ± 0.0091e	1.801 ± 0.0092a
F_Cd + GSH200	0.077 ± 0.0044d	0.042 ± 0.0021ef	0.119 ± 0.0065e	1.836 ± 0.0135a
F_GSH100	0.140 ± 0.0054a	0.128 ± 0.0031a	0.268 ± 0.0023a	1.094 ± 0.0697e
F_GSH200	0.144 ± 0.0032a	0.125 ± 0.0024a	0.269 ± 0.0008a	1.156 ± 0.0474de
M_CK	0.145 ± 0.0028a	0.114 ± 0.0027b	0.259 ± 0.0012ab	1.265 ± 0.0531cde
M_Cd	0.064 ± 0.0024e	0.038 ± 0.0014f	0.103 ± 0.0038f	1.674 ± 0.0036a
M_Cd + GSH100	0.106 ± 0.0079c	0.063 ± 0.0042d	0.169 ± 0.0094d	1.701 ± 0.1536a
M_Cd + GSH200	0.119 ± 0.0107b	0.071 ± 0.0058c	0.190 ± 0.0091c	1.690 ± 0.2415a
M_GSH100	0.148 ± 0.0028a	0.111 ± 0.0049b	0.259 ± 0.0021ab	1.342 ± 0.0857bc
M_GSH200	0.141 ± 0.0024a	0.109 ± 0.0064b	0.250 ± 0.0040b	1.299 ± 0.1013 cd

Data are presented as mean ± SD (n = 3). Different lowercase letters indicate significant differences (P < 0.05, Tukey’s test) among treatments and different sexes for the same parameter

Cd accumulation

Cd accumulation was substantially higher in females (82.97 ± 0.75 mg·kg⁻¹) than in males (61.80 ± 0.10 mg·kg⁻¹) following Cd exposure (P < 0.05; Fig. 8A). The application of 100 mg L⁻¹ exogenous GSH significantly reduced tissue Cd content, with a greater reduction observed in males (32.58%) than in females (8.36%).

Fig. 8.

Physiological and biochemical responses of male and female M. polymorpha to Cd stress and exogenous GSH. A Cd content. B Hydrogen peroxide (H₂O₂) content. C Malondialdehyde (MDA) content. D Endogenous GSH content. E Total flavonoid content. F–H Activities of antioxidant enzymes: F Superoxide dismutase (SOD), G Catalase (CAT), and H Peroxidase (POD)

Oxidative damage

Cadmium stress triggered a significant increase in oxidative markers in both sexes (P < 0.05; Fig. 8B, C). Compared to controls, H₂O₂ content increased by 69.48% in females and 95.75% in males under Cd stress alone, while MDA levels rose by 73.22% and 47.82%, respectively.

Exogenous GSH application alleviated this oxidative damage in a concentration-dependent manner. Under the Cd + GSH100 treatment, H₂O₂ levels decreased by 34.87% in females and 25.07% in males compared to the Cd-only group, with concurrent reductions in MDA content of 16.21% and 31.31%. The alleviating effect was enhanced at 200 mg L⁻¹ GSH (Cd + GSH200), where H₂O₂ levels dropped by 49.45% in females and 37.14% in males, and MDA content decreased by 34.04% and 33.97%.

A distinct sexual dimorphism was observed. Across all Cd-exposed conditions, male plants maintained significantly higher H₂O₂ content than females (P < 0.05). For MDA, while no significant sexual difference was found under Cd stress alone, females exhibited 34.85% higher MDA content than males under the Cd + GSH100 treatment (P < 0.05).

Non-enzymatic antioxidant systems response

To further validate the findings from the metabolic pathway analyses and examine the responses of two key non‑enzymatic antioxidants, we measured the endogenous GSH content and TFC.

Cd stress itself significantly induced the accumulation of endogenous GSH in both sexes (P < 0.05; Fig. 8D). The induction was more pronounced in males, reaching 0.703 ± 0.056 μmol g⁻¹ (a 258.7% increase over the control), compared to 0.414 ± 0.027 μmol g⁻¹ in females (a 72.5% increase). Exogenous GSH application under Cd stress further enhanced endogenous GSH pools in a dose-dependent manner. Males maintained significantly higher GSH levels than females under all Cd-exposed conditions. GSH treatment alone did not alter endogenous levels.

Cd stress exerted opposite effects on TFC between the sexes (Fig. 8E). Compared to controls, TFC decreased significantly in females (−35.23%) but increased in males (+ 36.85%). Consequently, under Cd stress alone, male TFC was approximately 109.45% higher than in females. Exogenous GSH application modulated these stress-induced changes. In females, both Cd + GSH treatments significantly increased TFC (by 76.08% and 73.75%) relative to the Cd-only group, bringing it closer to control levels. In males, the Cd + GSH100 treatment reduced the stress-elevated TFC by 24.69%, whereas the higher concentration (Cd + GSH200) had no significant effect.

Enzymatic antioxidant system response

Cd stress significantly increased the activities of SOD, CAT, and POD in both sexes (P < 0.05; Fig. 8F–H). The increases in females were 56.62% 28.49%, and 200.99%, respectively. In males, the increases were 348.78%, 63.92%, and 165.75%. Males exhibited a stronger induction of SOD and CAT activities than females.

Exogenous GSH further enhanced antioxidant enzymes activities under Cd stress. In females, compared to the Cd-only treatment, the addition of 100 mg L⁻¹ GSH (Cd + GSH100) elevated SOD, CAT, and POD activities by a further 110.36%, 35.86%, and 70.38% A higher GSH concentration (Cd + GSH200) resulted in even greater increases of 642.99%, 65.67%, and 71.43%. SOD activity in females showed the highest sensitivity to increasing GSH concentration. In males, the Cd + GSH100 treatment increased SOD, CAT, and POD activities by 349.22%, 4.42%, and 46.87% relative to the Cd-only group. However, compared to the Cd + GSH100 treatment, the magnitude of the GSH-induced increase was relatively lower under the Cd + GSH200 treatment, with rises of 229.01%, 2.96%, and 29.48%.

Under the Cd + GSH100 treatment, males maintained significantly higher SOD and CAT activities than females (by 250.69% and 22.50%), while POD levels were comparable between sexes. In contrast, under the Cd + GSH200 treatment, male SOD activity was 27.28% lower than in females, and male POD activity was 27.98% lower, while CAT activity remained comparable.

Discussion

Sexual dimorphism in cadmium accumulation and physiological resilience

Our results demonstrate a pronounced sexual dimorphism in the response of M. polymorpha to Cd stress. Females exhibited significantly higher tissue Cd accumulation compared to males, which was accompanied by more severe phenotypic damage. Specifically, Cd stress triggered a substantial decline in Chl a and b contents in both sexes, with females experiencing a greater reduction (Chl a: 75.34%; Chl b: 80.95%) than males (Chl a: 55.86%; Chl b: 66.67%). Transcriptomic analysis revealed that DEGs under Cd stress were significantly enriched in photosynthesis-related pathways, confirming at the molecular level that the female photosynthetic apparatus is more sensitive to Cd toxicity (Fig. 3A, C; Supplementary Fig. S2). This vulnerability was reflected in a 60.23% reduction in total Chl content and more extensive oxidative damage, evidenced by a 73.22% increase in MDA levels, compared to a 47.82% increase in males. In contrast, males maintained lower internal Cd concentrations and exhibited more robust inherent antioxidant defenses, specifically higher activities of SOD and CAT.

The non-enzymatic antioxidant system also displayed sex-dependent patterns. Although Cd stress induced endogenous glutathione accumulation in both sexes, the response was more robust in males, who achieved a 258.7% increase over controls compared to a 72.5% increase in females. Moreover, Cd stress exerted opposite effects on TFC, which decreased in females but increased in males. Both sexes accumulated osmoregulatory compounds as an adaptive response to osmotic imbalance, yet with distinct preferences: females preferentially accumulated soluble proteins (SP) and soluble sugars (SS), whereas males showed marked increases in SP and proline (Pro) (Supplementary Fig. S4).

Divergent metabolic reprogramming: flavonoids and tyrosine

Multi-omics integration revealed that sexual dimorphism in Cd tolerance is driven by distinct metabolic fluxes. Consistent with findings in Sorghum bicolor [40], flavonoid biosynthesis was highly mobilized under stress. In females, the suppression of downstream genes like CHI leads to the massive accumulation of naringenin chalcone (log₂FC = 3.88). Given the high antioxidant capacity of naringenin chalcone, its accumulation likely serves as a specialized chemical defense to compensate for the reduction of downstream derivatives (apigenin and naringenin) [41]. Conversely, males specifically upregulate F3’H to channel metabolic flux toward quercetin. As a classic flavonol with a free 3-OH group, quercetin exhibits superior antioxidant activity compared to apigenin or luteolin and functions as an effective metal chelator [42]. Moreover, it has been shown to alleviate stress in various plants by scavenging ROS [43]. As a potent antioxidant and metal chelator, quercetin biosynthesis represents a key component of the male-specific defense architecture.

We identified tyrosine metabolism as a critical, yet previously under-characterized, responsive pathway in M. polymorpha. (Fig. 5A). The downregulation of PPO and DDC along with a significant reduction in dopamine—a known antioxidant, may represent a characteristic metabolic signature of Cd stress in this species. We propose that this metabolic inhibition reflects an active reprogramming of resources under stress. Since phenylalanine is a shared precursor for both tyrosine and flavonoid biosynthesis, carbon flux may be preferentially diverted toward flavonoid biosynthesis, thereby limiting the availability of carbon skeletons for the tyrosine/dopamine branch. The more severe dopamine reduction in males is intriguing, given their higher overall Cd tolerance. The stronger inhibition observed in males may be linked to their greater investment in quercetin synthesis, which is consistent with our results from the correlation analysis of key metabolites (Supplementary Fig. S3).

The central role of glutathione in Cd detoxification

Our study confirms that GSH metabolism is a core conserved pathway in the response of M. polymorpha to Cd stress, consistent with observations in Solanum nigrum [31] and Celosia argentea [44]. At the transcriptional level, females mount a more intensive response, with sharper upregulation of synthesis genes (e.g., GCL, GSS) and key enzymes of the pentose phosphate pathway (e.g., G6PD) that supply NADPH for GSH recycling. However, prolonged Cd stress naturally induced endogenous GSH accumulation, particularly in males (Fig. 8B). This discrepancy between transcriptional response and final metabolite abundance likely arises from sex-specific post-transcriptional regulation, metabolic flux distribution, or differential GSH turnover rates [45, 46]. For instance, a lower rate of GSH oxidation consumption or slower degradation rate. Our pharmacological intervention showed that exogenous GSH effectively mitigated toxicity by reducing Cd accumulation (a 32.58% reduction in males versus 8.36% in females) and by enhancing antioxidant enzyme activities. Interestingly, female SOD activity was highly sensitive to GSH dosage, suggesting that exogenous thiols can partially rescue the less efficient female antioxidant system.

Mechanisms of GSH-mediated alleviation on physiological function

GSH is a central metabolite in the plant antioxidant defense system, playing key roles in cell differentiation, ROS scavenging, thiol-disulfide exchange, and the biosynthesis of PCs [39]. Within the complex detoxification network activated under Cd stress, GSH not only directly chelates free Cd²⁺ but also serves as the precursor for PCs. These peptides form stable complexes with Cd, which are subsequently compartmentalized into the vacuole via ABC transporters, effectively reducing cytosolic Cd toxicity [47]. Beyond chelation, GSH application significantly alleviated Cd-induced phenotypic damage and reversed chlorophyll degradation (Fig. 7, Table 1). The alleviation of Cd-induced photosynthetic pigment loss by exogenous GSH has also been reported in Brassica campestris [48]. Notably, the restorative effect of GSH on photosynthetic pigments was more prominent in females: the recovery magnitudes of Chl a, Chl b, and total chlorophyll were higher in females than in males. Moreover, the Chl a/b ratio increased further in GSH-treated females, suggesting an improvement in their light-use efficiency [49]. This indicates that GSH may regulate photosynthetic metabolic processes in female plants more effectively than in male plants.

Although Cd is not redox-active, it indirectly triggers the overproduction of ROS—including O₂•⁻, H₂O₂, and •OH—by displacing redox-active metals (e.g., Cu, Fe) from proteins and activating NADPH oxidases, ultimately leading to oxidative damage of proteins, DNA, and membrane lipids [50]. In this study, we observed significant elevations in H₂O₂ and MDA levels in both male and female M. polymorpha under Cd stress (Fig. 8 B, C), signifying pronounced lipid peroxidation under oxidative stress. To counteract this, both sexes exhibited significantly enhanced activities of antioxidant enzymes, specifically SOD, CAT, and POD (Fig. 8 F–H), aligning with reports in Stachys lanata [51] and Solanum tuberosum [38]. Notably, male thalli exhibited significantly higher activities and greater increases of SOD and CAT compared to females (P < 0.05), suggesting a stronger enzymatic antioxidant capacity under Cd stress. Interestingly, despite having lower MDA content than females, males maintained higher H₂O₂ levels under Cd stress. We speculate that this paradoxical observation may be explained by kinetic imbalance: the exceptionally high SOD activity in males rapidly converts O₂•⁻ to H₂O₂ at a rate that exceeds the processing capacity of CAT and POD, even though both enzymes are upregulated. This transient H₂O₂ accumulation may itself serve as a signaling molecule to prime additional defense responses, as has been observed in other plant stress systems [52, 53].

The mechanism of exogenous GSH action extends beyond direct chemical detoxification. A pivotal piece of evidence is that GSH treatment significantly bolsters SOD, CAT, and POD activities. If GSH acted solely as a direct ROS scavenger, a reduction in enzymatic antioxidant demand (negative feedback) would be expected upon decreased oxidative load, rather than an enhancement. The observed upregulation of enzyme activities under stress strongly suggests that exogenous GSH likely functions as a redox signaling molecule, priming the cellular defense system and thereby amplifying endogenous enzymatic antioxidant capacity. Multiple studies support that exogenous GSH enhances the plant antioxidant defense system, lowers ROS levels, and improves tolerance to various abiotic stresses [35, 36, 54]. Our study further reveals a sexual dimorphism in this signaling-priming effect. After applying a high concentration of GSH (200 mg·L⁻¹), males exhibited a synchronized decline in both antioxidant activities (SOD, POD) and oxidative markers (H₂O₂, MDA), indicating the successful restoration of redox homeostasis. In contrast, females maintained elevated enzyme activities to counteract the stress. This divergence implies that GSH-mediated signaling is more efficient in males, facilitating a more rapid transition to metabolic stability.

GSH can enhance Cd tolerance in plants by promoting the phenylpropanoid biosynthesis pathway. Our study shows that co-treatment with exogenous GSH and Cd markedly induced the accumulation of total flavonoids in M. polymorpha, an effect that was particularly pronounced in female thalli (Fig. 8E). This indicates that the GSH-mediated alleviation of Cd toxicity in females may rely more substantially on the accumulation of flavonoid non-enzymatic antioxidants.

Under HM stress, osmotic adjustment is a key mechanism for plants to maintain cellular water homeostasis and mitigate stress-induced damage. Studies in Gossypium hirsutum and Stachys lanata have shown that Cd stress induces the accumulation of osmolytes to alleviate toxicity [51, 55]. Similarly, our study demonstrates that Cd stress triggers the active accumulation of osmoregulatory compounds—including SS, SP, and Pro—in both female and male M. polymorpha as an adaptive response to osmotic imbalance (Supplementary Fig. S4). Specifically, females exhibited significant increases in SP and SS, whereas males showed marked rises in SP and Pro. Exogenous GSH treatment further revealed sexually dimorphic adjustment patterns: females preferentially accumulated more SS and SP, likely synergistically enhancing osmotic protection and energy supply; males relied more on Pro, enabling coupled osmotic adjustment and reactive ROS scavenging.

Based on these findings, we propose a working model for the divergent defense strategies of male and female M. polymorpha under Cd stress (Fig. 9): Female plants employ a “homeostasis-reinforcing” strategy centered on the robust upregulation of GSH synthesis and regeneration pathways to sustain a large, active GSH pool for Cd chelation and redox buffering, supplemented by naringenin chalcone accumulation. In contrast, males adopt an “inducible-sensitive” strategy, prioritizing the synthesis of the multifunctional quercetin and maintaining a baseline antioxidative enzyme system with high inducibility, which can be potently primed by low-dose exogenous GSH.

Fig. 9.

Integrated Model of Sexual Dimorphism in M. polymorpha Defense Strategies Under Cd Stress

In summary, this study deciphers the molecular basis of sexually dimorphic responses to heavy metal stress in an early land plant, offering a theoretical foundation for developing sex-oriented phytoremediation and stress-regulation technologies. However, several limitations remain to be addressed. Future studies employing GSH synthesis inhibitors (e.g., buthionine sulfoximine, BSO), key gene knockout or overexpression lines, and metabolic flux analysis (MFA) will be essential to precisely validate the central role of GSH metabolism and the intricate resource allocation among these divergent pathways.

Conclusion

This study provides a comprehensive landscape of sex-specific adaptation to heavy metal stress in the dioecious model plant M. polymorpha. We conclude that: Sexual dimorphism is pervasive at the transcriptional, metabolic, and physiological levels, with females acting as stronger bioaccumulators and males exhibiting higher tolerance. Metabolic flux is sex-dependent, characterized by divergent flavonoid partitioning and differential tyrosine metabolism suppression. GSH is a critical mediator of Cd resilience. Exogenous GSH application provides a potent strategy for alleviating oxidative damage and reducing metal uptake, though its efficacy is modulated by the sex of the plant. These findings highlight the necessity of considering sexual dimorphism in plant ecological studies and provide a molecular foundation for developing sex-aware phytoremediation strategies.

Materials and methods

Plant materials

Wild gemmae of male and female M. polymorpha were collected from the campus of Sichuan University (104°5′18"E, 30°37′44"N) and identified by Professor Li Wang. Gemmae were surface‑sterilized with 0.1% NaClO for 60 s and cultured on 1/2 B5 solid medium [22] at 22 °C under a 24 h light cycle (light intensity 70 µmol·m⁻² s⁻¹) for approximately one month. Uniform‑sized gametophytes were then transplanted into 150‑mm Petri dishes mixed with nutrient soil and vermiculite in a 3:1 ratio and irrigated with deionized water, and acclimatized for one week under the same growth conditions. The experimental soil was purchased from Stanley Agricultural Group Co., Ltd. (Shandong, China).

Cd stress and exogenous GSH treatments

For transcriptomic profiling (acute response, 96 h): Acclimatized male and female M. polymorpha plants were sprayed every 48 h with 10 mL of either 100 mg·L⁻¹ CdCl₂ solution or distilled water (control) for 96 h. Thalli were harvested 96 h after the initial treatment, immediately flash-frozen in liquid nitrogen, and stored at − 80 °C until analysis. The concentration and exposure duration were selected based on previous studies that induced clear stress responses without causing lethality [27]. Four groups were established: female control (F_0), female Cd-treated (F_96), male control (M_0), and male Cd-treated (M_96), with three biological replicates per group. For metabolomic profiling and physiological assays (chronic response, 20 d): Plants were subjected to the same treatment regimen but for 20 d. Thalli were then harvested, immediately flash-frozen in liquid nitrogen, and stored at − 80 °C for metabolomic analysis and physiological measurements.

For exogenous GSH intervention: Based on our preliminary results [56] and the previous literature [51], 100 and 200 mg·L⁻¹ GSH were selected. Plants were divided into six groups: (1) control (CK); (2) Cd (100 mg·L⁻¹ CdCl₂); (3) GSH100 (100 mg·L⁻¹ GSH); (4) GSH200 (200 mg·L⁻¹ GSH); (5) Cd + GSH100 (100 mg·L⁻¹ CdCl₂ + 100 mg·L⁻¹ GSH); and (6) Cd + GSH200 (100 mg·L⁻¹ CdCl₂ + 200 mg·L⁻¹ GSH). Each plant received 10 mL of the respective solution every 48 h for 20 d, after which thalli were harvested for physiological and biochemical analyses.

RNA sequencing and transcriptomic analysis

Total RNA was extracted from male and female M. polymorpha using TRIzol reagent. RNA concentration and integrity were evaluated using a NanoDrop2000 spectrophotometer and agarose gel electrophoresis. Library construction and sequencing (Illumina HiSeq 2500) were performed by Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China). Raw reads were processed with fastp (v0.23.2) [57] to obtain clean reads, which were then aligned to the M. polymorpha reference genome GCA_003032435.1 (https://www.ncbi.nlm.nih.gov/genome/3220?genome_assembly_id=372112) using HISAT2 (v2.2.1) [58]. Gene expression levels were quantified using featureCounts (v2.0.1) [59] and normalized using TPM (transcripts per million) and TMM (Trimmed Mean of M-values) methods. Differential expression analysis was performed with DESeq2 (v1.22.1) [60] using the criteria: |log₂(FoldChange)|> 1 and adjusted p-value (padj) < 0.05. GO and KEGG enrichment analyses were conducted using clusterProfiler (v3.8.1) [61]. The RNA-seq data are available in the NCBI Sequence Read Archive (SRA) under BioProject PRJNA1100982.

Metabolomic analysis

The UPLC-QTOF-MS metabolomic data were obtained from our previous study [27] and are available in the EMBL-EBI MetaboLights database under accession code MTBLS9998. Metabolites were identified by comparing m/z values, retention times, and fragmentation patterns with authentic standards and the Human Metabolome Database (HMDB). DAMs were identified according to the criteria: P < 0.05 and |log₂(FoldChange)|> 1. GO and KEGG enrichment analysis of DAMs was performed using MetaboAnalyst 5.0.

Quantitative real-time PCR (RT-qPCR) validation

RT-qPCR was performed to validate transcriptomic data. First-strand cDNA was synthesized from 1 μg total RNA using HiScript III RT SuperMix (Vazyme). RT-qPCR was conducted on a Bio-Rad CFX96 system using SYBR Green qPCR Premix. The PCR program was: 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The MpActin gene was used as the internal reference. Relative gene expression levels were calculated using the 2^⁻ΔΔCt method. Primer sequences are listed in Supplementary Table S14.

Physiological and biochemical measurements

Total chlorophyll was extracted with 95% ethanol and quantified by measuring absorbance at 665 nm and 649 nm using an enzyme-linked immunosorbent assay (ELISA) reader, following the method described by Yang et al. [56] Cd content was determined by inductively coupled plasma mass spectrometry (ICP-MS) at Wela Detection Technology Co., Ltd. (Guizhou, China). The activities of antioxidant enzymes (SOD, CAT, and POD), the contents of osmotic adjustment substances (Pro, SS, and SP), oxidative stress indicators(H₂O₂), membrane lipid peroxidation indicators (MDA), along with the content of non-enzymatic antioxidant substances (GSH and TFC), were measured strictly using assay kits from Comin Biotechnology Co., Ltd. (Jiangsu, China) according to the manufacturer’s instructions; specific methods are detailed in Supplementary Table S15.

Statistical analysis

Data are presented as mean ± standard deviation (SD) of three biological replicates. Statistical significance was determined by one-way analysis of variance (ANOVA) followed by Tukey’s HSD test (P < 0.05) using SPSS 26.0. Graphs for physiological and biochemical data were generated using GraphPad Prism (v10.6.0). The results of GO and KEGG enrichment analyses and correlation analysis were visualized using the R packages ggplot2 (v4.5.1).

Abbreviations

HM

Heavy metal

CHS

Chalcone synthase

F3’H

Flavonoid 3’-hydroxylase

SOD

Superoxide dismutase

CAT

Catalase

POD

Peroxidase

Cd

Cadmium

PCs

Phytochelatins

MT

Metallothionein

TPM

Transcripts per million

TMM

Trimmed Mean of M-values

DEGs

Differentially expressed genes

DAMs

Differential accumulated metabolites

ANOVA

One-way analysis of variance

NCBI

National Center for Biotechnology Information

C4H

Cinnamic acid 4-hydroxylase

HCT

Shikimate O-hydroxycinnamoyltransferase

C3’H

P-coumaroyl shikimate 3’ hydroxylase

CHI

Chalcone isomerase

F3H

Flavanone 3-hydroxylase

map00350

Tyrosine metabolism pathway

TAT

Tyrosine aminotransferase

HPD

4-Hydroxyphenylpyruvate dioxygenase

DD

Dopa decarboxylase

PPO

Polyphenol oxidase

map00480

Glutathione metabolism pathway

GSR

Glutathione reductase

IDH

Isocitrate dehydrogenase

6PGD

6-Phosphogluconate dehydrogenase

G6PD

Glucose-6-phosphate dehydrogenase

OPLAH

5-Oxoprolinase

GCL

Glutamate-cysteine ligase

GSS

Glutathione synthase

GST

Glutathione S-transferase

GSTK1

Glutathione S-transferase kappa 1

ODC

Ornithine decarboxylase

DHAR

Dehydroascorbate reductase

GPX

Glutathione peroxidase

GGT

Gamma-glutamyltranspeptidase

FNS

Flavone synthase

ROS

Reactive oxygen species

GSH

Glutathione

MDA

Malondialdehyde

Pro

Proline

SS

Soluble Sugar

SP

Soluble Protein

TFC

Total Flavonoid Content

Authors’ contributions

Conceptualization, X.Y. and L.W.; methodology, X.Y., J.Z.; software, X.Y.; validation, L.Z., L.Y.; formal analysis, X.Y., J.Z.; investigation, X.Y., J.Z., C.G., P.Y.; resources, L.W., X.B.; data curation, X.Y.; writing—original draft preparation, X.Y., J.Z.; writing—review and editing, J.Z., X.Y., L.W.; visualization, X.Y.; supervision, L.W.; project administration, L.W.; funding acquisition, L.W. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Guizhou Chishui Alsophila National Nature Reserve Administration Bureau (Grant No. 24H0344).

Data availability

The transcriptomic data generated in this study are publicly available in the NCBI SRA database under the BioProject accession number PRJNA1100982. The metabolomic profiling datasets are deposited in the EMBL-EBI MetaboLights repository with the accession code MTBLS9998. All plant materials used in this study are available from the corresponding author, upon reasonable request.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.