Professor Wu Yougen’s team from Nanfan College of Hainan University revealed the three-fold mechanism of glutathione (GSH) alleviating cadmium (Cd) stress of patchouli:
Regulation of photosynthetic pigments (increase chlorophyll content and enhance photosynthesis), activation of antioxidant system (increase SOD, POD and other enzyme activities, remove reactive oxygen species), and regulation of metabolic pathways (reduce cadmium toxicity through glycerophospholipid metabolism and flavonoid biosynthesis, promote the formation of cadmium-glutathione complex and achieve vacuole compartmentalization to resolve toxicity).
This finding not only provides a new theoretical basis for the study of plant stress resistance, but also provides an important reference for the cultivation of medicinal plants in cadmium-polluted areas:
Exogenous addition of glutathione can improve plant stress resistance, optimize the accumulation of medicinal ingredients, and reduce cadmium residue, thus ensuring the safety and quality of medicinal materials, and contributing to green agriculture and sustainable development.
The research results were published on March 10, 2025 in the Journal of Hazardous Materials (IF:12.2), the TOP journal of the 1st region of the Chinese Academy of Sciences in the field of Environment.
Abstract
Cadmium (Cd) pollution is an increasing threat to plant growth. Although glutathione (GSH) has been shown to have the potential to alleviate cadmium stress, the specific mechanism by which it alleviates cadmium stress in Pogostemon cablin has not been clarified.
In this study, four groups were set up: control group (CK), cadmium stress group (Cd), glutathione treatment group (GSH) and glutathione + cadmium stress group (GSH+Cd), and the changes of physiological parameters and enzyme activities were systematically analyzed.
results showed that photosynthetic pigment regulation:
The contents of chlorophyll a, b and carotenoids in the glutathione treated group were about 20% higher than those in the other groups, which was consistent with the phenomenon that plants could enhance the protection mechanism of photosynthetic system under cadmium stress.
Antioxidant system response:
The activity of antioxidant enzymes in cadmium-stressed group decreased by about 15%, while glutathione intervention significantly increased the activities of superoxide dismutase (SOD) and peroxidase (POD), and alleviated oxidative damage by removing reactive oxygen species (ROS).
Molecular mechanism analysis:
Integrated transcriptome and metabolome analysis revealed that glutathione alleviates cadmium toxicity by regulating glycerophospholipid metabolism (involving key genes dgkA1 and dgkA2) and flavonoid biosynthesis (key genes CCoAOMT1-4), and flavonoids can reduce the bioavailability of cadmium through chelation.
Metabolic pathway synergy:
Glutathione-activated members of the glutathione-S-transferase (GST) gene family are significantly upregulated, promoting the formation of the Cdma-glutathione complex and achieving detoxification through vacuole compartmentalization.
Conclusion: Glutathione alleviates cadmium stress of patchouli through three mechanisms:
(1) Enhance photosynthetic pigment synthesis to maintain energy metabolism;
(2) Enhance antioxidant enzyme activity and inhibit ROS accumulation;
(3) Regulate the metabolic pathway of glycerophospholipids and flavonoids to achieve cadmium detoxification.
This study provided a theoretical basis for agronomic regulation of patchouli cultivation in cadmatic-polluted areas.
Plant physiological response
The application of glutathione significantly promoted the growth of cadmium-stressed and non-stressed plants.
In order to better understand the effects of different treatments on leaf growth, stomatal structure, fresh weight and dry weight of leaves were measured and analyzed.
Scanning electron microscope (SEM) analysis showed that the stomata structure of patchouli leaves was severely damaged by cadmium stress, and the stomata were closed and flat.
In contrast, glutathione treatment improved stomatal pore size, while GSH+Cd treatment partially preserved stomatal structure, giving it a more raised and complete appearance.
At the same time, compared with the control group (CK), the fresh weight of leaves was significantly reduced.
Glutathione treatment significantly promoted plant growth, resulting in a significant increase in fresh weight.
Compared with cadmium treatment alone, plants treated with GSH+Cd showed less leaf damage and experienced partial growth recovery.
Quantitative analysis showed that the application of glutathione significantly enhanced the biomass and chlorophyll content of plants.
Compared with the control group (CK), the fresh weight and dry weight of plants treated with glutathione increased by 28.8% and 23.3%, respectively, while the fresh weight and dry weight of plants exposed to cadmium stress decreased by 23.9% and 32.5%, respectively.
Cadmium stress also reduced total chlorophyll content by 11.4%.
It is worth noting that the fresh weight and dry weight of plants treated with GSH+Cd were significantly higher than those treated with cadmium.
The chlorophyll content of GSH+Cd treatment group increased by 34.7%, and the chlorophyll a, b and carotenoid increased by 33.2%, 38.2% and 18.6%, respectively.
Glutathione pretreatment also improved chlorophyll fluorescence parameters of the control group and cadmium-stressed plants.
Specifically, Fv/Fm ratio, Y(II), qP and ETR in GSH+Cd treatment group increased by 10.8%, 78.3%, 81.7% and 23.8%, respectively, compared with cadmium treatment group, while qN decreased by 9%.
These results highlight the ability of glutathione to alleviate cadmium-induced stress and promote plant growth and health by enhancing plant physiological and photosynthetic performance.
Oxidative damage and antioxidant enzymes
Under cadmium stress, ROS levels in leaves of patchouli significantly increased, and the contents of H₂O₂, O₂⁻ and MDA increased by 46.6%, 32.8% and 280%, respectively.
GSH+Cd pretreatment reduced the contents of H₂O₂, O₂⁻ and MDA by 14.5%, 7.8% and 24.04%, respectively, compared with the cadmium treatment group.
The activities of key antioxidant enzymes in the leaves of Pogostemon cablin were evaluated, including SOD, CAT, POD, APX, GST, GR and GPX, which play a crucial role in clearing ROS and protecting plant cells from oxidative damage.
Cadmium exposure significantly reduced the activity of these enzymes, with SOD activity reduced by 43.5%, CAT activity decreased by 17%, POD activity decreased by 45.8%, and APX activity decreased by 66.5%.
It is worth noting that GSH+Cd pretreatment significantly enhanced the activity of key antioxidant enzymes, SOD activity increased by 27.5%, CAT activity increased by 15.7%, POD activity increased by 46%, and APX activity increased by 68.7%, compared with the cadmium treatment group.
Cadmium stress significantly increased the levels of GST, GR, GPX and glutathione by 58%, 235.6%, 42.5% and 94%, respectively, compared with control group.
Compared with the cadmium treatment group, the non-enzymatic antioxidant activity of the GSH+Cd treatment group was also significantly enhanced.
These results highlight the protective role of glutathione in heavy metal-induced oxidative stress, suggesting its potential in improving plant stress resistance under such conditions.
Comparative analysis of leaf transcriptome of patchouli
In order to elucidate the detoxification mechanism of glutathione under cadmium stress, RNA sequencing of cablin leaves was carried out.
After processing the raw sequencing data and removing the low-quality reads, the clean reads for the three samples were 37.03 million, 41.2 million, and 38.32 million, respectively.
Sequencing indicators showed high data quality, with GC content averaging 45%, Q20 over 99.7%, and Q30 over 98.5%.
All clean read sequences were aligned with the patchouli reference genome, with an alignment efficiency of about 97.5%.
Biological replications showed strong agreement with a correlation coefficient of over 0.8, confirming the reliability of sequencing data in subsequent analyses.
Principal component analysis (PCA) showed that the three groups of samples were significantly grouped, with the first and second principal components (PC1 and PC2) explaining 28.46% and 18.59% of the variance, respectively, for a total variance of 47.05%.
These results showed that there were significant differences in gene expression between the different treatment groups.
Compared with the control group, 7265, 4974 and 3914 differentially expressed genes (DEGs) were identified in the cadmium, glutathione and GSH+Cd treated groups, respectively.
There were 4170, 2603 and 2082 up-regulated DEGs in the cadmium, glutathione and GSH+Cd treatment groups, respectively.
Venn diagram analysis further revealed that 1178 DEGs were co-expressed in all three treatments, while 2544 DEGs were co-expressed in comparisons of cadmium and control and glutathione and control.
Comparative analysis of non-targeted metabolome of patchouli leaves
A total of 656 metabolites were identified by non-targeted metabolomics analysis of patchouli under different treatments.
Based on their structural characteristics, these metabolites are classified into 16 different chemical classes.
The main categories include lipids and lipid molecules (49.08%), organic acids and their derivatives (12.06%), phenylpropenes and polyketones (11.63%), organic heterocyclic compounds (8.23%), organic oxygen compounds (7.8%), benzo compounds (4.96%) and organic nitrogen compounds (1.84%), etc.
Heat maps showed the clustering patterns of metabolites across all samples, reflecting differences in metabolite expression profiles in the control group (CK), cadmium (Cd), glutathione, and GSH+Cd treatments.
Principal component analysis (PCA) showed a significant separation between the treatment and control groups, with the first two principal components (PC1 and PC2) explaining about 50% of the total variance.
These results showed that the metabolite profiles of the treated samples were significantly different from those of the control group.
The trend of metabolites in leaves of patchouli under different treatments was investigated by K-means cluster analysis.
Based on similar expression trends, metabolites are divided into six subclasses.
Subclasses 1 and 3 contained the most metabolites, with 125 and 127, respectively.
Subclass 1 showed the highest median metabolite level under glutathione treatment, while subclass 3 showed a higher median metabolite level under GSH+Cd treatment.
Subclass 4 (82 metabolites) and subclass 6 (102 metabolites) showed the highest median values in the control group.
To evaluate the effects of different treatments on the metabolites of patchouli, supervised orthogonal partial least squares discriminant analysis (OPLS-DA) was used.
The OPLS-DA model was constructed, the treatment group was compared with the control group, and the variable importance projection (VIP) score was calculated.
VIP scores are crucial for identifying differential metabolites in patchouli samples.
The model showed strong separation ability, with both R²Y and Q² values exceeding 0.80, indicating that the model had a strong ability to distinguish patchouli samples.
The metabolite differences between the control group and each treatment group were analyzed using the following criteria: Log₂ (fold change) ≥1 or ≤−1, VIP≥1, p <0.05.
Analysis results identified 95, 53 and 80 difference-accumulating metabolites (DAMs) between control group and cadmium (CK vs Cd), control group and glutathione (CK vs GSH), and control group and GSH+Cd (CK vs GSH+Cd), respectively.
A total of 161 DAMs were identified in all samples, 19 of which were co-expressed in all treatment groups.
These DAMs are classified into 12 chemical classes based on their structure.
Lipids and lipid molecules accounted for the largest proportion (37.89%), followed by organic acids and their derivatives (18.63%), phenylpropylene and polyketones (13.04%) and so on.
Compared with the control group, most lipids and lipid molecules were down-regulated in all treatment groups, and the decrease was most significant in the cadmium treatment group.
Compared with the cadmium treatment group, the levels of lipids and lipid molecules in the glutathione and GSH+Cd treatment groups were up-regulated.
Organic acids and their derivatives were most abundant in the cadmium-treated group and significantly down-regulated in the control group, while levels in the glutathione and GSH+Cd treatments were between the cadmium and control groups.
Compared with the control group, phenylpropylene and polyketones were significantly up-regulated in glutathione treatment, but significantly down-regulated in cadmium and GSH+Cd treatment.
Organic heterocyclic compounds, benzo compounds, alkaloids and their derivatives were significantly up-regulated in all three groups, and the up-regulation was most significant in the cadmium group compared with the control group.
In general, DAMs in glutathione group were significantly up-regulated compared with control group, while DAMS in cadmium group were significantly down-regulated for most compounds, with the exception of organic acids and their derivatives and organic heterocyclic compounds.
The response of the GSH+Cd group was intermediate between the control group and the cadmium group.
RNA-seq combined with metabolomics analysis of patchouli
In order to explore the relationship between gene expression and metabolic regulation, the co-expression networks of differentially expressed genes (DEGs) and differentially accumulated metabolites (DAMs) in patchouli were analyzed.
The correlation between DEGs and DAMs is shown in a nine-quadrant diagram. The results showed no significant difference in the fifth quadrant.
In quadrants 1, 2, and 4, gene expression levels were significantly higher than metabolite accumulation, indicating that gene expression was up-regulated while metabolite levels remained unchanged or down-regulated.
In quadrants 6, 8, and 9, gene expression levels were lower than metabolite accumulation, indicating that gene expression was down-regulated while metabolite levels remained stable or increased.
Quadrants 3 and 7 showed a consistent trend of differential expression between genes and metabolites, suggesting that metabolite accumulation may be positively regulated by gene expression.
Notably, 27 metabolites were identified as being positively regulated by 416 genes.
Subsequent KEGG enrichment analysis showed that these DEGs and DAMs were co-enriched in pathways related to glycerophospholipid metabolism and flavonoid biosynthesis.
To further investigate the effects of glutathione and Cd on plant gene expression and metabolite accumulation, we analyzed the interactions between identified genes and metabolites involved in glycerophospholipid metabolism and flavonoid biosynthesis.
In the glycerophospholipid metabolic pathway, two metabolites – 1-stearoyl-SN-glycerol-3-phosphate choline and LysoPA(18:2 (9Z:12Z)/0:0) – are regulated by 19 genes each.
The former metabolites showed a significant positive correlation with related genes, while the latter showed a significant negative correlation.
In the flavonoid biosynthesis pathway, two metabolites – epigallocatechin and 3,4,5, 7-tetrahydroxyflavone – are regulated by five and four genes, respectively.
Epigallocatechin was significantly positively correlated with regulatory genes, while 3,4,5, 7-tetrahydroxyflavone was significantly negatively correlated.
These results highlight the co-expression of DEGs and DAMs in key metabolic pathways after glutathione-mediated cadmium stress relief, especially in glycerophospholipid metabolism and flavonoid biosynthesis.
Genes and metabolites involved in metabolism of glycerophospholipids and flavonoids
Through integrated transcriptomic and metabolomic analysis, it was found that glutathione treatment significantly affected glycerol phospholipid metabolism and flavonoid biosynthesis in leaves of patchouli.
A total of 23 DEGs were identified by KEGG pathway mapping, of which 4 genes were up-regulated and 19 genes were most strongly expressed in the control group (CK).
Specifically, 11 genes were associated with glycerophosphate metabolism, resulting in the production of 1, 2-diacyl-SN-glycero-3-phosphate, of which 9 genes were most strongly expressed in the control group.
Three genes regulate the conversion of 1, 2-diacyl-SN-glycerol-3-phosphate to 1, 2-diacyl-sn-glycerol, in which dgkA-1 and dgkA-2 are expressed most strongly in the GSH+Cd treatment group.
Seven genes related to phosphatidylethanolamine metabolism were most strongly expressed in the control group.
These findings highlight the role of glutathione in regulating glycerophospholipid metabolism under cadmium stress.
conclusion
In this study, a number of physiological parameters, including dry weight, chlorophyll A and b, carotenoids, and antioxidant enzyme activities, were determined by applying cadmium (Cd) stress to vanilla leaves and subsequent application of exogenous glutathione.
The results showed that Cd stress inhibited the synthesis of photosynthetic pigments and decreased the enzyme activity in the leaves, while glutathione effectively restored the photosynthetic pigment level and enzyme activity, thus maintaining the photosynthetic function of the leaves.
Transcriptome and metabolome analysis showed that Cd mainly affected hormone signaling pathways and starch and sucrose metabolism in leaves.
In contrast, exogenous glutathione enriched differentially expressed genes (DEGs) in starch and sucrose metabolism and phenylalanine biosynthesis pathways.
Differential accumulation metabolites (DAMs) are also enriched in lipids, phenylalanine and other compounds.
Glutathione was found to protect starch and sucrose metabolism and enhance phenylalanine metabolism under Cd stress.
Transcriptome and metabolome are co-enriched in glycerophospholipid metabolism and flavonoid biosynthesis pathways.
The genes dgkA1, dgkA2 and CCoAOMT1-4 were identified as key regulators of glycerophospholipid metabolism and flavonoid biosynthesis in glutathione-mediated Cd stress relief, thereby supporting normal plant growth.
