Cysteine-mediated modulation of the glyoxalase system and HSP90 proteins enhances high-temperature stress tolerance in Arabidopsis thaliana
Selda Durmuşoğlu, Dilek Ünlüer, Aykut Sağlam, Asim Kadıoğlu
TL;DR
This study shows that cysteine helps Arabidopsis plants tolerate high temperatures by boosting the glyoxalase system and HSP90 proteins, reducing oxidative damage and improving physiological health.
Contribution
The study reveals a novel synergistic role of cysteine with the glyoxalase system and HSP90 proteins in enhancing plant thermotolerance.
Findings
Cysteine treatment under high-temperature stress increases relative water content and chlorophyll levels in Arabidopsis.
Cysteine boosts glyoxalase enzyme activity and reduces oxidative damage markers like TBARS and H₂O₂.
Cysteine enhances gene expression of glyoxalase system and HSPs, improving thermotolerance in Arabidopsis.
Abstract
High-temperature (HT) stress poses a major threat to plant growth and physiological functions by disrupting cellular homeostasis and metabolic processes. Despite extensive studies, the molecular and physiological mechanisms underlying plant adaptation to HT stress remain incompletely understood. This study investigates the role of cysteine (CYS), a thiol-containing amino acid, in enhancing high-temperature tolerance in Arabidopsis thaliana (A. thaliana) through the regulation of heat shock protein 90 (HSP90) and the glyoxalase (GLX) system. Our research demonstrates that CYS treatment under HT stress significantly enhances key physiological parameters, including relative water content (RWC), and total chlorophyll levels while reducing oxidative damage markers like thiobarbituric acid reactive substances (TBARS), and hydrogen peroxide (H₂O₂). In this study, findings from A. thaliana…
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Figure 6- —Karadeniz Technical University
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TopicsPlant Stress Responses and Tolerance · Advanced Glycation End Products research · Plant responses to water stress
Introduction
Environmental stresses significantly impact the growth, development, productivity, and overall health of plants significantly, with high-temperature (HT) stress emerging as a prominent challenge, especially in the context of global climate change (Goswami et al. 2022). Climate changes in recent years have led to an increase in temperatures, placing great stress on plants and negatively affecting plant life by worsening their normal physiological and biochemical processes, including growth, development, and productivity (Shapira et al. 2021). HT stress affects plants at multiple cellular levels, disrupting cellular activities, metabolism, gene expression, reproduction, and overall yield. The resultant damage is extensive, leading to protein denaturation, disruption of biomembranes, oxidative stress, osmotic imbalance, and the accumulation of harmful compounds like methylglyoxal (MG) (Sezgin Muslu and Kadıoğlu 2021a; Ahmad et al. 2022; Cáceres et al. 2024). Addressing these challenges, it is essential to gain a deeper understanding of the physiological responses and molecular pathways that govern plant tolerance to stress. Such knowledge is vital for developing effective strategies to enhance plant resilience and ensure sustainable agricultural productivity amidst changing environmental conditions.
The growing concern over climate change has intensified research into compounds that can mitigate the damaging effects of HT stress (Gautam et al. 2024). Among these approaches, scientists have explored strategies including the application of nutrients, amino acids, and plant hormones, all of which have shown promise in alleviating HT stress and promoting plant health (Jahan et al. 2021; Raza et al. 2023; Guo et al. 2023). Thiol-containing substances are of particular interest due to their strong antioxidant properties and their ability to enhance tolerance to abiotic stress (Terzi and Yıldız 2021).
Cysteine (CYS), a thiol-containing amino acid, stands out for its critical role in the synthesis of sulfur-containing compounds such as glutathione (GSH), a key antioxidant that protects plants under stressful conditions (Feng et al. 2023; Mishra et al. 2024). Research has demonstrated that CYS can help mitigate the adverse effects of various abiotic stresses, including salinity, heavy metal exposure, and drought (Erdal and Türk 2016; Hussein and Alshammari 2022). Despite the promising benefits of CYS in managing various environmental stresses, the specific interactions between CYS, the glyoxalase (GLX) system, and heat shock protein 90 (HSP90) proteins under HT stress conditions remain unclear. Identifying the protective mechanisms of CYS and how it affects these essential molecular pathways could help close a significant gap in the literature in this field. A deeper investigation into these interactions could provide valuable insights into how CYS may enhance plant resilience to HT, potentially offering innovative strategies for safeguarding crops in an increasingly warming planet.
The GLX system is a critical component of plant defence mechanisms against abiotic stress, particularly through its role in detoxifying MG, a highly toxic byproduct that accumulates under stressful conditions such as HT (Mudalkar et al. 2017; Altaf et al. 2024; Mohanan et al. 2024). MG poses a serious threat to cellular integrity by reacting with essential macromolecules like proteins, DNA, and lipids, leading to widespread cellular damage. The GLX system comprises two primary enzymes, Glyoxalase I (GLXI) and Glyoxalase II (GLXII), that work together in a GSH-dependent pathway to convert MG into the less harmful compound D-lactate, thereby protecting cells from its cytotoxic effects (Sezgin Muslu and Kadıoğlu 2021b; Xu et al. 2023). D-lactate dehydrogenase (D-LDH) plays a crucial role in detoxifying MG by processing D-lactate, the final product of the glyoxalase pathway (Alam et al. 2024). Additionally, the Glyoxalase III (GLXIII) enzyme, part of the DJ-1 protein superfamily, provides an alternative route for MG detoxification that does not rely on GSH or metal ions (Parvin et al. 2019; Talaat and Todorova 2022). Several isoforms of GLXI and GLXII encoded by the A. thaliana genome highlight the importance of these enzymes in the plant’s ability to tolerate stress (Schmitz et al. 2017). Some of these isoforms ensure the effective detoxification of MG and help maintain the delicate balance of GSH, which are essential for the plant’s resilience to environmental stressors (Kwon et al. 2013; Nahar et al. 2015; Lewandowska et al. 2019).
HSPs (heat shock proteins) are a group of proteins widely present in various organisms, including fungi, animals and plants (Lindquist 1986). These proteins are essential in managing the cellular response to HT stress, where their expression levels, particularly HSP transcripts, are significantly increased (Mumtaz et al. 2023; Durmusoglu et al. 2024). This upregulation is crucial for the HT stress response within living cells. HSPs are categorized into several families based on their molecular weight, such as HSP110, HSP90, HSP70/HSP80, HSP60, and small molecular HSPs (sHSP) (Horwitz 1992). Initially, the sHSP family was thought to function solely as molecular chaperones, but further research has revealed that other HSP families, including HSP90, HSP70, and HSP60, also perform molecular chaperone functions. Of them, HSP90’s function in responses to stress received significant attention, with research focusing on how it contributes to the body’s ability to cope with stressors and how it can be used as a therapeutic target in stress-related conditions. While the involvement of A. thaliana HSP90 in stress responses has been established in earlier studies, recent research suggests that HSP90 may play a broader role in various biological stress responses. The HSP90 family is a highly conserved molecular chaperone that plays a vital role in maintaining protein homeostasis, particularly under stress conditions (Prasinos et al. 2005; Ticha et al. 2020). They assist in the proper folding, stabilisation, and prevention of protein aggregation when plants are exposed to environmental stresses. In A. thaliana, seven distinct HSP90 proteins are found across different cellular compartments. Among these, four cytosolic isoforms—HSP90.1, HSP90.2, HSP90.3, and HSP90.4—are particularly crucial in mediating the plant’s response to stress (Krishna and Gloor 2001; Zhou et al. 2020). Research has highlighted that specific isoforms, such as AtHSP90.3 and AtHSP90.2, are instrumental in enhancing the plant’s tolerance to high-temperature and other abiotic stresses, underscoring the importance of HSP90s in stress adaptation (Xu et al. 2010; Song et al. 2009).
The MG detoxification pathway has been reported to be associated with HSPs in some animal cells. Studies have shown an association between MG and HSPB6, one of the sHSPs (Sudnitsyna and Gusev 2017). In addition, MG negatively affects the structure of HSPs such as HSP40, HSP70, and HSP90 by disrupting the ubiquitin-proteasome system, which is required for the degradation of damaged proteins (Bento et al. 2010). These findings suggest that the MG detoxification pathway plays a role in the regulation of HSPs in some animal disease states. In plants, some studies emphasize the relationship between GSH and HSPs (Kumar and Chattopadhyay 2018). However, the relationship between the GLX system and HSPs in plants and the potential interactions between CYS and HSPs under HT stress and their combined effects on the GLX system have not yet been fully elucidated.
This research examines the role of CYS in promoting HT tolerance of A. thaliana. The study is based on the hypothesis that CYS is a key player in enhancing plant resistance by modulating the GLX system and specifically through its interaction with HSP90 proteins, critical components of plant stress response mechanisms. We investigated in detail the effect of CYS on the activity of GLX enzymes, which are vital for detoxifying harmful by-products such as MG accumulated under stress conditions. We also assessed the regulatory role of CYS on these key molecular pathways by analyzing the expression levels of genes linked to both HSP90 and the GLX system, especially under HT stress conditions.
Although the benefits of CYS in managing various environmental stresses are promising, the specific interactions between CYS, the GLX system, and HSP90 proteins under HT stress are not yet fully understood. In this study, we aim to reveal the effect of CYS on these pathways and discover novel protective properties of CYS that may contribute to stress tolerance. Further investigation of these interactions could provide valuable insights into how CYS can enhance plant resistance to HT stress, contributing to the development of innovative strategies to mitigate the adverse effects of rising global temperatures on agricultural production.
Materials and methods
Plant materials and growth conditions
The seeds used in this study were A. thaliana ecotype Col-0 CS1093 (NASC ID: N1093), hsp90.1 SALK_007614 (NASC ID: N507614), and hsp90.4 SALK_036835C (NASC ID: N674476), encoding HSP90.1 and HSP90.4. These seeds were provided by the Nottingham Arabidopsis Stock Center (NASC).
Seeds of Col-0 and mutant plants were stratified at 4 °C for 48 h to break dormancy (Tocquin et al. 2003). After stratification, seeds were sterilized with 15% bleach (v/v) for 5 min, rinsed thoroughly three times with sterile deionized water, and placed onto plates containing 1X Murashige and Skoog (MS) medium with vitamins (Murashige and Skoog 1962) (Duchefa Biochemie, NLHaarlem), 0.5% w/v Plant Tissue Culture Agar (Neogen Culture Media), and 1% w/v sucrose. The seeds were then grown in plant growth chambers under the following conditions: 16-hour light / 8-hour dark photoperiod (100 µmol m^− 2^ s^− 1^ light intensity), 24 °C during the day and 18 °C at night, with 70% humidity.
CYS, GLXI inhibitor and stress treatments
5-day-old seedlings were transferred to an appropriate treatment media. For CYS treatment, a modified version of Kim’s method was used to apply different concentrations of CYS (0, 50, 100, 150, and 250 µmol) after 5 days of growth (Kim et al. 2022). HT tolerance was evaluated using Vidya’s method with slight modifications (Vidya et al. 2018).
To assess HT tolerance, 21-day-old seedlings were exposed to 38 °C for 2 h, recovered at 22 °C for 2 h, followed by a final exposure to 45 °C for 1 h. All HT treatments were performed in the dark. The plants were placed in a growth chamber for a 3-day recovery period before scoring (Lajkó et al. 1997; Vidya et al. 2018).
At the end of the 3rd day, 25-day-old seedlings were harvested. The concentration of 150 µM CYS was selected for subsequent experiments based on our preliminary trials, in which a range of cysteine concentrations (0, 50, 100, 150, and 200 µM) was evaluated. Among these, 150 µM consistently resulted in the most favorable physiological outcomes, including reduced membrane damage, improved water status, enhanced survival rate, and higher total chlorophyll content. The experimental groups are outlined in Table 1.
Table 1. Experimental groups and treatment conditions for Col-0 and mutant A. thaliana plantsTreatment groupDescription1Control (CK)MS medium without CYS2High-temperature Stress (HT)35 °C/42 °C without CYS3Cysteine (CYS)150 µmol CYS without HT stress4Cysteine/High-temperature Stress (CYS + HT)150 µmol CYS + 35 °C/42°C HT
The 25-day-old seedlings were photographed, harvested, frozen, and stored at − 80 °C for subsequent biochemical and molecular analyses. The experimental groups used in the design are detailed in Table 1.
For the GLX inhibitor treatment, Col-0 and hsp90.4 mutant seedlings were treated at 5 µM GLXI inhibitor (S-p-bromobenzylglutathione cyclopentyl diester, BBGD, SML1306, Merck) supplemented in MS medium (Thornalley et al. 1996). Control groups (with or without CYS) did not include the inhibitor. The plants were grown on this medium until they were 21 days old. HT stress treatments were applied as described above. Phenotypic defects were imaged, and total chlorophyll content and thiobarbituric acid reactive substances (TBARS) were measured. GSH-independent GLX system-related assays were conducted 72 h after stress treatment.
Survival rate and RWC
In an HT survival assay, following the method of Chen et al. (2022), 21-day-old seedlings were subjected to HT treatment and allowed to recover for three days. Survival was assessed based on etiolation: fully, partially, or non-etiolated. The survival rate was calculated using the following formula:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ \frac{{{\text{number of non}} - {\text{etiolated seedlings}} + \frac{1}{2}{\text{number of partially etiolated seedlings}}}}{{{\text{total number of seedlings}}}} $$\end{document}A method described by Castillo (1996) was employed to calculate the RWC of the leaves. According to this method, firstly, the fresh weight (FW) of the leaves was determined, then, to obtain the turgor weight (TW), the leaves were soaked in double distilled water for 4 h. Subsequently, the leaves were desiccated at 70 °C for a day to obtain the dry weight (DW). Finally, the RWC of the leaves was calculated using the following formula:
\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$ {\text{RWC }}\left( \% \right) = \left( {\frac{{{\mathrm{FW}} - {\mathrm{DW}}}}{{{\mathrm{TW}} - {\mathrm{DW}}}}} \right) \times 100 $$\end{document}Total chlorophyll content assay
The chlorophyll content was measured using a UV-Vis spectrophotometer following the method outlined by Hiscox and Israelstam (1979). Leaf samples were homogenized in acetone, centrifuged, and absorbance was measured at 645 nm and 663 nm. The total chlorophyll content was expressed in nmol g^− 1^ FW.
TBARS and H2O2 assessment
The amount of TBARS in leaf tissues was measured using the Heath and Packer (1968) method, which involves measuring absorbance at 532 and 600 nm with a specific extinction coefficient. H_2_O_2_ levels were determined using the method described by Velikova et al. (2000). Samples were homogenized in TCA, mixed with potassium phosphate buffer and KI, and absorbance was measured at 390 nm against an H_2_O_2_ standard. Results were expressed in µmol g^− 1^ FW.
Analysis of GSH, MG and activities of GLX system enzymes
GSH content of leaves was determined according to Tietze (1969). Leaf samples were pulverized in liquid nitrogen and extracted with meta-Phosphoric acid (HPO_3_) and ethylenediaminetetraacetic acid (EDTA). The supernatant from this extraction was used to measure GSH through a reaction mixture and observe absorbance changes at 412 nm. GSH concentration was calculated using a standard curve.
MG content was measured based on the protocol by Yadav et al. (2005). Leaf samples were extracted in perchloric acid (HClO_4_), chilled, and centrifuged. The clear supernatant was then treated with o-phenylenediamine and HClO₄, and the derivatized MG was assessed at 335 nm. MG levels were quantified using a standard curve.
To assess enzyme activity, leaf samples were homogenized in potassium phosphate buffers and centrifuged to obtain supernatants for enzyme assays. Total soluble protein was quantified using a bovine serum albumin standard (Bradford 1976). GLXI (EC: 4.4.1.5) activity was measured according to Hasanuzzaman et al. (2011) by the increase in absorbance at 240 nm, using a reaction mixture of potassium phosphate buffer, magnesium sulfate, GSH, and MG. The activity was calculated using an extinction coefficient of 3.37 mM^− 1^.cm^− 1^. According to Principato et al. (1987), GLXII (EC: 3.1.2.6) activity was determined by monitoring GSH formation at 412 nm in a mixture of Tris-HCl buffer, DTNB, and SLG. The activity was calculated using an epsilon coefficient of 13.6 mM^− 1^.cm^− 1^.
RNA isolation and RT-PCR analysis and gene expression assay
To perform gene expression analysis, 0.1 g of leaf samples were used to collect 2 µg of cDNA using the Favorprep total RNA isolation kit. A cDNA Reverse Transcription Kit from Applied Biosystems was employed for reverse transcription. RT-qPCR was performed with Eva Green qPCR Supermix and gene-specific primer sets (see Table S1) using the CFX Connect Real-Time PCR System from Bio-Rad. Actin (ACT) was utilized as the reference gene.
Statistical analyses
The experiments were conducted three times, with six biological replicates for each repetition. The results were presented as means with the corresponding standard deviation. Statistical analysis was performed using the Duncan Multiple Comparison test (One-way ANOVA) in SPSS software for Microsoft Windows (Ver. 23.0, SPSS Inc., USA) to determine the significant differences between the means. A significance level of 5% (P < 0.05) was considered for all treatments.
Results
Exogenous application of CYS to A. thaliana seedlings under HT stress
To investigate the effect of CYS on HT stress tolerance, 21-day-old Col-0 seedlings were subjected to two stages of HT treatment. High temperature stress treatment conditions and phenotypic appearance of plants 3 days after treatment are shown in Fig. 1A. These seedlings were cultivated according to the conditions detailed in the Materials and Methods sections. The initial analysis focused on determining RWC, survival rate, total chlorophyll, MDA, and H_2_O_2_ levels in the Col-0 plants.
A 3-day recovery period was applied to assess the survival rate of A. thaliana plants following HT stress. CYS + HT treatment resulted in a 70% survival rate, demonstrating a substantial improvement compared to HT stress alone (50%), while the CYS-alone and control (CK) groups exhibited near-complete survival (Fig. 1B).
Fig. 1. Exogenous CYS involves HT tolerance in A. thaliana.** A** For HT treatment, 21-d-old seedlings were incubated at designated HT treatment schemes. The image shows the plants 3 days after the treatment.** B** The seedling survival rate was calculated for the Col-0 seedlings. The effect of CYS on RWC** C**, Total Chlorophyll content** D**, TBARS content** E**, and H_2_O_2_ content** F** in Col-0 seedlings under HT treatments. CK (Control), HT (High-Temperature Stress), CYS (Cysteine), CYS + HT (Cysteine + High-Temperature Stress). Data represent means ± SE of three replicates. By Duncan’s test, different letters indicate significant differences (P < 0.05) amongst the treatments
Although relative water content in the CYS + HT group did not fully reach the levels observed in the CYS-alone and CK groups, it was significantly improved compared to HT-stressed plants. Under normal conditions, both the CYS and CK groups exhibited nearly identical water content, indicating that CYS alone does not disturb plant hydration. In contrast, plants subjected to HT stress experienced a 19% decline in RWC relative to the CK group. Notably, CYS + HT-treated plants maintained 15.26% higher water content compared to the HT group. (Fig. 1C).
Under HT stress conditions, the total chlorophyll content in plants showed a 29.05% reduction compared to the CK group. However, treatment with CYS led to a substantial increase of 49.26% in chlorophyll content. Notably, the total chlorophyll content in the CYS + HT group was 33.53% higher than in HT-stressed plants and showed only a slight reduction compared to the CYS-alone and CK groups (Fig. 1D).
To evaluate oxidative damage induced by HT, the levels of TBARS and H₂O₂ were measured in plants with and without CYS treatment. TBARS levels in the CYS + HT group were moderately higher than those observed in the CK and CYS-alone groups, both of which maintained minimal lipid peroxidation under non-stress conditions. TBARS, an indicator of lipid peroxidation, increased by 42.5% in HT-stressed plants compared to the CK group. However, in the CYS + HT group, TBARS content decreased by 7.6% relative to the HT condition alone (Fig. 1E).
Additionally, the accumulation of H_2_O_2_, a common response to cellular stress, increased by 77.76% under HT stress compared to the control. In the CYS + HT group, H_2_O_2_ levels remained higher than those in the CK and CYS-alone groups, which maintained low basal ROS levels under non-stress conditions. Importantly, H_2_O_2_ levels in the CYS + HT group were 30.82% lower than in plants exposed to HT stress alone (Fig. 1F).
The role of CYS in regulation of GLX system components and gene expression in A. thaliana under HT stress
In this study, T-DNA insertion mutants (hsp90.1 and hsp90.4) and Col-0 seedlings were subjected to the two-stage HT treatment described earlier (Fig. 1A). To test the hypothesis that CYS enhances plant resistance by modulating the GLX system through interactions with HSP90 proteins, biochemical analyses were conducted on Col-0, hsp90.1, and hsp90.4 mutants to measure GLX enzyme activities. For this purpose, key components of the GLX system, including MG and GSH content, were analyzed in mutants and Col-0 seedlings.
The concentration of MG was observed to increase under HT stress conditions across all experimental plant groups when compared to their respective controls (CK). Notably, in Col-0 seedlings, the MG levels in the CYS + HT group showed a significant decrease of 11.84% compared to the HT group. However, in the mutant seedlings, MG levels increased by approximately 7.6% in hsp90.1, while hsp90.4 showed only a slight decrease within the same treatment groups (Fig. 2A).
In response to HT stress, all plant groups exhibited elevated GSH content compared to the CK group. In Col-0 seedlings, treatment with CYS resulted in a 36.92% increase in GSH levels relative to the CK group. However, this treatment did not significantly alter GSH levels in the mutant groups. Furthermore, no significant differences were observed in GSH levels between the CYS + HT and HT-only groups in mutant seedlings (Fig. 2B).
Fig. 2. Effects of CYS on the GSH-Dependent MG scavenging pathway under HT stress.** A** MG content,** B** GSH content,** C** GLXI Activity, (D) GLXII Activity. CK (Control), HT (High-Temperature Stress), CYS (Cysteine), CYS + HT (Cysteine + High-Temperature Stress). Data represent means ± SE of three replicates. By Duncan’s test, different letters indicate significant differences (P < 0.05) amongst the treatments
Under HT stress, GLXI and GLXII enzyme activities increased in all plant groups, with Col-0 exhibiting a more pronounced response compared to mutant lines. Specifically, GLXI activity in Col-0 seedlings increased by 41.02% under HT stress compared to the control, while CYS treatment alone led to a 13.17% increase. In Col-0 plants subjected to both HT stress and CYS treatment, GLXI activity showed a further 8.64% increase relative to the HT-only group. However, in hsp90.1 and hsp90.4 mutants, CYS treatment alone did not significantly alter GLXI activity. Similarly, GLXII activity increased across all plant groups under HT stress, with a 20.32% rise in Col-0 plants compared to the control, while CYS treatment under HT stress further enhanced GLXII activity by 5.99% in Col-0. In contrast, the increase in GLXII activity was less pronounced in hsp90.1 and hsp90.4 mutants, and CYS treatment did not induce a statistically significant change compared to the HT-only group, with a slight decrease in enzyme activity observed. These findings suggest that Col-0 plants exhibit a stronger GLX system response under HT stress, potentially contributing to enhanced MG detoxification, while hsp90.1 and hsp90.4 mutants display a diminished enzymatic response to CYS treatment (Fig. 2C and D). The differences between the mutant groups were insignificant, indicating that the effectiveness of CYS in enhancing GLX system enzyme activities under HT stress was largely dependent on the presence of HSP90.1 and HSP90.4.
To further investigate these findings, we performed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis on the GLX gene family (GLXI.1,* GLXI.2*,* GLXI.3*,* GLXII.2*,* GLXII.4*,* GLXII.5)* in A. thaliana. These genes are directly involved in glyoxalase activity and contribute to the observed changes in GLXI and GLXII enzyme activities (Borysiuk et al. 2022). In Col-0 plants, CYS treatment under HT stress significantly induced the expression of GLXI.1,* GLXI.2*,* GLXI.3*,* GLXII.2*, and GLXII.4, with increases of 1.3-, 1.4-, 1.1-, 1.1-, and 1.9-fold, respectively, compared to the HT group. Conversely, in the hsp90.1 mutant, mRNA levels of these genes were generally reduced, particularly in the GLXII family, and CYS treatment did not result in significant differences compared to HT stress alone (Fig. 3). In the CYS-only group of hsp90.1 plants, no notable induction was detected. Similarly, in the hsp90.4 mutant, the expression levels of GLXI.1,* GLXII.2*, and GLXII.4 were significantly decreased, with no notable changes upon CYS treatment under HT stress compared to HT stress alone. However, the CYS-only group of hsp90.4 plants showed a slight increase in GLXI.2 expression, although this change was not statistically significant (Fig. 3). Notably, the mRNA level of GLXII.5 did not significantly decrease in either mutant compared to Col-0 (Fig. S1).
Fig. 3. Effect of exogenous CYS on expression profiles of GLX1.1,* GLX1.2*,* GLX1.3*,* GLXII.2*,* GLXII.4*,* DJ-1 A*,* DJ-1D* and D-LDH in Col-0, hsp90.1, and hsp90.4 mutants under HT stress. CK (Control), HT (High-Temperature Stress), CYS (Cysteine), CYS + HT (Cysteine + High-Temperature Stress). Data represent means ± SE of three replicates. By Duncan’s test, different letters indicate significant differences (P < 0.05) amongst the treatments
Interestingly, the mRNA abundance of DJ-1 A and DJ-1D, which are part of the GLXIII glyoxalase family, remained largely unchanged with CYS treatment under HT stress, suggesting that the GSH-dependent pathway primarily mediates the stress tolerance induced by CYS in the presence of HSP90.1 and HSP90.4 (Fig. 3). In the CYS-alone group, DJ-1 A expression slightly increased in Col-0 but not in the mutants while DJ-1D levels in the mutants remained low, consistent with HT stress results. However, DJ-1D expression significantly decreased in the hsp90.1 and hsp90.4 mutants compared to Col-0 across all groups, despite no observed changes under CYS treatment during HT.
Additionally, the expression of D-LDH, which catalyzes the conversion of D-lactate (the end product of the GLX system) to pyruvate, was significantly higher in Col-0 plants compared to the mutants. Following CYS treatment under HT stress, the D-LDH transcript was markedly induced in Col-0 compared to HT stress alone, suggesting an increase in MG degradation via enhanced D-lactate production and conversion by the D-LDH enzyme. In the CYS-alone group, D-LDH expression increased in Col-0 but not in the mutants. In contrast, the absence of HSP90.1 and HSP90.4 resulted in significantly reduced D-LDH transcript levels in the mutants compared to Col-0. Moreover, in the mutants, the CYS + HT treatment did not lead to significant changes in transcript levels under HT stress, unlike in Col-0 (Fig. 3).
The effect of the disruption of GSH-dependent GLX system on cysteine’s HT response in the absence of HSP90.4
In this study, to investigate the GSH-dependent GLX system’s role in CYS-mediated tolerance under HT stress, Col-0 and hsp90.4 seedlings were treated with 5 µM GLXI inhibitor under HT stress. This represents the first time a GLXI inhibitor has been applied to A. thaliana for this purpose (Fig. 4A–D). GLX I inhibitors such as BBGD inhibit GLX enzyme activity, leading to intracellular accumulation of MG, which can be cytotoxic at high levels (Alhujaily 2024). Considering the results of the inhibitor treatment, plants treated with 5 µM GLXI inhibitor were smaller than plants growing under normal conditions, although all plants in the control plant groups were healthy (Fig. 4A–D). The application of GLXI inhibitor resulted in more significant phenotypic defects in hsp90.4 plants compared to Col-0 plants indicating that the GSH-dependent GLX system plays a role in mitigating HT stress, particularly when HSP90.4 is functional. Also, when GLXI and GLXII enzyme activities were inhibited by GLXI inhibitor, the CYS treatment did not produce any positive effects under HT stress, as was the case in the groups without GLXI inhibitor treatment. This suggests that CYS enhances plant tolerance to HT stress by targeting the GSH-dependent GLX system enzymes GLXI and GLXII (Fig. 4C and D).
To provide quantitative support for the phenotypic data observed in the images, chlorophyll content and MDA levels were measured in the plants. Chlorophyll and MDA analyses were conducted on Col-0 plants to assess the impact of 5 µM GLXI inhibitor under HT stress and CYS treatment (Fig. 4E and F). The results revealed that plants treated with GLXI inhibitor had lower chlorophyll content and higher MDA levels compared to untreated plants.
Fig. 4. Effects of GLXI inhibitor treatment on Col-0 and mutant plants with and without CYS under HT stress.** A** Col-0 treated with 0 µM GLXI inhibitor** B** Col-0 treated with 5 µM GLXI inhibitor,** C** hsp90.4 mutant treated with 0 µM GLXI inhibitor,** D** hsp90.4 mutant treated with 5 µM GLXI inhibitor,** E** Total chlorophyll content,** F** MDA content. CK (Control), HT (High-Temperature Stress), CYS + HT (Cysteine + High-temperature Stress). Data represent means ± SE of three replicates. By Duncan’s test, different letters indicate significant differences (P < 0.05) amongst the treatments
In summary, the images effectively demonstrate the protective role of CYS and the negative effect of GLXI inhibition. The contrast between Col-0 and hsp90.4 mutants highlights the specific role of HSP90.4 in regulating the GLX detoxification system to provide stress tolerance.
CYS-mediated regulation of HSP expression under HT stress in A. thaliana
In the present study, we investigated the response of heat shock genes to CYS treatment under HT stress in both Col-0 and mutant A. thaliana plants. Specifically, we examined the expression of HSP21,* HSP70*,* HSP90.1*,* HSP90.4*, and HSP101 genes. The results have indicated that all HSPs exhibited elevated expression levels under HT stress compared to the control, with the most significant increases observed in HSP70 and HSP90.1. In the CYS + HT group, the expression levels of HSP70,* HSP90.1*, and HSP90.4 in Col-0 plants were further elevated by 1.67-fold, 1.19-fold, and 1.49-fold, respectively, compared to plants exposed to HT stress alone (Fig. 5). In the CYS-alone group of Col-0 plants, HSP70 expression increased compared to CK, while HSP90.1 and HSP90.4 showed only slight changes. However, no statistically significant changes were detected in the expression of HSP21 and HSP101 (Fig. S2).
In the hsp90.1 mutant, unlike in Col-0, CYS treatment did not lead to an increase in the expression of HSP70 or HSP90.4 in the CYS + HT group when compared to HT stress alone. In contrast, in the hsp90.4 mutant, CYS treatment resulted in increased expression of HSP70, while no such increase was observed for HSP90.1 transcripts. In both mutants, the CYS-alone treatment did not notably alter HSP expressions. Additionally, we measured the expression levels of HSP90.4 and HSP90.1 specifically in the mutant plants to verify these findings. The analysis confirmed that the transcript levels of these genes were significantly reduced in the mutants, with no notable differences between the various treatment groups.
Fig. 5. Changes induced by CYS treatment on the expression levels of HSP70,* HSP90.1*, and HSP90.4 genes in Col-0 and mutant plants under HT stress. CK (Control), HT (High-temperature stress), CYS (Cysteine), CYS + HT (Cysteine + High-Temperature stress). Data represent means ± SE of three replicates. By Duncan’s test, different letters indicate significant differences (P < 0.05) amongst the treatments
Discussion
Recent research has increasingly concentrated on the effects of elevated temperatures, driven by global warming, on plant health (Sadiq et al. 2020; Tian et al. 2023). Among them, the impact of CYS on antioxidant mechanisms within plants under various abiotic stresses and its role in conferring stress tolerance to the plant has garnered significant attention (Mijiti et al. 2022; El-Bassiouny et al. 2024). Despite this interest, the protective effects of CYS against HT stress in plants the role of the GLX system and HSP90 proteins in this process, and also the relationship between CYS application, GLX system, and HSPs remain somewhat unclear.
CYS enhances HT tolerance in A. thaliana
In the present study, CYS-treated A. thaliana plants exhibited higher survival, improved water retention, and increased chlorophyll content compared to untreated plants, highlighting CYS’s role in mitigating HT-induced oxidative damage. The CYS + HT group improved over HT alone in all evaluated metrics, proving CYS reduces stress. The CYS + HT group recovered significantly, although not as much as CK or CYS-alone. CYS + HT may have somewhat greater TBARS and H₂O₂ than CK and CYS groups due to residual stress. These elevations do not reduce mitigation compared to HT alone. Notably, TBARS can rise independently of H₂O₂ due to other ROS like hydroxyl radicals or stress-triggered lipid peroxidation pathways (Gill and Tuteja 2010; Farmer and Mueller 2013). Despite no significant rise in H₂O₂, the CYS + HT group had higher TBARS levels, suggesting that lipid peroxidation is not completely dependent on it. Advanced glycation end products (AGEs) production from MG buildup causes lipid peroxidation and ROS generation (Yadav et al. 2021). This suggests that CYS limits MG buildup through the glyoxalase pathway, reducing oxidative damage. The CYS + HT group had greater chlorophyll despite moderate TBARS levels, suggesting maintained photosynthetic structures. This suggests that partial lipid oxidation does not decrease photosynthetic capacity (Awasthi et al. 2015). These findings support the literature that CYS increases plant resistance by maintaining photosynthetic efficiency and lowering water loss under stress (Gautam et al. 2022; Hussein and Alshammari 2022; Galleguillos et al. 2023).
CYS boosts GLX enzyme and gene expression levels in the presence of HSP90.1/90.4
Our experiments show that exogenous CYS under HT stress lowers MG levels and increases GLXI and GLXII activity in Col-0 plants, detoxifying the GLX system in A. thaliana. CYS did not significantly influence enzyme activity in hsp90.1 and hsp90.4 mutants, indicating the relevance of HSP90 proteins in GLX system activation by CYS (Li et al. 2020; Parvin et al. 2020; Gambhir et al. 2022). CYS reduced MG and increased GLXI and GLXII activity in Col-0 seedlings, detoxifying them. CYS did not impact MG levels or enzyme activity in hsp90.1 and hsp90.4 mutants, showing that these proteins are necessary for cysteine’s GLX system involvement. Despite markedly reduced GLXI and GLXII activities in hsp90.1 and hsp90.4 mutants, MG levels remained relatively stable, likely due to multiple compensatory mechanisms such as heat-induced metabolic slowdown limiting MG biosynthesis, activation of alternative detoxification enzymes, increased MG sequestration into protein adducts, and functional redundancy among other HSP90 isoforms that help maintain proteostasis and stress adaptation under HT (Kaur et al. 2014; Mostofa et al. 2015). Additionally, recent findings reveal that CYS treatment in A. thaliana under HT stress increases GSH levels, a critical cofactor in the GLX system responsible for detoxifying MG.
Our findings reveal that CYS treatment upregulates key GLXI and II-related genes, boosting MG detoxification in Col-0 plants and leading to improved stress resilience and reduced oxidative damage under HT stress. Even without HT stress, CYS alone moderately induced the expression of certain GLX genes (GLXI.1,* GLXI.3*,* GLXII.4*) in Col-0, but not in hsp90.1 or hsp90.4, showing that these isoforms are needed for CYS-mediated regulation. Similar patterns were observed for DJ-1 A,* DJ-1D*, and D-LDH expression, where CYS-induced increases occurred only in Col-0, further supporting the dependency of these responses on functional HSP90.1 and HSP90.4. The hsp90.1 and hsp90.4 mutants lack this upregulation and incur higher physiological damage, highlighting HSP90’s importance in CYS activating the GLX system and improving stress tolerance in A. thaliana. DJ-1 A and DJ-1D, GLXIII genes, have steady mRNA levels with CYS administration, suggesting that the GSH-dependent system mediates CYS-induced stress tolerance under HT. However, DJ-1D expression is significantly reduced in hsp90 mutants, indicating HSP90’s regulatory influence in this pathway under HT. CYS + HT treatment also significantly induces D-LDH gene expression, which converts D-lactate to pyruvate, in Col-0 but not in mutants. This shows that CYS may increase stress tolerance by increasing GSH-dependent GLX enzymes and D-lactate breakdown via HSP90, helping MG detoxification under HT stress.
Disruption of GSH-dependent GLX system affects cysteine’s HT response
The research aimed to examine the effects of a GLXI inhibitor on A. thaliana Col-0 and hsp90.4 seedlings under HT stress, specifically investigating its impact on plant responses to such conditions. Col-0 plants treated with CYS maintained higher chlorophyll content and lower MDA levels despite GLXI inhibition. These findings confirm that the GLX system plays a critical role in CYS-mediated HT tolerance (Fig. 4A–F). The use of the GLXI inhibitor confirmed the critical role of the GLX system in conferring CYS-induced protection against HT stress. However, the protective role of CYS was absent in seedlings treated with GLXI inhibitor, suggesting that cysteine’s efficacy is dependent on the GSH-dependent GLX system (Fig. 4C and D).
CYS-Mediated modulation of HSP and GLX system interactions under HT stress
Key HSP genes were investigated to see how CYS affects HSP expression during HT stress. HSP90.1,* HSP21*,* HSP90.4*,* HSP101*, and HSP70 dramatically increased in both Col-0 and mutant plants under HT stress, consistent with prior findings. The higher HSP expression in mutants suggests a compensatory activation of other HSPs to offset the absence of specific isoforms. CYS selectively elevated HSP90.1,* HSP90.4*, and HSP70 expression in Col-0 plants, improving stress tolerance. HSP21 and HSP101 remained unchanged, showing CYS’s specificity. CYS failed to stimulate HSP70 in hsp90.1 mutants, demonstrating its importance for this response, although HSP70 was increased in hsp90.4 mutants. These findings show that CYS and key HSPs work together to manage HT stress. Kumar and Chattopadhyay (2018) demonstrated that the BZIP10 transcription factor activates the HSP90.1 promoter in response to elevated GSH levels, leading to increased HSP90.1 expression. Our results suggests that exogenous CYS administration increases GSH, which upregulates HSP90.1 and HSP90.4 expression. This increased expression stimulates GLX system enzymes under HT stress, promoting A. thaliana thermotolerance. Overall, heat shock gene expression study shows that CYS therapy upregulates essential HSPs, increasing stress adaption. Kumar and Chattopadhyay (2018) demonstrated that the BZIP10 transcription factor activates the HSP90.1 promoter in response to higher GSH levels, leading to an increase in HSP90.1 expression. Based on literature findings, CYS increased GSH levels, which elevated HSP90.1 and HSP90.4 expression and improved A. thaliana thermotolerance by boosting GLX system enzyme activities (Fig. 6B). Using our data and related literature, we constructed a suitable model (Fig. 6A and B).
Fig. 6A working model that the GLX system regulates plant resilience in response to HT stress together with CYS, HSP90.1, and HSP90.4. After HT treatments cytosolic MG significantly increased. CYS administration causes an increase in the amount of GSH. Increased GSH induces the accumulation of HSP90.1 through the BZIP10 transcription factor (Kumar and Chattopadyay2018) and also HSP90.4. HSP90.1 and HSP90.4 increase the activity of the GLX system by stimulating GLXI.1,* GLXI.2*,* GLXI.3*,* GLXII.2*,* GLXII.4* and GLXI.1,* GLXII.2*,* GLXII.4*, respectively through CYS, resulting in rapid degradation of increased MG and thermotolerance in the plant. In addition, HSP90.1 and HSP90.4 together stimulate DJ-1D, which is involved in the GSH-independent GLX system, in a CYS-independent manner and contribute to the degradation of MG. Dashed arrows indicate a direct or indirect effect of cysteine. Orange color indicates HSP90.1 and purple color indicates HSP90.4 (color figure online)
Conclusion
This study demonstrates that exogenous CYS enhances thermotolerance in Arabidopsis thaliana by modulating multiple protective mechanisms. CYS treatment alleviated HT-induced oxidative stress by improving relative water content, maintaining chlorophyll levels, and reducing H_2_O_2_ accumulation and lipid peroxidation. At the molecular level, CYS promoted the detoxification of MG through increased activity and expression of glyoxalase enzymes (GLXI and GLXII), a response that was dependent on the presence of HSP90 proteins. In hsp90.1 and hsp90.4 mutants, the CYS-induced activation of the GLX system was abolished, indicating a regulatory role of HSP90s in this pathway. Moreover, CYS selectively upregulated HSP70,* HSP90.1*, and HSP90.4 transcripts in wild-type plants under heat stress, while the absence of HSP70 induction in the hsp90.1 mutant confirmed the hierarchical regulation of heat shock proteins. These findings provide a comprehensive model of an integrated stress response pathway and identify the CYS-HSP90-GLX axis as a promising target for genetic and biochemical strategies aimed at developing climate-resilient crops.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary Material 1
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