JAC1 Promotes Thermotolerance in Arabidopsis by Limiting Heat-Induced H2O2 Accumulation and Protecting PGLP1 from Sulfenylation-Mediated Inhibition

TL;DR

The JAC1 protein helps plants survive heat stress by reducing harmful hydrogen peroxide and protecting a key enzyme involved in photorespiration.

Contribution

JAC1 is newly identified as a positive regulator of thermotolerance through its role in H2O2 homeostasis and PGLP1 sulfenylation inhibition.

Findings

JAC1 overexpression improves plant survival under long-term heat stress.

JAC1 limits H2O2 accumulation and prevents PGLP1 sulfenylation during heat stress.

PGLP1 activity is crucial for thermotolerance, and JAC1 functions upstream of PGLP1.

Abstract

Heat stress is a major constraint on plant growth and productivity, and excessive accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS), especially hydrogen peroxide (H2O2), is a primary cause of heat-induced cellular damage. Photorespiration becomes accelerated at high temperature and generates the toxic metabolite 2-phosphoglycolate (2PG), whose accumulation is prevented by the first photorespiratory enzyme 2-phosphoglycolate phosphatase (PGLP1). Here, we identify the auxilin-like J-domain protein JAC1 (AT1G75100) as a positive regulator of thermotolerance in Arabidopsis thaliana (A. thaliana). JAC1 transcripts were rapidly induced by heat treatment, and loss-of-function jac1 mutants (jac1-1 and jac1-2) were hypersensitive to long-term heat stress (38 °C, 7 d), whereas three independent JAC1 overexpression lines (#2, #4, #5) exhibited enhanced survival. Consistent with a role in ROS homeostasis, heat-treated jac1 mutants showed stronger DAB staining, consistent with higher apparent H2O2 levels, whereas JAC1 overexpression lines showed weaker staining. Although PGLP1 abundance (mRNA and protein) was unchanged among genotypes, heat treatment (38 °C, 12 h) caused a marked reduction in leaf PGLP1 enzymatic activity in jac1 mutants and a significant increase in JAC1 overexpression lines, leading to corresponding changes in 2PG content. PGLP1 sulfenylation, a H2O2-dependent post-translational modification that inhibits PGLP1, was detectable only after heat and was enhanced in jac1 mutants but suppressed in JAC1 overexpression lines. Genetic manipulation of PGLP1 supported that PGLP1 functions downstream of JAC1: overexpressing PGLP1 in jac1-1 rescued heat sensitivity, whereas knocking down PGLP1 in the JAC1 overexpression background reversed thermotolerance. Together, our results support a model in which JAC1 maintains heat-induced H2O2 homeostasis, thereby limiting PGLP1 sulfenylation, sustaining PGLP1 activity and preventing toxic 2PG accumulation during heat stress.

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1. Introduction

Rising global temperatures and the increasing frequency of heat waves are threatening crop production and ecosystem stability [1,2]. At the cellular level, heat stress perturbs membrane integrity, protein homeostasis and photosynthetic electron transport, and it frequently triggers the overproduction of reactive oxygen species (ROS) [3,4,5,6]. While ROS can function as signals [7,8], excessive ROS—particularly hydrogen peroxide (H_2_O_2_)—causes oxidative damage to proteins, lipids and nucleic acids, ultimately compromising plant survival [9,10].

High temperature also reshapes chloroplast metabolism by lowering Rubisco CO_2_ selectivity, thereby favoring the oxygenation reaction and accelerating photorespiration [11]. Photorespiration is essential because it prevents the accumulation of 2-phosphoglycolate (2PG), a potent inhibitor of central carbon metabolism and a highly toxic compound for plants [12,13]. Accordingly, disruption of 2PG detoxification (e.g., pglp1) is lethal in Arabidopsis thaliana [12]. The first committed step of photorespiration is catalyzed by 2-phosphoglycolate phosphatase (PGLP1), which dephosphorylates 2PG to glycolate. Beyond its metabolic role, PGLP1 is subject to redox regulation. Previous work demonstrated that heat stress promotes chloroplastic H_2_O_2_ accumulation and triggers PGLP1 sulfenylation, which inhibits PGLP1 enzymatic activity and results in 2PG over-accumulation; conversely, maintaining PGLP1 activity enhances thermotolerance [14].

JAC1 (J-DOMAIN PROTEIN REQUIRED FOR CHLOROPLAST ACCUMULATION RESPONSE 1; AT1G75100) encodes an auxilin-like J-domain protein originally characterized as an essential component of phototropin-mediated chloroplast positioning [15,16,17]. JAC1 is thought to act as a co-chaperone for Hsc70 and is implicated in actin-dependent chloroplast movement. J-domain proteins typically stimulate Hsp70/Hsc70 ATPase activity and support proteostasis, which is crucial for stress tolerance including heat stress [18]. Although JAC1 has been intensively studied in light responses, emerging evidence suggests broader roles in stress-associated processes, including oxidative stress and photooxidative stress responses under specific conditions [19,20].

Given the close interconnection between chloroplast function, ROS homeostasis and heat-induced photorespiratory constraints, we hypothesized that JAC1 might contribute to thermotolerance by modulating ROS accumulation and protecting critical photorespiratory enzymes. To test this, we analyzed thermotolerance phenotypes of jac1 loss-of-function mutants and multiple independent JAC1 overexpression lines, quantified heat-induced H_2_O_2_ accumulation, and examined PGLP1 activity, 2PG accumulation and PGLP1 sulfenylation. Finally, we performed genetic epistasis experiments by altering PGLP1 expression in jac1 and JAC1 overexpression backgrounds. These analyses reveal a previously unrecognized role of JAC1 in safeguarding photorespiration under heat stress via a ROS–PGLP1–2PG axis.

2. Results

2.1. Heat Induces JAC1 Expression and JAC1 Positively Regulates Thermotolerance

To determine whether JAC1 participates in the heat stress response, we first examined JAC1 transcript dynamics under high temperature. RT–qPCR analysis revealed that JAC1 mRNA was rapidly induced by heat treatment at 38 °C: transcript abundance increased ~3-fold after 6 h, further increased at 12 h, and remained at a comparable level at 24 h (Figure 1A). These kinetics indicate that JAC1 is a heat-responsive gene.

Next, we assessed thermotolerance using a long-term heat stress regime (38 °C for 7 d) followed by recovery at 23 °C. Compared with wild type, both jac1 loss-of-function mutants (jac1-1 and jac1-2) displayed markedly increased heat sensitivity, characterized by a pronounced decrease in survival rate after recovery (Figure 1B–D). In contrast, three independent JAC1 overexpression lines (#2, #4 and #5) exhibited significantly higher survival rates than wild type (Figure 1B–D). Together, these results establish JAC1 as a positive regulator of Arabidopsis thermotolerance.

2.2. JAC1 Attenuates Heat-Induced H2O2 Accumulation in Leaves

Because excessive H_2_O_2_ accumulation is a major contributor to heat-induced injury, we investigated whether JAC1 affects ROS homeostasis under heat stress. Leaves were subjected to short-term heat treatment (38 °C for 12 h) and stained with DAB to visualize H_2_O_2_ accumulation. Under control conditions (23 °C), DAB staining was weak across all genotypes. After heat treatment, DAB staining intensities increased in all lines, but the magnitude differed markedly among genotypes (Figure 2A). Both jac1-1 and jac1-2 exhibited substantially stronger DAB staining than wild type, consistent with higher apparent H_2_O_2_ levels. Conversely, JAC1 overexpression lines (#2, #4 and #5) showed weaker staining than wild type, consistent with lower apparent H_2_O_2_ levels (Figure 2A). Semi-quantitative image analysis confirmed that heat-induced DAB intensities were significantly higher in jac1 mutants and significantly lower in JAC1 overexpression lines relative to wild type (Figure 2B). These data support a role of JAC1 in restricting heat-triggered H_2_O_2_ over-accumulation.

2.3. JAC1 Regulates Heat-Dependent PGLP1 Activity and 2PG Homeostasis Without Altering PGLP1 Abundance

Previous work demonstrated that H_2_O_2_-dependent sulfenylation inhibits PGLP1 activity under heat stress, leading to toxic 2PG accumulation and reduced thermotolerance [14]. We therefore tested whether the altered H_2_O_2_ accumulation observed in jac1 mutants and JAC1 overexpression lines translates into changes in PGLP1 activity and 2PG levels.

Under non-stress conditions (23 °C), leaf PGLP1 activity did not differ significantly among wild type, jac1-1, jac1-2 or the three JAC1 overexpression lines (Figure 3A). After heat treatment (38 °C for 12 h), however, PGLP1 activity was significantly reduced in jac1-1 and jac1-2 compared with wild type, whereas all three JAC1 overexpression lines showed significantly higher PGLP1 activity than wild type (Figure 3A). Consistent with these activity changes, 2PG contents remained low and comparable across genotypes at 23 °C but diverged sharply after heat stress: jac1 mutants accumulated significantly more 2PG, while JAC1 overexpression lines accumulated significantly less 2PG than wild type (Figure 3B).

To determine whether these differences were caused by altered PGLP1 expression, we measured PGLP1 transcript abundance and protein accumulation. RT–qPCR showed that PGLP1 mRNA levels were not significantly different among genotypes under either control or heat conditions (Figure S1A). Likewise, immunoblot analysis revealed similar PGLP1 protein abundance across all lines before and after heat stress (Figure S1B). Thus, JAC1 modulates PGLP1 enzymatic activity and 2PG homeostasis during heat stress without detectably changing PGLP1 transcript or protein levels.

2.4. JAC1 Modulates Heat-Triggered PGLP1 Sulfenylation

The absence of PGLP1 abundance changes suggested that post-translational regulation contributes to the JAC1-dependent differences in PGLP1 activity. We therefore examined PGLP1 sulfenylation using a biotin-switch assay. No sulfenylated PGLP1 signal was detected under control conditions (23 °C) in any genotype. After heat treatment (38 °C for 12 h), sulfenylated PGLP1 was readily detectable in wild type, jac1 mutants and JAC1 overexpression lines (Figure 4A). Importantly, the sulfenylation signal was significantly stronger in jac1-1 and jac1-2 than in wild type, whereas all three JAC1 overexpression lines exhibited significantly weaker sulfenylation signals (Figure 4A,B). These results mirror genotype-dependent differences in H_2_O_2_ accumulation and PGLP1 activity (Figure 2 and Figure 3), supporting a model in which JAC1 is associated with reduced heat-induced PGLP1 sulfenylation, thereby maintaining higher PGLP1 activity and preventing 2PG over-accumulation.

2.5. Genetic Manipulation of PGLP1 Supports an Upstream Role of JAC1 in Thermotolerance

To examine whether JAC1-mediated thermotolerance involves modulation of PGLP1 activity, we altered PGLP1 expression in jac1-1 and 35S::JAC1 genetic backgrounds and evaluated the resulting biochemical and phenotypic outputs relative to WT. In the jac1-1 background, three independent jac1-1/35S::PGLP1 lines (#1, #4 and #7) displayed a strong increase in PGLP1 transcripts (~3-fold of WT; Figure 5A) accompanied by markedly elevated PGLP1 activity under both mock conditions and after heat treatment (38 °C for 12 h) (Figure 5B). Consistent with enhanced enzymatic capacity, heat-induced 2PG accumulation was significantly reduced in jac1-1/35S::PGLP1 lines compared with WT (Figure 5D), and these lines showed survival comparable to WT in the long-term thermotolerance assay (38 °C for 7 d followed by recovery) (Figure 5C,E), aligning with the heat-sensitive trend observed for jac1 mutants (Figure 1C,D). Conversely, antisense suppression of PGLP1 in the 35S::JAC1 #2 background (35S::JAC1/asPGLP1 #3, #4 and #9) reduced PGLP1 transcripts to ~0.3-fold of WT (Figure 5A), lowered PGLP1 activity after heat treatment and increased 2PG accumulation relative to WT (Figure 5B,D). Notably, 35S::JAC1/asPGLP1 lines exhibited reduced survival under long-term heat stress (Figure 5C,E), contrasting with the enhanced survival of JAC1-overexpressing plants observed earlier (Figure 1C,D). Together, these reciprocal genetic manipulations support PGLP1 as a key downstream effector within the JAC1-regulated heat-response pathway and are consistent with a JAC1–H_2_O_2_–PGLP1–2PG module during heat stress (Figure 5F).

3. Discussion

Heat tolerance is increasingly important for plant productivity, yet many thermotolerance mechanisms converge on preserving metabolic function under oxidative stress [1,2]. This study identifies JAC1 as a heat-inducible positive regulator of thermotolerance and reveals a mechanistic route by which an auxilin-like J-domain protein supports heat resistance through redox control of photorespiration. Notably, while recent work established that heat-induced chloroplastic H_2_O_2_ can sulfenylate and inhibit PGLP1 to modulate thermotolerance [14], the upstream genetic factors that tune this oxidative pressure remained unclear; our results identify JAC1 as a positive regulator that constrains heat-associated H_2_O_2_ accumulation and thereby safeguards PGLP1 activity and 2PG homeostasis. Although JAC1 has been investigated mainly in phototropin-mediated chloroplast positioning [15,17], we show that JAC1 substantially contributes to survival under prolonged high temperature, with opposite phenotypes in jac1 mutants versus multiple independent JAC1-overexpression lines.

A practical implication of our findings is that JAC1 contributes to restraining the extent of heat-induced H_2_O_2_ accumulation, a common driver of cellular injury during heat stress [4,5,21]. The consistent DAB staining differences across genotypes indicate that JAC1 shifts ROS balance under heat, either by limiting ROS formation and/or enhancing detoxification, thereby maintaining H_2_O_2_ within a range compatible with signaling while avoiding excessive oxidative damage [6,10]. It should be noted that DAB staining reflects bulk tissue accumulation and cannot assign the H_2_O_2_ signal to a specific compartment; thus, the observed genotype-dependent changes likely represent an altered H_2_O_2_ steady state shaped by integrated production and detoxification across multiple cellular sites [22,23,24]. Consistent with the extensive literature, heat-associated H_2_O_2_ can be shaped by chloroplastic electron transport [25], peroxisomal photorespiratory metabolism and catalase capacity [26,27], and apoplastic RBOH-dependent ROS signaling [28,29,30]. Nevertheless, the close correspondence between H_2_O_2_ differences and the downstream redox-dependent regulation of a chloroplast-localized photorespiratory enzyme suggests that JAC1 meaningfully influences the oxidant environment relevant to photorespiration under heat.

Crucially, the downstream consequences of JAC1-dependent H_2_O_2_ control are mechanistically interpretable and agriculturally relevant: JAC1 limits PGLP1 sulfenylation, maintains PGLP1 activity, and prevents 2PG over-accumulation during heat stress [14]. Because PGLP1 abundance did not change among genotypes, redox-dependent modification offers an efficient means to tune photorespiratory capacity without requiring slower transcriptional reprogramming. Preventing 2PG build-up is particularly important given its toxicity and potential to disrupt central carbon metabolism [12,13]; thus, safeguarding PGLP1 provides a direct metabolic rationale for improved survival under heat.

The genetic manipulations of PGLP1 further support the functional hierarchy implied by the biochemical data. Increasing PGLP1 expression in a jac1-1 background improved metabolic outputs (higher PGLP1 activity, lower 2PG) and enhanced survival under heat, whereas reducing PGLP1 expression in a JAC1-overexpression background shifted metabolism in the opposite direction and weakened thermotolerance. Together, these results highlight PGLP1 as a key effector through which JAC1 confers heat resistance, framing a JAC1–H_2_O_2_–PGLP1–2PG module that links redox balance to photorespiratory robustness. From an application perspective, the conservation and tractability of this module, together with the centrality of ROS–photorespiration coupling during heat stress [9,11], suggest that targeting the JAC1–PGLP1 axis could provide a rational route to improve thermotolerance in crops.

4. Conclusions

JAC1 is heat inducible and promotes Arabidopsis thermotolerance. By restraining heat-induced H_2_O_2_ accumulation, JAC1 limits PGLP1 sulfenylation, sustains PGLP1 activity, and prevents toxic 2PG over-accumulation during heat stress. Genetic analyses support PGLP1 as a key downstream effector of JAC1, establishing a JAC1–H_2_O_2_–PGLP1–2PG regulatory module that protects plants from heat-induced damage.

5. Materials and Methods

5.1. Plant Materials and Growth Conditions

Arabidopsis thaliana (A. thaliana) ecotype Columbia-0 (Col-0) was used as the wild-type control. The jac1-1 and jac1-2 mutants were obtained as T-DNA-tagged lines [15] from the Arabidopsis Biological Resource Center (ABRC, The Ohio State University, Columbus, OH, USA). Homozygous plants were identified by PCR, and the confirmed T3 generation was used for experiments. Three independent JAC1 overexpression lines (35S::JAC1 #2 #4 #5) were used. For epistasis analysis, three independent PGLP1 overexpression lines in the jac1-1 background (jac1-1/35S::PGLP1 #1, #4, and #7) and three independent PGLP1 knockdown lines in the 35S::JAC1 #2 background (35S::JAC1/as-PGLP1 #3, #4, and #9) were generated. Plants were grown in soil at 23 °C under long-day conditions (16 h light/8 h dark) with ~150 μmol m^−2^ s^−1^ white light.

5.2. Heat Stress Treatments

For biochemical and physiological assays (DAB staining, PGLP1 activity, 2PG quantification, immunoblotting, sulfenylation assay and RT–qPCR unless otherwise indicated), 3-week-old soil-grown plants were transferred to 38 °C for 12 h under the same photoperiod and light intensity. For JAC1 transcript time-course analysis, plants were transferred to 38 °C for 0, 6, 12 or 24 h, and leaf tissues were sampled at each time point. For thermotolerance phenotyping, plants were transferred to 38 °C for 7 d, followed by recovery at 23 °C for 4 d. Plants that produced new green leaves after recovery were counted as surviving individuals. Unless otherwise specified, ~150 plants were used per biological replicate.

5.3. Sampling Strategy

For enzyme activity, metabolite profiling, protein extraction, sulfenylation assay and RT–qPCR, each biological replicate consisted of pooled rosette leaves collected from multiple individual plants grown and treated in parallel. Immediately after harvest, samples were frozen in liquid nitrogen and stored at −80 °C.

5.4. DAB Staining and Image Quantification

Heat-induced H_2_O_2_ accumulation was evaluated by 3,3′-diaminobenzidine (DAB; Beyotime Biotech Inc., Songjiang, Shanghai, China) staining as previously reported [31]. Briefly, leaves were incubated in freshly prepared staining solution (1 mg mL^−1^ DAB, 10 mM Na_2_HPO_4_, 0.1% [v/v] Tween-20) for 8 h at 30 °C in the dark, and then rinsed several times with 70% ethanol to remove chlorophyll. Leaf images were captured using a digital camera (Nikon Corporation, Tokyo, Japan). The relative intensity of DAB staining was semi-quantitatively analyzed using Photoshop CS6 (Adobe, San Jose, CA, USA) (Adobe) by measuring mean grayscale values in defined leaf areas and normalizing to the wild-type control measured in parallel. Images were converted to 8-bit grayscale and the same leaf region was measured across samples; higher grayscale intensity corresponded to stronger DAB deposition. At least 10 leaves were analyzed per genotype per biological replicate.

5.5. Plasmid Construction and Plant Transformation

JAC1 and PGLP1 coding sequences were amplified from Arabidopsis cDNA and cloned into pCAMBIA1300S under the control of the CaMV 35S promoter for constitutive expression. For PGLP1 knockdown, an antisense fragment of PGLP1 (as-PGLP1) was cloned into pBI121 in the antisense orientation. All constructs were verified by sequencing (GenScript Biotech Corporation, Nanjing, Jiangsu, China) and introduced into Agrobacterium tumefaciens strain GV3101. Arabidopsis transformation was performed by the floral dip method [32]. Transgenic plants were selected on appropriate selection medium, and independent homozygous T3 lines were identified by resistance segregation and confirmed by RT–qPCR. Primers used are listed in Table S1.

5.6. RNA Extraction and RT–qPCR

Total RNA was extracted from leaves using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with RNase-free DNase I (Invitrogen, Carlsbad, CA, USA). First-strand cDNA synthesis was performed using ReverTra Ace (TOYOBO Co., Ltd., Osaka, Japan). qPCR was conducted on a Bio-Rad CFX96 system (Bio-Rad Laboratories, Hercules, CA, USA) using SYBR Green I dye (Beyotime Biotech Inc., Songjiang, Shanghai, China) with the following program: 95 °C for 3 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 45 s. PP2AA3 (At1g13320) and EIF4A1 (At3g13920) were used as internal reference genes as previously validated for Arabidopsis qPCR normalization [33,34]. Data acquisition and analysis were performed using CFX Manager software (version 3.1; Bio-Rad Laboratories, Hercules, CA, USA). All RT–qPCR experiments included three independent biological replicates and three technical repeats. Primers used are listed in Table S1.

5.7. Protein Extraction and Immunoblot Analysis

Total proteins were extracted from frozen leaf tissues in denaturing extraction buffer containing protease inhibitors. Protein concentration was determined by Bradford assay [35]. Equal amounts of protein were separated by SDS–PAGE and transferred to PVDF membranes. PGLP1 protein was detected using rabbit anti-PGLP1 antibody [14]; anti-Actin (M20009, Abmart, Shanghai, China) (M20009, Abmart) was used as a loading control. Signals were visualized by chemiluminescence and quantified by densitometry when required.

5.8. PGLP1 Activity Assay

Leaf PGLP activity was determined following the previously reported protocol [12]. Briefly, frozen leaves were homogenized in ice-cold extraction buffer (10 mM HEPES-NaOH, pH 7.0, 0.1 mM EDTA, 5 mM MgCl_2_ and 10% glycerol) and clarified by centrifugation (12,000× g, 10 min, 4 °C). The enzymatic reaction was initiated by adding 2PG (final 1 mM) to the extract in assay buffer (50 mM HEPES-NaOH, pH 7.0, 5 mM MgCl_2_) and incubated at 30 °C for 10 min. Reactions were stopped with 0.5 M HCl, and released inorganic phosphate was quantified using a malachite green colorimetric reagent (A_620_). Activity was calculated from a KH_2_PO_4_ standard curve and expressed as nmol Pi min^−1^ mg^−1^ protein. Assay linearity was confirmed by varying protein amount and incubation time; all measurements were performed within the linear range.

5.9. Measurement of 2PG Content

Leaf 2-phosphoglycolate (2PG) content was quantified by LC-MS/MS essentially as described in our previous work [14] and based on established protocols for photorespiratory intermediates [36]. Briefly, for each biological replicate, rosette leaves pooled from multiple plants were harvested, immediately frozen in liquid nitrogen, and ground to a fine powder. Metabolites were extracted from frozen leaf powder using a pre-chilled solvent system (ethanol:formic acid:water, 3:71:26, v/v/v), clarified by centrifugation (12,000× g, 10 min, 4 °C), dried under vacuum, resuspended in acetonitrile, and filtered (0.22 um) prior to analysis. LC-MS/MS was performed on a Q-Exactive mass spectrometer (Thermo Scientific, Waltham, MA, USA) with negative electrospray ionization. Separation was achieved on a zwitterionic hydrophilic interaction chromatography column (5 um, 2.1 × 150 mm, Merck, Rahway, NJ, USA) using 10 mM ammonium acetate in water (A) and acetonitrile (B) at a constant flow rate of 0.35 mL min-1. A multistep gradient was applied (0–1 min, 20% A; 1–5 min, 20–50% A; 5–8 min, 50% A; 8–9 min, 50–20% A; 9–15 min, 20% A; v/v). Source settings were: capillary temperature 320 °C, spray voltage 3.2 kV, sheath gas 40, auxiliary gas 15, and S-lens RF 55. Full MS spectra were acquired at 70,000 resolution (FWHM). 2PG abundance was quantified using an external calibration curve generated with authentic 2PG standards (2-phosphoglycolic acid; Sigma-Aldrich, St. Louis, MO, USA; CAS 13147-57-4) and normalized to fresh weight. No isotope-labeled internal standard was used.

5.10. Biotin-Switch Assay for Detection of PGLP1 Sulfenylation

PGLP1 sulfenylation was assessed using a biotin-switch approach as previously reported [14]. In brief, proteins were extracted under conditions that preserve redox modifications. Free thiols and persulfides were blocked with methyl methanethiosulfonate (MMTS; Beyotime Biotech Inc., Songjiang, Shanghai, China). Sulfenic acids (–SOH) were selectively reduced to thiols using sodium arsenite, and the newly generated thiols were labeled with N-[6-(biotinamido)hexyl]-3′-(2′-pyridyldithio) propionamide (biotin-HPDP; Beyotime Biotech Inc., Songjiang, Shanghai, China). Biotin-labeled proteins were enriched using streptavidin–agarose (Beyotime Biotech Inc., Songjiang, Shanghai, China) and sulfenylated PGLP1 was detected by immunoblotting with anti-PGLP1 antibody.

5.11. Statistical Analysis

All experiments were performed with at least three independent biological replicates unless otherwise stated. Data are presented as mean ± SD. All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple-comparisons test, or by Student’s t-test when only two groups were compared. Different letters indicate significant differences at p < 0.05.