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Article

Age-Dependent Differences in Leaf Sulfur Assimilation and Relationship with Resistance to Air Pollutant SO2

by
Jinxia Feng
1,
Luyi Wang
1,
Wenxin Liu
2,
Ying Gao
1 and
Xianchong Wan
1,*
1
Institute of Ecological Conservation and Restoration, Chinese Academy of Forestry, Beijing 100091, China
2
College of Pharmacy and Life Science, Jiujiang University, Jiujiang 332005, China
*
Author to whom correspondence should be addressed.
Forests 2025, 16(10), 1582; https://doi.org/10.3390/f16101582
Submission received: 25 July 2025 / Revised: 24 September 2025 / Accepted: 30 September 2025 / Published: 14 October 2025
(This article belongs to the Section Forest Meteorology and Climate Change)
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Abstract

Two poplar varieties with different resistance to sulfur dioxide were subjected to different concentrations of SO2 fumigation treatment. Young and mature leaves of Purui poplar (resistant) vs. 74/76 poplar (susceptible) were used to measure the changes in the activity of enzymes and metabolite content. Among the five key enzymes involved in sulfur metabolism and sulfur metabolites, APR, SO enzyme, GSH, and sulfate content have the greatest impact on young leaves of Purui, followed by 74/76 young leaves. The results show that for both Purui and 74/76 poplar, young leaves have stronger sulfur metabolism ability than mature leaves, indicating that young leaves have stronger SO2 resistance. Purui has stronger sulfur metabolism ability than 74/76 poplar, especially reflected in their young leaves. The comparison between young and mature leaves, as well as the comparison between resistant and susceptible varieties, mutually confirms that sulfur metabolism in leaves is an important mechanism for sulfur dioxide resistance.

1. Introduction

Sulfur dioxide (SO2) is a major air pollutant that is predominantly absorbed by plant leaves through stomata and dissolves on the aqueous phase of the cell surface or in the cytoplasm, producing bisulfite and sulfite ions [1]. These sulfites have high phytotoxicity to plants [2] and pose a threat to plants through the formation of reactive oxygen species (ROS) and other free radicals formed during the oxidation of sulfite to sulfate or interference with physiological processes caused by acidification and sulfite decomposition [3]. If sulfite persists in plant cells for a considerable period of time, the plant will be subjected to severe stress and potential damage. In order to maintain activity and even survival, plants are equipped with effective stress protection mechanisms [4]. These ions are metabolized in the plant, either through sulfur assimilation to form cysteine, glutathione, and other sulfur-containing compounds or through enzymatic or non-enzymatic oxidation to sulfates [5,6,7,8,9,10]. Therefore, the reduction or oxidation of sulfite is considered to represent the detoxification of bisulfite and sulfite.
The enzyme sulfite oxidase (SO) catalyzes the conversion from sulfite (SO32−) to sulfate (SO42−) in the peroxisome [9,11,12]. The latter may then be reduced by APS reductase (APR) to sulfite (SO32−) [13]. When plants are exposed to sulfur dioxide in the atmosphere, it can be assumed that the sulfate–sulfite cycle driven by APR and SO is used to fine-tune sulfur distribution, which is also used for sulfite detoxification [14]. Sulfite is subsequently reduced to sulfide (S2−) by sulfite reductase (SiR). Sulfide combines with O-acetylserine (OAS) to form cysteine (Cys), which is the precursor to all sulfur-containing organic compounds. Serineacetyltransferase (SAT) synthesizes the Cys precursor O-acetylserine (OAS), and O-acetylserine (thiol) lyase (OASTL) exchanges the acetate of OAS for sulfide. An overall scheme is shown in Figure 1.
Therefore, the tolerance level toward acute SO2 stress depends on the capacity for sulfite detoxification, leading to the formation of sulfate and/or the synthesis of large amounts of GSH by the reductive pathway, which serves as a storage reservoir for reduced sulfur [10] and also an antioxidant to alleviate oxidative stress [15]. Thus, specific biochemical processes and the relevant enzyme activities are among crucial factors in SO2 susceptibility, which can explain its high intra- and interspecific variability [16].
SO activity has been proven to play a crucial role in protecting plants from SO2/sulfite toxicity [17,18]. SO overexpression lines accumulated relatively more sulfate and showed almost no necrosis under higher SO2 concentration condition [17]. There is a close correlation between resistance to sulfur dioxide/sulfite and SiR expression levels. and plants with reduced SiR expression exhibit higher sensitivity than the wild type, manifested in significant leaf necrosis and chlorophyll bleaching [19]. The reduction or oxidation pathways that seem to jointly regulate sulfite detoxification in Arabidopsis [12,14] also exist in poplar [16]. In addition, the relatively high SO2 tolerance of a poplar is also related to a high capacity for sulfate accumulation and thiol synthesis [16]. Moreover, our experimental results have also shown that resistant varieties have stronger resistance to sulfur dioxide than susceptible varieties, which is reflected in their different biochemical metabolisms [20].
Previously, people have noticed that new leaves have stronger resistance to sulfur dioxide than mature leaves [21,22,23], and the new leaves can release more hydrogen sulfide [23]. The production of hydrogen sulfide in sulfur metabolism is related to sulfite reductase and APR activity. But these key enzymes have not been compared between new leaves and mature leaves. Resistance of cucurbit leaves to acute exposure to SO2 varies with the leaf position on the plant axis, gradually decreasing from top to bottom. This resistance gradient to SO2 is not due to avoidance, as resistant leaves actually have a significantly higher SO2 absorption rate than susceptible leaves [21]. It has also been found that the degree of stomatal opening is not related to the damage caused by sulfur dioxide [22,24]. Bressan et al. [21] infer the existence of a resistance mechanism based on biochemical and developmental control, which plays a role after SO2 enters the leaf. Young leaves have stronger metabolism capacity of sulfur compared with mature leaves of a hybrid poplar; however, it is not known whether the differential capacity could be related to the SO2 resistance [25]. Biochemical comparisons of mature and young leaves with such differences in resistance should be helpful in determining the biochemistry of SO2 toxicity. However, to our surprise, such comparative studies have received little attention to date.
A comprehensive comparison of the enzymes involved in thioredoxin and its metabolites can further elucidate the mechanism underlying the difference in sulfur dioxide resistance between young and mature leaves. Comparisons between young and mature leaves, as well as comparisons between sulfur dioxide-resistant and -susceptible varieties, are used to mutually confirm that sulfur metabolism in leaves is an important mechanism for sulfur dioxide resistance. The main aims of the present study were (1) to determine the critical level for SO2 toxicity and thus susceptibility of two poplar varieties with different resistance to sulfur dioxide under controlled environmental conditions, (2) to identify the important detoxification mechanism, (3) to compare young and mature leaves, as well as resistant and susceptible varieties, to mutually confirm that sulfur metabolism in leaves is an important mechanism for sulfur dioxide resistance.

2. Materials and Methods

2.1. Plant Materials

This experiment was performed with one-year-old cuttings of two poplar varieties, Populus × euramericana CV. ‘Purui’, a resistant variety, and Populus × euramericana CV. ‘74/76’, a susceptible variety. The cuttings were grown in a greenhouse (26 ± 5 °C) until 15–18 expanded leaves were produced and then 48 plants per variety of relatively similar size were selected and moved to a growth chamber for one week to acclimatize to the conditions: 25/16 °C (day/night), 16 h light, 125 ± 5 μmol m−2 s−1 at plant level with a photosynthetic photon flux density (PPFD, with fluorescent light), and 60% relative air humidity. The pots were wrapped in airtight plastic to avoid SO2 absorbance by the soil substrate.

2.2. Experimental Design

In each SO2 concentration treatment, six plants of each variety were placed in a Plexiglass box (40 × 50 × 90 cm3) that was set in the above-mentioned growth chamber. The plants were exposed to SO2 using an experimental fumigation system referring to the design by Randewig et al. [14]. The detailed conditions and process were described in the previous report [20].
In comparison to the results of the SO2 exposure study with different SO2 concentrations (0.65, 0.8, 1.0, 1.2 μL L−1) for approximately 3 days performed by Randewig et al. [16], the experiment in the present study was conducted with two poplar varieties exposed to 0.7, 1.4, and 2.1 μL L−1 SO2 for 5 h in a Plexiglass enclosure [20]. Leaves, representing two discrete leaf age classes, were investigated. The first three newly fully unfolded leaves of the plant from the top were designated as new leaves, while the 11th–13th leaves of the plant from top to bottom were designated as mature leaves. In order to compare the resistance to SO2 through visible damage symptoms between different varieties or leaf ages, 6 plants per variety were subjected to each of 4 different concentrations of SO2 for 5 h. The entire leaf area and the cut-off necrotic leaf areas (by visual estimation) were quantified using a leaf area meter (LI-3100; Li-Cor, Lincoln, NE, USA). The leaves with necrotic leaf areas exceeding 30% of the leaf area were referred to as damaged leaves.

2.3. Determination of Enzyme Activities

Frozen leaves were used to create extracts with the Plant Total Protein Extraction Kit (Sigma, St. Louis, MO, USA). Protein content and enzyme activities were detected using a microplate ELISA reader (SpectraMax 190, Molecular Devices, LLC. San Jose, CA, USA). Protein quantification was carried out with the method of Bradford [26]. For calibration, a standard curve was made with different amounts of bovine serum albumin (Sigma-Aldrich), and the calibration curve was y = 47.814x − 0.6793.
(1)
Sulfite reductase (SiR) activity was determined using the method described by Randewig et al. and Hartmann et al. [14,25]. The detailed conditions and process were described in a previous report [20].
(2)
O-acetylserine (thiol) lyase (OAS-TL) and serine acetyltransferase (SAT) activity was measured by using the method described by Randewig et al. and Hartmann et al. [14,25]. The detailed conditions and process were described in a previous report [20].
(3)
For adenosine 5′-phosphosulfate reductase (APR) activity measurements, frozen leaf material (100 mg) was ground in liquid nitrogen and homogenized in 3 mL extraction buffer [100 mM mono-/dipotassium phosphate buffer (pH 7.7) with 10 mM DTT, 10 mM Na2SO3, 5 mM sodium EDTA, 0.5 mM AMP, 1% Triton X-100, 10 mM L-Cys, and 2% polyvinylpyrrolidone (PVP40)]. The extract was centrifuged for 10 min at 12,000× g at 4 °C. The enzyme reaction solution contained 1 mL extract and 3 mL reaction buffer [50 mM Tris-HCl buffer (pH = 7.2) with 12 μmol/L Na2SO3, 1.5 μmol/L K3Fe(CN)6, 1.2 μmol/L AMP and 24 μmol/L EDTA] [27]. The light absorption was read at a wavelength of 420 nm.
(4)
Sulfite oxidase (SO) activity was determined using the method by Randewig et al. [14,16]. Enzyme protein was extracted as described previously [20]. For SO activity measurement, 200 μL of the extract was mixed with 200 μL Tris acetate buffer, pH 7.25. Thereafter, 100 μL of 0.5 mM sulfite was added to start the reaction. A solution containing formaldehyde and acid fuchsin was used to stop the enzyme reaction. The color changes caused by SO-mediated sulfate formation were observed at a wavelength of 580 nm [18].

2.4. Metabolite Determination

(1)
Quantification of thiols
The thiol content was determined by a high-pressure liquid chromatography (HPLC) system (Waters 2695 HPLC, Waters Co., Milford, MA, USA) [28]. The thiol was extracted and analyzed as described previously [20].
(2)
Quantification of sulfate
Leaf SO42− content was quantified using anion exchange chromatography (Dionex ICS-3000 Dionex Co., Sunnyvale, CA, USA) [17]. Oven-dried leaf material (0.1 g) was ground and homogenized in 1 mL deionized water including 100 mg PVPP. The extract was shaken for 1 h at 4 °C, boiled for 15 min, and then centrifuged for 10 min at 15,000× g at 4 °C. The supernatant was centrifuged again for 5 min and diluted 20 times. Sulfate was quantified using a calibration curve (y = 0.0721x − 0.0885, R2 = 0.9989) of increasing sulfate concentration (0–120 ppm).

2.5. Principal Component Analysis (PCA)

Principal component analysis (PCA) was performed on the measured enzyme activities and metabolite data to identify major sources of variation. The data were log2-transformed and Z-score-standardized prior to analysis. PCA was conducted using the ‘prcomp’ function in R software (v4.2.2). The analysis aimed to identify the major patterns of variations among the 16 treatment combinations (2 varieties × 2 leaf ages).

2.6. Statistical Analyses

Statistical analyses were performed using SAS 10.0 (SAS Institute Inc., Cary, NC, USA). Data were subjected to two-way ANOVA (with variety and treatment or leaf age and treatment as factors) to detect differences between treatments (SO2 exposure and controls) and between different poplar varieties. Duncan’s test was used for multiple comparisons. The significant difference was set at α = 0.05. Before ANOVA, data were tested for normality with the Shapiro–Wilcoxon test and were transformed using the natural log when necessary.

3. Results

3.1. Visible Symptoms of Injury in Response to SO2 Exposure

Under a high concentration of SO2 (2.1 μL L−1 SO2), the susceptible variety, P. × euramericana cv. ‘74/76’, exhibited extensive damage to leaves induced by SO2 (Figure 2), while the resistant variety, P. × euramericana cv. ‘Purui’, had no extensive leaf damage, although there were necrotic spots on mature leaves. Here, the leaves with necrotic leaf areas exceeding 30% of the leaf area were designated as severely damaged leaves. The severely damaged leaves of the susceptible variety accounted for 25 ± 4.8 percent of the total leaves. The moderate concentration of 1.4 μL L−1 SO2 caused phenotypical symptoms of injury with small necrotic spots on the leaves of the susceptible variety but not on the leaves of the resistant variety. The lower concentration of 0.7 μL L−1 SO2 did not induce visible symptoms for either the resistant or susceptible variety. Resistance of poplar leaves to acute exposure to SO2 varied with the leaf position on the plant axis, gradually decreasing from the apex downward. The visible symptoms of injury occurred on mature leaves of Purui poplar, while the young leaves remained visibly unaffected under the high concentration of SO2 (2.1 μL L−1 SO2). A similar scenario occurred in the susceptible variety at the moderate concentration of 1.4 μL L−1 SO2.

3.2. Variation in Enzyme Activities in Leaves

With the increase in SO2 concentration, except for APR in mature leaves of 74/76 poplar, the five enzymes involved in sulfur metabolism initially increased. However, as the concentration of SO2 increased to a high concentration of SO2 (2.1 μL L−1 SO2), the activity of all enzymes decreased except for SO in the young leaves of Purui. The activities of SO, APR, and SiR in young leaves were significantly higher than those in mature leaves (p < 0.05). The activities of SAT and OAS-TL in the young leaves of the control (without SO2 treatment) seedlings were relatively low. After treatment with low concentrations of SO2 (0.7 μL L−1 SO2), the activities of these two enzymes increased in both young and mature leaves. As the concentration of SO2 further increased, the activity of the two enzymes in young leaves rapidly decreased, while the activity in mature leaves remained relatively unchanged (Figure 3). The activities of SAT and OAS-TL in young leaves of Purui poplar were lower than those in the mature leaves (p < 0.05), except under the low concentration of SO2 (0.7 μL L−1 SO2), where there were no significant differences in SAT and OAS-TL between young and mature leaves. As for 74/76 poplar, SAT activity in young leaves was relatively lower than that in mature leaves. There were no significant differences in OAS-TL between young and mature leaves of 74/76 poplar across all concentrations of SO2.
With exposure to different sulfur dioxide concentrations, SO activity in young leaves of Purui poplar was significantly (p < 0.05) higher than that of 74/76 poplar (Supplementary Material Figure S1). However, there was no difference in SO activity in mature leaves between the two poplar varieties. There was also no significant difference in SAT activity in either young or mature leaves between the two poplar varieties subjected to SO2 treatments, but only when the SO2 concentration was 2.1 μL L−1 SO2 was the enzyme activity in mature leaves of Purui poplar significantly (p < 0.05) higher than that in mature leaves of 74/76 poplar. The activities of SiR, APR, and OAS-TL in mature leaves of Purui poplar were generally higher than those in mature leaves of 74/76 poplar when the plants were subjected to different concentrations of SO2. Especially for SiR and APR, their activities in mature leaves of Purui poplar were all significantly (p < 0.05) higher than those of 74/76 poplar when the plants were subjected to all concentrations of SO2, and their activities in young leaves of Purui poplar were significantly (p < 0.05) higher than those of 74/76 poplar when the plants were subjected to higher concentrations of SO2.

3.3. Variation in Sulfur Metabolites in Leaves

For the Purui poplar variety, the content of sulfate ion (SO4−2) in both young and mature leaves increased with the increase in SO2 concentration, and the content of sulfate ion in young leaves was significantly (p < 0.05) higher than that in the mature leaves under all four different SO2 concentrations, with increases of 93%, 58%, 64%, and 76%, respectively (Figure 4). For the 74/76 poplar variety, the sulfate ion content in young leaves was slightly higher than that in mature leaves across different concentrations of SO2. The concentrations of sulfate ion in the young leaves of Purui poplar were significantly (p < 0.05) higher than those of 74/76 poplar young leaves under all four different SO2 concentrations, with increases of 60%, 28%, 97%, and 97%, respectively (Supplementary Material Figure S2). For the mature leaves of the two poplars, there was no significant difference in the content of sulfate ions between Purui poplar and 74/76 poplar under the ambient environment.
The content of glutathione (GSH) in young leaves of Purui poplar significantly increased under SO2 fumigation, reached the highest level when the SO2 concentration was 1.4 μL L−1 SO2, and then slightly reduced with the further increase of SO2 concentration (Figure 4). The variation pattern of GSH content in mature leaves of Purui poplar with SO2 concentration was similar to that in the young leaves. When the SO2 concentration was 0.7 μL L−1 SO2, the GSH content in mature leaves reached its highest level. The GSH content in young leaves of Purui poplar was significantly (p < 0.05) higher than that in the mature leaves under all four different SO2 concentrations, with increases of 45%, 30%, 91%, and 154%, respectively. For the 74/76 poplar variety, there was no difference in GSH content in young leaves across different concentrations of SO2. However, the content of GSH in mature leaves of 74/76 poplar had a similar change pattern to that in mature leaves of Purui poplar with the SO2 concentration. The GSH content in young leaves of 74/76 poplar was significantly (p < 0.05) higher than that in the mature leaves under all four different SO2 concentrations, except under 0.7 μL L−1 SO2 treatment, where there was no significant different in GSH content between young and mature leaves.
Under the control condition, there was no significant difference in GSH concentration in young leaves between the two varieties. With the fumigation of SO2, the concentrations of GSH in the young leaves of Purui poplar were significantly higher than those of 74/76 poplar under the other three SO2 concentrations (Supplementary Material Figure S2). Both varieties had similar responses in GSH content in mature leaves to SO2 treatments; however, the concentrations of GSH in Purui poplar mature leaves were significantly (p < 0.05) higher than those in 74/76 poplar mature leaves across all SO2 concentrations, with increases of 64%, 62%, 54%, and 31%, respectively.
For both varieties, the content of cysteine (Cys) in young leaves increased with the increase in SO2 concentrations with a similar change pattern (Supplementary Material Figure S2). There were no differences in the content of cysteine in young leaves of Purui poplar or 74/76 poplar under any SO2 concentration.

3.4. Major Variation Patterns of Enzymes Activities and Sulfur Metabolites

The principal component analysis was performed based on data for the activity of five enzymes and the content of sulfate and glutathione in four groups (young leaves vs. mature leaves; resistant vs. susceptible varieties). Principal component 1 can reflect 55.4% of the total difference, and principal component 2 can reflect 26.4% of the total difference, totaling 81.8%, indicating strong representativeness of the data and reflecting the differences very well (Figure 5). The separation differences between the four groups (mature leaves of Purui, mature leaves of 74/76, young leaves of Purui, and young leaves of 74/76) are significant, and the differences on the surface are always significant. Especially, the young leaves of Purui are the farthest. APR, SO enzyme, GSH, and sulfate content contribute greatly to principal component 1 and have the greatest impact on young leaves of Purui, followed by 74/76 young leaves. SAT and OASTL enzymes have the greatest contribution to principal component 2 and have the greatest impact on mature leaves of Purui, while SIR enzyme has the greater impact on mature leaves of 74/76.

4. Discussion

In the present study, the SO activity in young leaves of Purui poplar increased after SO2 exposure, but not in those of 74/76 poplar (Figure 2). In mature leaves, the SO activity in both Purui poplar and 74/76 poplar showed an increase after exposure to SO2, but their overall activity was relatively low compared to that in young leaves. SiR activity showed an increase in both Purui poplar and 74/76 poplar, but the enzyme activity in mature leaves was lower than in their young leaves. Apparently, SO and SiR in Purui poplar are complementary enzymes keeping sulfite below the toxic sulfite level [19,29]. Previously, it was reported that resistance to SO2 mainly relies on reduction detoxification rather than oxidation detoxification [23], as the oxidation process forms reactive oxygen species (ROS) and other free radicals, which themselves have a stress effect on plants, although the oxidation process can rapidly reduce the content of toxic sulfite ions. This may be the reason why many plants adopt reduction detoxification. However, in this experiment, oxidation and reduction in Purui poplar occur simultaneously, with the oxidation process increasing sulfate and the reduction process increasing GSH production. As an antioxidant, GSH has a relieving effect on peroxidation. The young leaves in the resistant variety have high levels of GSH, especially under SO2 fumigation, which can ensure timely alleviation of peroxidation reactions, while the susceptible variety has lower GSH content in its leaves and thus has to rely mainly on reduction detoxification. In this study, the content of sulfate and GSH in the young leaves of Purui poplar increased with the increase of sulfur dioxide concentration, which is similar to the previous research results, where exposure to SO2 led to a significant increase in sulfates in young leaves, and the maximum increase in GSH at 1.0 μL L−1 SO2 was approximately 150% compared to the control [16]. These studies also confirm that there may be two detoxification pathways for Purui poplar. One is to oxidize toxic sulfites into non-toxic sulfates and store them in vacuoles, and the other is to convert sulfites in leaves into reduced sulfur ions for the synthesis of sulfur-containing compounds [11,30]. Previous studies have shown that the two detoxification pathways exist in Arabidopsis thaliana [14,31].
In the results of this study, when Purui poplar plants were subjected to a 1.4 μL L−1 SO2 stress environment, there was a significant difference in SO activity between the leaves of SO2-treated Purui poplar and the CK. This means that at this time, the leaves of Purui poplar extensively converted toxic sulfites into sulfates. However, there was no significant difference in sulfate content between the young leaves of SO2-treated Purui poplar and the CK. For the young leaves of Purui poplar, only when the sulfur dioxide fumigation concentration reached 2.1 μL L−1 was there a significant difference in sulfate ion content in the leaves. Meanwhile, the SO activity at 0.7 μL L−1 SO2 reached a significant difference from CK. This may indicate that the sulfate obtained by SO oxidation in Purui poplar is not only stored in vacuoles but also enters the process of sulfur assimilation, in which the sulfate might be changed to reduced S-contained organic substances, such as GSH and Cys. The simultaneous increase in APR activity also confirms the activity of sulfur assimilation. For mature leaves, when subjected to a SO2 stress environment, SO activity and sulfate ions significantly increase synchronously, which may be due to the slow synthesis of sulfur-containing compounds in mature leaves leading to the accumulation of sulfate ions.
GSH plays an important role in regulating the redox balance in plants. Its main functions include serving as a mobile reduced sulfur reservoir in plants, acting a reducing agent in enzymatic reactions, and participating in resisting or reducing oxidative stress damage caused by various adverse environmental factors to plants [9,17]. After sulfur dioxide enters the plant body, it can lead to the production of reactive oxygen species and other free radical ions [32,33,34], which can cause changes in plant metabolism, most of which are seriously harmful to plants [17]. GSH has the function of clearing reactive oxygen species and protecting plants from SO2 hazards [19]. This study indicates that when Purui poplar is subjected to sulfur dioxide fumigation stress, excessive sulfur dioxide undergoes a series of oxidation–reduction processes to synthesize the stress-resistant compound GSH. Purui poplar has a higher GSH content than 74/76 poplar, and both Purui poplar and 74/76 poplar have higher GSH content in young leaves compared to mature leaves. On the other hand, GSH in the young leaves of 74/76 poplar did not respond to sulfur dioxide treatment, which means that 74/76 poplar does not have this detoxification ability.
Randewig et al. [14] exposed wild-type and SO-overexpressing Arabidopsis thaliana to sulfur dioxide and found that APR regulates the sulfate sulfite cycle in plant sulfur assimilation pathways. The APR-catalyzed reaction is a crucial step in the process of sulfate assimilation [7]. The sulfate in the plant is very stable, and in order to continue synthesizing subsequent compounds such as GSH, it is necessary to activate the stable state of sulfate. At this point, the catalytic action of APR is required, and the sulfite obtained from the catalytic reaction is a sulfur donor of sulfur-containing compounds in the plant [7]. The results of this study indicate that when Purui poplar plants are subjected to sulfur dioxide stress, they upregulate the activity of APR in their leaves to accelerate the assimilation pathway of sulfate. APR works synergistically with SO to accelerate the synthesis of GSH in response to stress damage. When the concentration of sulfur dioxide in the environment reaches 1.4 μL L−1, the activity of APR in Purui poplar is significantly higher than that in 74/76 poplar, indicating that the sulfur assimilation pathway in Purui poplar is faster and has a higher tolerance to sulfur dioxide stress when subjected to stress. This study also demonstrates that young leaves of plants have higher tolerance than mature leaves. Plants exposed to sulfur dioxide stress significantly increased the activity of APR in the leaves of Purui poplar, while the activity of APR in the leaves of 74/76 poplar showed a decreasing trend. Previously, it was reported that when plants were exposed to sulfur dioxide stress, their APR decreased, for example, in studies on beech [35], spruce [36], and Arabidopsis [14]. APR reduces the content of sulfite in the body by inhibiting the reduction of sulfate, thereby reducing its toxic effects and playing a negative-feedback-regulatory role in the synthesis of sulfur-containing compounds [37,38,39]. The changes in APR activity within the leaves of 74/76 poplar in this study are consistent with known research results, while the opposite is true for Purui poplar. APR is a key enzyme that determines the activity of sulfur assimilation pathways in plants and ultimately determines the amount of sulfur synthesized into glutathione and proteins [12].
Sulfite reductase (SiR) is also a key enzyme in the process of sulfur assimilation, reducing sulfites to S2−. Studies have shown that reduced SiR activity in Arabidopsis or tomato plants means that they cannot cope with leaf tissue damage caused by exogenous sulfites [40,41]. This indicates that SiR can reduce the toxic effects of sulfites on plants by reducing them to CyS. Obviously, SO and SiR work together to keep sulfites below species-specific toxic level [19,29]. In the present study, the accumulation of GSH resulted from a strong increase in SO and SiR activities in young leaves of Purui poplar after SO2 exposure. However, SiR activity only increased in young leaves of 74/76 poplar, while the GSH remained unchanged after SO2 exposure.
In this study, the difference in SAT activity between the leaves of Purui poplar and 74/76 poplar was not significant, while the activity of SAT in mature leaves was significantly higher than that in young leaves. This may be because not only does the activity of SAT in the leaves of Purui poplar rely on the activation of OAS, but excessive accumulation of OAS in mature leaves actually inhibits SAT activity. OAS-TL showed significant differences between Purui poplar and 74/76 poplar under mild stress (0.7 μL L−1), but no significant difference was observed with a further increase in sulfur dioxide fumigation concentration. This may indicate that although SiR can convert sulfites into S2− necessary for the synthesis of CyS, OAS-TL is still needed to convert inorganic sulfur into the sulfur-containing compound CyS. On the surface, SAT and OAS-TL are not directly related to leaf age or the variety’s ability to resist SO2. However, there is a complex coordination or balance between these enzymes involved in sulfur metabolism.

5. Conclusions

APR, SO enzyme, GSH, and sulfate content have the greatest impact on young leaves of Purui, followed by 74/76 young leaves. The results show that for both Purui and 74/76 poplar, young leaves have stronger sulfur metabolism ability than mature leaves, indicating that young leaves have stronger SO2 resistance. Purui has a stronger sulfur metabolism ability than 74/76 poplar, which is especially reflected in their young leaves. The comparison between young and mature leaves and the comparison between resistant and susceptible varieties mutually confirm that sulfur metabolism in leaves is an important mechanism for sulfur dioxide resistance. This makes Purui a promising variety for planting in SO2-polluted environments.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/f16101582/s1, Figure S1: Activities of important sulfur metabolism enzymes in P. × euramericana cv. ‘Purui’ and P. × euramericana cv. ‘74/76’ in response to 0.7, 1.4, and 2.1 µL L−1 SO2 fumigation for 5 h; Figure S2: Sulfur metabolites in the control and SO2-fumigated (0.7, 1.4, and 2.1 µL L−1 SO2 for 5 h) P. × euramericana cv. ‘Purui’ and P. × euramericana cv. ‘74/76’ poplars.

Author Contributions

X.W. and J.F. designed the study and analyzed the data. J.F., L.W. and W.L. performed the experiments. J.F. and L.W. prepared figures and/or tables and wrote drafts of the article. Y.G. performed the PCA. X.W. and J.F. wrote the final manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Fundamental Research Funds of CAF (CAFYBB2024ZA014), and Research Funds of Institute of Ecological Conservation and Restoration, CAF (STSTC202306).

Data Availability Statement

The data presented in this study is available on request from the cor- responding author.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
PuruiPopulus × euramericana CV. ‘Purui’
74/76Populus × euramericana CV. ‘74/76’
SOsulfite oxidase
SiRsulfite reductase
SATserine acetyltransferase
OASO-acetylserine
OASTLO-acetylserine (thiol) lyase
APRadenosine 5′-phosphosulfate reductase
GSHglutathione
Cyscysteine

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Figure 1. Scheme of sulfate–sulfite cycle in plants.
Figure 1. Scheme of sulfate–sulfite cycle in plants.
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Figure 2. Visible symptoms of injury induced by SO2. Left is the resistant variety, P. × euramericana cv. ‘Purui’, and right is the susceptible variety, P. × euramericana cv. ‘74/76’. Both varieties were subjected to 2.1 μL L−1 SO2 for 5 h.
Figure 2. Visible symptoms of injury induced by SO2. Left is the resistant variety, P. × euramericana cv. ‘Purui’, and right is the susceptible variety, P. × euramericana cv. ‘74/76’. Both varieties were subjected to 2.1 μL L−1 SO2 for 5 h.
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Figure 3. Activities of important sulfur metabolism enzymes. (A) SO activity; (B) APR activity; (C) SiR activity; (D) SAT activity; (E) OAS-TL activity. Means and standard errors (n = 3) of enzyme activities are shown, and the unit of enzyme activity (nmol/min/mg protein). Different letters indicate significant differences among the treatments within the same poplar variety (p < 0.05). The different letters in brackets indicate significant differences between young and mature leaves of the same poplar variety under the same treatment (p < 0.05). SO, sulfite oxidase; APR, adenosine 5′-phosphosulfate reductase; SIR, sulfite reductase; SAT, serine acetyltransferase; OASTL, O-acetylserine (thiol) lyase; ‘Purui’ stands for P. × euramericana cv. ‘Purui’; and ‘74/76’ stands for P. × euramericana cv. ‘74/76’.
Figure 3. Activities of important sulfur metabolism enzymes. (A) SO activity; (B) APR activity; (C) SiR activity; (D) SAT activity; (E) OAS-TL activity. Means and standard errors (n = 3) of enzyme activities are shown, and the unit of enzyme activity (nmol/min/mg protein). Different letters indicate significant differences among the treatments within the same poplar variety (p < 0.05). The different letters in brackets indicate significant differences between young and mature leaves of the same poplar variety under the same treatment (p < 0.05). SO, sulfite oxidase; APR, adenosine 5′-phosphosulfate reductase; SIR, sulfite reductase; SAT, serine acetyltransferase; OASTL, O-acetylserine (thiol) lyase; ‘Purui’ stands for P. × euramericana cv. ‘Purui’; and ‘74/76’ stands for P. × euramericana cv. ‘74/76’.
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Figure 4. Sulfur metabolites in the control and SO2-fumigated samples (0.7, 1.4, and 2.1 µL L−1 SO2 for 5 h). (A) SO42− content; (B) GSH content. Means and standard errors (n = 3) of sulfur metabolites are shown, and the unit of enzyme activity (µmol/g fresh weight). Different letters indicate significant differences among the treatments within the same poplar variety (p < 0.05). The different lowercase letters in brackets indicate significant differences between young and mature leaves of the same poplar variety under the same treatment (p < 0.05). ‘Purui’ stands for P. × euramericana cv. ‘Purui’; ‘74/76’ stands for P. × euramericana cv. ‘74/76’.
Figure 4. Sulfur metabolites in the control and SO2-fumigated samples (0.7, 1.4, and 2.1 µL L−1 SO2 for 5 h). (A) SO42− content; (B) GSH content. Means and standard errors (n = 3) of sulfur metabolites are shown, and the unit of enzyme activity (µmol/g fresh weight). Different letters indicate significant differences among the treatments within the same poplar variety (p < 0.05). The different lowercase letters in brackets indicate significant differences between young and mature leaves of the same poplar variety under the same treatment (p < 0.05). ‘Purui’ stands for P. × euramericana cv. ‘Purui’; ‘74/76’ stands for P. × euramericana cv. ‘74/76’.
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Figure 5. Principal component analysis (PCA) of 5 enzymes and 2 sulfur metabolites. M. 107 refers to mature leaves of 74/76 variety, M.P refers to mature leaves of Purui variety, Y. 107 refers to mature leaves of 74/76 variety, and Y.P refers to mature leaves of Purui variety.
Figure 5. Principal component analysis (PCA) of 5 enzymes and 2 sulfur metabolites. M. 107 refers to mature leaves of 74/76 variety, M.P refers to mature leaves of Purui variety, Y. 107 refers to mature leaves of 74/76 variety, and Y.P refers to mature leaves of Purui variety.
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Feng, J.; Wang, L.; Liu, W.; Gao, Y.; Wan, X. Age-Dependent Differences in Leaf Sulfur Assimilation and Relationship with Resistance to Air Pollutant SO2. Forests 2025, 16, 1582. https://doi.org/10.3390/f16101582

AMA Style

Feng J, Wang L, Liu W, Gao Y, Wan X. Age-Dependent Differences in Leaf Sulfur Assimilation and Relationship with Resistance to Air Pollutant SO2. Forests. 2025; 16(10):1582. https://doi.org/10.3390/f16101582

Chicago/Turabian Style

Feng, Jinxia, Luyi Wang, Wenxin Liu, Ying Gao, and Xianchong Wan. 2025. "Age-Dependent Differences in Leaf Sulfur Assimilation and Relationship with Resistance to Air Pollutant SO2" Forests 16, no. 10: 1582. https://doi.org/10.3390/f16101582

APA Style

Feng, J., Wang, L., Liu, W., Gao, Y., & Wan, X. (2025). Age-Dependent Differences in Leaf Sulfur Assimilation and Relationship with Resistance to Air Pollutant SO2. Forests, 16(10), 1582. https://doi.org/10.3390/f16101582

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