1. Introduction
Heavy metal pollution, particularly in soil, is a significant ecological issue that poses risks to human health [1]. Pb is the most common heavy metal and ranks as the most hazardous among 20 priority pollutants [2]. As a nonessential element, excessive Pb inhibits plant growth, reduces photosynthetic efficiency, disrupts cell membranes, and impairs PSII function [3]. Additionally, it is often found in coexistence with polycyclic aromatic hydrocarbons (PAHs) in soil, with about 90% of PAHs found in this environmental medium [4,5]. Pyrene, a type of PAH, is a priority pollutant due to its persistence and high toxicity. It can be absorbed by plants, affecting their antioxidant and photosynthetic systems and limiting biomass accumulation [6,7]. Importantly, the co-occurrence of heavy metals and PAHs can lead to interactive cytotoxic effects, posing significant challenges for plants in contaminated soil [4,8].
Photosynthesis is crucial for plant growth but is sensitive to contamination. This sensitivity makes it a common focus for studying how plants adapt to adverse conditions [9]. Recently, the use of chlorophyll fluorescence assays has grown due to their noninvasive nature, sensitivity, speed, and ease of operation [10]. Under Pb stress, mulberry seedlings show a reduced net photosynthetic rate (PN), stomatal conductance (gs), transpiration rate (E), and intercellular CO2 concentration (Ci), along with a decreased maximal quantum yield of PSII (Fv/Fm), actual photochemical quantum efficiency of PSII (ΦPSII), and electron transport rate (ETR), while non-photochemical quenching (NPQ) increases [11]. Similarly, tomato plants exposed to pyrene stress exhibit significant declines in PN, gs, E, Ci, Fv/Fm, ΦPSII, and the photochemical quenching coefficient (qP) [12].
The impact of soil co-stressed with heavy metals and PAHs has been extensively researched. However, the findings from these studies are inconsistent. For example, under co-stress conditions of Cd and pyrene, Festuca arundinacea shows higher biomass accumulation compared to when exposed to the individual stress of Cd [8]. Conversely, pyrene significantly increased Cu toxicity in Brassica juncea [13]. Moreover, pyrene mitigated growth inhibition caused by Cu in maize [14] and Pb in ryegrass [15]. However, it could not alleviate Cd toxicity in maize [16]. Thus, the response of plants to co-stress requires further analysis to understand the potential synergistic or antagonistic effects of these pollutants.
Clonal plants, commonly found in natural habitats, benefit from clonal integration, which allows for resource sharing among interconnected ramets (or offshoots) through their stolons (or rhizomes) [17]. This integration is a crucial adaptive strategy that helps clonal plants respond to environmental stress, significantly enhancing the fitness of individual ramets [18]. Zoysia japonica (Zoysia), a resilient clonal plant with an extensive root system, can survive in challenging environments [19]. Our research group has previously studied the clonal integration of Zoysia under heterogeneous nutrient conditions, discovering that this integration can significantly enhance PN, E, and gs in connected stressed ramets [20]. Additionally, it can increase the activity of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), while reducing malondialdehyde (MDA) levels [21]. Similar studies indicate that clonal integration can improve photosynthesis in stressed ramets under various stress conditions, such as changes in water and nitrogen availability [22], salinity stress [23], and submergence [24].
Resource distribution in natural environments varies, and pollutants also exhibit heterogeneous distributions [25]. Pollution levels can vary significantly after soil heavy metal contamination; some ramets may thrive in unpolluted microhabitats while remaining connected to those in contaminated areas [26]. Similarly, the distribution patterns of PAHs are heterogeneous in contaminated environments [27]. Moreover, considering the significant overlap between heavy metal and PAH sources and sinks [28], it is reasonable to suggest that clonal plants frequently encounter situations analogous to those in heterogeneous heavy metal soils. Specifically, some ramets may thrive in unpolluted microhabitats, while others face PAH pollution or are in areas contaminated by both heavy metals and PAHs. Previous studies have mainly focused on the advantages of clonal integration in the context of heterogeneous heavy metal pollution [17,29,30,31]. However, the effects of clonal integration in clonal plants subjected to heterogeneous PAH contamination, particularly when occurring simultaneously with heavy metal pollutants, remain largely unexplored.
In recent years, rapid industrial and agricultural modernization has led to severe soil contamination by heavy metals and PAHs [1,4]. Although considerable research has examined the effects of co-contamination on plant responses, the specific impacts on clonal plants under simultaneous exposure to these stressors remain underexplored. Most studies have focused on clonal integration related to heavy metal stress, highlighting a gap in understanding how clonal plants respond to both Pb and pyrene. This study aims to fill this gap by investigating the clonal integration of photosynthesis in Zoysia under heterogeneous Pb+Pyrene stress. We measured the biomass, chlorophyll content, photosynthetic parameters, chlorophyll fluorescence, MDA levels, and antioxidant enzyme (SOD, CAT, POD) activities in connected and disconnected ramets. By comparing the performance of clonal integration under heterogeneous combined stress and individual stress, we aim to gain deeper insights into the mechanisms of clonal integration.
2. Materials and Methods
2.1. Plant Materials and Experimental Treatments
The experiment was conducted at the Eco-Experimental Garden of Liaoning University, where soil was collected and mixed with sand in a ratio of 1:1 (Wsoil: Wsand). The basic properties of the mixed soil were as follows: pH, 7.23; total organic carbon, 8.8%; available nitrogen, 110 mg·Kg−1; available phosphorus, 62.5 mg·Kg−1; available potassium, 220 mg·Kg−1; Mg, 22 mg·Kg−1; Cr, 0.13 mg·Kg−1; Ni, 0.02 mg·Kg−1; Cu, 2.8 mg·Kg−1; Pb, 8.2 mg·Kg−1; Zn, 15.3 mg·Kg−1; Mn, 3.3 mg·Kg−1; and Cd, 0.05 mg·Kg−1.
Clonal ramets of similar size were selected, each containing two rooted ramets connected by a stolon. A small opening was made at the top of each plastic pot for the stolons. Soil moisture was maintained above 75% of field capacity through regular watering with distilled water. Additionally, a compound fertilizer containing 6.0% total organic nitrogen, 35% organic carbon, and 4% P2O5 (provided by Shenyang Zhongze New Fertilizer Co., Ltd., Shenyang, China) was applied every 14 days.
After a two-week recovery period, plastic pots were transferred to the garden. The garden’s environmental conditions were maintained at 18.5 ± 2.0 °C to 30.5 ± 2.0 °C, with a relative humidity of 60 ± 5%. After a normal growth period of 30 days in the garden, half of the clonal ramets were randomly chosen for stolon severance. After a 7-day recovery period, connected and disconnected ramets were treated with three heterogeneous stresses (Pb, pyrene, Pb+Pyrene). We adopted the soil contamination method from Lu et al. [8], which demonstrated excellent soil Pb recovery (101.2 ± 3.52%) and pyrene recovery (96.3 ± 6.32%). Using this method, we conducted soil contamination with Pb at 2.0 g·kg−1 and pyrene at 0.2 g·kg−1. The Pb (99%) and pyrene (98%) used in the experiment were sourced from Macklin Biochemical Co., Ltd. (Shanghai, China). This resulted in a total of 13 experimental treatments, as illustrated in Figure 1. For connected ramets, treatments included the following: no heterogeneous stress (CNH); no Pb stress (CPb−) and Pb stress (CPb+); as well as no pyrene stress (CPy−) and pyrene stress (CPy+); alongside no Pb+Pyrene stress (CPP−) and Pb+Pyrene stress (CPP+). Similarly, disconnected ramets underwent treatments of no Pb (DPb−) and Pb stress (DPb+), no pyrene (DPy−) and pyrene stress (DPy+), and no Pb+Pyrene (DPP−) and Pb+Pyrene (DPP+). Each treatment was replicated three times. After 4 days of treatment, ramets were harvested separately and weighed.
2.2. Determination of Chlorophyll Content
Chlorophyll content was measured using a chlorophyll meter (SPAD-502, Konica Minolta, Inc., Tokyo, Japan). The SPAD value was recorded as a relative indication of the total chlorophyll content in the leaves.
2.3. Determination of Photosynthetic Parameters
PN, gs, Ci, and E were measured with a portable photosynthesis meter (Li-6400, Li-Cor, Lincoln, NE, USA) at a light intensity of 1000 μmol·m−2·s−1 and a CO2 concentration of 400 μmol·mol−1 at 09:00 A.M. to 11:00 A.M. Water-use efficiency (WUE) = PN/E, and carboxylation efficiency (CE) = PN/Ci [32].
2.4. Determination of Chlorophyll Fluorescence Parameters
Chlorophyll fluorescence parameters were measured using the Li-6400. The leaves were dark adapted for 30 min, and then the initial fluorescence (F0) and maximum fluorescence (Fm) were measured. After that, the leaves were exposed to natural light for 60 min to activate them, and the steady-state fluorescence (Fs), maximum fluorescence (Fm′), and minimum fluorescence (F0′) were measured. The other fluorescence parameters were then calculated using the following formulas: Fv/Fm = (Fm − F0)/Fm; PSII potential activity, Fv/F0 = (Fm − F0)/F0; PSII effective quantum efficiency, Fv′/Fm′ = (Fm′ − F0′)/Fm′; ФPSII = (Fm′ − Fs)/Fm′; ETR = 0.5 × 0.85 × ФPSII × PPFD; qP = (Fm′ − Fs)/(Fm′ − F0′); and NPQ = (Fm − Fm′)/Fm′ [11,33].
2.5. Determination of Lipid Peroxidation and Antioxidant Enzyme Activity
Lipid peroxidation was estimated by measuring the content of MDA [34]. The content of MDA and the activities of SOD, POD, and CAT were determined using ELISA kits purchased from Jiangsu Meimian Industrial Co., Ltd. (Nanjing, China). The procedure and determination method were performed according to the instructions provided with the ELISA kit.
Leaf samples (1.0 g) were ground with liquid nitrogen, and then 9 mL of pre-cooled phosphoric acid buffer solution (pH = 7.3) was added. The mixture was centrifuged at 2500 rpm for 20 min at 4 °C. Finally, the supernatant was stored at −20 °C as the crude enzyme extract.
The ELISA kit operates on a double antibody sandwich method. Purified plant MDA/SOD/POD/CAT was placed in the microporous plate to form solid-phase antibodies. The crude enzyme extract was added to the microporous plate as the antigen. Then, horseradish peroxidase-conjugated second antibody was added to the microporous plate. Antigens and enzyme-labeled specific antibodies were combined on the solid-phase complex, and the unbound ones were removed after thorough washing. The substrate TMB was added to develop color, and then 50 μL of stop solution was added as a stop solution and finally appeared yellow. The optical density (OD) value of the samples at 450 nm was detected using a microplate reader (Infinite F50, Tecan Co., Ltd., Männedorf, Switzerland). The standard curve was established according to the OD values and concentrations of the standard samples, allowing for the calculation of MDA content and the activities of SOD, POD, and CAT through a linear regression equation.
2.6. Statistical Analysis
In this study, each experiment was independently repeated three times. Significant differences were determined using one-way analysis of variance (ANOVA) with the SPSS statistics (19.0) software. A significance level of p ≤ 0.05 was used to indicate significant differences, which were determined through the LSD test and two-tailed Student’s t-test. Figures were created using OriginPro 2021 and PROcreate programs.
4. Discussion
This study explores the impact of heterogeneous Pb and/or pyrene stress, revealing that the degree of growth inhibition varied among the stressed ramets. Importantly, clonal integration significantly increased the biomass accumulation of connected stressed ramets compared to disconnected ones. Additionally, pollution stress significantly reduces photosynthetic efficiency; however, this negative impact is mitigated through this integration. The ability to maintain photosynthesis can effectively support the resource demands of clonal plants in polluted environments. However, under conditions of combined heterogeneous pollution, the adaptive benefits of clonal integration are diminished.
Chlorophyll is crucial for photosynthesis but can be negatively impacted by abiotic stress. Maintaining high chlorophyll levels is essential for photosynthesis under stress [12]. This study found that clonal integration significantly increased SPAD values of stressed ramets. These results align with previous research, which showed that clonal integration under heterogeneous environments increases chlorophyll levels [35,36,37].
The most apparent sign of pollutant stress is a decrease in PN, which is directly related to carbon fixation efficiency and biomass accumulation [38]. Our data clearly showed that heterogeneous stresses cause a significant reduction in PN, while clonal integration alleviates this decline. Photosynthetic impairment often results from either stomatal factors, non-stomatal factors, or a combination of both [39]. Our study found that stressed ramets significantly reduced gs, Ci, E, while showing an increase in WUE, suggesting that stomatal closure is one of the factors contributing to the decline of PN. We speculated that toxic stress damages the root system’s ability to absorb, transport, and store water and nutrients, leading to changes in leaf water content and turgor, and consequently, stomatal closure [40,41]. Observations suggest that clonal integration in connected stressed ramets increases gs, Ci, and E, mitigating the stomatal factors leading to PN reduction.
Our results indicated that the decrease in Ci was less pronounced than that in gs and E, alongside a decline in CE, suggesting that CO2 availability is not the only limiting factor for photosynthesis. Instead, non-stomatal factors, particularly the reduced photochemical activity of PSII, are likely the primary constraints [11,42]. Chlorophyll fluorescence parameters, such as Fv/Fm and ΦPSII, effectively assess PSII function and the effects of environmental stress. Fv/Fm indicates potential quantum efficiency, while ΦPSII reflects actual efficiency, both revealing damage to PSII [43]. Stressed ramets exhibited significant reductions in Fv/Fm and ΦPSII under heterogeneous stresses, but clonal integration mitigated these declines, highlighting its protective role for PSII reaction centers.
In addition, increases in Fv/Fm are linked to reduced energy loss as heat [44]. Plants utilize protective mechanisms such as NPQ to dissipate excess excitation energy as heat [45]. Our data showed that heterogeneous stresses significantly increased NPQ, whereas clonal integration reduced this increase. A lower NPQ suggests better absorption and utilization of light energy, although it does not directly assess changes in PSII operating efficiency [46,47]. Conversely, Fv′/Fm′ estimates the maximum quantum efficiency of PSII photochemistry in illuminated leaves when QA is maximally oxidized [47]. Stressed ramets demonstrated significant reductions in Fv′/Fm′, but clonal integration alleviated this impact.
F0 indicates fluorescence when PSII reaction centers are open, while qP reflects energy consumed during photosynthesis [44]. Heterogeneous stresses decreased F0 and qP, but clonal integration significantly alleviated this impairment. An increase in F0 and a decrease in qP indicate an over-reduced state of the plastoquinone pool, inhibiting the photosynthetic electron transport chain [48]. ETR, a key indicator of electron transport at the PSII reaction center [47], was negatively affected by heterogeneous stresses; however, clonal integration mitigated this impact. Pollutant toxicity can close or inactivate the PSII system, limiting electron flow for photosynthetic carbon metabolism. Clonal integration alleviated these toxic effects, enhancing the photosynthetic efficiency of connected stressed ramets.
Additionally, Fv/F0, indicating the efficiency of the PSII oxygen-evolving complex (OEC), serves as a sensitive endpoint for assessing photosynthesis in ecotoxicological studies [49]. In stressed ramets, heterogeneous stresses resulted in a decrease in Fv/F0, which is the chlorophyll fluorescence parameter most affected by heterogeneous stress and shows the most significant enhancement due to clonal integration. This indicates that heterogeneous stress can severely damage the structure and activity of the OEC in stressed ramets. However, clonal integration can significantly mitigate this damage.
Metabolic inhibition is another major non-stomatal factor [39]. Oxidative stress negatively affects plant growth. If oxidative stress is not counteracted by the antioxidant system, it can lead to cell membrane damage, protein oxidation, and lipid peroxidation, producing MDA [50]. Antioxidant enzymes mitigate oxidative stress in plants under abiotic conditions, protecting the photosynthetic apparatus and maintaining photosynthetic efficiency [51]. In our study, stressed ramets showed elevated MDA due to heterogeneous stresses; however, clonal integration reduced this increase by enhancing SOD activity and lessening the decline in CAT and POD activities. This suggests that the antioxidant system cannot completely alleviate oxidative damage caused by heterogeneous Pb and/or pyrene stress, but clonal integration mitigates the damage to ramets.
Clonal-integration intensity and the degree of sharing depend on the environmental conditions to which the respective ramets are exposed [18]. A fundamental aspect of a heterogeneous environment is contrast, as minimal differences between patches are essential for promoting resource sharing. When patch contrast is too low, clonal plants may perceive their environment as homogeneous, which diminishes the likelihood of effective clonal integration [52]. Conversely, in heterogeneous environments with higher contrast, the effects of integration are often more pronounced [23,30,52]. However, under severe stress conditions, plants may adopt a strategy of low assistance [18,31].
Our study found that clonal integration can promote growth, but its benefits vary under different heterogeneous stress conditions. Specifically, under heterogeneous Pb+Pyrene stress, the positive effects on connected stressed ramets were minimal. Conversely, the growth, photosynthetic activity, and antioxidant systems of disconnected stressed ramets were most significantly impacted. This suggests that, although the contrast of heterogeneous Pb+Pyrene stress was highest, the promoting effects of clonal integration were notably limited, likely due to the high toxicity of the Pb+Pyrene mixture impacting adaptability. Although heavy metals and PAHs can form cation-π interactions that reduce their bioavailability [4], the ability of Pb and PAHs to form such interactions is relatively weak [53]. As a result, in mixtures of Pb+Pyrene, the reduction in toxicity due to decreased bioavailability is minimal. Additionally, Pb and pyrene may enhance each other’s absorption. Heavy metals and PAHs can damage plant roots by altering the fluidity and lipophilicity of root cell membranes and the ionic balance within cells, making the compromised roots more susceptible to absorbing other pollutants [8,53,54]. Furthermore, heavy metal ions absorbed by plants can be converted into both low-toxicity and high-toxicity forms. However, when PAHs are present, the transformation of heavy metal ions is more likely to favor the production of high-toxicity forms [55]. Additionally, heavy metals can inhibit the mineralization of PAHs within plants [56]. This interaction increases the damage caused by Pb+Pyrene in cells, ultimately affecting the adaptability of clonal integration.
Clonal integration enhanced the growth of stressed ramets, aligning with previous reports. However, it also impacted the growth of donor ramets (unstressed ramets), which contradicts findings from prior meta-analyses suggesting that clonal integration does not affect the growth of donor ramets [57]. Our results indicate that clonal integration influenced not only the biomass accumulation of donor ramets (CPb−, CPy−, and CPP−) but also their photosynthetic efficiency and antioxidant enzyme activity. This finding is consistent with earlier studies on clonal integration under heterogeneous Pb [58] and Cu [59] stress, which noted costs incurred by donor ramets. Notably, in our study, we observed that under the highly toxic Pb+Pyrene treatment, the costs for donor ramets (connected unstressed ramets) were greater than those under individual Pb or pyrene treatments. This, combined with the low benefits associated with clonal integration in the Pb+Pyrene treatment, resulted in the costs of clonal integration for the whole clone exceeding the benefits. Consequently, we identified a phenomenon where clonal plants exhibit high benefits and low costs in low-toxicity environments, while in high-toxicity environments, they display low benefits and high costs. Due to the limitations of our experimental conditions and the stage of this research, we cannot currently determine the underlying reasons for this phenomenon. It may be related to changes in the forms of toxic substances, alterations in absorption and transport capabilities, or impacts on key cellular functions in complex toxic environments, highlighting the need for further investigation into these aspects.
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Yichen Liu www.mdpi.com
