Exogenous Hydrogen Sulfide Alleviates the Toxicity of Cu²⁺ Stress on the Growth Physiology and Quality Components in Camellia sinensis L.

Abstract

Cu²⁺ stress impairs the growth and metabolism of tea plants, further reducing the quality of tea leaves. As a gas signaling molecule, H₂S participates in various physiological processes and improves the stress resistance of plants. Here, the role of H₂S in the response of tea plants to Cu²⁺ stress was investigated by analyzing the effects of Cu²⁺ stress on phenotypes, photosynthetic parameters, physiological indicators, and quality component contents with H₂S or PAG (H₂S synthase inhibitor) pretreatment. It was indicated that exogenous H₂S reduced Cu accumulation and alleviated the inhibition of growth physiology mediated by Cu²⁺ stress in tea plants, mitigated the leaf cell ultrastructure damage, and increased the biomass, root activity, chlorophyll content, and photosynthetic capacity of tea plants under Cu²⁺ stress. Additionally, exogenous H₂S activated the antioxidant system by increasing antioxidant enzyme activities and non-enzymatic antioxidant contents and enhanced the production of endogenous H₂S by promoting LCD activity, thereby improving the antioxidant capacity of tea plants under Cu²⁺ stress. Meanwhile, the application of H₂S effectively alleviated the reduction in the quality components of tea leaves under Cu²⁺ stress. Above all, exogenous H₂S can enhanced tea plants’ tolerance to Cu²⁺ stress, providing a technical reference for the management of tea plants.

1. Introduction

Copper (Cu) is one of the abundant heavy metal elements in nature, and it is an essential micronutrient for plant growth and is involved in a variety of metabolic reactions during plant growth and development [1]. However, the tolerance of plants to Cu is poor, and generally, a content of Cu in plants exceeding 20 mg/kg causes toxicity and stress, which can even lead to serious plant death [2,3]. Zhai et al. [4] found that high concentrations of Cu²⁺ caused reduced root vigor, increased relative membrane permeability of leaves and roots, and increased malondialdehyde (MDA) contents in wheat seedlings. Xiao et al. [5] found that the root length, seedling length, and biomass of cereal seedlings were inhibited to different degrees under Cu²⁺ stress.

The damage caused by Cu²⁺ stress to roots is obvious because the root system is the main organ of the plant that is directly poisoned by heavy metals. Cu²⁺ stress has been found to cause damage to root tips, the shortening of root crowns, and the inhibition of root growth and elongation [6,7,8]. In addition, both the cell membrane and organelle membrane ultrastructural systems of plants are damaged to some extent under Cu²⁺ stress. The ultrastructure of the root tip cells of wheat seedling under copper stress showed that the cell wall and cell membrane suffered varying degrees of destruction, which caused an increase in the intercellular space and the disappearance of some organelles [9]. Furthermore, Cu also participates in the synthesis of plant chlorophyll and other pigments, and Cu²⁺ stress can alter the plant chlorophyll content, mainly manifested in the destruction of chloroplasts and inhibition of chlorophyll synthesis and causing chlorophyll damage [10]. Zhang et al. [11] found that the chlorophyll content of Perennial Ryegrass showed a significant downward trend as the concentration of Cu²⁺ stress increased. Meanwhile, Cu²⁺ stress can also cause changes in photosynthetic parameters. The research of Zhang et al. [12] indicated that the transpiration rate (Trmmol, Tr) and stomatal conductance (Conductance, Cond) of navel orange leaves increased under 0.1 μmol/L Cu²⁺ treatment, while under Cu²⁺ stress (≥5.0 μmol/L), the photosynthetic efficiency, net photosynthetic rate (photosynthetic rate, Pn), light saturation point, Tr, and Cond of that all decreased.

Plants subjected to Cu²⁺ stress produce large amounts of reactive oxygen species (ROS), which are mainly scavenged and counteracted by enzymatic antioxidant systems, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and glutathione reductase (GR), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and monodehydroascorbate reductase (MDHAR) in the AsA–GSH cycle, while synergizing with non-enzymatic antioxidant systems in the body, such as soluble phenols, ascorbic acid (AsA), reduced glutathione (GSH), proline (Pro), vitamin E (VE), coenzymes, and sulfhydryl compounds, to maintain normal cellular metabolism [13]. It has been reported that GSH contains special sulfhydryl groups that can complex with Cu²⁺ to form chelates with lower toxicity to plants and that GSH is also reductive and can be easily reduced to oxidized glutathione (GSSG), which allows for the direct scavenging of ROS [14]. Moreover, the sulfhydryl group of GSH can be coupled with the electrophilic groups of endogenous or exogenous harmful substances under the catalysis of glutathione transferase, GST, increasing its hydrophobicity, making it easier to pass through the cell membrane and be excreted from the body, thereby achieving detoxification [15]. Similarly, it has been shown that under Cu²⁺ stress, Pro can act as a cellular osmoregulator, reacting with hydroxyl radicals to reduce the cellular accumulation of hydroxyl radicals and maintain the cellular structure and protein stabilization, resulting in undamaged subcellular structures [16].

In recent years, due to the improvement of tea economic benefits, tea farmers have extensively used chemical fertilizers, pesticides, organic fertilizers, etc., to increase tea production. These agricultural materials contain a certain amount of the Cu element, which leads to an increase in the Cu content in the soil and an increase in Cu accumulation within the tea plant [Camellia sinensis (L.) O. Kuntze] [17]. Therefore, research on the impact of Cu on tea plants has received more attention. The Cu element in tea plants generally originates from the soil, water, and atmosphere, and the tea plant mainly absorbs Cu through the roots, which is further transported, distributed, and accumulated in different tissues [18]. Cu²⁺ stress endangers the growth and development of tea plants and causes a series of toxic symptoms [19]. It has previously been observed that the activities of SOD, POD, and CAT in the roots and leaves of tea plants show a tendency to increase first and then decrease with an increase of the concentration of Cu²⁺ stress [20]; the photosynthetic rate, Tr, and chlorophyll content of tea plant leaves first increases and then decreases when affected by Cu²⁺ stress [17]; the content of tea polyphenols, catechins, theanine, and caffeine and other quality indicators of tea plant leaves also showed a tendency to increase and then decrease under different concentrations of Cu²⁺ stress [17]. In addition, studies on two cultivars of tea plants [Camellia sinensis (L.) O. Kuntze cvs. Chinary and Assamica] in response to 100 mΜ CuSO₄ stress found that chlorophyll and protein contents were reduced and that the activities of glutathione biosynthesis enzyme (γ-ECS) and glutathione synthase (GSHS) were elevated in both cultivars, whereas the production of ROS under Cu²⁺ stress was more apparent in Assamica than in Chinary, and the Pro content was higher in Chinary than in Assamica [21].

As an important signaling molecule, hydrogen sulfide (H₂S) is involved in a variety of physiological processes of plants, such as seed germination, seedling biomass, morphogenesis, and photosynthesis [22]. It can also enhance plant responses to biotic and abiotic stresses, improve stress resistance, and reduce the harm of stress [23]. A certain physiological concentration of H₂S promotes root development, and it was found that H₂S promoted the number of growing lateral roots, total root length, and total root surface area in the root system of peach seedlings, which were significantly inhibited by treatment with an H₂S scavenger [24]. Che, et al. [25] investigated the regulation of photosynthesis in Spinacia oleracea seedlings and found that H₂S could increase the chlorophyll content of Spinacia oleracea. However, high concentrations of H₂S destroyed the structure of chloroplasts and thus had an inhibitory effect on photosynthesis in plants [25]. In addition, H₂S can be involved in the plant stress response to heavy metals, including Al, Cu, Cd, Cr, As, Zn, Pb, Ni, etc., and mitigate the effects of heavy metal stress on plant growth [26]. Zhu et al. [27] showed that exogenous H₂S pretreatment could reduce Al³⁺ uptake in rice roots by decreasing the expression of the Al³⁺ transporter protein gene OsNramp5 and reduced pectin methyl esterase activity and pectin and hemicellulose contents in the rice roots, which in turn lowered the negative charge and reduced the binding of Al³⁺ in the cell wall. At the same time, exogenous H₂S pretreatment significantly increased the expression of citric-acid-synthesis genes in rice, which contributed to the increase in the release of citric acid, thus mitigating the Al³⁺ toxicity on root elongation; H₂S pretreatment also increased the activities of antioxidant enzymes, such as SOD, APX, CAT, and POD, in the rice root system, and significantly reduced the contents of MDA and H₂O₂, thus alleviating the disruption of Al³⁺ to the membranes in rice.

Plants can produce endogenous H₂S, and the enzymatic degradation of cysteine (Cys) to produce H₂S is considered the main pathway [28]. Cysteine desulphydrases (CDes) comprise L- and D-cysteine desulphydrases (L/D-CDes), which disintegrate L/D-cysteine (L/D-Cys) to H₂S, ammonia (NH₃), and pyruvate, respectively. While L-Cys is the main form of Cys present in plants, it was also found that the activity of L-cysteine desulphydrase (L-CDes, LCD) in Arabidopsis thaliana was about 2–3 times higher than the activity of D-cysteine desulphydrase (D-CDes, DCD) [29]. Therefore, LCD is important for endogenous hydrogen sulfide synthesis in plants. However, the low level of H₂S in plants does not allow them to perform their functions more efficiently, and the problem of the H₂S supply can be solved by exogenous H₂S donors. Currently, H₂S donors are generally two kinds of inorganic sulfides, sodium hydrosulfide (NaHS) and sodium sulfide (Na₂S), which can release H₂S in an aqueous solution within a few seconds [30]. H₂S in the body exists in the form of gaseous H₂S molecules (1/3) and in the form of HS⁻ (2/3). H₂S and HS⁻ produced by H₂S donors can be synthesized and decomposed in solution in a dynamic equilibrium state, which ensures the content of H₂S molecules in the body and serves to stabilize the pH value [23,31]. And a reverse physiological function analysis of H₂S function can use H₂S scavengers and inhibitors: scavenger hypotaurine (HT), inhibitor DL-propargylglycine (PAG), carboxymethoxylamine hemihydrochloride, hydroxylammonia, and CDes catabolism products C₃H₃KO₃ and NH₃, etc. [32].

The tea plant is one of the three major beverage crops. With the improvement in the consumption level and the development of the tea industry, the quality and safety of tea products are receiving more and more attention. It is also worth noting that industrial pollution emissions, improper agricultural fertilization, pesticide residues, and soil acidification can lead to increased soil Cu contents in tea gardens, thus subjecting the tea plant to Cu²⁺ stress. Cu²⁺ stress can inhibit the growth and metabolism of tea plants, thereby affecting the quality and yield of tea. In addition, tea plants may excessively absorb and accumulate Cu in Cu-contaminated tea gardens, leading to excessive Cu contents in tea leaves. The long-term intake of tea with a high Cu content causes Cu poisoning in the human body, damaging the liver, kidney, and nervous system functions [33]. However, Cu is also an essential trace nutrient element for plant growth and development [1]. Therefore, analyzing the protective mechanism of tea plants in excessive Cu environments is of great significance for balancing the advantages and disadvantages of Cu, ensuring the normal growth of tea plants, and ensuring the safe production of tea. Currently, research on tea plants and the Cu element has mainly focused on the dissolution rate of Cu²⁺ after tea processing, the absorption and accumulation characteristics of Cu²⁺ by tea plants, and the impact of Cu²⁺ on tea plant growth [34,35,36]. However, the physiological characteristics of tea plants under Cu²⁺ stress and the pathways of Cu²⁺ stress alleviation still need to be studied. In addition, H₂S can enhance the resistance of plants to heavy metal stress, but there are few studies on H₂S gas signal molecules in tea plants and whether exogenous H₂S can alleviate Cu²⁺ stress in tea plants. In this study, we explored the mitigating effect of H₂S on the response of tea plants to Cu²⁺ stress and its impact on the main quality components of tea leaves, so as to provide a scientific basis and theoretical support for the daily cultivation and management of tea plantations and the protection of tea plants against Cu²⁺ stress.

2. Materials and Methods

2.1. Plant Materials

The annual seedlings of C. sinensis cv. ‘Zhongcha 108’ (Nanjing Ya Run Tea Co., Ltd., Nanjing, China) with a height of 15–20 cm and good health were used in this study. Tea seedlings with the same growth were selected and hydroponically cultured using culture solution (pH 5.5) prepared according to the method of Ghanati et al. [37] and replaced every five days. After 30 d of hydroponics, seedlings were pretreated with 100 μM Na₂S·9H₂O (H₂S)/1.0 mM PAG (PAG)/untreated culture solution (0) for 15 d and then treated with 0.5 mM CuSO₄·5H₂O (0.5 Cu)/1.0 mM CuSO₄·5H₂O (1.0 Cu)/untreated culture solution (0) for 15 d. The specific treatment programs are listed in

Table 1. Specific treatment programs of Camellia sinensis.

Abbreviation	Treatment
CK (0 + 0)	Untreated culture solution (0) pretreatment for 15 days, followed by untreated culture solution (0) treatment for 15 days.
H2S + 0	100 μM Na2S·9H2O (H2S) pretreatment for 15 days, followed by untreated culture solution (0) treatment for 15 days.
PAG + 0	1.0 mM PAG (PAG) pretreatment for 15 days, followed by untreated culture solution (0) treatment for 15 days.
0 + 0.5 Cu	Untreated culture solution (0) pretreatment for 15 days, followed by 0.5 mM CuSO4·5H2O (0.5 Cu) treatment for 15 days.
H2S + 0.5 Cu	100 μM Na2S·9H2O (H2S) pretreatment for 15 days, followed by 0.5 mM CuSO4·5H2O (0.5 Cu) treatment for 15 days.
PAG + 0.5 Cu	1.0 mM PAG (PAG) pretreatment for 15 days, followed by 0.5 mM CuSO4·5H2O (0.5 Cu) treatment for 15 days.
0 + 1.0 Cu	Untreated culture solution (0) pretreatment for 15 days, followed by 1.0 mM CuSO4·5H2O (1.0 Cu) treatment for 15 days.
H2S + 1.0 Cu	100 μM Na2S·9H2O (H2S) pretreatment for 15 days, followed by 1.0 mM CuSO4·5H2O (1.0 Cu) treatment for 15 days.
PAG + 1.0 Cu	1.0 mM PAG (PAG) pretreatment for 15 days, followed by 1.0 mM CuSO4·5H2O (1.0 Cu) treatment for 15 days.

. The whole hydroponics and treatment processes were carried out with a light cycle of 12 h/12 h, relative humidity of 75–80%, temperature of 25 °C/22 °C, and light intensity of 30,000 lx. After the treatment, the tea seedlings were washed clean with deionized water and photographed to record their phenotypes.

2.2. Cu Content Determination

The young leaves (one bud and two leaves), mature leaves (three to six leaves), stems, and roots after treatment were dried to a constant weight, ground into a powder, and passed through a 0.5 mm sieve. According to the method of dry ashing [38], the dried samples were digested with HNO₃-HClO₄ (v:v = 4:1) mixed acid, and then, the Cu content in each part was determined via ICP-OES (iCAP 7400, Thermo Fisher Scientific Inc., Waltham, MA, USA).

2.3. Biomass and Root Activity Determination

The treated tea seedlings were washed and drained of surface water and weighed in units of 4 seedlings for the fresh weight and then immediately baked to a constant weight and weighed for the dry weight. Finally, the fresh weight (FW) and dry weight (DW) of individual tea seedlings were calculated. The root tips (about 1.0 cm) of tea seedlings after each treatment were randomly taken, mixed, and then quickly used to determine root activity using the triphenyltetrazolium chloride (TTC) method [39].

2.4. Ultrastructural Observation of Leaf Cells

On the second and third mature leaves of the tea seedlings, leaf pieces (approximately 2.0 mm × 2.0 mm in size) were quickly cut in the middle of the leaf (3.0 mm from the veins) with a clean blade. The leaf pieces were quickly placed in 2.5% glutaraldehyde fixative, vacuum pumped, and fixed at 0–4 °C for 24 h. Then, the samples were rinsed with phosphate buffer (pH 7.2) 3 times, fixed in 1.0% osmic acid for 1 h, washed in distilled water 3 times, treated in 50%, 70%, 80%, and 100% acetic acid for 10 min, respectively, and replaced with acetone for 24 h. The ultrathin slices (thickness ≤ 100 nm) of the samples were prepared longitudinally using an ultrathin cryosectioner (EM UC7, Leica Microsystems Inc., Wetzlar, Germany), and the samples were observed and images were collected under a transmission electron microscope (HT7700, Hitachi, Ltd., Tokyo, Japan).

2.5. Chlorophyll Content and Photosynthetic Index Determination

The contents of chlorophyll a, chlorophyll b, and total chlorophyll were determined and calculated according to the methods of Xiang et al. [40]. The photosynthetic indexes of the second and third mature leaves were measured using a portable photosynthesizer (Li-6400, LI-COR Inc., Lincoln, NE, USA). The photosynthetic indexes included Pn, Cond, Tr, and the intercellular CO₂ concentration (Ci). The light source in the leaf chamber was LED red and blue light, the temperature of the leaf chamber was 25 ± 2 °C, the lamp was set at 1000.0 μmol/(m²·s), the flow was set at 500.0 μmol/s, and the air flow was kept relatively stable.

2.6. Antioxidant-System-Associated Indicator Determination

The leaves and roots of tea seedlings from each treatment were collected and immediately stored at −80 °C. The malondialdehyde (MDA) content was measured using the TBA method [41]. The content of Pro was determined using the 2.5% acid ninhydrin method [42]. The activity of SOD was determined using a superoxide dismutase detection kit (NBT riboflavin microplate method) (NO. R22262; Yuanye Biotechnology Co., Ltd., Shanghai, China). The activity of POD was determined using a peroxidase kit (guaiacol microplate method) (NO. R30311; Yuanye Biotechnology Co., Ltd., Shanghai, China). The activity of CAT was determined using a catalase kit (ammonium molybdate microplate method) (NO. R22072; Yuanye Biotechnology Co., Ltd., Shanghai, China). The GSH content was measured using a reduced glutathione content detection kit (BC1170; Solarbio Technology Co., Ltd., Beijing, China). The GSSG content was measured using an oxidized glutathione content detection kit (BC1180; Solarbio Technology Co., Ltd., Beijing, China). The activity of GST was determined using a glutathione S-transferase activity detection kit (BC0350; Solarbio Technology Co., Ltd., Beijing, China). The GR activity was assayed using a glutathione reductase activity detection kit (BC1160; Solarbio Technology Co., Ltd., Beijing, China).

2.7. LCD Activity Determination

The activity of LCD was measured with the frozen samples of the treated leaves and roots using a cysteine desulfurase kit (MBE21193; Jiancheng Bioengineering Institute, Nanjing, China).

2.8. Component Determination

According to the method of the international standard ISO 1572:1980 [43], all the fresh leaves, after treatment, were collected and made into dry samples of tea leaves for the detection of quality components. The tea polyphenol content was determined using the forintol colorimetric method according to international standard ISO 14502-1:2005 [44]. The catechin content was determined using the High Performance Liquid Chromatography (HPLC) method according to the international standard ISO 14502-2:2005 [45]. The free amino acid content was determined using ninhydrin colorimetric method according to national standard GB/T 8314-2013 [46]. The caffeine content was determined using HPLC method according to the international standard ISO 10727:2002 [47].

2.9. Statistical Analysis

The experimental data were analyzed using Excel 2016 software (version 16.0.4226.1003, Microsoft Corporation, WA, USA), IBM SPSS Statistics (version 25.0, SPSS Inc, Chicago, IL, USA), and GraphPad Prism software (version 8.0, GraphPad Software, San Diego, CA, USA). The significant differences of results were determined based on an analysis of variance (ANOVA) and Duncan’s multiple range test (p < 0.05) and marked with different letters.

3. Results

3.1. The Cu Contents

The accumulation of Cu in different tea plant tissues showed the following: roots > stems > mature leaves > young leaves (Figure 1). Cu accumulation in the young leaves (Figure 1A), mature leaves (Figure 1B), stems (Figure 1C), and roots (Figure 1D) of tea plants under Cu²⁺ treatment was significantly higher compared to CK. Compared with the 0.5 mM Cu²⁺ treatment concentration, only Cu in young leaves was significantly increased under the 1 mM Cu²⁺ treatment (Figure 1A), while Cu in mature leaves, stems, and roots was significantly decreased (Figure 1B–D). In the absence of exogenous Cu²⁺, H₂S pretreatment resulted in a significant increase in Cu accumulation in the leaves (Figure 1A,B) and roots (Figure 1D) but had no significant effect on Cu accumulation in the stems (Figure 1C), whereas PAG pretreatment resulted in a significant increase in the Cu content in young leaves (Figure 1A) and roots (Figure 1D) and no significant change in Cu accumulation in the stems (Figure 1C) and mature leaves (Figure 1B). Under exogenous 0.5 mM Cu²⁺ treatment, H₂S pretreatment resulted in significantly higher Cu accumulation in young leaves (Figure 1A), but the Cu content in mature leaves, roots, and stems all decreased significantly after H₂S pretreatment (Figure 1B–D); meanwhile, PAG pretreatment resulted in significantly higher Cu contents in young leaves (Figure 1A) and roots (Figure 1D) and significantly lower Cu contents in mature leaves (Figure 1B) and stems (Figure 1C), whereas the Cu content in stems was still significantly higher than that in the H₂S pretreatment group (Figure 1C). Under exogenous 1.0 mM Cu²⁺ treatment, H₂S pretreatment resulted in a significant decrease in the Cu content in young leaves (Figure 1A) and a significant increase in mature leaves (Figure 1B) and roots (Figure 1D); meanwhile, PAG pretreatment resulted in a significant decrease in the Cu content in young leaves (Figure 1A) and a significant increase in the Cu content in mature leaves (Figure 1B), stems (Figure 1C), and roots (Figure 1D).

3.2. The Growth and Root Activities of Tea Plants

The phenotypes under each treatment indicated that the growth of the tea plant was significantly inhibited by exogenous Cu²⁺ stress (Figure 2). Under 0.5 mM Cu²⁺ treatment, the leaves of tea seedlings were significantly reduced compared with CK, and the roots were dark yellow and brown with a few new roots (Figure 2B,E); under 1 mM Cu²⁺ treatment, the leaves were more severely shed and turned yellow, the roots were brown and thin, and there was almost no new root growth (Figure 2H). When no exogenous Cu²⁺ treatment was applied, H₂S treatment significantly promoted the growth of tea plants, with more leaves, more root branches, more lateral roots, and significantly longer root lengths (Figure 2A). Under the same concentration of Cu²⁺ stress, the number of tea seedling roots treated with exogenous H₂S was also increased (Figure 2D,G). However, under PAG treatment, the leaves fell off extremely easily, the leaf color lost its green color, and yellow leaves appeared, which were subjected to severe stress damage (Figure 2C,F,I).

From the biomass of tea seedlings under each treatment, it can be seen that exogenous Cu²⁺ treatment led to a significant decrease in the fresh and dry weights of young and mature leaves, a decrease in the root biomass, but not significant, and no significant change in the stem biomass (

Table 2. Effects of exogenous H₂S on the fresh weight and dry weight in tea plants under Cu²⁺ stress.

Treatment	Young Leaf		Mature Leaf		Stem		Root
	Fresh Weight (g)	Dry Weight (g)	Fresh Weight (g)	Dry Weight (g)	Fresh Weight (g)	Dry Weight (g)	Fresh Weight (g)	Dry Weight (g)
H2S + 0	0.53 ± 0.10 a	0.20 ± 0.05 a	1.4 ± 0.13 a	0.50 ± 0.07 a	1.3 ± 0.10 a	0.53 ± 0.03 a	1.38 ± 0.15 a	0.23 ± 0.01 a
CK	0.45 ± 0.07 bc	0.19 ± 0.01 a	1.04 ± 0.12 b	0.39 ± 0.05 b	0.99 ± 0.03 cde	0.4 ± 0.02 bcd	0.88 ± 0.24 b	0.09 ± 0.03 bc
PAG + 0	0.28 ± 0.01 de	0.11 ± 0.01 bc	0.98 ± 0.21 b	0.33 ± 0.06 b	0.93 ± 0.11 de	0.39 ± 0.03 bcd	0.65 ± 0.04 bc	0.08 ± 0.01 cd
H2 S + 0.5 Cu	0.36 ± 0.07 cd	0.13 ± 0.01 b	0.54 ± 0.03 c	0.2 ± 0.02 c	1.14 ± 0.11 bc	0.47 ± 0.05 ab	0.84 ± 0.03 b	0.12 ± 0.00 b
0 + 0.5 Cu	0.25 ± 0.05 de	0.09 ± 0.02 bc	0.44 ± 0.13 cd	0.15 ± 0.06 cd	1.05 ± 0.06 cd	0.42 ± 0.02 bc	0.70 ± 0.04 bc	0.06 ± 0.00 cd
PAG + 0.5 Cu	0.24 ± 0.06 de	0.09 ± 0.01 bc	0.33 ± 0.06 de	0.13 ± 0.03 cde	0.92 ± 0.15 de	0.38 ± 0.04 cd	0.48 ± 0.11 c	0.04 ± 0.01 d
H2 S + 1.0 Cu	0.27 ± 0.08 de	0.09 ± 0.02 bc	0.21 ± 0.01 ef	0.06 ± 0.00 def	1.06 ± 0.15 cd	0.44 ± 0.08 bc	0.71 ± 0.21 bc	0.08 ± 0.03 cd
0 + 1.0 Cu	0.20 ± 0.06 e	0.07 ± 0.02 c	0.19 ± 0.04 ef	0.06 ± 0.01 ef	1.00 ± 0.08 cde	0.41 ± 0.02 bcd	0.55 ± 0.10 c	0.07 ± 0.02 cd
PAG + 1.0 Cu	0.17 ± 0.02 e	0.06 ± 0.01 c	0.06 ± 0.02 f	0.02 ± 0.01 f	0.82 ± 0.02 e	0.33 ± 0.01 d	0.50 ± 0.10 c	0.05 ± 0.02 cd

Note: each value represents the mean ± SD (n = 3). Different lowercase letters indicate significant differences among treatments (Duncan’s multiple range test; p < 0.05).

). Without Cu²⁺ stress, the fresh weight of young leaves and the fresh and dry weight of mature leaves, stems, and roots under H₂S pretreatment were significantly higher than those in the CK group (Table 2). Under Cu²⁺ stress, H₂S pretreatment increased the fresh and dry weights of young leaves, mature leaves, stems, and roots to varying degrees, and H₂S pretreatment significantly increased the dry weights of roots under 0.5 mM Cu²⁺ treatment (Table 2). However, without Cu²⁺ stress, PAG pretreatment significantly reduced the dry weight and fresh weight of young leaves (Table 2); with exogenous Cu²⁺ treatment, the dry and fresh weights of each tissue of tea plant decreased under PAG pretreatment, but not significantly (Table 2).

The root activities of tea seedlings under each treatment were determined, and the results showed that exogenous Cu²⁺ significantly inhibited the root activity of the tea plant (Figure 3). H₂S pretreatment significantly increased the root activity with or without exogenous Cu²⁺ application; especially, the root activity in the H₂S + 0.5 Cu group was 114.01% higher than that in 0 + 0.5 Cu group, and the root activity in the H₂S + 1.0 Cu group was 77.07% higher than that in the 0 + 1.0 Cu group (Figure 3). In the absence of exogenous Cu²⁺, PAG pretreatment significantly reduced the root activity; with exogenous Cu²⁺ stress, root activity decreased but not significantly under PAG pretreatment (Figure 3).

3.3. The Ultrastructural Changes in Leaf Cells

The ultrastructure of tea plant leaf cells under different treatments was investigated (Figure 4). In the absence of exogenous Cu²⁺, the chloroplast structure in the leaf cells pretreated with H₂S was complete and oval, the chloroplast membrane was clearly visible, the matrix lamella was closely arranged along the longitudinal direction of the chloroplast, there were no osmiophilic granules in the chloroplasts, and starch granules were contained in the cells (Figure 4A); a small number of osmiophilic granules appeared in the leaf cells after PAG pretreatment (Figure 4C). In the CK group, the chloroplast membranes and cell membranes were clearly visible, and chloroplasts were oval and close to the inner side of cell membrane, and their matrix lamellae were closely arranged along the longitudinal direction of chloroplasts (Figure 4B). The chloroplast membranes of leaf cells under the stress of exogenous 0.5 mM and 1.0 mM Cu²⁺ were still clearly visible, chloroplasts were complete, matrix lamellae were tightly arranged, and more osmiophilic granules appeared (Figure 4E,H), whereas chloroplasts were deformed and larger vacuoles appeared after treatment with 1.0 mM Cu²⁺ (Figure 4H). Under 0.5 mM and 1.0 mM Cu²⁺ stress, a small number of osmiophilic granules and complete and oval chloroplast structures were observed in leaf cells pretreated with exogenous H₂S (Figure 4D,G), but small vacuoles appeared in leaf cells of the H₂S + 1.0 Cu treatment group (Figure 4G). In the PAG + 0.5 Cu treatment group, the chloroplast structure of leaf cells was relatively complete and began to dissolve, with a large number of osmiophilic granules in chloroplasts (Figure 4F); in the PAG + 1.0 Cu treatment group, the chloroplast envelope in the leaf cells was destroyed, the chloroplasts were free in the leaf cells, the matrix lamellae structure was disordered and fractured, and there were almost no starch granules, containing a large number of osmiophilic granules (Figure 4I).

3.4. The Changes in the Photosynthetic System in Tea Plants

The level of the chlorophyll content directly affects the photosynthesis ability of plants and the quality of tea products (Figure 5). It was found that exogenous Cu²⁺ stress of 0.5 mM or 1.0 mM significantly decreased the contents of chlorophyll a, chlorophyll b, and total chlorophyll (Figure 5A–C), and the higher the concentration of Cu²⁺ stress, the greater the degree of a decline in these contents (Figure 5). Under 0.5 mM and 1.0 mM Cu²⁺ stress, the chlorophyll a and total chlorophyll contents in tea leaves pretreated with H₂S were significantly increased compared with those in the Cu²⁺ stress treatment group (Figure 5A,C); under 0.5 mM Cu²⁺ stress, the chlorophyll b content was significantly increased after H₂S pretreatment, but the change in the chlorophyll b content was not significant under H₂S + 1.0 Cu treatment (Figure 5B). The contents of chlorophyll a, chlorophyll b, and total chlorophyll in the PAG + 0.5 Cu and PAG + 1.0 Cu treatment groups were significantly lower than those in the same concentration of Cu²⁺ stress treatment group (Figure 5A–C).

The ratio of chlorophyll a/b in tea plant increased with the increase in the Cu²⁺ stress concentration. Under the stress of exogenous Cu²⁺, the ratio of chlorophyll a/b of tea plants had no significant change after H₂S pretreatment (Figure 5D). Under treatment with 0.5 mM Cu²⁺, the ratio of chlorophyll a/b increased significantly after PAG pretreatment compared with the same concentration of Cu²⁺; under 1.0 mM Cu²⁺, there was no significant change in the PAG + 1.0 Cu treatment group compared with the 0 + 1.0 Cu treatment group (Figure 5D).

In addition, the photosynthetic indexes were also measured, and the results showed that with the increase in the Cu²⁺ stress concentration, Pn of leaves significantly decreased (Figure 5E). Under 0.5 mM Cu²⁺ stress, Pn after H₂S pretreatment was significantly higher than that in the 0 + 0.5 Cu group; under 1.0 mM Cu²⁺ stress, there was no significant difference in Pn between the H₂S + 1.0 Cu group and the 0 + 1.0 Cu group (Figure 5E). However, regardless of whether there was exogenous Cu²⁺ treatment, the Pn of the PAG pretreatment group was significantly lower than that of the group with 0 pretreatment (Figure 5E).

Moreover, the leaf Cond significantly decreased with the increase in the Cu²⁺ stress concentration (Figure 5F). Under exogenous Cu²⁺ treatment, the Cond significantly increased under H₂S pretreatment (Figure 5F). Compared with the CK or 0 + 1.0 Cu group, the Cond of PAG + 0 or PAG + 1.0 Cu treatment groups significantly decreased, while the difference between the PAG+ 0.5 Cu treatment group and the 0+ 0.5 Cu group was not significant (Figure 5F).

With the increase in the Cu²⁺ stress concentration, the Ci of leaves significantly increased (Figure 5G). Under no external Cu²⁺ stress and external 1.0 mM Cu²⁺ stress, H₂S pretreatment resulted in a significant decrease in Ci (Figure 5G). Under the application of exogenous Cu²⁺ stress, the Ci of the PAG pretreatment group significantly increased compared to that of groups with other pretreatments (Figure 5G).

Compared to the groups without Cu²⁺ treatment, the Tr of leaves significantly decreased after external Cu²⁺ treatment (Figure 5H). With or without exogenous Cu²⁺ treatment, Tr significantly increased under H₂S pretreatment (Figure 5H), whereas the Tr was significantly decreased in the PAG + 0 or PAG + 1.0 Cu treatment groups as compared to the CK or 0 + 1.0 Cu group (Figure 5H).

3.5. The Changes in the Antioxidant System in Tea Plants

The effect of H₂S on the antioxidant system of tea leaves in response to Cu²⁺ stress was studied (Figure 6). The MDA content in leaves significantly increased with an increasing Cu²⁺ stress concentration (Figure 6A). In the absence of exogenous Cu²⁺ stress, H₂S pretreatment significantly reduced the MDA content, while PAG pretreatment resulted in a significant increase in the MDA content (Figure 6A). Under exogenous 0.5 mM Cu²⁺ stress, H₂S pretreatment also significantly reduced the MDA content (Figure 6A). Meanwhile, under exogenous 1.0 mM Cu²⁺ stress, the MDA content with H₂S pretreatment was not significantly different from that of the 0 + 1.0 Cu group but significantly lower than that with PAG pretreatment (Figure 6A).

With the increase in the Cu²⁺ stress concentration, the content of Pro in leaves significantly increased (Figure 6B). Under 0.5 mM Cu²⁺ stress, the Pro content in leaves pretreated with H₂S significantly decreased compared to the 0 + 0.5 Cu group, while there was no significant difference in Pro content between leaves pretreated with H₂S and those with 0 pretreatment under no exogenous Cu²⁺ treatment or 1.0 mM Cu²⁺ stress (Figure 6B). The Pro content of leaves pretreated with PAG was significantly higher than that of other pretreatment groups under either no exogenous Cu²⁺ treatment or 1.0 mM Cu²⁺ stress, whereas the difference between the PAG + 0.5 Cu group and 0 + 0.5 Cu group was not significant (Figure 6B).

Under exogenous Cu²⁺ treatment, the SOD activities of leaves and roots was obviously enhanced under 0.5 mM Cu²⁺ stress and significantly decreased under 1.0 mM Cu²⁺ stress (Figure 6C,D). Under the stress of 0.5 mM Cu²⁺, both exogenous H₂S pretreatment and PAG pretreatment significantly increased the SOD activity in leaves and roots, and the activity after PAG pretreatment was significantly higher than that with H₂S treatment (Figure 6C,D). In contrast, there was no significant effect of H₂S pretreatment or PAG pretreatment on the SOD activity in the leaves and roots under 1.0 mM Cu²⁺ stress (Figure 6C,D).

The CAT activity in leaves and roots was significantly increased under 0.5 mM Cu²⁺ stress, while it was significantly reduced under 1.0 mM Cu²⁺ stress (Figure 6E,F). The effect of H₂S pretreatment on leaf CAT activity was not significant, but H₂S pretreatment significantly increased CAT activity in roots under 0.5 mM Cu²⁺ stress (Figure 6E,F). Under 1.0 mM Cu²⁺ stress, PAG pretreatment had no significant effect on CAT activity in both leaves and roots; however, under no external Cu²⁺ treatment or the exogenous application of 0.5 mM Cu²⁺ stress, CAT activity in leaves and roots in the PAG pretreatment group was markedly higher than that in 0 pretreatment group and H₂S pretreatment group (Figure 6E,F).

Similarly, the activity of POD in leaves and roots significantly increased compared to the CK group under 0.5 mM Cu²⁺ stress, but it significantly decreased under 1.0 mM Cu²⁺ stress (Figure 6G,H). H₂S pretreatment significantly enhanced POD activity in leaves only under 1.0 mM Cu²⁺ stress and had no significant effect on leaf POD activity under other concentrations of Cu²⁺ treatment and POD activity in roots (Figure 6G,H). Under 0.5 mM Cu²⁺ stress, PAG pretreatment resulted in a significant increase in POD activity in both the leaves and roots; with 1.0 mM Cu²⁺ stress, PAG pretreatment had no significant effect on POD activity in leaves and roots (Figure 6G,H).

Furthermore, GR activity in leaves and roots was also significantly increased under 0.5 mM Cu²⁺ stress and significantly suppressed under 1.0 mM Cu²⁺ stress (Figure 7A,B). With no exogenous Cu²⁺ treatment or 0.5 mM Cu²⁺ stress, leaf GR activity was evidently increased by H₂S pretreatment (Figure 7A). Meanwhile, PAG pretreatment had no significant effect on GR activity in leaves (Figure 7A). H₂S pretreatment significantly enhanced the GR activity in roots with or without exogenous Cu²⁺ stress (Figure 7B). However, PAG pretreatment significantly elevated GR activity in roots only under 0.5 mM Cu²⁺ stress, and the extent of the elevation was significantly lower than that in the H₂S pretreatment group (Figure 7B).

In addition, the relevant indexes of the non-enzymatic antioxidant system of tea plants under each treatment were also determined. The GST activities in leaves and roots were significantly increased under exogenous 0.5 mM Cu²⁺ stress and then significantly decreased with the 1.0 mM Cu²⁺ stress (Figure 7C,D). After exogenously applied Cu²⁺ stress, H₂S pretreatment resulted in a significant elevation in GST activity in leaves and roots, and PAG pretreatment resulted in the obvious inhibition of GST activity in leaves and roots (Figure 7C,D).

The content of GSH in leaves and roots also increased significantly under 0.5 mM Cu²⁺ stress and decreased significantly under 1.0 mM Cu²⁺ stress (Figure 7E,F). H₂S pretreatment resulted in significantly higher GSH contents in leaves and roots under 0, 0.5, and 1.0 mM Cu²⁺ treatments (Figure 7E,F). PAG pretreatment resulted in a significant decrease in the GSH content in leaves under all concentrations of Cu²⁺ treatments, as well as that in roots under 0 and 0.5 mM Cu²⁺ treatments, whereas it had no significant effect on GSH contents in roots under 1.0 mM Cu²⁺ stress (Figure 7E,F).

The GSH/GSSG ratio is the main dynamic indicator of the redox state of cells, reflecting the redox state of tea plants under Cu²⁺ stress. Increasing concentrations of Cu²⁺ alone did not significantly alter GSH/GSSG in leaves and roots (Figure 7G,H). After H₂S pretreatment, the GSH/GSSG of leaves increased but not significantly, while that of roots was sensibly enhanced under 0.5 mM Cu²⁺ stress (Figure 7G,H). After PAG pretreatment, the changes in the GSH/GSSG in the leaves and roots were not obvious (Figure 7G,H).

3.6. The Changes in LCD Activities in Tea Plants

The activities of LCD in tea seedlings under each treatment were examined, as this is the key synthetic enzyme for endogenous H₂S production. The results showed that the LCD activities of leaves and roots were significantly increased under exogenous 0.5 mM Cu²⁺ stress and significantly decreased under 1.0 mM Cu²⁺ stress (Figure 8A,B). When treated with exogenous Cu²⁺, the LCD activities of leaves markedly increased under H₂S pretreatment (Figure 8A); the root LCD activities under H₂S pretreatment significantly increased under 0.5 mM Cu²⁺ stress and also ascended but, not significantly, under 1.0 mM Cu²⁺ stress (Figure 8B). Under PAG pretreatment, the LCD activities in both leaves and roots were significantly inhibited under 0.5 mM Cu²⁺ stress, while there was no significant change in those with PAG pretreatment under 1.0 mM Cu²⁺ stress (Figure 8A,B).

3.7. The Changes in Main Components in Tea Plants

In this study, the change in the main components of tea seedlings under each treatment was monitored. The results revealed that the tea polyphenol content showed a decreasing trend with an increasing Cu²⁺ treatment concentration and was significantly reduced under 1.0 mM Cu²⁺ stress (Figure 9A). The content of tea polyphenols in tea plants pretreated with H₂S without exogenous Cu²⁺ treatment was significantly higher than other treatments (Figure 9A). Under Cu²⁺ stress of 0.5 mM or 1.0 mM, the tea polyphenol content in leaves pretreated with H₂S was higher than that in the 0 pretreatment group, but not significantly (Figure 9A). The tea polyphenol contents in PAG pretreatment groups were all not markedly lower than those in the 0 pretreatment groups (Figure 9A).

The total catechins gradually decreased with the increase in the Cu²⁺ concentration (

Table 3. Effects of exogenous H₂S on catechin components in the leaves of tea plants under Cu²⁺ stress.

Treatment	GC (Gallocatechin) (%)	EGC (Epigallocatechin) (%)	C (Catechin) (%)	EC (Epicatechin) (%)	EGCG (Epigallocatechin Gallate) (%)	ECG (Epicatechin Gallate) (%)	GCG (Gallocatechin Gallate) (%)	Ester Catechin (%)	Total Catechins (%)
H2S + 0	0.8 ± 0.01 abc	2.91 ± 0.06 a	0.25 ± 0 ab	1.11 ± 0.04 a	4.3 ± 0.17 a	0.39 ± 0 a	0.73 ± 0.01 a	5.42 ± 0.18 a	10.48 ± 0.16 a
CK	0.81 ± 0.03 ab	2.92 ± 0.41 a	0.24 ± 0 cd	0.93 ± 0.09 bc	3.68 ± 0.42 bc	0.38 ± 0.01 a	0.62 ± 0.06 b	4.68 ± 0.48 bc	9.57 ± 1.02 ab
PAG + 0	0.82 ± 0.01 a	2.44 ± 0.22 bc	0.25 ± 0 a	0.94 ± 0.05 bc	3.95 ± 0.13 ab	0.37 ± 0.01 ab	0.59 ± 0.04 bc	4.91 ± 0.13 b	9.37 ± 0.23 b
H2 S + 0.5 Cu	0.8 ± 0.02 abc	2.98 ± 0.23 a	0.23 ± 0 e	1.09 ± 0.08 a	3.04 ± 0.35 de	0.39 ± 0.01 a	0.68 ± 0.05 a	4.11 ± 0.4 d	9.22 ± 0.72 bc
0 + 0.5 Cu	0.79 ± 0.02 bc	2.65 ± 0.18 ab	0.24 ± 0 d	1.04 ± 0.02 ab	3.28 ± 0.14 cde	0.36 ± 0.03 b	0.7 ± 0.02 a	4.34 ± 0.2 cd	9.05 ± 0.41 bc
PAG + 0.5 Cu	0.76 ± 0.01 c	2.45 ± 0.03 bc	0.24 ± 0 cd	0.99 ± 0.02 abc	3.43 ± 0.07 cd	0.38 ± 0 ab	0.59 ± 0.01 bc	4.4 ± 0.08 bcd	8.84 ± 0.07 bc
H2 S + 1.0 Cu	0.78 ± 0.03 bc	2.72 ± 0.06 ab	0.23 ± 0 e	1.06 ± 0.17 ab	2.92 ± 0.12 e	0.37 ± 0 ab	0.68 ± 0.03 a	3.97 ± 0.09 d	8.76 ± 0.28 bc
0 + 1.0 Cu	0.76 ± 0.01 c	2.42 ± 0.18 bc	0.24 ± 0 d	0.87 ± 0.02 c	3.3 ± 0.25 cde	0.38 ± 0.01 ab	0.55 ± 0.02 c	4.22 ± 0.27 cd	8.52 ± 0.46 bc
PAG + 1.0 Cu	0.77 ± 0.03 bc	2.2 ± 0.27 c	0.24 ± 0 bc	0.88 ± 0.06 c	3.26 ± 0.35 cde	0.37 ± 0.01 ab	0.56 ± 0.05 bc	4.2 ± 0.4 cd	8.29 ± 0.76 c

Note: ester catechin content (%) is the sum of total EGCG, ECG, and GCG, and the total catechin content (%) is the sum of total GC, EGC, C, EC, EGCG, ECG, and GCG. Each value represents the mean ± SD (n = 3). Different lowercase letters indicate significant differences among treatments (Duncan’s multiple range test; p < 0.05).

). Under each concentration of Cu²⁺ treatment, the total catechins of tea leaves in the H₂S pretreatment group were higher than those in the 0 pretreatment group, but not significantly; the total catechins in the PAG pretreatment group were lower than those in the 0 pretreatment group, which were also not significant (Table 3). Additionally, compared with the CK group, the exogenous application of 0.5 mM Cu²⁺ stress resulted in a significant increase in the gallocatechin gallate (GCG) content, a significant decrease in the epicatechin gallate (ECG) content, a decrease, but not significant, in gallocatechin (GC), epigallocatechin (EGC), epigallocatechin gallate (EGCG), and ester catechin contents, an increase, but not obvious, in the epicatechin (EC) content, and no change in the catechin (C) content (Table 3). Under 1.0 mM Cu²⁺ stress, the contents of GC, EGC, and GCG decreased significantly, while the contents of EC, EGCG, and ester catechins decreased, but not significantly, while the contents of C and ECG did not change (Table 3). Furthermore, in the absence of exogenous Cu²⁺, the contents of C, EC, EGCG, GCG, and ester catechins in tea leaves increased significantly after exogenous H₂S pretreatment, while the content of GC decreased significantly, and the changes in EGC and ECG contents were not obvious (Table 3). Under 0.5 mM Cu²⁺ treatment, the ECG content increased significantly, the C content decreased markedly, and other catechin changes were not obvious in tea leaves pretreated with H₂S; under 1.0 mM Cu²⁺ treatment, the contents of EC and GCG increased significantly, the C content decreased significantly, and other changes were unremarkable in the H₂S pretreatment group (Table 3).

Under both 0.5 mM and 1.0 mM Cu²⁺ stress, the free amino acid contents of tea leaves were significantly lower compared to the groups without exogenous Cu²⁺ treatment (Figure 9B). The free amino acid contents of leaves pretreated with H₂S were significantly enhanced in the absence of exogenous Cu²⁺ treatment or under 0.5 mM Cu²⁺ stress; meanwhile, those pretreated with PAG were significantly diminished under no exogenous Cu²⁺ or exogenous 1.0 mM Cu²⁺ stress (Figure 9B).

Compared with the CK group, the contents of caffeine of leaves were significantly reduced under 0.5 mM and 1.0 mM Cu²⁺ stress (Figure 9C). In the absence of exogenous Cu²⁺ treatment or in the presence of exogenous 0.5 mM Cu²⁺ stress, the caffeine content in leaves significantly increased after H₂S pretreatment (Figure 9C). The caffeine contents in the leaves pretreated with PAG significantly decreased when there was no external Cu²⁺ treatment and significantly increased under exogenous 0.5 mM Cu²⁺ stress (Figure 9C). Under exogenously applied 1.0 mM Cu²⁺ stress, neither H₂S nor PAG pretreatment had a significant effect on leaf caffeine contents (Figure 9C).

4. Discussion

An excessive concentration of Cu²⁺ is detrimental to plant growth and metabolism [48]. H₂S, as a gas transmitter, can participate in various physiological and biochemical processes in plants and has a positive impact on alleviating abiotic stress in plants [49]. In this study, the effects of exogenous H₂S on the response of tea plants to Cu²⁺ stress and its impact on the main quality components of tea leaves were investigated.

4.1. H₂S Pretreatment Reduces Cu Accumulation in Tea Plants Under Cu²⁺ Stress

Firstly, Cu²⁺ stress caused a large amount of Cu to accumulate in tea plants, and the accumulation increased with an increasing Cu²⁺ concentration (Figure 1). Reducing plant uptake of heavy metals is one of the important mechanisms to mitigate the effects of heavy metal stress on plant growth [26]. It was reported that exogenous H₂S could reduce the absorption of cadmium (Cd) by the roots of Malus hupehensis under Cd stress [50]. This study also found that H₂S pretreatment reduced the total Cu content in tea plants under 0.5 mM Cu²⁺ stress, with a significant decrease in Cu accumulation in mature leaves, stems, and roots (Figure 1B,C,D), thereby responding to the inhibitory effect of Cu²⁺ stress on tea plant growth.

4.2. H₂S Pretreatment Alleviates the Toxicities of Cu²⁺ Stress on the Growth Physiology of Tea Plants

Moreover, it was shown that exogenous H₂S played a positive role in alleviating the inhibitory effect of Cu²⁺ stress on growth in tea plants. Cu²⁺ stress resulted in a decrease in the number of aboveground leaves, inhibition of root growth, destruction of the leaf ultrastructure, reduction in the leaf chlorophyll content, and a decrease in photosynthesis capacity, and these effects of stress were particularly significant at a concentration of 1.0 mM Cu²⁺ (Figure 2, Figure 3, Figure 4 and Figure 5). Here, it was found that H₂S pretreatment significantly alleviated the inhibitory effect of Cu²⁺ stress on the growth of tea plants, which was mainly manifested as alleviating the damage to the ultrastructure of leaf cells, increasing the biomass of all parts of the tea seedlings, as well as the root activity, and enhancing the chlorophyll content, net photosynthetic rate, stomatal conductance, and transpiration rate of the leaves (Figure 2, Figure 3, Figure 4 and Figure 5, Table 2). However, exogenous PAG application significantly exacerbated the inhibition of tea plant growth mediated by Cu²⁺ stress (Figure 2, Figure 3, Figure 4 and Figure 5, Table 2). There are a large number of studies showing that exogenous H₂S can effectively alleviate the growth and development of plants under abiotic stress. Zheng et al. [51] pointed out that H₂S could improve the root activity of tomato under salt stress; Qian et al. [52] found that exogenous H₂S promoted the root length and biomass of oilseed rape (Brassica napus L.) under Al stress, which were consistent with the results of this research (Figure 3, Table 2). Moreover, Chen, Wu, Wang, Zheng, Lin, Dong, He, Pei, and Zheng [25] found that H₂S increased the chlorophyll content, light saturation point, and maximum net photosynthetic rate of Spinacia oleracea. Kaya et al. [53] reported that H₂S enhanced the chlorophyll content of wheat under Cd stress. The results of this study also demonstrated that exogenous H₂S pretreatment could alleviate the inhibitory effect of Cu²⁺ stress on the chlorophyll content and photosynthetic capacity (Figure 5), suggesting that H₂S could respond to Cu²⁺ stress by regulating changes in the photosynthetic system of tea plant.

4.3. H₂S Pretreatment Enhances Antioxidant Stress Activation in Tea Plants Under Cu²⁺ Stress

Additionally, our study revealed that exogenous H₂S pretreatment alleviated the excessive production of MDA and Pro under Cu²⁺ stress, enhanced the activities of antioxidant enzymes (SOD, CAT, POD), and improved the action of the glutathione antioxidant system, including an increased GSH content, elevated GR and GST activities, and increased GSH/GSSG in tea plants (Figure 6 and Figure 7). Nomani et al. [54] found that exogenous 200 µM H₂S restored the physiological and biochemical characteristics of Artemisia annua under Cu²⁺ stress by reducing plant lipid peroxidation and increasing antioxidant enzyme activities. Shi et al. [55] found that NaHS pretreatment modulated the metabolism of three antioxidant enzymes, CAT, POD, and GR, as well as the redox state of non-enzymatic glutathione in Cynodon dactylon, further alleviating the abiotic-stress-induced ROS burst and cell damage. In addition, Shan et al. [56] also showed that NaHS pretreatment upregulated the GR activity of maize leaves and regulated the redox state of AsA and GSH, improving the antioxidant capacity and alleviating the oxidative damage of maize seedlings under salt stress. Similarly, Guo et al. [57] found that the exogenous application of NaHS improved the tolerance of Salix matsudana Koidz to Cd by increasing the content of the intracellular non-enzymatic antioxidant GSH. In this research, it was suggested that H₂S could respond to the damaging effects of Cu²⁺ stress by alleviating the production of ROS and regulating the activities of antioxidant enzymes and the contents of non-enzymatic antioxidants. However, the 1.0 mM Cu²⁺ stress might have damaged the tea plants too much and exceeded the resistance threshold of the antioxidant system. Combining our results with previous studies, we suggest that H₂S can promote the reduction of GSSG to GSH and increase the GSH content by increasing the GR activity of tea plant under Cu²⁺ stress and further catalyze the coupling of harmful substances with GSH by increasing the activity of GST in response to the toxic effect of Cu²⁺ stress on tea plants.

Furthermore, this study showed that the application of exogenous H₂S could enhance the LCD activity of tea leaves and roots under Cu²⁺ stress (Figure 8), suggesting that exogenous H₂S may promote the production of endogenous H₂S in response to the toxicity of Cu²⁺ on tea plants. Previous studies have demonstrated that alteration of the endogenous H₂S content in plants under heavy metal stress may be related to the mechanism of plants’ responses to stress. Shen et al. [58] showed that H₂S induced the thiol group R-SH sulfhydrylation of LCD 1 in guard cells of Arabidopsis thaliana under drought stress, which further activated its activity, resulting in the generation of large amounts of endogenous H₂S in a short period of time.

4.4. H₂S Pretreatment Compensates for the Quality Loss of Tea Plants Caused by Cu²⁺ Stress

Finally, it was found that Cu²⁺ stress caused a significant decrease in the contents of tea polyphenols, free amino acids, caffeine, and various catechin fractions in tea leaves, and H₂S pretreatment alleviated the decrease in the contents of these quality components in tea leaves due to Cu²⁺ stress (Figure 9, Table 3). Liu et al. [59] found that the total phenolic content and flavonoid content of leaves in the seedling stage and the free amino acid content of leaves in the spike stage were significantly increased after exogenous H₂S treatment in Avena nuda under saline and alkaline stress, suggesting that H₂S could regulate the abiotic stress of Avena nuda by promoting the contents of osmoregulatory substances in vivo. Tea polyphenols, catechins, free amino acids, and caffeine, which are secondary metabolites in tea leaves, can participate in the osmoregulation of tea plants [60]. Moreover, the accumulation of osmoregulatory substances and the improvement of antioxidant capacity are important mechanisms of plant adaptation to abiotic stresses [61]. Therefore, we inferred that exogenous H₂S could participate in osmoregulation of the tea plant by increasing the contents of these quality components in tea leaves, in response to the effects of Cu²⁺ stress. In addition, tea polyphenols, catechins, amino acids, and caffeine are the four important chemical components that form the quality of tea leaves, and their contents in tea leaves directly affect the results of the sensory evaluations, nutritional value, and economic value of tea [60,62].The decrease in the contents of these quality components under Cu²⁺ stress will lead to a reduction in tea quality, while the application of exogenous H₂S effectively reduced the change in and loss of tea quality.

5. Conclusions

In summary, excessive Cu²⁺ inhibited the growth and development of tea plants, ultimately affecting tea yield and quality. However, 100 μM exogenous H₂S alleviated the toxic effects of excessive Cu²⁺ on tea plants and had a positive impact on their growth metabolism, as well as the quality component formation. In response to Cu²⁺ stress, exogenous H₂S significantly reduced Cu accumulation and activated the antioxidant systems and LCD activities, effectively mitigating the damage of Cu²⁺ stress on root growth, the leaf ultrastructure, photosynthesis, etc., and alleviating the negative effects of Cu²⁺ stress on the quality components in tea plants. Thus, we recommend using exogenous H₂S as a low-cost and environmentally friendly biological regulator for the soil remediation of Cu-contaminated tea gardens and stress-resistant cultivation management of tea plants in the future, avoiding tea garden abandonment and promoting sustainable development of the tea industry. And through the regulation of exogenous H₂S, the accumulation of Cu in tea leaves was effectively reduced, and the quality components were effectively maintained, helping to ensure the safe production of tea and guaranteeing its quality. This study not only has direct guiding significance for cultivation management and the safe production of tea plants but also provides new directions for the breeding of stress-resistant tea plant varieties in the future, which can also provide theoretical references for the physiological regulation of other crops under heavy metal stresses.