Exogenous Caffeine (1,3,7-Trimethylxanthine) Application Diminishes Cadmium Toxicity by Modulating Physio-Biochemical Attributes and Improving the Growth of Spinach (Spinacia oleracea L.)

Abstract

Leafy vegetables usually absorb and retain heavy metals more readily than most of the other crop plants, and thus contribute ≥70% of the total cadmium (Cd) intake of humans. Caffeine mediates plant growth and has proved to be beneficial against pathogens and insects. Therefore, it was hypothesized that foliar applications of caffeine could alter metabolism and reduce Cd toxicity in spinach (Spinacia oleracea L.). Seven-day old spinach seedlings were provided with Cd (0, 50, and 100 µM) stress. Caffeine (0, 5, or 10 mM) foliar spray was given twice at after 20 days of seeds germination with an interval of one week. In results, Cd stress reduced photosynthetic pigments biosynthesis, increased oxidative stress, imbalanced nutrient retention, and inhibited plant growth. On the other hand, the caffeine-treated spinach plants showed better growth owing to the enhanced biosynthesis of chlorophylls, better oxidative defense systems, and lower accumulation and transport of Cd within the plant tissues. Furthermore, caffeine application enhanced the accumulation of the proline and ascorbic acid, but reduced MDA and H₂O₂ contents and Cd in plant leaves, and ultimately improved mineral nutrition of spinach plants exposed to different Cd regimes. In conclusion, exogenous application of caffeine significantly diminishes Cd stress by modulating physiological, biochemical, and growth attributes of spinach (Spinacia oleracea L.)

1. Introduction

Soil is a necessary growth medium and a great source of nutrients for all kinds of crop cultivation [1]. It is readily available to plants with a high root-shoot translocation rate [2]. The widespread sources of Cd include wastewater and sewage sludge produced by industries and mining discharge [3]. Cd enters into the food chain by the excessive use of untreated wastewater for irrigation, phosphate fertilizers, pesticides, effluents from metal synthesis industries, heating systems, power stations, waste incinerators, and mining [4]. Cd has a higher sedimentation rate in soils along with a high translocation rate from soil to plant root-shoot system. The extra uptake of Cd interferes with the uptake and transport of essential nutrients, resulting in imbalanced plant nutrient contents. [5]. The mechanism of Cd-induced toxicity is the competitive Cd absorption at the root surface owing to its structural similarity with nutrient cations such as zinc and phosphorous [6]. In addition, Cd uses the same trans-membrane carriers being used for Ca²⁺, Fe²⁺, Mg²⁺, Cu²⁺, and Zn²⁺ ions; thus, it replaces Zn, Ca, and Fe from the prosthetic group of various proteins and brings imbalances in plant physiological processes [7]. Cd induces plant disorders which include chlorosis, necrosis, and decline in plant growth via decreasing the rate of cell division and inhibition of metabolic processes (respiration, nitrate, and carbon assimilation) which may ultimately cause plant death. Being phytotoxic in nature, Cd involves in the reduction in photosynthetic pigment biosynthesis and interferes with the formation of pigment–lipoproteins complexes, which result in less functional photosystem I (PSI) and reduced photosynthetic rate [8]. Even though it is a non-redox active metal, Cd causes oxidative stress, damages cell membranes, and changes the enzymes’ catalytic sites. Moreover, Cd has a high affinity for thiolates, which gives rise to the reduction in Cd toxic effects on plants. Cd converts thiolates to the inactive form via binding with sulfhydryl groups (-SH). Metal uptake in the plants’ edible parts has negative impacts on the consumers’ health. Cadmium intake through vegetable consumption contributed ≥70% of human exposure to Cd [9].

Plant hormones improve stress tolerance via the regulation of different in planta physio-chemical processes and their crucial role encourages a search for plant growth regulators. Alkaloids, a class of secondary metabolites, comprise larger groups of nitrogen-containing compounds in plants that perform various functions such as defense against bacteria, insects, and fungi as well as allelopathic activity. Caffeine is a purine alkaloid present naturally in more than 80 plants species. It is a dietary component of the human diet and attains attention due to its antimicrobial, fungicidal, anti-herbivoral, and allelopathic activity. The critical role of caffeine in chemical defense against biotic stressors (fungi and insects) has been well-documented [10,11]. Furthermore, caffeine imparts an allelopathic behavior to the patron plants, thus giving them selective benefits via suppressing the nearby competing plants and/or soil pathogens [12]. Important plant metabolites produced during caffeine degradation by conventional oxidative purine catabolism pathway include xanthine, uric acid, allantoin, NH₃, and CO₂ [13]. In addition to its role in nitrogen assimilation, the purine degradation pathway was found to be a central player for plant modulations under stress conditions [14]. For example, allantoin serves as a transportable nitrogen-rich compound and protects plants from abiotic stresses via enhancing antioxidants enzymes activities such as superoxide dismutase [15] or through a synergistic incitement of abscisic acid up-regulation [14]. Similarly, supplementation of medium with uric acid helped Arabidopsis plants to alleviate the drought-induced negative effects via enhancing the chlorophyll content and increasing plant biomass [16].

The fact that the pest repellent effects of caffeine are associated with its stimulatory role in the regulation of defense-related genes [17] as well as the limited knowledge about caffeine’s role in abiotic stress tolerance of vegetables/crops urged us to conduct the current study. It was hypothesized that the exogenous caffeine application could modulate the internal biochemical patterns and cause intricate changes to reduce heavy metal stress in spinach. So, the objective of the current study was to study the possible physio-biochemical mechanisms by which caffeine could alter Cd uptake and the Cd accumulation to edible parts of spinach. The results obtained could be helpful to provide a safer technology for enhancing vegetable production through crop cultivation in Cd-contaminated areas.

2. Materials and Method

2.1. Experiment Design and Growth Analysis

A completely randomized design trial was run in the Botanical Garden of Government College University, Faisalabad, Pakistan. Spinach seeds (collected from Ayub Agricultural Research Institute, Faisalabad, Pakistan) were grown in plastic pots (9 inches width and 9.5 inches length), with 5.0 kg soil in each pot, under natural environment conditions having 68 ± 5% humidity with day and night temperature of 29 and 20 °C, respectively. Thinning was performed after seeds germination (7 days after seed sowing) to maintain 5 seedlings per pot. Fertilizers (urea 2.19 g, DAP 1.36 g, and potash 2.40 g per 5.0 kg soil pot⁻¹) were then applied. Seedlings were irrigated with Cd(NO₃)2-contaminated water having 0, 50 µM (5.6 mg kg⁻¹ soil) or 100 µM (11.2 mg kg⁻¹ soil) Cd after seed germination. Plants were irrigated as per crop requirement. Caffeine (Mol. wt. 194.19 g) solution was prepared using distilled H₂O. Our preliminary experiments revealed that caffeine solutions of 5 and 10 mM were more relevant to be used in the present study and indicated that 10 mM caffeine was the maximum concentration that showed the best effect and could significantly alleviate Cd stress impacts. A 0.5 L diluted caffeine (5 or 10 mM) solution (or double-distilled H₂O for control) was sprayed on 20 plants (4 replicates) twice after a one-week interval. Plants (40 days old) were harvested, and growth measurements were recorded. The plants were rinsed thoroughly with distilled H₂O to eliminate soils particles. The plants were dried in the air, and fresh weights measurements were taken immediately using an analytical balance. Dry weights (shoot and root separately) were noted after oven drying at 70 °C for 48 h.

2.2. Soil Analysis

Soil samples were dried in air, sieved by a 2 mm sieve. Various soil properties such as the size of soil particles [18], EC, pH, sodium absorption ratio [19], and ammonium bicarbonate diethylene triamine pentaacetic acid (AB-DTPA) [20] were recorded. The soil properties included sand (48%), clay (36%), silt (16%), EC (0.811 dS m⁻¹), pH (7.65), Ca mg kg⁻¹ (7.71), Na mg kg⁻¹ (9.46), K mg kg⁻¹ (1.34), Cd mg kg⁻¹ (0.83), Pb mg kg⁻¹ (6.64), Zn mg kg⁻¹ (4.93), Fe mg kg⁻¹ (50.34), and Mn mg kg⁻¹ (6.37).

2.3. Chlorophyll and Gas Exchange Parameters

Plant chlorophylls content was recorded by dipping 0.5 g of leaves samples in acetone (80% v/v) at 4 °C overnight, followed by grinding and then recording reading at different wavelengths (663, 645, and 480 nm) by spectrophotometer [21]. The chlorophyll contents were expressed as mg g⁻¹ plant FW. The stomata conductance and rate of photosynthesis were recorded from the second uppermost entirely expanded leafy using IRGA at 10.00–12.00 a.m.

2.4. Determination of Lipid Peroxidation (Malondialdehyde Content), Hydrogen Peroxide, and Electrolyte Leakage

Malondialdehyde (MDA) content was measured using the protocol of Carmack and Horst [22]. Approximately, 0.5 g of fresh leaf sample was homogenized with trichloro-acetic acid (TCA) solution followed by vigorous vortexing. A total of 4 mL of the mixture was added into 4 mL of 2-thiobarbituric acid (in 20% TCA) and incubated for 30 min at 95 °C. The mixture was vortexed and cooled, and the absorbance was taken at 532 and 600 nm. The method of Velikova et al. [23] was used to measure the level of hydrogen peroxide (H₂O₂) in plant leaves by homogenizing 0.5 g of fresh leaves in 5 mL of trichloro-acetic acid solution. The reaction mixture was then centrifuged for 15 min and 0.5 mL of the supernatant was taken in a fresh test tube. Thereafter, 0.5 mL of potassium phosphate buffer (pH 7) and 1 mL of potassium iodide were added. The mixture was then vortexed vigorously and the absorbance was noted at 390 nm and H₂O₂ content was finally expressed as µmol g⁻¹ FW. The method of Dionisio-Sese and Tobita [24] was used to calculate the electrolyte leakage (EL) in plant leaves. In brief, the leaf was cut into small pieces and dipped in test tubes with deionized H₂O. Initial EC₁ reading and final EC₂ value of the solution were noted by extracting the plant sample at 32 °C for 2 h. The final EC2 reading was taken after the autoclaving samples for 20 min at 25 °C. The given formula was used to calculate EL.

EL = (EC_1/EC₂) × 100

2.5. Measurement of Superoxide Dismutase (SOD), Peroxidase(POD), and Catalase(CAT) Activities

Leaf sample was ground in liquid nitrogen and standardized in 0.5 M phosphate buffer (pH 7.8). Catalase contents were estimated following the method of Aebi [25]. The enzyme activity was estimated by mixing plant extract with 0.75 M H₂O₂ and absorbance of wavelength was noted at 240 nm using a spectrophotometer and expressed in U/mg protein. POD activity was estimated by mixing 100 μL of leaf extract with 50 mM phosphate buffer of pH 7.8, 20 mM guaiacol, and 40 mM H₂O₂ in test tubes. The reaction mixture was then used to measure POD activity by noting absorbance at 470 nm [26] and the enzyme activity was expressed in U/mg protein. To estimate the SOD activity, 50 μL of leaf extract was mixed with 50 mM phosphate buffer (pH 7.8), 1.3 μM vitamin B2 (riboflavin), 50 μM NBT (Nitro Blue Tetrazolium), 13 mM methionine, and 0.1 mM EDTA in the test tube. The reaction mixture was covered with aluminum foil placed in a chamber under 30 W light. After 15 min, SOD activities were recorded at 560 nm [26] and were expressed in U mg⁻¹ protein.

2.6. Determination of Ascorbic Acid and Proline

To measure the plant ascorbic acid contents, the protocol of Mukherjee and Choudhuri [27] was used. Half gram of fresh leaves samples was homogenized with trichloroacetic acid (10 mL) and filtered. Four mL of supernatant of plant leaf extract was taken in test tubes and one drop of 10% thiourea, 2 mL of 2% diphenyl hydrazine, and 5 mL of H₂SO₄ (80%) were added. The absorbance was measured at 530 nm using a spectrophotometer and expressed as mg g⁻¹ FW.

Proline contents in the plant sample were measured following the procedure of Bates et al. [28]. A total of 0.5 g of leaf samples was ground with 10 mL sulfosalicylic acid (3%), filtered with Whatman No. 2 filter paper. In test tubes, 2 mL of the filtered solution, 2 mL of acid ninhydrin along 2 mL of glacial acetic acid were added and these test tubes were placed in a water bath for 1 h at 80 °C. The tubes were then kept in an ice bath to stop the reaction. Four mL of toluene was then added in test tubes and mixed strongly for 15–20 s with the help of a test tube mixer and readings were noted at absorbance (520 nm).

2.7. Concentration of Cd in Plants

Cadmium concentration in plants samples was checked using the protocol proposed by Rehman et al. [29]. In brief, 0.5 g of shoot and root, separately, was digested in HNO₃ and HClO₄ (4:1, v/v) in a conical flask overnight. The homogenate was then placed on a hot plate for about 8–10 h at 350 °C. Afterward, samples were run on AAS for the estimation of Cd.

2.8. Statistical Analyses

Four biological replicates were used for the experimental analysis and the data obtained were subject to the analysis of variance using IBM SPSS. The difference among means was scored by employing HSD Tukey’s test (p ≤ 0.05). Logarithmic or inverse transformations were performed for data normalization, where necessary, before data analysis.

3. Results

3.1. Plant Growth

Plant growth characteristics are useful indicators to assess the Cd toxicity effects on plants. High accumulation of heavy metal/Cd in root zone may result in lower uptake of water and thus reduces plant fresh weight. Meanwhile, the toxic impact of Cd on plant photosynthetic machinery leads to less dry biomass production.

To reveal the influence of caffeine on spinach growth under Cd stress, plant growth attributes were recorded. Foliar application of caffeine exerted positive effects on the studied growth attributes of spinach grown under Cd stress (Figure 1). Spinach seedlings grown under 50 µM cadmium nitrate (Cd) stress and without foliar application of caffeine showed a remarkable reduction (26.5%) in the shoot fresh weight, in comparison with control plants. The reduction in shoot fresh weights was more significant (44.3%) upon the addition of 100 µM Cd in the absence of caffeine application. Foliar application of caffeine (5 mM) resulted in significant increases in shoot fresh weight (i.e., 6, 10, and 16% under 0, 50, and 100 µM Cd, respectively), as compared with controls. As compared to controls, the maximum increase was 29% in shoot fresh weight and was recorded with 10 mM caffeine spray despite the presence of 100 µM Cd in the irrigated water. Application of 10 mM of caffeine induced significant increases of 14, 19, and 30% in root fresh weight under Cd stress levels of 0, 50, and 100 µM, respectively, in comparison with the control plant. Exogenous caffeine resulted in higher plant dry mass. Moreover, significant increases of 20, 17, and 24% in shoot dry mass, relative to control, were recorded in 10 mM caffeine-treated plants grown under 0, 50, and 100 µM Cd, respectively. The impact of Cd stress on leaf morphology and area was obvious. Cadmium stress resulted in a significant reduction in leaf area (Figure 1); however, caffeine application recovered the plants from Cd-induced toxic effects as revealed by increases in leaf area. Increasing caffeine concentration was correlated with increased leaf area, and the maximum increase of 41% in leaf area was noted in 10 mM caffeine-treated plants under 100 µM Cd stress, in comparison with control plants (Figure 1).

3.2. Photosynthetic Pigments

Plant photosynthetic apparatus is a major target under Cd stress. The main pigment of photosynthesis in most plants is chlorophyll involved in the light-harvesting process. Estimation of leaf chlorophyll contents is beneficial for foreseeing photosynthetic rate and harmful or beneficial effects of any element. The present study indicated that the application of caffeine for plants grown with or without Cd stress resulted in altered photosynthetic pigments content. Chlorophyll a content was significantly increased up to 15, 15, and 21% in 0, 50, and 100 µM Cd stressed plants treated with 5 mM caffeine, respectively, in comparison to control plants which showed chlorophyll content of 1.7, 1.3, and 0.9 mg g⁻¹ FW. As compared to control, a maximum increase of 39% in chl. a was recorded in 10 mM caffeine-treated plants grown under 100 µM Cd. The application of 5 mM caffeine significantly enhanced chlorophyll b content, in comparison to control. A maximum increase of 33% in chl. b was observed in 10 mM caffeine-treated plants grown under 100 µM Cd, as compared to control. Plants sprayed with 10 mM of caffeine showed non-significant decreases of 4, 14, and 5% in chlorophyll a/b under Cd stress levels of 0, 50, and 100 µM, respectively, in comparison with relevant controls (Figure 2).

3.3. Stomatal Conductance and Plant Photosynthetic Efficiency

In the current study, foliar treatment of caffeine caused modulations in spinach plants under Cd stress in terms of gas exchange attributes, i.e., stomatal conductance and photosynthetic rate. Under 100 µM Cd, spinach plants showed a maximum decrease of 60.4% in stomatal conductance, in comparison with the control plant. Moreover, the exogenous application of 10 mM caffeine significantly increased the stomatal conductance up to 18, 27, and 27% in plants grown under the Cd stress levels of 0, 50, and 100 µM, respectively, in comparison with the controls.

The application of caffeine (5 or 10 mM) resulted in significant increases in photosynthetic rate despite the absence or presence of Cd (50 or 100 µM). Additionally, the application of 5 mM caffeine caused increases of 10, 14, and 25% in the photosynthetic rate of spinach plants grown under Cd stress levels of 0, 50, and 100 µM, respectively, as compared to control plants. The maximum increase of 53% was recorded in the photosynthetic rate of 10 mM caffeine-treated plants grown under 100 µM Cd (Figure 3 and Figure 4) [30,31].

3.4. Hydrogen Peroxide and MDA Content

Being a non-redox metal, Cd cannot carry a single transfer of an electron and does not produce reactive oxygen species (ROS) such as singlet oxygen, hydrogen peroxide, and superoxide anion, though it interferes with antioxidant defense systems and causes oxidative stress. Cd also causes several disorders such as leakage of ions, oxidation of lipids and protein, redox imbalance, and cell membrane denaturation. ROS cause the oxidation of polyunsaturated fatty acids in several membranes in the cell in a process known as “lipid peroxidation,” which changes the structure and function of the cell membrane. This process can be used as an indicator for oxidative stress by the analysis of the content of malondialdehyde (MDA) produced from the decomposition of the by-product of lipid peroxidation in plant cells. Hydrogen peroxide (H₂O₂) and MDA contents were determined to evaluate the influence of caffeine on oxidative damage in Cd-stressed spinach plants. Hydrogen peroxide contents were significantly reduced by 31, 13, and 13% in roots and 27, 16, and 19% in leaves of spinach plants treated with 10 mM caffeine under Cd stress levels of 0, 50, and 100 µM, respectively, as compared to control plants. The foliar spray of caffeine (5 mM) to spinach plants significantly reduced MDA content by 21, 5, and 6% in roots and 33, 4, and 8% in leaves of spinach plants under Cd stress of 0, 50, and 100 µM, respectively, in comparison with the control plant. A maximum significant decrease of 36 and 57% in MDA content was recorded in 10 mM caffeine-treated plant roots and leaves, respectively, in the absence of Cd. Plants treated with 5 mM caffeine showed a significant reduction in electrolyte leakage by 15, 11, and 8% in roots and 18, 6, and 5% in leaves of spinach plant under Cd stress levels of 0, 50, and 100 µM Cd, respectively, in comparison with the controls. A significant decrease of 13 and 12% was recorded, respectively, in roots and leaves of spinach plants treated with 10 mM caffeine under 100 µM Cd stress, in comparison with the relevant controls (Figure 5).

3.5. Activity of Antioxidants Enzymes

Plants have evolved various defense systems to survive under stressful circumstances. The antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) act as elements of plant enzymatic defense systems. These enzymes detoxify ROS and reduce the stress-induced toxic effects. Superoxide dismutase (SOD), a metalloprotein, is the main scavenger that catalyzes the dismutation of superoxide towards H₂O₂. H₂O₂ is also harmful to cells which should be detoxified further to oxygen and water through POD and CAT. The activities of antioxidant enzymes were recorded in spinach plants grown under Cd stress with or without foliar application of caffeine. The results showed significant increases in antioxidants upon caffeine application. Superoxide dismutase activity was significantly increased up to 12, 7, and 7% in roots and 14, 12, and 9% in leaves of spinach plants treated with 5 mM caffeine under Cd stress levels of 0, 50,and 100 µM, respectively, in comparison with the relevant controls. Maximum significant increases of 15 and 19% were observed in 10 mM caffeine-treated spinach plant roots and leaves, respectively, under 100 µM of Cd, in comparison with the respective controls. Application of 5 mM caffeine enhanced CAT activity by 20, 9, and 12% and 15, 10, and 6% in roots and leaves of spinach plant grown under Cd stress levels, in comparison with the control plant. The maximum significant increases of 21 and 15% in CAT activity were recorded in plant roots and leaves, respectively, treated with 10 mM caffeine under 100 µM Cd stress, in comparison with the control plant. The foliar spray of 5 mM caffeine significantly enhanced POD activity by 19, 12, and 9% in roots and 12, 9, and 13% in leaves of spinach plant grown under Cd stress levels of 0, 50, and 100 µM, in comparison with the control plant. Moreover, the application of 10 mM caffeine recorded a maximum level of 18 and 21% in POD activity of spinach plant roots and leaves, respectively, grown under 100 µM Cd, in comparison with the control plant (Figure 6).

3.6. Determination of Ascorbic Acid and Proline

Proline is a non-enzymatic antioxidant that could alleviate the toxic effects of ROS in plants under adverse environmental conditions. Proline acts as an effective osmolyte that accumulates in plant cells to maintain the cellular osmotic balance under metal stress. Proline accumulation in the plant cells protects cell membranes from oxidative damages.

The results revealed significant increases of 11, 7, and 8% and 7, 11, and 11% in ascorbic acid contents in roots and leaves of spinach plants treated with 5 mM caffeine under Cd stress levels of 0, 50, and 100 µM, in comparison with the respective controls. Furthermore, 10 mM caffeine-treated spinach plant roots and leaves showed a significant increase of 17 and 19% in ascorbic acid contents, under 100 µM of Cd stress, in comparison with the control plant. Application of 10 mM caffeine significantly enhanced proline content by 49, 31, and 17% in roots and 40, 20, and 22% in leaves of spinach plants grown under Cd stress levels of 0, 50, and 100 µM, respectively, in comparison with the respective controls (Figure 7).

3.7. Cd Concentration

Cadmium is a freely absorbed and quickly translocated heavy metal that is transported from the root cortex to the shoots. Application of 5 mM caffeine reduced Cd content by 13, 24, and 13% in roots and 19, 11, and 17% in leaves of spinach plants grown under Cd stress levels of 0, 50, and 100 µM, in comparison with the respective controls. A maximum decrease of 27 and 21% was observed in roots and leaves of 10 mM caffeine-treated spinach plants grown under 100 µM Cd stress level, in comparison with the control plant (Figure 8).

4. Discussion

Cadmium is a highly toxic heavy metal and has negative impacts on plant growth, which eventually leads to plant death. A decrease in plant growth due to Cd stress has been documented in tomato [32], mungbean [33], cotton [34], wheat [35], and maize [36]. The result of the current study indicated that Cd stress reduced spinach growth and biomass. It seems likely that Cd exposure to plants brought changes in the rate of cell division, cell growth, and cellular structure, resulting in lower water uptake as well as impacting the photosynthetic efficiency. Cadmium has strong absorption and translocation patterns in plants which result in impaired transpiration rate through stomatal closure [37] as well as reduced spinach biomass as recorded in the current study. Exogenous application of caffeine resulted in increased plant height, growth, and biomass. A group of metabolites which might protect plants from oxidative damage of several biotic and abiotic stresses are ureide metabolism intermediates which produce during caffeine catabolism. Ureides are formed by the oxidative degradation of purines. The xanthosine form xanthine changes to uric acid which is then metabolized by allantion synthase (ALNS) and uricase to allantion. Allantion further hydrolyzes by enzyme allantionase to form allantoate and then whole degradation of allantoate generates nitrogen as four molecules of ammonia, along with CO₂ and glyoxylate (one molecule) (Werner et al. 2010) [38]. Ureides are well known as a vital nitrogen enriched compound which perform a major role in storage and transport of nitrogen in legumes (Coleto et al. 2014) [39]. The significance of ureide catabolism is not only attributed to their capability to recycle nitrogen from purine but also to their potential role as a reactive ROS scavenger (Brychkova et al. 2008, Takagi et al. 2016) [40,41]. Similarly, the stimulatory role of caffeine in increasing the plant height of Capsicum annum L. was previously reported [16]. Exogenous use of uric acid, a metabolite produced during caffeine breakdown, improved the fresh and dry weight of Arabidopsis plants grown under drought stress conditions [42]. The increase in plant growth might be due to the positive effects of caffeine on the biochemical and metabolic processes associated with plant growth and development.

Chlorophyll is the primary photosynthetic pigment in plants and plays a key role in regulating stress tolerance [43,44,45,46,47,48]. Cd stress induces the peroxidation process which disturbs thylakoid membrane structure and function and eventually reduces the chlorophyll content. It has been also reported that Cd inhibits chlorophyll biosynthesis [43]. In the current study, the foliar application of caffeine enhanced the chlorophyll content in spinach plants grown under Cd stress. The evidence indicated that the application of purine alkaloids could alleviate the damages caused by heavy metal stress via promoting the synthesis of chlorophyll in plants such as Lycopersicon esculentum [49], Solanum melagina [50], and Sedum alfredii [51]. Similarly, caffeine application enhanced the chlorophyll content in Cucumis sativus L. [52]. Moreover, it has been described that the exogenous application of allantoin, a nitrogen-rich compound present in plants as an intermediary metabolite of purine metabolism, reduced the level of O⁻² and H₂O₂ in the leaves of Arabidopsis thaliana L. accompanied by an increase in chlorophyll content [14,53]. The foliar spray of caffeine reduced oxidative stress levels and ultimately enhanced chlorophyll content in spinach plants grown under Cd stress conditions. This might be due to the elevated level of xanthine and allantoin after caffeine application.

Cadmium stress causes the inhibition of chlorophyll pigments biosynthesis. Photosynthetic pigments, particularly Chl a and Chl b, play a key role in the absorption and transmission of light energy. In the current study, the contents of Chl a and Chl b were reduced after Cd exposure, which might be due to replacing Zn²⁺, Fe²⁺, and Mg or forming an association with—SH of enzymes involved in chlorophyll biosynthesis [54]. Photosynthetic rate and stomatal conductance are indicators of plant responses to stress and represent the main indices to estimate damage caused by heavy metals [55]. The current study revealed that Cd stress reduced the efficiency of spinach plants in terms of gas exchange attributes. This decrease in photosynthetic rate might be due to the reduction in stomatal conductance. Thus, the reduced photosynthetic rate might be linked to the lower availability of CO₂ as a result of stomatal closure as reported earlier [56]. Moreover, Cd stress affects electron transport and fixation of CO₂ in photosynthesis which limits chlorophyll synthesis, resulting in photosynthesis inhibition [57]. Moreover, the increased level of H₂O₂ recorded in the current study results in a reduced rate of photosynthesis (i.e., slow Calvin cycle) due to oxidative bursts which may significantly reduce plant growth and development [58]. However, caffeine foliar spray enhanced the leaf gas exchange capability of spinach plants, as indicated by better stomatal conductance and photosynthetic rate. Similarly, caffeine application promoted the opening of stomata in Vicia faba L. plants [59]. Additionally, salt stress reduced the photosynthetic rate in sugar beet plants [30]. However, the application of allantoin mitigated the inhibition of photosynthetic pigments and activity of RuBP and induced the photosynthetic rate in sugar beet plants grown under salt stress [30]. Similar results were also recorded in cucumber plants under Cd stress [31].

Malondialdehyde, a byproduct of lipid peroxidation, is an indicator of oxidative damage in plants [60,61,62]. MDA content is enhanced in the plants grown under Cd²⁺, such as mustard and lettuce [63]. A high concentration of MDA was also observed in the leaves of Bruguiera gymnorrhiza L. plants exposed to different heavy metals [64]. Similarly, the accumulation of reactive oxygen species induced oxidative damage in plants [34]. In the current study, Cd stress enhanced the level of oxidative stress in spinach plants. Increased MDA content under imposed Cd stress depicts that the metal stress initiated oxidative stress, triggered membrane damage, and resulted in increased ions leakage. Similar findings were also reported by Hajaji et al. [65]. However, the application of caffeine reduced the extent of Cd toxicity. The exogenous application of caffeine reduced MDA content which can, in turn, alleviate plant cells destruction. Similarly, the application of allantoin reduced the levels of H₂O₂ and O⁻² in Arabidopsis thaliana L. and also mitigated the harmful effects on the environment [56,66].

Cadmium toxicity causes oxidative stress in plant cells through the production of reactive oxygen species [67], inhibition of enzymatic activity, and the increase in lipid peroxidation [6] as indicated by MDA content. However, the stimulation of antioxidant enzymes such as POD, CAT, and SOD plays a vital role in reducing this oxidative toxicity under different stress conditions [68,69,70,71]. SOD removes O⁻² and reduces membrane lipids peroxidation. CAT also removes O⁻² and H₂O₂ and reduces their activity in plants under Cd stress. The optimal activities of CAT, SOD, and POD in spinach plants, due to caffeine application, indicate the involvement of these antioxidants in minimizing Cd-induced damages in plants grown under Cd stress [72]. The current study indicated that the application of caffeine could alleviate the effects of reactive oxygen species by increasing the contents of antioxidants in spinach plants. Once caffeine is in plant cells, it undergoes catabolic pathways. Moreover, important plant metabolites are produced during caffeine degradation by the conventional oxidative purine catabolism pathway and include xanthine, allantoin, NH₃, and CO₂. Foliar application of caffeine activates the purine catabolism pathway and modulates the levels of endogenous xanthine and allantoin. Allantoin and allantoate are the key purine metabolites which scavenge reactive oxygen species and protect plants from oxidative damage, including Arabidopsis thaliana L. [14,40]. Allantoin is considered a potential scavenger for ROS and has a crucial role in plant response against stress conditions [41,73]. Similarly, it has been reported that allantoin might have direct or indirect impacts on ROS accumulation in plants [74]. The increase in proline content, under metal stress, is one of the first indications of stress sensing in plants [75]. The increase in proline content, recorded in the current study, is likely due to increased ROS synthesis under Cd stress as proline has a key role in the degradation of free radicals and is a scavenger of ROS species [72]. Similarly, Zhang et al. [76] indicated that Cd stress enhances proline content to alleviate the harmful effects of Cd stress in lettuce plants [76]. Moreover, it has been described that the accumulation of Cd varies among different plant species and is absorbed via symplastic and apoplastic pathways [77]. The current study revealed that caffeine application reduced Cd accumulation in spinach plants. The decrease in Cd content in plant shoots might be because caffeine application enhanced Cd accumulation in spinach roots and reduced the transport of Cd from the roots to the edible parts of plants.

5. Conclusions

Cadmium stress significantly reduced plant growth, chlorophyll content, and photosynthetic rate in spinach plants. On the other hand, the foliar application of caffeine enhanced the activities of antioxidant compounds and promoted plant growth. Caffeine application also reduced MDA and H₂O₂ contents by reducing Cd uptake of spinach roots. Furthermore, caffeine application stimulated the oxidative defense systems through up-regulating the activity of antioxidant enzymes, thereby improving Cd tolerance. The reduced Cd uptake and root-shoot translocation, due to the exogenous application of caffeine, reduced the threat to health upon human consumption. It was also evident that 10 mM caffeine application was better than the lower level of caffeine (5 mM). As little information is currently available for caffeine application as a plant growth modulator, further lab studies involving other food crops and molecular approaches are suggested to appraise the actual potential of caffeine usage for enhancing crop productivity.