Suppression effect of fertilizers on heavy metal stress in aquatic plants studied by the probe beam deflection/fluorescence quenching method

ABSTRACT

The suppression effect of fertilizers on heavy metal (HM) stress in aquatic plants was studied using the probe beam deflection/fluorescence quenching method. Egeria densa Planchon, Cu²⁺, and HYPONEX were used as model aquatic plants, HM ions, and fertilizers, respectively. The model aquatic plant was cultured in mixture solutions of 10⁻⁶ M Cu²⁺ and HYPONEX with different dilution ratios containing a fluorescent probe of 10⁻⁶ M Ru (II) complex (Tris (2,2’-bipyridyl) ruthenium (II) chloride). Change trends in both DO concentration and probe beam deflection with time at vicinities of within micrometers from the aquatic plant leaf surface were monitored in real time during both respiration and photosynthesis processes. The experimental results showed that the fertilizer HYPONEX suppressed the HM stress in aquatic plants caused by 10⁻⁶ M Cu²⁺. The lower the dilution ratio or the higher the concentration of the fertilizer, the greater the suppression of HM stress in aquatic plants. Additionally, the suppression of HM stress by the fertilizer during respiration appears to be more remarkable than that during photosynthesis.

INTRODUCTION

It is well known that global climate change and widespread environmental pollution give plants various environmental stresses and thus limit their growth and reduce productivity of agricultural crops (Riyazuddin et al., 2022). Heavy metal (HM) stress is particularly noticeable because it not only affects crop production but also leads to enrichment of HMs in plants, thereby threatening people’s lives and health via the food chain (Zhang et al., 2018; Ghori et al., 2019). Although some HM elements, such as Cu are essential for plant growth (Printz et al., 2016), a slightly higher than optimal level of Cu can detrimentally affect biochemical and physiological processes, including photosynthesis, nitrogen metabolism, mineral uptake, and other physiological activities in plants (Drążkiewicz et al., 2004; He et al., 2024). Therefore, it is important to explore and study the influence of HM stress on plant growth.

Most studies on HM stress in plants are based on either plant growth observation or field sampling and subsequent laboratory chemical analyses of HM contents or chemical species such as enzymes or lipids (Zhang et al., 2018; He et al., 2024). The former is based on visual changes in the color or size of plants, requiring not only rich experience but also time of days or even weeks. The latter usually uses modern analytical equipment such as inductively coupled plasma mass spectrometry (ICP-MS), atomic absorption spectrometry, omics, and molecular biology approaches to determine HM concentration in plants. Plant samples are usually decomposed or dissolved; thus real-time in situ monitoring is difficult. Gas exchange measurements of CO₂ and/or O₂ using an assimilation chamber (Sun et al., 2015; Muhammad et al., 2022) have also been used to study HM stress in plants because HM stress greatly inhibits photosynthesis (Clijsters et al., 1985; Kupper et al., 2008; Ghori et al., 2019). However, the measured CO₂ and/or O₂ concentrations in the assimilation chamber were spatially and temporally averaged and thus did not reflect real-time CO₂ and/or O₂ movements across the plant surface. It is also difficult to distinguish CO₂ and/or O₂ released or absorbed from different parts of the plant, e.g., leaves, stems, and roots.

Recently, we developed a novel highly sensitive method for studying HM stress in aquatic plants (Wu et al., 2018; Wu et al., 2021). The proposed method is based on the simultaneous monitoring of dissolved oxygen (DO)-quenched fluorescence and material movement-induced deflection of a probe beam at vicinities within micrometers from the aquatic plant surface. In this method, a focused laser probe beam was passed through the vicinity of an aquatic plant in a culture solution containing a fluorescent probe of 10⁻⁶ M Ru (II) complex (Tris (2, 2’-bipyridyl) Ru (II) chloride). The fluorescence of the Ru (II) complex in the vicinity was excited; and consequently, quenched by DO. Changes in the DO concentration in the vicinity were obtained by analyzing the quenched fluorescence intensity. At the same time, information on the material movements of the physiologically active species such as CO₂ and O₂ could be analyzed from the monitored defection signals of the probe beam. The changing trends of both the DO and deflection signals with time were opposite because material movements including O₂ movement across the plant surface were reversed during respiration and photosynthesis. The method is much more sensitive than conventional methods such as the assimilation chamber method, where the determined concentration changes of CO₂ or O₂ are spatially averaged and thus much lower than those at the vicinities of the plant surface, while the beam deflection/fluorescence quenching method probed the maximum concentration changes at the vicinities of the plant surface. The method also allows for distinguishing material movement across different organ surfaces, such as the roots, stems, and leaves of the plant (Wu et al., 2021).

In previous works, HM stress in aquatic plants was studied in a Ru (II) complex solution containing 10⁻⁶ M HM metal ions, such as Co²⁺, Ni²⁺, or Cu²⁺ (Wu et al., 2021). The experimental results showed that existence of the 10⁻⁶ M HM ions greatly altered the changing trends of both DO and deflection of the probe beam with time during photosynthesis and respiration because the HM stress greatly influenced the physiological activities and in turn the materials movements across the plant surface. Conversely, chemical species in real water environments are much more complex than the culture solution only containing the 10⁻⁶ M Ru (II) complex and 10⁻⁶ M HM ions. For example, real water environments contain various chemical components of fertilizers, which should affect the HM stress in aquatic plants. Therefore, the HM stress studied in previous experiments may be much different from the real one in water environments.

This work investigated the effects of fertilizers on the HM stress in aquatic plants by comparing the changing trends of both DO and beam deflection with time in the presence or absence of fertilizers. As in previous works, the aquatic plants E. densa and Cu²⁺ were used as model plants and HM ions, respectively. The well-known HYPONEX was used as a model fertilizer. Aquatic plants were cultured in mixture solutions of 10⁻⁶ M Ru (II) complex and 10⁻⁶ M Cu²⁺ containing HYPONEX at different dilution ratios. Changing trends in both DO concentration and deflection signals at the vicinities of aquatic plants with time in the presence or absence of HYPONEX during both photosynthesis and respiration processes were investigated and compared.

MATERIALS AND METHODS

HYPONEX original culture medium (HYPONEX Japan) was purchased from a plant-culture shop in Fukuoka city of Japan; and all chemicals were purchased from Fujifilm Wako Pure Chemical Cor. Japan without further purification. The HYPONEX original culture medium is recommended to be used with a dilution of 250-fold~2000-fold according to different plant species before use by the manufacturer. Stock solutions of 10⁻² M Ru (II) complex and 10⁻² M Cu²⁺ were prepared by dissolving certain amounts of Tris (2, 2’-bipyridyl) Ru (II) chloride and CuSO₄ ·5H₂O in 100 mL of distilled deionized water, respectively. The stock solutions were diluted to desired concentrations with either deionized distilled water or the diluted HYPONEX solutions and further mixed according to suitable mixing ratios for preparation of the culture solutions containing 10⁻⁶ M Cu²⁺, 10⁻⁶ M Ru (II) complex, and HYPONEX with certain dilution ratios. For example, a culture solution containing 10⁻⁶ M Cu²⁺, 10⁻⁶ M Ru (II) complex, and 500-fold diluted HYPONEX was prepared by adding 1 mL of 10⁻⁴ M Cu²⁺, 10⁻⁴ M Ru (II) complex, and 50-fold diluted HYPONEX into a 100 mL flask, followed by the addition of distilled deionized water to the flask markings. As controls, experiments in the 10⁻⁶ M Ru (II) complex solution without the addition of both Cu²⁺ and HYPONEX were also carried out.

The beam deflection/fluorescence quenching detection system was almost the same as the previous one (Wu et al., 2018; Wu et al., 2021). As illustrated in Fig. 1, the system was placed in a dark room with a window through which a red-blue light-emitting diode (LED; output power: 8 W; Luxour, Japan) with wavelengths of ~660 nm and ~460 nm illuminated the aquatic plants during the photosynthetic process. During respiration, the red-blue LED was switched off while the window of the dark room was closed. Approximately 3 cm of E. densa (bought from an aquarium shop in Fukuoka city of Japan) was placed in a culture dish (φ56 mm×15 mm) filled with 20 mL of culture solutions. The culture dish was placed on an X-Y-Z micro-stage (Edmund Optics) to adjust the distance between the focus point of the probe beam and the plant leaf surface. A semiconductor laser of 405 nm (output power: 3.0 mW, Sigma Koki, Japan) was used as the light source for both deflection and fluorescence measurements. The laser probe beam was reflected from a dichromic mirror and then focused to a middle vicinity of a leaf in the aquatic plant. Deflection of the probe beam was detected by a position sensor consisting of a bi-cell photodiode. The fluorescence transmitted back through the dichromic mirror was monitored using a photomultiplier tube (PMT). A commercial DO/Temperature sensor (pyro science GmbH) was also inserted into the culture dish to monitor the DO and temperature changes of the bulk culture solution. The monitored temperature, DO, deflection, and fluorescence intensity were concurrently input into a digital multimeter (Texio Technology Corporation, Japan) and recorded on a computer.

Fig. 1 Illustration of the probe beam deflection/fluorescence quenching detection system

Deflection signals and DO-quenched fluorescence intensities during both the respiration and photosynthesis processes were monitored first in the control, second in the culture solutions containing 10⁻⁶ M Cu²⁺ without, and third with HYPONEX. The monitored DO-quenched fluorescence intensities were transferred to DO concentrations at the vicinities of the plant leaves using the same calculation method as previously described (Wu et al., 2018; Wu et al., 2021).

Changes of DO concentration (ΔDO) and deflection (ΔDE) during the monitoring periods, which have been used as quantitative evaluation parameters of the method (Wu et al., 2023), were calculated as follows.

  

$$\mathrm{\Delta }\text{DO}={\text{DO}}_{\text{e}}-{\text{DO}}_{\text{b}}$$(1)

  

$$\mathrm{\Delta }\text{DE}={\text{DE}}_{\text{e}}-{\text{DE}}_{\text{b}}$$(2)

where DO_e, DO_b, DE_e, and DE_b are DO concentrations and deflection signals at the end and beginning of the monitoring period, respectively.

RESULTS AND DISCUSSION

Fig. 2 shows changes in both DO concentration and beam deflection signals with time during both the photosynthesis and respiration process in control and various culture solutions.

Fig. 2 Monitored results of DO (A and B) and deflection signals (C and D) during the photosynthesis (A and C) and respiration (B and D) processes. Light blue, green, blue, red-brown, gray, and yellow lines represent results for control, culture solutions containing both 10⁻⁶ M Cu and HYPONEX with dilution ratios of 250-, 500-, 1000-, and 2000-fold respectively, and culture solution containing 10⁻⁶ M Cu without HYPONEX, respectively

First, results of both DO concentrations and deflection signals in the control and culture solution containing 10⁻⁶ M Cu²⁺ without the existence of HYPONEX are compared; and they are expressed as light blue and yellow lines in Fig. 2, respectively. In the control experiments, the changing trends of both DO and deflection signals with time were opposite during photosynthesis and respiration process (light blue lines in Fig. 2). This occurred because the movements of material across the leaf surface during the photosynthesis were reverse to those during respiration. However, the changing trends of both DO and deflection signals with time during both photosynthesis and respiration became the same or similar in the culture solution containing 10⁻⁶ M Cu²⁺ without the existence of HYPONEX, as shown by yellow lines in Fig. 2. This was because the HM stress of 10⁻⁶ M Cu²⁺ greatly altered the physiological activities during both photosynthesis and respiration process (Wu et al., 2021). In particular, the presence of 10⁻⁶ M Cu²⁺ in the culture solution greatly inhibited photosynthesis and thus the DO at the vicinities of the leaf decreased with time, even during photosynthesis. These results agreed well with previously reported results (Wu et al., 2021).

Next, the monitoring results of both DO concentrations and deflection signals in the culture solutions containing 10⁻⁶ M Cu²⁺ and HYPONEX with different dilution ratios are compared; and they are also expressed in Fig. 2. Fig. 2-A shows that when the culture solutions contained 250-, 500-, 1000-, and 2000-fold diluted HYPONEX, DO concentrations at the vicinities of the leaves increased with time during the first 4000 s, 2200 s, 1000 s, and 100 s, respectively, and then began to decrease with time during photosynthesis. This indicated that the presence of HYPONEX suppressed the photosynthetic inhibition of 10⁻⁶ M Cu²⁺ at a certain period at the beginning of photosynthesis. The lower the dilution ratio or the higher the HYPONEX concentration, the longer the period of photosynthetic inhibition. In contrast, even with the maker-recommended minimum dilution ratio of 250-fold, the influence of the 10⁻⁶ M Cu²⁺ HM stress on photosynthesis could not be eliminated because the DO decreased with time after approximately 4000 s of photosynthesis. When the dilution ratio was the maker-recommended maximum of 2000-fold, the DO decreasing trend was similar to that without the existence of HYPONEX, except during the first 100 s. This suggested that the HYPONEX diluted as to 2000-fold gave little suppression on the 10⁻⁶ M Cu²⁺ HM stress in aquatic plants during photosynthesis.

Fig. 2-B shows changes in DO concentrations with time during respiration. When 2000-fold diluted HYPONEX existed in the culture solution containing 10⁻⁶ M Cu²⁺, the DO decreasing trend with time was similar to that in the 10⁻⁶ M Cu²⁺ culture solution without HYPONEX, except during the first 2000 s. In contrast, when 250-, 500-, and 1000-fold diluted HYPONEX was present in the culture solutions containing 10⁻⁶ M Cu²⁺, the DO decreasing trend with time became much slower than that in the 10⁻⁶ M Cu²⁺ without HYPONEX. In particular, for the culture solutions containing the 250- and 500-fold diluted HYPONEX, the DO decreasing trends after approximately 4200 s were almost the same as the control trends, indicating that the greater oxygen consumption in the aquatic plant in the presence of 10⁻⁶ M Cu²⁺ during the respiration process was restored to the control trend. These results suggested that the existence of 2000-fold diluted HYPONEX suppressed the 10⁻⁶ M Cu²⁺ HM stress only slightly during the first 2000 s, while the 1000-, 500-, and 250-fold diluted HYPONEX greatly suppressed the 10⁻⁶ M HM stress during respiration.

Fig. 2-C shows the changing trends of the deflection signals with time during photosynthesis. When the culture solutions contained 250-, 500-, and 1000-fold diluted HYPONEX, the deflection signals decreased with time during the first 4000, 2500, and 1500 s, respectively, and then increased with time during the photosynthesis process. This suggested that the presence of HYPONEX at dilution ratios of 250-, 500-, and 1000-fold suppressed the 10⁻⁶ M HM stress at a certain period of the beginning of photosynthesis. The lower the dilution ratio or the higher the concentration of HYPONEX, the longer the suppressed period. In contrast, the changing trend of the deflection signals with time in the 2000-fold diluted HYPONEX culture solution was almost the same as that without HYPONEX, suggesting that the 2000-fold diluted HYPONEX suppressed the 10⁻⁶ M Cu²⁺ HM stress during the photosynthesis process. These results agreed well with the DO monitoring results in Fig. 2-A.

Fig. 2-D shows the changing trends of the deflection signals with time during respiration. In comparison with the control, the culture solutions containing 10⁻⁶ M Cu without HYPONEX showed the fastest increase in deflection signals with time. The second and third fastest increasing deflection signals with time were the 10⁻⁶ M Cu culture solutions containing 2000- and 1000-fold diluted HYPONEX, respectively. The increasing trend of the deflection signals with time was almost the same or slightly slower than the control signals for the 10⁻⁶ M Cu culture solution containing 500- or 250-fold diluted HYPONEX. This also suggested that the presence of 500- or 250-fold diluted HYPONEX almost completely suppressed the 10⁻⁶ M Cu²⁺ HM stress during respiration, consistent with the DO results shown in Fig. 2-B.

Fig. 3 shows the relationships between ΔDO or ΔDE and dilution ratio of HYPONEX during both photosynthesis (A) and respiration (B). For the photosynthesis process, values of both the ΔDO and ΔDE in presence of 2000-fold diluted HYPONEX were close to those without HYPONEX, suggesting little suppression of the 2000-fold diluted HYPONEX on the 10⁻⁶ M Cu HM stress. When the dilution ratio was smaller than 2000-fold, both the ΔDO and ΔDE changed toward the control as the dilution ratio decreased. However, both the ΔDO and ΔDE could not reach the control level even when the dilution ratio was 250-fold. This meant that the 10⁻⁶ M Cu²⁺ HM stress in aquatic plants during the photosynthetic process could not be suppressed completely even in the presence of 250-fold diluted HYPONEX. In contrast, both the ΔDO and ΔDE changed toward the control with a decrease of the dilution ratio, and both the ΔDO and ΔDE reached to the control ones when the dilution ratio was 500- or 250-fold during the respiration process. This indicated that the 10⁻⁶ M Cu²⁺-induced HM stress in aquatic plants during respiration could be suppressed by the addition of 500- or 250-fold diluted HYPONEX. These results suggest that the suppressing effect of HYPONEX on HM stress during respiration is more remarkable than that during the photosynthesis process.

Fig. 3 Relationships between ΔDO/ΔDE and the dilution ratio of HYPONEX added to culture solutions during photosynthesis (A) and respiration (B) process

In aquatic plants, Cu²⁺ HM stress includes (Mir et al., 2021; Chen et al., 2022): 1) photosynthesis inhibition caused by excess Cu²⁺, which inhibits chlorophyll biosynthesis and damages photosystem II (PSII); 2) oxidative damage to cellular components like lipids, proteins, and DNA by reactive oxygen species which are generated by excess levels of Cu²⁺; 3) enzyme inhibition caused by binding of excess Cu²⁺ to various enzymes; and 4) nutrient deficiencies generated by excess Cu²⁺-caused interrupting of uptake and transport of essential nutrients. Two reasons are considered to explain the suppression of HM stress in aquatic plants by the fertilizer HYPONEX in the culture solution. The uptake of Cu²⁺ by aquatic plants from the culture solution and the excess Cu²⁺ inside aquatic plants were suppressed because organic components in HYPONEX both in the culture solution and uptake or absorption in aquatic plants might have chelated or complexed with excess Cu²⁺ in both the culture solution and aquatic plants. Another is that the HYPONEX provided nutrients to the aquatic plants, thereby enhancing their anti-HM stress ability. The details remain to be investigated.

It is concluded that the fertilizer HYPONEX suppressed the HM stress of the 10⁻⁶ M Cu²⁺. Moreover, the higher the concentration of the fertilizer, the more remarkable the HM stress was suppressed in the aquatic plants. In addition, the suppression effect on HM stress during respiration appears to be more remarkable than that during photosynthesis. The method can explore the suppression effect of fertilizer or other additives on HM stress in aquatic plants in a few hours; thus, it is a faster method compared with conventional methods. Moreover, it provides detailed insights into how the HM ions in the culture solution influence the movement of materials, including O₂ movements across the plant leaf surface during both respiration and photosynthesis, nondestructively and noninvasively, which cannot be achieved by traditional methods. The method is also able to offer a clear understanding of how a fertilizer affects HM stress at different stages by thorough analysis of both DO and deflection changes during photosynthesis and respiration. It is expected to be a novel and powerful tool to study not only HM stress but also other environmental stresses in plants.

ACKNOWLEDGEMENTS

This work was partly supported by Grant-in Aid for Scientific Research (No. 24550109 and No. 20K05572) from the Japan Society for the Promotion of Science.

CONFLICTS OF INTEREST

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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