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Performance of chlorophyll a fluorescence parameters in Lemna minor under heavy metal stress induced by various concentration of copper


The objective of the present investigation was to understand the efficacy of chlorophyll fluorescence analysis and to identify the specific photosynthetic parameters for early and rapid detection of Cu-induced HM-stress in plants. Aquatic angiosperm Lemna minor was exposed to various concentrations (0–40 µM) of Cu. We observed that the FV/FO (Efficiency of the water-splitting complex on the donor side of PSII), quantum yield for electron transport, and quantum yield of primary photochemistry were decreased however, dissipated quantum yield was increased with Cu concentration. ABS/CSM, TRO/CSM, ETO/CSM and maximum quantum yield were displayed the dose–response relationship under Cu stress. Performance indexes were increased initially due to the beneficial effects of Cu at lower concentration while decreased significantly (p ≤ 0.05) at highest concentration of Cu. The outcomes of the present research revealed that the ChlF analysis is very sensitive tool that can be used to determine the toxicity of heavy metals in plants.


In nature, plants are continually exposed to abiotic and biotic stresses. Heavy metals (HMs) like Hg, Cu, Pb, Zn, Ni, Co, Mn, As, etc. have been accumulating in soils for a long time due to anthropogenic activity such as the use of chemical fertiliser, sewage, industrial and smelting wastes1. HMs are non-biodegradable elements that cannot be eliminated from the environment through natural processes. Some of them are said to be immobile, that unable to move from the site where they have accumulated, others are referred to as mobile because they can be taken up by the root system of plants via diffusion, endocytosis, or metal transporters2,3,4,5,6,7,8,9,10,11,12,13. However, some metals, such as zinc, copper (Cu), nickel, etc. are important micronutrients that must be absorbed in small amounts as cofactors for several enzymes. Besides these, some HMs found in pesticides such as Cd, Pb, Hg etc. have no beneficial properties and become harmful when their concentration exceeds a threshold limit14,15,16,17,18. These HMs may or may not be essential for the proper growth of plants but accumulate in the plants (as a natural accumulator) from soil and water19,20,21. Metal accumulation rates and plant tolerance vary from species to species22. Some of the HMs become more toxic than others causing chlorosis, stunted growth, root browning, and mortality are some of the apparent indicators of HM toxicity in plants23, 24.

Cu (Cu) is an important element in plants that serves several functions at the physiological and molecular levels. However, the excessive levels of it might constitute a risk to the survival of plants. Cu is a key component of plastocyanin and cytochrome oxidase that are essential for photosynthesis and respiration which have a crucial function in plant carbon assimilation and ATP generation25, 26 Cu-stressed plants exhibit a variety of visible symptoms, including chlorosis, stunted development, ion leakage and reduced root growth27. Excessive levels of Cu in plants can lead to oxidative stress that causes severe damage to membranes and macromolecules, as well as having a negative impact on many metabolic pathways28. Neelima and Reddy investigated the effects of Cu in Solanum melongena seeds and revealed that excess Cu reduces germination, seedling length, and root number29. All these consequences are extremely harmful to the plant.

Chlorophyll a fluorescence (ChlF) is a commonly used method to detect plant stress conditions in plant research, frequently in association with other morphological, chemical, and physiological variables30,31,32,33,34,35,36,37,38,39,40. Chl a fluorescence (ChlF) is the natural phenomenon describing the dissipation and heat radiation or re-emission of the portion of absorbed energy which is not utilised to drive photosynthesis23, 41,42,43,44,45,46. ChlF measurement provides information about changes in photosynthetic efficiency and heat dissipation47, 48. It is an incredibly simple, non-invasive, extremely sensitive, rapid, and accurate method and providing a quantitative assessment of oxygenic photosynthesis37. Plants exposed to HM ions disrupt photosynthesis as a result of a single or cumulative event of HM interaction with protein which increase the rate of ROS generation and which replaces essential kations in protein active centers28.

Some HM ions, for example, Cu, Hg, Cd, Zn, or Ni can replace the core Mg ion in chlorophyll molecules, resulting in chlorophyll-metal complexes and a reduction in PSII quantum efficiency49,50,51. Apart from evaluating specific parameters, of which the FV/FO and FV/FM are the most well-known and extensively utilised, the interpretation of double normalised curves using the JIP test is becoming increasingly popular in environmental research practices42. Plots are formed using data collected at a high sampling rate within a second of the dark-adapted leaf being exposed to light, as the independent variable on a logarithmic timeline. On such plots, inflection points (J-I-P) are noticed when the recorded fluorescence increases which provide the foundation for inferences regarding the photosynthetic apparatus' structure and function. the O-J-I-P transient is prime source of observed variations in the efficiency of the chlorophyll antenna in capturing light energy and transfer to plastoquinone QA (the electron acceptor) is the only limitation of photochemical conversion in PSII52, 53. Even though, ChlF there are years of in-depth expertise, valid interpretations of ChlF data still require more research54. ChlF measurement has become a simple, effective, and dependable technique for outdoor environmental research to improve knowledge and current technology42, 43, 45, 55,56,57,58,59.

HM, pollution is becoming more prevalent in the environment, demanding rapid and effective solutions for metal remediation. The use of metal-accumulating plants for remediation has recently given rise to a new technology known as phytoremediation60. An ideal hyperaccumulator plant species must meet two requirements for this technology to be viable are HM tolerance and accumulation. Consequently, a better knowledge of the metal tolerance mechanism(s) is critical for the development of effective phytoremediation techniques61. The chlorophyll a fluorescence has long been used to measure the effects of environmental stress on plants, because they provide a quick approach to determine injury in the absence of visual signs62, 63. Therefore, the ability to identify the toxic effects before any morphological symptoms can be seen makes phytoremediation an extremely effective method for identifying metal-tolerant plants.

Duckweeds have high potential to grow under HM stress because of their potential to bioremeidte HMs through either by rhizofilteration or phytotransformation. Therefore, besides use in bioremediations, duckweeds serve a rich source of essential HMs such Cu and Zn for improving feed efficiency of animals64.

Chlorophyll (Chl) a fluorescence signals have become one of the potent indicators for early detection of HMs in soil and aquatic bodies25, 65, 66. In the present study, we used the chlorophyll (Chl) a fluorescence transient to investigate the effects of HM stress induced by various concentration of Cu in L. minor plants grown in a nutrient medium.

Materials and methods

Plant material and growth condition

L. minor plants were collected from the region of Ayad river located at Udaipur, India (24° 35′ 14.97′′ N, 73° 42′ 38.75′′ E) (As per the Biological Diversity act, 2002 of National Biodiversity Authority of India, the Indian researchers neither require prior approval nor need to give prior intimation to SBB for obtaining biological resource for conducting research67). The plant was identified by Dr. Vineet Soni based on the morphological characteristics (oval shaped fronds, 2–5 fronds remained together, presence of three nerves in each frond and cylindrical root sheath with two lateral wings). The collected fronds (stock culture) were maintained in plastic (PVC) aquariums in Jacob culture media as per the OECD guideline of 200268. The stock culture and Cu treated plants were kept in controlled conditions at 150–230 µmol/m2/s (PAR) by using white fluorescent light, 14:10 h light: dark cycle, and 25/20 °C day/night temperature. This medium consisted of the following: Stock solution (A): Ca (NO3)2, 60.0 g/L, Stock solution (B): MgSO4·7H2O, 102.0 g/L; KNO3, 100.0 g/L; KH2PO4, 14.0 g/L, Stock solution (C): H3BO4, 0.300 g/L; MnCl2·4H2O, 0.3145 g/L; ZnSO4·7H2O, 0.0356 g/L; Na2MoO4·2H2O, 0.0118 g/L, Stock solution (D): CuSO4·5H2O, 0.0125 g/L; FeEDTA (Ethylenediaminetetraacetate acid), 1.8520 g/L. Stock solutions were kept in a refrigerator and growth media prepared by adding 10 mL of each stock to 1 L of distilled water and then adjusting the pH 6.0 using NaOH or HCl69.

Cu exposure

For the ChlF experiment ~ 30 two or three-fronded, healthy plants (300 mg) were taken from stock culture and transferred to glass bottle containing 250 mL of growth medium without EDTA and exposed to various concentrations of CuSO4·5 H2O (Sigma Aldrich, C8027, ≥ 98%) (0, 10, 20, 30, and 40 μM) for 24 h. The metal exposure experiments were performed according to procedure described by Teisseire and Guy using EDTA free growth medium since it is a chelating agent and alter the metal adsorption process in plants (can increase the bioavailability of metal)70. Control plants were grown under both EDTA and Cu free growth medium. The experiment glass bottles were placed in a controlled environment as described above.

Chlorophyll a fluorescence transient

ChlF was measured using a plant efficiency analyser (Handy PEA fluorimeter, Hansatech instruments Ltd. England). Before measurement fronds were dark-adapted for 50–60 min at 26 °C. Thereafter, ChlF signals were analysed with the Biolyzer v.3.0.6 software (developed by Laboratory of Bioenergetics, University of Geneva, Switzerland). The experiments were done in six replicates and repeated three times to ensure the results. JIP-test method has been developed by which several selected phenomenological and biophysical parameters quantifying the PSII and PSI behaviors are calculated. Several parameters can be derived from the polyphasic ChlF rise (OJIP curve) that provide information about photosynthetic fluxes41, 71,72,73. Abbreviations, formulas, and definitions of the JIP-test parameters used in the current study are presented in Table 1.

Table 1 Principal component analysis of chlorophyll a flourescence parameters of L. minor under various concentration of Cu.

Principal component analysis (PCA), grid correlation matrix and heat map

The relations between the selected JIP-test parameters were tested by Principal Component Analysis. ChlF parameter was selected for the PCA analysis to classify the variables that show the maximal fluctuations. Dimension 2 (PC 2) described the maximum of the variability which accounted for 79.15% and dimension 1 (PC 1) accounted for 18.17%, respectively. The positive and negative correlation between the parameters also shows the variation of the parameters in the respective principal components (dimensions) (Table 2). The correlation between all ChlF parameters investigated in this paper were analysed through grid correlation matrix by using Python software which expressed between + 1 and − 1 with colour code. The calculated JIP parameters were also presented by the heat map, through normalizing them between 1 and 100 by using a color code green to red.

Table 2 Abbreviations, formulas, and definitions of the JIP-test parameters.

Statistical analysis

Statistical analysis was performed using analysis of variance (ANOVA), followed by a Tukey HSD test (p = 0.05) using XLSTAT 2020. Only significant values (p ≤ 0.05) of measurements are presented in figures. The heat map was prepared by normalizing the values and bringing them all to a range between 1 and 100 to provide an unbiased color code. Three color code combination of red for high (100%), yellow for medium (50%), and green for the lowest value (1%) was used to represent the heat map. The MS excel and CorelDraw software were used for calculation and designing of the heatmap. In addition, a principal component analysis (PCA) was conducted by eigenvalue decomposition of a data correlation matrix using OriginPro 2016. PCA was applied to find the patterns of the fluorescence parameter and variations in the experimental data. The 48-h lethal dose (LD50 and LD90) was determine by Probit Analysis using SPSS (22.0). Comparision of mortility ratios between experimental and control groups in the deferent concentrations was performed with Chi-square testing.


Cu stress significantly altered the growth and productivity of L. minor through the modulation of the photosynthetic process. In the present studies, impacts of Cu-induced HM stress on ChlF kinetics, specific energy fluxes, phenomenological energy fluxes, and performance indexes were studied in L. minor.

ChlF rise

ChlF rise of L. minor was measured after 24 h of Cu treatment and a typical OJIP induction curve was displayed when plotted on the logarithm time scale (Fig. 1). With increasing the Cu concentration, the fluorescence yield at various intermediary steps, such as J, I, and P was reduced. In control plants, two intermediate peaks FJ (chlorophyll fluorescence at 2 ms) and FI (chlorophyll fluorescence at 300 ms) were formed between FO and FM, ChlF increased continuously from initial (FO) to maximal (FM) fluorescence intensity in L. minor growing under control conditions. HM induced reduction in PSII photochemistry and electron transport activity were severe at the highest concentration of Cu.

Figure 1
figure 1

ChlF rises in L. minor plants exposed 24 h. to different concentrations of CuSO4 (0.0 µM to 40.0 µM) and O, J, I, and P indicate PSII rapid fluorescence transients.

Biophysical parameters derived by the ‘JIP-test’ equations’

FO and FM

The minimal fluorescence intensity (FO) and the maximum fluorescence intensity (FM) both are decreased with increasing the Cu concentration (Fig. 2A). Fluorescence intensity recorded at 50 µs is denoted as FO and at this time the all primary quinone acceptor (QA) is in the open (oxidized) state.

Figure 2
figure 2

Radar plots (A–C) showing various technical fluorescence parameters. Each line represents the average of 6 measurements per treatment. Asterisk denote the significance at p ≤ 0.05 level.

The maximum primary yield of photochemistry of PS I1 (FV/FO) are related with photosynthetic efficiency of plant and increased value of FV/FO indicates proper functioning of PSII. The FV/FO ratio (ratio between the rate constants of photochemical and nonphotochemical deactivation of excited Chl molecules) for L. minor plants decreased gradually at 10 µM (94.86% of control) and 30 µM (89.44% of control) concentration of Cu (Fig. 2A). Further, a significant decline in FV/FO ratio (43.58% of control) was recorded at a high level (40 µM) of Cu exposure as a result of a significant decrease in FV (41.00% of control) as shown in Figs. 2A and 3.

Figure 3
figure 3

Heat map represents relative variability of several photosynthesis-related parameters, obtained after using the JIP test for L. minor under Cu stress. Data are for different concentrations (0.0 µM to 40.0 µM), obtained after 24 h red is for lower value (1%), yellow for medium (50%), and green for the highest values (100%) All the data obtained were first normalized to bring the value of the parameters in the range of 1–100 to provide an unbiased colour code.

The relative variable fluorescence at 2 ms (J step) is denoted as VJ which is the measure of the fraction of primary quinone electron acceptor of PSII in its reduced state [QA − /QA(total)]74. At the lower level of HM stress, a slight reduction in the value of VJ was observed but with increasing the metal concentration to a high level the VJ value was increased to 224.74% of control (Figs. 2A, 3).

Complimentary Area (SM) is also an important parameter that is directly proportional to the number of reduction and oxidation of one QA- molecule during the fast OJIP transient or number of electrons passing through the electron transport chain75. The turnover number (N) is represented as the number of times QA becomes reduced and re-oxidized another time, till the FM (Maximum fluorescence intensity) is reached76,77,78,79. At severe Cu stress the increased value of turnover number (N) value was recorded (145.40% of control) which was also represented by PSI cyclic electron transport as photoprotection (Fig. 2A). The increased values of SM in L. minor under Cu exposure (131.40% of control at moderate HM treatment) displayed the reduced electron transport between these photosystems.

Quantum yield

The quantum yield of primary photochemistry FV/FM (φPo), which reflects the overall photosynthetic potential of active PSII reaction centers, was not affected by Cu-induced HM stress in plants. However, a slight decline in FV/FM was recorded at 40 µM Cu (Fig. 2C). A similar trend was observed in ET/ABS (φEo) (Figs. 2C, 3). The lowest values of φEo, approximately half of control, were recorded when L. minor was subjected to 40 µM Cu. In contrast, DI/ABS (φDo) remained almost the same until the exposure of 30 µM Cu and thereafter enhanced about two folds of the control level in plants grown in media containing 40 µM Cu (Figs. 2C, 3).

Specific energy flux (membrane model)

The specific energy fluxes such as absorption flux per reaction center (ABS/RC), trapped energy flux per reaction center (TRO/RC), electron transport flux per reaction center (ETO/RC), and dissipated energy flux per reaction center (DIO/RC) were analyzed to determine the photosynthetic performance of active PS II reaction centers of L. minor subjected to various concentrations of Cu (Figs. 3, 4). Up to 30 µM Cu, no significant variations in absorption flux per reaction center (ABS/RC) was recorded while at 40 µM, a remarkable enhancement in absorption potential of active reaction centers was recorded (162.83% of control) (Figs. 3, 4). A similar trend in TRO/RC was observed as shown in the heatmap (Fig. 3). TRO/RC remained almost constant up to 30 µM Cu and thereafter increased with increasing the severity of Cu-induced HM stress (129.68% of control). In contrast, no significant changes in electron transport flux per reaction center (ETO/RC) were recorded up to 20 µM Cu concentration while decreased at severe Cu stress (73.58% of control). On the contrary, the DIO/RC remained constant up to 30 µM Cu and then increased about three folds with the progression of Cu concentration (297.67% of control) as shown in Fig. 2B. The effects of Cu-induced HM stress on the specific energy fluxes (absorption flux per reaction center, trapped energy flux per reaction center, electron transport flux per reaction center, and dissipated energy flux per reaction center) are presented diagrammatically through thylakoid membrane models (Fig. 4). It is of interest to investigate if HM stress changes the ratio among antenna light-harvesting complex (ABS) and active PSII reaction centers (RC). According to the leaf pipeline model in severe Cu stress, there is more active RC and the higher value of specific energy flux (ABS/RC, TRO/RC, and DIO/RC) shows the increased ability of RC to the reduction of plastoquinone (Fig. 4).

Figure 4
figure 4

Thylakoid membrane model for specific energy fluxes (per reaction, RC) in L. minor fronds when subjected to various concentration of CuSO4, (A); control, (B); 10.0 µM, (C); 20.0 µM, (D); 30.0 µM and (E); 40.0 µM.

Phenomenological energy flux (leaf model)

Phenomenological energy fluxes mean absorption flux per cross-section (ABS/CS), trapped energy flux per cross-section (TRO/CSM), electron transport flux per cross-section (ET/CS), and dissipated energy flux per cross-section (DIO/CSM) significantly modulated by Cu-induced HM stress in L. minor. Absorption flux per cross-section (ABS/CSM) did not alter with increasing concentrations of Cu up to 30 µM (Fig. 5). The lowest values of ABS/CSM, TRO/CSM and ETO/CSM were noticed at the highest concentration of Cu (92.09%, 73.34% and 41.61% of control respectively). Absorption potential per cross-section significantly declined when plants were treated with 30 µM Cu for 48 h. TRO/CSM reduced remarkably up to 30 µM Cu and thereafter declined up to 50% with increasing concentration of Cu (Fig. 5).

Figure 5
figure 5

Energy pipeline leaf model of phenomenological fluxes (per cross section, CS) in L. minor fronds when subjected to various concentration of Cu, (A); control, (B); 10.0 µM, (C); 20.0 µM, (D); 30.0 µM and (E); 40.0 µM.

Electron transport efficiency of plants was found tolerant to Cu-induced HM stress. A high concentration of Cu (40 µM) extremely reduced the electron transfer system in thylakoid membranes (Fig. 5). DIO/CSM was increased significantly (p ≤ 0.05) at 30 µM Cu treatment and after that decreased slightly).

KP and KN

De-excitation rate constants for nonphotochemical reaction (KN) increased under Cu stress and at severe stress conditions KN value approaches 198.46% of the control (Figs. 2C, 3). While de-excitation rate constants for photochemical reaction (KP) lowered slightly (86.48% of control) at 40 µM Cu concentration.

Performance index

Overall effects of Cu-induced HM stress on various photosynthetic parameters are presented in the form of a radar plot (Fig. 2). To analyze the effects of Cu-induced HM stress on overall photosynthesis performance, PIABS and PICS were determined in L. minor exposed to various intensities of Cu stress. Cu stress led to a significant effect on the performance index on absorption basis (PIABS) and performance index of PS II and PS I (PICS) in L. minor. PIABS and PICS continuously increased with increasing concentration of Cu up to 30 µM, and then declined sharply with the progression of Cu-induced HM stress. The lowest performance index on absorption basis (PIABS) and performance index of PS II and PS I (PICS) were recorded in plants cultivated on media containing 40 µM Cu (Figs. 2C, 3).

The PCA results displayed that Dim 1 and Dim 2 represented 97.32% of the variation in the ChlF parameter under Cu induced HM stress in L. minor (Fig. 6). The loadings for ABS/CSO, ETO/RC, TRO/CSO, ETO/CSO, FV/FM, PICS and PIABS are in quadrate I and IV while, TRo/RC, ABS/RC, DIO/RC, DIO/CSO and FO/FM are accounted in quadrate II and III. All treatments, except 40 µM, are located in quadrate I and IV. The loading arrow of 40 µM is longer than others in all quadrates. Thus, the higher concentration of Cu was significantly affecting the major JIP parameters located in quadrate II and III. The mild Cu stress up to 20 µM was less toxic as compared to severe stress and plants performed better which was described by performance index parameters in quadrate IV (Fig. 6).

Figure 6
figure 6

The principal component analysis with four Cu treatment conditions. The PCA is based on the chlorophyll fluorescence data. Arrows represent the Chlorophyll a fluorescence parameter on the corresponding dimensions (PC 1 and PC2), where PC 2 expressed most of the variability in the data.

In Table 3, values of the lethal dese responsible for 50% mortality (LD50) and 90% mortality (LD90), calculated by probit analysis with 95% probability level, are given. The LD50 and LD90 values of Cu was 25.70 µM and 87.80 µM respectively.

Table 3 The acute 48–h LD50 values of Cu and their confidance limits in L. minor according to Logit analysis.


Many studies on the plant’s physiological changes under various HM stress have been reported. These studies indicated that plants have developed a series of mechanisms to protect themselves from these adverse environmental threats. ChlF analyses have been shown to detect complex biochemical alteration in photosynthetic apparatus in a vast range of plant species, including both terrestrial and aquatic80. The present investigation shows the Cu induced changes in various fluorescence parameters of photosystem II in duckweed L. minor.

ChlF rise

Excess energy enhanced the utilisation capacity of plants in extreme environments, after which photoprotective systems quenched the extra Chl radical, and the extra energy was dissipated as heat79, 81, 82. The reduction in ChlF from PSII is being used to quantify these mechanisms, which are jointly referred to as non-photochemical quenching (NPQ)41, 83, 84. The J-I and I-P, and the J step still appeared at the severe Cu stress (40 µM), indicating the tolerance capacity of the plant85. The O-J is a photochemical phase and J-I-P is a thermal phase, these are two characteristics of OJIP transient and presented three various reduction processes of the electron transport chain52, 74, 86, 87. The photochemical phase (O-J) is mainly light-dependent and comprises information regarding antenna size and connection between PSII reaction centers88, 89. Further, the reduction in remaining ETC is denoted by the thermal phase (J-I-P) rise90.

Biophysical parameters derived by the ‘JIP-test’ equations’

The values of minimal fluorescence intensity are an important parameter and can provide insight in the irreversible damage of PSII, associated with light-harvesting complex II (LHCII) and hindering the electron transfer on the reduced side of PSII83, 91. Because of conformational changes in the D1 protein under Cu stress, which cause changes in the characteristics of PSII electron acceptors, decreasing FM under HM stress may be related to less efficient PSII activity83.

In determining the maximum primary yield of photochemistry, FV/FO is a parameter that accounts for simultaneous variations in FM and FO45. The decreased values of FV/FO in fronds under Cu stress show the alteration in the electron transport rate to the primary electron acceptors from PSII and a reduction in the number and size of the reaction center. Martinazzo et al. and Janka et al. also reported the environmental stress-induced decrease in the FV/FO ratio in different plant species92, 93. The increased level of relative variable fluorescence under Cu treatment indicates that the electron transfer at the donor side of PSII was affected. The affected FV/FO can be due to the modified unquenchable fluorescence (FO) that altered the energy relay from antenna complex to reaction center94. According to PCA analysis the quantum yield was positively correlated with the electron transport per cross-section while negatively correlated (Fig. 6) with FO/FM located in the opposite direction of the PCA loading plot, which was also confirmed by the Correlation matrix (Fig. 7). Another possibility of reduced maximum primary yield under Cu stress can be the substitution of central atom of chlorophyll molecule, Mg by Cu. This substitution can hinders photosynthetic light-harvesting in the affected chlorophyll molecules95.

Figure 7
figure 7

Grid correlation matrix shows the correlation between all calculated chlorophyll a fluorescence parameter with color code.

The “JIP test” of fluorescence transient in photosynthetic organisms, subjected to abiotic stress revealed a marked decrease in φPo96. The slight reduction in φPO might be due to a decrease in PSII photochemical efficiency resulting from Cu stress (in most higher plants having usually a value in the range of 0.78–0.8497). In the light condition, a reduction in the maximum quantum yield of PSII (φPo) shows that HM stress inhibits the redox reaction following QA and causes a delay in electron transport between QA and Q90. These parameters are very important and provide relevant information on electron transport activity at the PSII acceptor sites. The present finding suggested that Cu treatment reduces the electron transport at the PSII acceptor site in L. minor.

Energy pipeline models (membrane and leaf model), presented in Figs. 4 and 5, have displayed that various sites in PSII are sensitive to several environmental stresses98,99,100. Based on present results, TRO/CSM and ETO/CSM decreased with increasing the Cu concentration because active RCs are converted into inactive or closed (dark circle in model) RCs consequently decreasing the trapping efficiency and electron transport from PSII74, 81, 101. PCA biplot shown ETO/CSM, DIO/CSM are positively correlated, which is also observed by grid correlation matrix (Figs. 6, 7). The ABS/RC is determined by taking the total amount of photons absorbed by Chl molecules throughout all RCs by the total number of active RCs58. The ratio of active/inactive RCs influences it, and as the number of active centers rose, the ABS/RC ratio reduced. TRO/RC is the maximum rate at which an exciton is captured by the RC, resulting in a decrease in QA. An increase in this ratio indicates that all the QA has been reduced83. Reduction in ETO/RC describes that the re-oxidation of reduced QA through electron transport in an active RC is decreased because a greater number of the active RC are available, hence it only reflects the activity of active RCs. Figure 4 demonstrates a reduction in per active RC electron transport but an overall increase in electron transport. The ratio of total dissipation of un-trapped excitation energy from all RCs to the number of active RCs is defined as DIO/RC. Dissipation arises as heat, fluorescence, and energy transfer to other systems and the ratios of active/inactive RCs also have an impact. Due to the effective utilisation of energy by the active RCs, the ratio of total dissipation to the number of active RCs (DIO/RC) is not very impacted102, 103.

The FV/FM ratio = (FM − FO)/FM is an important JIP parameter that represents the conversion efficiency of primary light energy in the PS II reaction center and is used as a stress indicator in a large number of photosynthetic studies53, 82, 87. However, since it is dependent on FO and FM fluorescence levels, this quantitative parameter is not usually sensitive enough to detect alteration across samples. Srivastava et al. employed the performance index (PI), a novel, more responsive, and significant parameter to measure photosynthesic efficiency under stress104. The performance index, PI, is derived using three (or four) components based on reaction center density, trapping efficiency, and electron transport efficiency, in the same way as a Goldman equation105. As a result, if any of these components is affected by stress, the effect will be visible in the performance index, which has a higher sensitivity. Performance index (PIABS) is calculated on an energy absorption basis while the performance index on cross-section (PICS) is obtained by multiplying the performance index on absorption basis PIABS, by the phenomenological energy flux, ABS/CS = Fo (or FM): and the value of PIabs and PCS significantly lowered in a plant grown under Cu stress (Fig. 2C). PIABS are decreased due to reduced activity of the RC so the overall activity of the RC is decreased41, 83, 106 based on results in this study and statistical models (PCA and Correlation matrix) some of the important JIP parameters such as Phenomenological energy flux (ABS/CSM, TRO/CSM and ETO/CSM), maximum quantum yield (φPO), Performance index per absorbance (PIABS) and per cross-section (PICS) are displayed the dose–response relationship under Cu stress. Probit analysis is usually used in toxicology to determine the relative toxicity of chemicals to living organisms107. Copper LD50 values (Table 3) demonstrated that this molecule can be considered as highly toxic to L. minor. Copper phytotoxicity was assessed through the visible symptoms of toxicity and determination of the concentration that results in a 50% reduction in the growth of L. minor (LD 50). According to Teisseire & Guy (2000), CuSO4 at 10 μM was inhibitory for L. minor (Teisseire and Guy). However, some plant species tolerate this element at concentrations higher than those used in medium cultures. Our study indicated that, L. minor was sensitive to copper for concentrations ≥ 25 µM108. This is caused by the different duckweed species used and by the different test conditions, especially concerning the nutrient media as well as by the methods of evaluation (Appenroth et al.109).


In the present study, the efficacy of ChlF kinetics in the detection of Cu-induced HM stress was analysed in L. minor. Treatment of lower concentration of Cu (0.0–20.0 µM) had mild negative effect on photosynthesis. As the Cu is an essential micronutrient and plays a vital role in many biochemical processes, hence under moderate metal concentration the L. minor performed normally without any deleterious effect. A typical OJIP curve was obtained which shows that the plant efficiently used the solar energy for photosynthesis which is expressed in the term of increased active reaction center and performance index. In contrast, at higher Cu concentration (30.0–40.0 µM), the OJIP curve has been flattened due to a reduction in electron transport towards PSI (P700), and a major portion of absorbed energy was dissipated in the form of heat because of an increased number of the inactive reaction center. Conclusively, phenomenological energy flux (ABS/CSM, TRO/CSM and ETO/CSM), maximum quantum yield (φPO), performance indexes (PIABS and PICS) are powerful indicators of HM stress in plants and can be used for rapid detection of HM-induced water pollutant.Additionally, the key OJIP parameters screened in this paper could be a good tool for the rapid detaction of primary mode of action of HM on the photosynthetic apparatus in L. minor.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.


  1. Aydinalp, C. & Marinova, S. The effects of heavy metals on seed germination and plant growth on alfalfa plant (Medicago sativa). Bulg. J. Agric. Sci. 15, 347–350 (2009).

    Google Scholar 

  2. Öztürk, M. A. Plants and Pollutants in Developed and Developing Countries (Ege University, 1989).

    Google Scholar 

  3. Ali, I. & Jain, C. K. Advances in arsenic speciation techniques. Int. J. Environ. Anal. Chem. 84, 947–964 (2004).

    CAS  Article  Google Scholar 

  4. Krzesłowska, M. The cell wall in plant cell response to trace metals: Polysaccharide remodeling and its role in defense strategy. Acta Physiol. Plant. 33, 35–51 (2011).

    Article  CAS  Google Scholar 

  5. Ali, I., Gupta, V. K., Khan, T. A. & Asim, M. Removal of arsenate from aqueous solution by electro-coagulation method using Al-Fe electrodes. Int. J. Electrochem. Sci. 7, 1898–1907 (2012).

    CAS  Google Scholar 

  6. Ali, I., ALOthman, Z. A. & Sanagi, M. M. Green synthesis of iron nano-impregnated adsorbent for fast removal of fluoride from water. J. Mol. Liq. 211, 457–465 (2015).

    CAS  Article  Google Scholar 

  7. Suhail, M. & Ali, I. Advanced spiral periodic classification of the elements. Chem. Int. 3, 220–224 (2017).

    Google Scholar 

  8. Ali, I. et al. Artificial neural network modelling of amido black dye sorption on iron composite nano material: Kinetics and thermodynamics studies. J. Mol. Liq. 250, 1–8 (2018).

    CAS  Article  Google Scholar 

  9. Sabir, M. et al. Phytoremediation: Mechanisms and adaptations. Soil Remediat. Plants Prospect. Challenges 85, 85–105 (2014).

    ADS  Google Scholar 

  10. Dehghani, M. H., Sanaei, D., Ali, I. & Bhatnagar, A. Removal of chromium (VI) from aqueous solution using treated waste newspaper as a low-cost adsorbent: Kinetic modeling and isotherm studies. J. Mol. Liq. 215, 671–679 (2016).

    CAS  Article  Google Scholar 

  11. Alharbi, O. M. L. et al. Health and environmental effects of persistent organic pollutants. J. Mol. Liq. 263, 442–453 (2018).

    CAS  Article  Google Scholar 

  12. Basheer, A. A. New generation nano-adsorbents for the removal of emerging contaminants in water. J. Mol. Liq. 261, 583–593 (2018).

    CAS  Article  Google Scholar 

  13. Burakova, E. A. et al. Novel and economic method of carbon nanotubes synthesis on a nickel magnesium oxide catalyst using microwave radiation. J. Mol. Liq. 253, 340–346 (2018).

    CAS  Article  Google Scholar 

  14. Gough, L. P. Element Concentrations Toxic to Plants, Animals and Man (1979).

  15. Adriano, D. C. Trace Elements in the Terrestrial Environment (Springer, 2013).

    Google Scholar 

  16. Guecel, S. et al. Studies on trace metals in soils and plants growing in the vicinity of copper mining area-Lefke, Cyprus. Fresenius Environ. Bull. 18, 360–368 (2009).

    Google Scholar 

  17. Ashraf, M., Ozturk, M. & Ahmad, M. S. A. Toxins and their phytoremediation. In Plant Adaptation and Phytoremediation (eds Ashraf, M. et al.) 1–32 (Springer, 2010).

    Chapter  Google Scholar 

  18. Sharma, S. et al. Adsorption of Rhodamine B dye from aqueous solution onto acid activated mango (Magnifera indica) leaf powder: Equilibrium, kinetic and thermodynamic studies. J. Toxicol. Environ. Health Sci. 3, 286–297 (2011).

    Google Scholar 

  19. Ozturk, M. & Turkan, I. Heavy metal accumulation by plants growing alongside the motor roads: A case study from Turkey. In Plants as Biomonitors Indic. Heavy Met. Terr. Environ. 515–522 (1993).

  20. Aksoy, A. & Öztürk, M. A. Phoenix dactylifera L. as a Biomonitor of Heavy Metal Pollution in Turkey (1996).

  21. Ali, I. & Aboul-Enein, H. Y. Speciation of arsenic and chromium metal ions by reversed phase high performance liquid chromatography. Chemosphere 48, 275–278 (2002).

    CAS  PubMed  Article  ADS  Google Scholar 

  22. Mukherjee, A., Agrawal, S. B. & Agrawal, M. Heavy metal accumulation potential and tolerance in tree and grass species. In Plant Responses to Xenobiotics (eds Singh, A. et al.) 177–210 (Springer, 2016).

    Chapter  Google Scholar 

  23. Ashraf, S., Ali, Q., Zahir, Z. A., Ashraf, S. & Asghar, H. N. Phytoremediation: Environmentally sustainable way for reclamation of heavy metal polluted soils. Ecotoxicol. Environ. Saf. 174, 714–727 (2019).

    CAS  PubMed  Article  Google Scholar 

  24. Öztürk, M., Özözen, G., Minareci, O. & Minareci, E. Determination of heavy metals in of fishes, water and sediment from the Demirköprü Dam Lake (Turkey). J. Appl. Biol. Sci. 2, 99–104 (2008).

    Google Scholar 

  25. Singh, H., Kumar, D. & Soni, V. Copper and mercury induced oxidative stresses and antioxidant responses of Spirodela polyrhiza (L.) Schleid. Biochem. Biophys. Rep. 23, 100781 (2020).

    PubMed  PubMed Central  Google Scholar 

  26. Raj, S., Singh, H., Trivedi, R. & Soni, V. Biogenic synthesis of AgNPs employing Terminalia arjuna leaf extract and its efficacy towards catalytic degradation of organic dyes. Sci. Rep. 10, 9616 (2020).

    PubMed  PubMed Central  Article  ADS  CAS  Google Scholar 

  27. Bouazizi, H., Jouili, H., Geitmann, A. & El Ferjani, E. Copper toxicity in expanding leaves of Phaseolus vulgaris L.: Antioxidant enzyme response and nutrient element uptake. Ecotoxicol. Environ. Saf. 73, 1304–1308 (2010).

    CAS  PubMed  Article  Google Scholar 

  28. Yadav, S. K. Heavy metals toxicity in plants: An overview on the role of glutathione and phytochelatins in heavy metal stress tolerance of plants. S. Afr. J. Bot. (2010).

    Article  Google Scholar 

  29. Neelima, P. & Reddy, K. J. Interaction of copper and cadmium with seedling growth and biochemical responses in Solanum melongena. Nat. Environ. Pollut. Technol. 1, 285–290 (2002).

    CAS  Google Scholar 

  30. Percival, G. C., Keary, I. P. & Sulaiman, A.-H. An assessment of the drought tolerance of Fraxinus genotypes for urban landscape plantings. Urban For. Urban Green. 5, 17–27 (2006).

    Article  Google Scholar 

  31. Binder, W. D. & Fielder, P. Chlorophyll fluorescence as an indicator of frost hardiness in white spruce seedlings from different latitudes. New For. 11, 233–253 (1996).

    Google Scholar 

  32. Maxwell, K. & Johnson, G. N. Chlorophyll fluorescence—A practical guide. J. Exp. Bot. 51, 659–668 (2000).

    CAS  PubMed  Article  Google Scholar 

  33. Hermans, C. et al. Quality assessment of urban trees: A comparative study of physiological characterisation, airborne imaging and on site fluorescence monitoring by the OJIP-test. J. Plant Physiol. 160, 81–90 (2003).

    CAS  PubMed  Article  Google Scholar 

  34. Papageorgiou, G. C. et al. Eugene I. Rabinowitch: A prophet of photosynthesis and of peace in the world. Photosynth. Res. 141, 143–150 (2019).

    PubMed  Article  CAS  Google Scholar 

  35. Baker, N. R. & Rosenqvist, E. Applications of chlorophyll fluorescence can improve crop production strategies: An examination of future possibilities. J. Exp. Bot. 55, 1607–1621 (2004).

    CAS  PubMed  Article  Google Scholar 

  36. L’Hirondelle, S. J., Simpson, D. G. & Binder, W. D. Chlorophyll fluorescence, root growth potential, and stomatal conductance as estimates of field performance potential in conifer seedlings. New For. 34, 235–251 (2007).

    Article  Google Scholar 

  37. Murchie, E. H. & Lawson, T. Chlorophyll fluorescence analysis: A guide to good practice and understanding some new applications. J. Exp. Bot. 64, 3983–3998 (2013).

    CAS  PubMed  Article  Google Scholar 

  38. Salvatori, E. et al. Plant stress analysis: Application of prompt, delayed chlorophyll fluorescence and 820 nm modulated reflectance. Insights from independent experiments. Plant Physiol. Biochem. 85, 105–113 (2014).

    CAS  PubMed  Article  Google Scholar 

  39. Pollastrini, M. et al. Taxonomic and ecological relevance of the chlorophyll a fluorescence signature of tree species in mixed European forests. New Phytol. 212, 51–65 (2016).

    CAS  PubMed  Article  Google Scholar 

  40. Fusaro, L., Salvatori, E., Mereu, S. & Manes, F. Photosynthetic traits as indicators for phenotyping urban and peri-urban forests: A case study in the metropolitan city of Rome. Ecol. Indic. 103, 301–311 (2019).

    CAS  Article  Google Scholar 

  41. Kumar, D., Singh, H., Raj, S. & Soni, V. Chlorophyll a fluorescence kinetics of mung bean (Vigna radiata L.) grown under artificial continuous light. Biochem. Biophys. Reports 24, 100813 (2020).

    Article  Google Scholar 

  42. Stirbet, A. & Govindjee,. On the relation between the Kautsky effect (chlorophyll a fluorescence induction) and Photosystem II: Basics and applications of the OJIP fluorescence transient. J. Photochem. Photobiol. B Biol. 104, 236. (2011).

    CAS  Article  Google Scholar 

  43. Pogrzeba, M. et al. Relationships between soil parameters and physiological status of Miscanthus x giganteus cultivated on soil contaminated with trace elements under NPK fertilisation vs. microbial inoculation. Environ. Pollut. 225, 163–174 (2017).

    CAS  PubMed  Article  Google Scholar 

  44. Singh, H. et al. Tolerance and decolorization potential of duckweed (Lemna gibba) to C.I. Basic Green 4. Sci. Rep. 11, 10889 (2021).

    CAS  PubMed  PubMed Central  Article  ADS  Google Scholar 

  45. Tuba, Z. et al. Chlorophyll a fluorescence measurements for validating the tolerant bryophytes for heavy metal (Pb) biomapping. Curr. Sci. 98, 1505–1508 (2010).

    CAS  Google Scholar 

  46. Kumar, D., Singh, H., Bhatt, U., Soni, V. & Allakhverdiev, S. Effect of continuous light on antioxidant activity, lipid peroxidation, proline and chlorophyll content in Vigna radiata L.. Funct. Plant Biol. 49, 145 (2021).

    Article  CAS  Google Scholar 

  47. Baker, N. R. & Oxborough, K. Chlorophyll fluorescence as a probe of photosynthetic productivity. In Chlorophyll a Fluorescence (eds Papageorgiou, G. C. & Govindjee) 65–82 (Springer, 2004).

    Chapter  Google Scholar 

  48. Stirbet, A. et al. Chlorophyll a fluorescence induction in higher plants: Modelling and numerical simulation. J. Theor. Biol. 193, 131–151 (1998).

    CAS  Article  ADS  Google Scholar 

  49. Dietz, K.-J. & Pfannschmidt, T. Novel regulators in photosynthetic redox control of plant metabolism and gene expression. Plant Physiol. 155, 1477–1485 (2011).

    CAS  PubMed  Article  Google Scholar 

  50. Hall, J. L. Cellular mechanisms for heavy metal detoxification and tolerance. J. Exp. Bot. (2002).

    Article  PubMed  Google Scholar 

  51. Sharma, S. S. & Dietz, K. J. The relationship between metal toxicity and cellular redox imbalance. Trends Plant Sci. 14, 43–50 (2009).

    CAS  PubMed  Article  Google Scholar 

  52. Stirbet, A. & Govindjee,. Chlorophyll a fluorescence induction: A personal perspective of the thermal phase, the J-I–P rise. Photosynth. Res. 113, 15–61 (2012).

    CAS  PubMed  Article  Google Scholar 

  53. Kalaji, H. M. et al. Experimental in vivo measurements of light emission in plants: A perspective dedicated to David Walker. Photosynth. Res. 114, 69–96 (2012).

    CAS  PubMed  Article  Google Scholar 

  54. Schansker, G., Tóth, S. Z., Holzwarth, A. R. & Garab, G. Chlorophyll a fluorescence: Beyond the limits of the QA model. Photosynth. Res. 120, 43–58 (2014).

    CAS  PubMed  Article  Google Scholar 

  55. Dkabrowski, P., Pawluśkiewicz, B., Baczewska, A. H. & Oglkecki Pawełand Kalaji, H. Chlorophyll a fluorescence of perennial ryegrass (Lolium perenne L.) varieties under long term exposure to shade. Zemdirbyste-Agriculture 102, 305 (2015).

    Article  Google Scholar 

  56. Živčák, M. et al. Measurements of chlorophyll fluorescence in different leaf positions may detect nitrogen deficiency in wheat. Zemdirbyste-Agriculture 101, 437 (2014).

    Article  Google Scholar 

  57. Kalaji, H. & Nalborczyk, E. Gas exchange of barley seedlings growing under salinity stress. Photosynthesis 25, 197–202 (1991).

    Google Scholar 

  58. Rapacz, M., Sasal, M., Kalaji, H. M. & Kościelniak, J. Is the OJIP test a reliable indicator of winter hardiness and freezing tolerance of common wheat and triticale under variable winter environments? PLoS ONE 10, e0134820 (2015).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  59. Dkabrowski, P. et al. Delayed chlorophyll a fluorescence, MR 820, and gas exchange changes in perennial ryegrass under salt stress. J. Lumin. 183, 322–333 (2017).

    Article  CAS  Google Scholar 

  60. Baker, A. J. M. & Brooks, R. Terrestrial higher plants which hyperaccumulate metallic elements. A review of their distribution, ecology and phytochemistry. Biorecovery 1, 81–126 (1989).

    CAS  Google Scholar 

  61. Dan, T. V., KrishnaRaj, S. & Saxena, P. K. Metal tolerance of scented geranium (Pelargonium sp. ‘Frensham’): Effects of cadmium and nickel on chlorophyll fluorescence kinetics. Int. J. Phytoremediat. 2, 91–104 (2000).

    CAS  Article  Google Scholar 

  62. Bolhar-Nordenkampf, H. R. et al. Chlorophyll fluorescence as a probe of the photosynthetic competence of leaves in the field: A review of current instrumentation. Funct. Ecol. 3, 497–514 (1989).

    Article  Google Scholar 

  63. KrishnaRaj, S., Mawson, B. T., Yeung, E. C. & Thorpe, T. A. Utilization of induction and quenching kinetics of chlorophyll a fluorescence for in vivo salinity screening studies in wheat (Triticum aestivum vars. Kharchia-65 and Fielder). Can. J. Bot. 71, 87–92 (1993).

    Article  Google Scholar 

  64. Devlamynck, R. et al. Lemna minor cultivation for treating swine manure and providing micronutrients for animal feed. Plants 10, 1124 (2021).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  65. Dewez, D., Goltsev, V., Kalaji, H. M. & Oukarroum, A. Inhibitory effects of silver nanoparticles on photosystem II performance in Lemna gibba probed by chlorophyll fluorescence. Curr. Plant Biol. 16, 15–21 (2018).

    Article  Google Scholar 

  66. Whitacre, D. M. Reviews of Environmental Contamination and Toxicology Vol. 232 (Springer, 2014).

    Book  Google Scholar 

  67. National Biodiversity Authority. National Biodiversity Act, 2002 (MOEF, 2002).

    Google Scholar 

  68. OECD. Lemna sp. Growth Inhibition Test. Oecd Guidel. Test. Chem. 22 (2002).

  69. Dirilgen, N. Mercury and lead: Assessing the toxic effects on growth and metal accumulation by Lemna minor. Ecotoxicol. Environ. Saf. 74, 48–54 (2011).

    CAS  PubMed  Article  Google Scholar 

  70. Teisseire, H. & Guy, V. Copper-induced changes in antioxidant enzymes activities in fronds of duckweed (Lemna minor). Plant Sci. (2000).

    Article  Google Scholar 

  71. Strasser, R. J., Tsimilli-Michael, M. & Srivastava, A. Analysis of the chlorophyll a fluorescence transient. In Chlorophyll a Fluorescence (eds Papageorgiou, G. C. & Govindjee) 321–362 (Springer, 2004).

    Chapter  Google Scholar 

  72. Strasser, R. G. On the OJIP fluorescence transient in leaves and D1 mutants of Chlamydomonas reinhardtii. In Research in Photosynthesis: Proceedings of the IXth International Congress on Photosynthesis, Vol. 2, 29–32 (1992).

  73. Strasser, R. J., Srivastava, A. & Tsimilli-Michael, M. The Fluorescence Transient as a Tool to Characterize and Screen Photosynthetic Samples. Probing Photosynthesis: Mechanisms, Regulation and Adaptation (2000).

  74. Strasser, R. J., Srivastava, A. & Govindjee,. Polyphasic chlorophyll a fluorescence transient in plants and cyanobacteria. Photochem. Photobiol. 61, 32–42 (1995).

    CAS  Article  Google Scholar 

  75. Stirbet, A. Biology On the relation between the Kautsky effect (chlorophyll a fluorescence induction) and Photosystem II: Basics and applications of the OJIP fluorescence transient q. J. Photochem. Photobiol. B Biol. 104, 236–257 (2011).

    CAS  Article  Google Scholar 

  76. Bhatt, U. et al. Rehydration quickly assembles photosynthetic complexes in desiccation tolerant Riccia gangetica. Biomed. J. Sci. Tech. Res. 30, 23034–23037 (2020).

    Google Scholar 

  77. Bhatt, U., Singh, H., Kumar, D. & Soni, V. Rehydration induces quick recovery of photosynthesis in desiccation tolerant moss Semibarbula orientalis. J. Plant Sci. Res. 35, 183 (2019).

    Article  Google Scholar 

  78. Bhatt, U., Singh, H., Kumar, D., Strasser, R. J. & Soni, V. Severe leaf-vein infestation upregulates antioxidant and photosynthetic activities in the lamina of Ficus religiosa. Acta Physiol. Plant 44, 1–9 (2022).

    Article  CAS  Google Scholar 

  79. Soni, V., Keswani, K., Bhatt, U., Kumar, D. & Singh, H. In vitro propagation and analysis of mixotrophic potential to improve survival rate of Dolichandra unguis-cati under ex vitro conditions. Heliyon 7, e06101 (2021).

    PubMed  PubMed Central  Article  Google Scholar 

  80. Gonzalez-Mendoza, D. et al. Copper stress on photosynthesis of black mangle (Avicennia germinans). An. Acad. Bras. Cienc. 85, 665–670 (2013).

    CAS  PubMed  Article  Google Scholar 

  81. Tsimilli-Michael, M. Revisiting JIP-test: An educative review on concepts, assumptions, approximations, definitions and terminology. Photosynthetica 58, 275–292 (2020).

    CAS  Article  Google Scholar 

  82. Baker, N. Chlorophyll fluorescence: A probe of photosynthesis in vivo. Annu. Rev. Plant Biol. 59, 89–113 (2008).

    CAS  PubMed  Article  Google Scholar 

  83. Kalaji, H. M. et al. Frequently asked questions about in vivo chlorophyll fluorescence: Practical issues. Photosynth. Res. (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  84. Kalaji, H. M. et al. Chlorophyll a fluorescence as a tool to monitor physiological status of plants under abiotic stress conditions. Acta Physiol. Plant 38, 102 (2016).

    Article  CAS  Google Scholar 

  85. Panda, D., Rao, D. N., Sharma, S. G., Strasser, R. J. & Sarkar, R. K. Submergence effects on rice genotypes during seedling stage: Probing of submergence driven changes of photosystem 2 by chlorophyll a fluorescence induction OJIP transients. Photosynthetica 44, 69–75 (2006).

    CAS  Article  Google Scholar 

  86. Schansker, G. et al. Characterization of the 820-nm transmission signal paralleling the chlorophyll a fluorescence rise (OJIP) in pea leaves. Funct. Plant Biol. 30, 785–796 (2003).

    CAS  PubMed  Article  Google Scholar 

  87. Strasser, R. J., Tsimilli-Michael, M. & Srivastava, A. Analysis of the chlorophyll a fluorescence transient. In Chlorophyll a Fluorescence: A Signature of Photosynthesis (eds Papageorgiou, G. C. & Govindjee) 321–362 (Springer, 2004).

    Chapter  Google Scholar 

  88. Neubauer, C. & Schreiber, U. The polyphasic rise of chlorophyll fluorescence upon onset of strong continuous illumination: I. Saturation characteristics and partial control by the photosystem II acceptor side. Z. für Nat. C 42, 1246–1254 (1987).

    CAS  Article  Google Scholar 

  89. Schansker, G., Tóth, S. Z. & Strasser, R. J. Dark recovery of the Chl a fluorescence transient (OJIP) after light adaptation: The qT-component of non-photochemical quenching is related to an activated photosystem I acceptor side. Biochim. Biophys. Acta Bioenerg. 1757, 787–797 (2006).

    CAS  Article  Google Scholar 

  90. Schansker, G., Tóth, S. Z. & Strasser, R. J. Methylviologen and dibromothymoquinone treatments of pea leaves reveal the role of photosystem I in the Chl a fluorescence rise OJIP. Biochim. Biophys. Acta Bioenerg. 1706, 250–261 (2005).

    CAS  Article  Google Scholar 

  91. Goltsev, V. N. et al. Variable chlorophyll fluorescence and its use for assessing physiological condition of plant photosynthetic apparatus. Russ. J. Plant Physiol. (2016).

    Article  Google Scholar 

  92. Martinazzo, E. G., Ramm, A. & Bacarin, M. A. The chlorophyll a fluorescence as an indicator of the temperature stress in the leaves of Prunus persica. Braz. J. Plant Physiol. (2012).

    Article  Google Scholar 

  93. Janka, E., Körner, O., Rosenqvist, E. & Ottosen, C. O. High temperature stress monitoring and detection using chlorophyll a fluorescence and infrared thermography in chrysanthemum (Dendranthema grandiflora). Plant Physiol. Biochem. (2013).

    Article  PubMed  Google Scholar 

  94. DeEll, J. R., Kooten, O. V., Prange, R. K. & Murr, D. P. Applications of chlorophyll fluorescence techniques in postharvest physiology. Hortic. Rev. (Am. Soc. Hortic. Sci.) 23, 69–107 (2010).

    Article  Google Scholar 

  95. Küpper, H., Šetlík, I., Spiller, M., Küpper, F. C. & Prášil, O. Heavy metal-induced inhibition of photosynthesis: Targets of in vivo heavy metal chlorophyll formation. J. Phycol. 38, 429–441 (2002).

    Google Scholar 

  96. Tsimilli-Michael, M., Pêcheux, M. & Strasser, R. J. Vitality and stress adaptation of the symbionts of coral reef and temperate foraminifers probed in hospite by the fluorescence kinetics OJIP. Arch. Des. Sci. 51, 205–240 (1998).

    Google Scholar 

  97. Björkman, O. & Demmig, B. Photon yield of O 2 evolution and chlorophyll fluorescence characteristics at 77 K among vascular plants of diverse origins. Planta 170, 489–504 (1987).

    PubMed  Article  Google Scholar 

  98. Gautam, A., Agrawal, D., SaiPrasad, S. V. & Jajoo, A. A quick method to screen high and low yielding wheat cultivars exposed to high temperature. Physiol. Mol. Biol. Plants 20, 533–537 (2014).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  99. Mehta, P., Jajoo, A., Mathur, S. & Bharti, S. Chlorophyll a fluorescence study revealing effects of high salt stress on Photosystem II in wheat leaves. Plant Physiol. Biochem. 48, 16–20 (2010).

    CAS  PubMed  Article  Google Scholar 

  100. Zushi, K., Kajiwara, S. & Matsuzoe, N. Chlorophyll a fluorescence OJIP transient as a tool to characterize and evaluate response to heat and chilling stress in tomato leaf and fruit. Sci. Hortic. (Amsterdam) 148, 39–46 (2012).

    CAS  Article  Google Scholar 

  101. Zushi, K. & Matsuzoe, N. Using of chlorophyll a fluorescence OJIP transients for sensing salt stress in the leaves and fruits of tomato. Sci. Hortic. (Amsterdam) 219, 216–221 (2017).

    CAS  Article  Google Scholar 

  102. Grieco, M., Suorsa, M., Jajoo, A., Tikkanen, M. & Aro, E. M. Light-harvesting II antenna trimers connect energetically the entire photosynthetic machinery—Including both photosystems II and I. Biochim. Biophys. Acta Bioenerg. (2015).

    Article  Google Scholar 

  103. Heber, U., Soni, V. & Strasser, R. J. Photoprotection of reaction centers: Thermal dissipation of absorbed light energy vs charge separation in lichens. Physiol. Plant. 142, 65–78 (2011).

    CAS  PubMed  Article  Google Scholar 

  104. Srivastava, A. et al. Greening of peas: Parallel measurements of 77 K emission spectra, OJIP chlorophyll a fluorescence transient, period four oscillation of the initial fluorescence level, delayed light emission, and P700. Photosynthetica 37, 365–392 (1999).

    CAS  Article  Google Scholar 

  105. Goldman, D. E. Potential, impedance, and rectification in membranes. J. Gen. Physiol. 27, 37–60 (1943).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  106. Kalaji, H. M. et al. Frequently asked questions about chlorophyll fluorescence, the sequel. Photosynth. Res. 132, 13–66 (2017).

    CAS  PubMed  Article  Google Scholar 

  107. Hassan, J. & Tabarraei, H. Toxicity of copper on rainbow trout: Lethal concentration or lethal dose evaluation. Environ. Sci 11, 98–102 (2015).

    CAS  Google Scholar 

  108. Kellaf, N. & Zardoui, M. Growth, photosynthesis and respiratory response to copper in Lemna minor: A potential use of duckweed in biomonitoring. J. Environ. Health Sci. Eng. 7, 299–306 (2010).

    Google Scholar 

  109. Appenroth, K.-J., Krech, K., Keresztes, A., Fischer, W. & Koloczek, H. Effects of nickel on the chloroplasts of the duckweeds Spirodela polyrhiza and Lemna minor and their possible use in biomonitoring and phytoremediation. Chemosphere 78, 216–223 (2010).

    CAS  PubMed  Article  ADS  Google Scholar 

  110. Stirbet, A. & Govindjee,. Chlorophyll a fluorescence induction: Understanding the thermal phase, the JIP rise. Photosyn. Res 113, 15–61 (2012).

    CAS  Article  Google Scholar 

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The authors thank the Mohanlal Sukhadia University, India for providing laboratory facilities. Authors are also grateful to Prof. Reto J. Strasser, University of Geneva, Switzerland for his help in the analysis of chlorophyll fluorescence data.

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H.S. conceived the original screening and research plans; V.S. supervised the experiments; H.S.; D.K. and V.S. performed the experiments; H.S. designed the experiments and analyzed the data; H.S. conceived the project and wrote the article with contributions of all the authors; V.S. supervised and completed the writing and agrees to serve as the author responsible for contact and ensures communication.

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Correspondence to Vineet Soni.

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Singh, H., Kumar, D. & Soni, V. Performance of chlorophyll a fluorescence parameters in Lemna minor under heavy metal stress induced by various concentration of copper. Sci Rep 12, 10620 (2022).

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