Cultures of the great duckweed, Spirodela polyrhiza (L.) Schleiden (RDSC Clone ID 5634), originally sourced from a drainage canal in Nature Park Kopački Rit, Croatia were grown in a modified Steinberg nutrient solution (Steinberg, 1946) under controlled aseptic conditions in 3-L culture flasks. The nutrient solution was changed every 7 to 10 days and was composed of 3.46 mM KNO₃, 1.25 mM Ca(NO₃)₂, 0.66 mM KH₂PO₄, 0.072 mM K₂HPO₄, 0.41 mM MgSO₄, 1.94 μM H₃BO₃, 0.63 μM ZnSO₄, 0.18 μM Na₂MoO₄, 0.91 μM MnCl₂, 2.81 μM Fe(III)EDTA, 1 and 0.22 EDTA; pH was 5.5 ± 0.1(ISO, 2017). The photoperiod in a plant growth chamber was set to 16-/8-h light/dark, and light intensity was 120 μmol·s⁻¹·m⁻² (TLD and CWL 36W; Philips). Temperature was set to 21/25 °C in the dark/light.

For pre-cultivation, cultures were started with a single two-frond colony (mother and daughter fronds) in 6-well cell culture plates (Guangzhou Jet Bio-Filtration Co., Ltd., China), with one colony per well. After 72 h of pre-cultivation, colonies with four fully developed fronds were inoculated into 10 ml of a nutrient solution with different cadmium concentrations on a new set of 6-well culture plates. Pre-cultivation and experiment conditions were the same as cultivation conditions regarding photoperiod, light intensity, and temperature. Growth conditions during pre-cultivation enabled an approximately doubling time of 2.5 days as required by standard protocols (ISO, 2005, 2017). Also, growth rates of S. polyrhiza stock cultures were monitored 14 days before the start of the experiment to check the validity and meet the conditions of the standard protocols. The cadmium chloride anhydride (≥99 %; Fluka Chemie, Switzerland) exposure series applied in a modified Steinberg (StMo) medium included the following nominal concentrations of Cd: 0 (C0, control), 7 (C1), 14 (C2), 28 (C3), 56 (C4), 111 (C5), 222 (C6), 445 (C7), and 890 μM (C8). The chosen concentration range allowed for measurements of stronger immediate and short-term effects and enabled better differentiation among the fronds. Since the great duckweed develops offspring vegetatively from meristematic pockets at the frond's proximal end, the maternal frond is the oldest and is designated as MF, mother frond; two new offspring develop from it: DF₁, the first daughter frond and DF₂, the second daughter frond. The granddaughter frond (GDF) originates from the first daughter frond. All measurements were made on individual fronds, separated before measurement gently by hand, to determine Cd's effect on differently aged and developed plants. Also, measurements were made after 6, 24, and 72 h of exposure to different Cd concentrations.

Carotenoids (Car x+c), chlorophyll a (Chl_a), and chlorophyll b (Chl_b) of different S. polyrhiza fronds were determined spectrophotometrically from 0.1 g of fresh weight samples in three replicates. Plant samples were extracted with pure acetone containing magnesium hydroxycarbonate (0.5% w/v). The extract was centrifuged (18,000 g, 4°C, and 15 min), and after several re-extractions, absorbances were recorded at 470, 644.8, and 661.6 nm (Analytik Jena, Specord 40). Pigment contents were calculated according to Lichtenthaler and Buschmann (2001):

Chlorophyll fluorescence was measured in every frond, in the four-frond colony, control, and treatment groups after 6, 24, and 72 h of exposure. Each measurement was performed on six replicates with a Handy PEA⁺continuous-excitation type chlorophyll fluorescence analyzer (Hansatech, United Kingdom). The plants were previously adapted to the dark for 30 min. The transient was induced with a red-light pulse of 3,000 μmol·m⁻²·s⁻¹ and analyzed using the JIP-test (Strasser and Strasser, 1995; Stirbet et al., 2014; Tsimilli-Michael, 2020). For a detailed evaluation of the OK, OJ, JI, and IP phases, transient curves were normalized as relative variable fluorescence at time t as follows: Vt=(Ft-F0)(Fm-F0)=ΦF(t), where Φ_F(t) is the fluorescence yield. Kinetic differences were calculated from the relative variable fluorescence by subtracting the transient of stressed and control plants. For detailed definitions and explanations of the JIP test parameters, refer to Supplementary Table 1 (Stirbet and Govindjee, 2011; Kalaji et al., 2016, 2017; Stirbet et al., 2018; Tsimilli-Michael, 2020).

Oxygen exchange rate was measured with a Clark-type oxygen electrode and a Chlorolab2⁺ system (Hansatech, United Kingdom) at 21°C. Free-floating species, like S. polyrhiza, take up most of the carbon needed via gaseous CO₂ from the air. Under laboratory conditions, in a carbonate-free medium, 63% of the carbon used by these plants in photosynthesis comes from the atmosphere. However, duckweeds take 86% carbon from aqueous inorganic carbon (Filbin and Hough, 1985; Landolt and Kandeler, 1987). Therefore, to estimate O₂ exchange rates accurately, we used a 0.1 M bicarbonate buffer (15 ml 0.1 M K₂CO₃/85 ml 0.1 M NaHCO₃, adjusted to pH 8.9) that provided a constant saturating CO₂ concentration in which S. polyrhiza fronds were placed (Walker, 1987). Under continuous stirring, changes in oxygen concentration in the dark and light (at 120 μmol·m⁻²·s⁻¹ of photosynthetically active radiation) were recorded. Net O₂ exchange rates were calculated per phase by determining the slope of O₂ exchange curves over 5 min of dark and light periods in three consecutive repetitions (30 min). The net result of oxygen evolution was observed as the sum of all oxygen fluxes between the buffer medium and S. polyrhiza fronds (here, the buffer was the substrate for gas exchange). These fluxes included gross O₂ production (the rate of water splitting at PSII), the oxygenation of Rubisco, mitochondrial respiration, photorespiration (if present), and all other metabolic processes (e.g., the Mehler reaction and nitrate reduction). Oxygen uptake rate during the dark was an estimate of mitochondrial respiration (dark respiration). Both oxygen evolution and uptake rate represent metabolic rates. All the measurements were replicated three times, and rates were normalized to plant sample fresh weight (μmole O₂ g⁻¹FW·h⁻¹).

Anatomical observations were performed on mother and daughter plants exposed to C1 (7 μM Cd) and C3 (28 μM Cd) concentrations after 6, 24, and 72 h of exposure with a transmission electron microscope (TEM) and after 72 h with a light microscope. For light microscopy, cutoffs of S. polyrhiza fronds were fixated (24 h, 4°C) with 1 % glutaraldehyde in a 50-mM phosphate buffer (pH = 6.8). Gradual dehydration was carried out at room temperature in 1-h steps with 2-methoxyethanol, 96 % ethanol, n-propanol, and n-butanol. Pre-infiltration was performed for 24 h with equal parts of an infiltration solution and n-butanol. Dehydrated specimens were embedded into a Leica Historesin Embedding kit. Semi-thin (3 μm) sections were stained with a Lugol solution to detect differences in starch distribution and observed with a light microscope (Carl Zeiss 106; Jena, Germany) and Moticam 350.

For TEM, a primary fixation of S. polyrhiza frond tiny cutoffs was performed for 60 min with 1% glutaraldehyde in a 50-mM cacodylate buffer (pH 7.2) while gently evacuating to remove air in the tissue. After rinsing with a cacodylate buffer for 3 × 10 min, post-fixation was carried out in 1% OsO₄ dissolved in a 50-mM cacodylate buffer (8–12 h, in the dark at 4°C). Gradual dehydration was performed by passing the samples through increasing ethanol concentrations and propylene oxide at the end. Dehydrated specimens were embedded into Spurr's propylene oxide and epoxy resin mixture in three 2–3 h infiltration steps (2:1, 1:1, 1:2) while gently agitating vials at room temperature. The specimens were then placed into fresh Spurr's mixture for 4–5 h at 40°C, replaced with a pure new resin, and, finally, 3–4 h later, allowed to polymerize at 60–65°C for 48 h. Ultra-thin (80 nm) sections were cut with a Leica EM UC7 ultramicrotome (Leica Microsystems, Vienna, Austria) and post-stained with 0.02 M lead citrate dissolved in fresh distilled water containing 0.16 M sodium hydroxide (3 min) and 2% uranyl acetate dissolved in distilled water (10 min). The sections were examined in Zeiss Libra 120 Plus TEM (Carl Zeiss AG, Oberkochen, Germany). Micrographs were taken with an XF416 4k camera (Tietz Video and Image Processing Systems GmbH, Gauting, Germany).

To determine how S. polyrhiza pigment content responds to Cd treatment during 72 h of exposure and the amount of variation in pigment content accounted for by frond age, we conducted a two-way analysis of covariance (ANCOVA) with frond age as a controlling variable (covariate). When necessary, variables were log-transformed to meet the statistical assumptions of the ANCOVA. The mean difference between treatment and control was calculated for each tested parameter to assess similarities in response to Cd between different generations of fronds. As the calculation of the mean difference does not consider the standard deviation within the groups, Hedges effect size was calculated. Effect size is a relevant interpretation of an estimated magnitude of an effect from effect statistics or, in other words, a quantitative measure based on overall standard deviation (both control and treatment). The bigger the effect size, the higher the increase/decrease in the treatment's specific parameter (Hedges and Olkin, 1985). The statistical significance (acquired by t-test and confidence interval statistics) of the differences between treatment and control in individual parameters coincides with Cd treatment's high to very high effect. By combining the estimation of effect size and confidence interval statistics, we obtained more information than p-value testing alone provides (Nakagawa and Cuthill, 2007). Presentation and interpretation of effect size reduced the prevailing misinterpretation of null-hypothesis and p-value testing and improved scientific results, providing a meaningful evaluation of treatment effect and its comparison among fronds and exposure times.

Furthermore, principal component analysis (PCA), a multivariate statistical technique, was conducted to reduce a large set of Chl fluorescence parameters to the most informative ones (Goltsev et al., 2012; Kalaji et al., 2017). Also, PCA was conducted to investigate a combined effect of cadmium concentrations and frond age on the structure of variability among fronds and correlations between Chl fluorescence parameters with pigment contents. Factor loadings on the first two principal components (PCs) were shown after oblique (direct oblimin) rotation. At the same time, a hierarchical k-means clustering algorithm on main features was used to obtain optimal cluster solutions (Bussotti et al., 2020). Data presentations were made in Excel and statistical analyses were conducted with Statistica 13.5 (© 1984–2018 TIBCO Software Inc.).

A four-frond colony of the great duckweed S. polyrhiza represents generations of different plant ages: mother, daughter, and granddaughter plants. Plant pigment content changes with age, so if we control the age response, we will improve the likelihood of finding a significant two-way interaction if one exists. Therefore, the results of the two-way ANCOVA model explained 74 to 83% of the variability in pigment content and their ratios (Table 1). Cd concentrations, exposure time, and their significant interaction indicated that the response rate differs between treatments, thus bringing important information explaining the variability of chlorophyll concentrations. The duration of Cd exposure accounted for the most variation in pigment content as the most influential variable; as shown in Table 1, the Fisher F-statistic and effect size are highest for the time variable. A more detailed overview of the statistical analysis results is given in Table 2.

Since time controls the response of Chl content to cadmium treatments and not the frond age (Table 1), based on pigment content and statistical analysis of measured values, a multilayer perceptron model (MLP) algorithm with a three-layer structure illustrated S. polyrhiza response. The surface response 3D plot for the relationship between time and concentration-dependent pigment content in Figure 1 shows that high concentration and longer exposure time resulted in highest reduction in pigment content. The inclined profile of surface plots implies the interacting effect of both factors. Since the ANCOVA showed that fronds do not account for significant variability in chlorophyll content, frond age was used as an input variable with summation in Chl and activation function in Car. The observed decrease in pigment content could be related to either reduction in the rate of chlorophyll biosynthesis by Cd inhibition of protochlorophyllide reductase or chlorophyll degradation (Ci et al., 2010; Song et al., 2019; Franić et al., 2020; Janeeshma et al., 2021). However, carotenoid contents behaved slightly differently in fronds of different ages. Their reduction rate depended on exposure time, Cd concentration, and frond age (Table 1 and Figure 1). Therefore, a response surface plot in Figure 1 shows the relationship between Car content and the ratio Chl/Car with S. polyrhiza frond age. The oldest MFs exhibited a more significant and faster reduction of total carotenoids as Cd concentrations were increased. There was also a subsequent increase in total carotenoids with increase in cadmium concentration, most likely due to pigment degradation and accelerated senescence caused by Cd (Krupa and Moniak, 1998; Tkalec et al., 2008).

The polyphasic rise of Chl fluorescence induction kinetics in S. polyrhiza exposed for 6, 24, and 72 h to Cd concentrations ranging from 0 to 890 μM was monitored to localize the action sites of Cd stress in the photosynthetic electron transport chain. For simplification, only the results obtained after 72 h of Cd exposure were selected for graphical presentation in Supplementary Figure 2. However, to reveal the time sequence of events, differential curves of Chl fluorescence transient normalized between O-K, O-J, and I-P steps were shown after 6, 24, and 72 h of Cd exposure (Supplementary Figures 3–5). The effect of cadmium stress on electron transport at the donor and acceptor sides of PSII and PSI was determined from quantitative data of JIP-test parameters. The mean differences of these parameters between treatments and the control are shown in Figures 2–4.

The results show how cadmium affects different photosynthetic electron transport segments relative to their structure and function. Connectivity between reaction centers and their antenna complexes was expressed as an L-band of Chl fluorescence transient normalized between O-K steps (Stirbet and Govindjee, 2011). The L-band's positive deviations increased their amplitude with Cd concentration, exposure time, and frond age (Supplementary Figure 3). A higher amplitude of L-band in mother plants meant a more significant loss of antenna connectivity due to Cd stress. Moreover, the decrease in the number of active reaction centers within the chlorophyll bed by increasing cadmium concentrations was confirmed by a more significant increase in antenna size estimation or ABS/RC in older mature plants (MF and DF₁, Figure 2A). Younger plants had a similar rise in ABS/RC (from 10 to 20 %) regardless of how much Cd they were exposed to (C1–C5). This increase meant either active reaction centers were inactivated or the apparent antenna size increased. Either way, in low concentrations and younger fronds, a slight rise in ABS/RC was probably a protective mechanism from photooxidative damage and excess in absorbed light energy (Yusuf et al., 2010).

In the MFs, an increase in ABS/RC was accompanied by increased trapped photon flow (TR₀/RC, V_K) with the same trend. An increase in TR₀/RC was accompanied by a decrease in the maximum quantum yield of PSII (ϕP₀), indicating that the probability of a trapped photon ending up in the RC and causing a photochemical event decreases with increase in Cd concentration and with greater effect size in the MFs than in the DFs. This increase in trapping and reduction of maximum quantum yield at higher concentrations was probably due to the rise in the number of inactive centers, which dissipated the total energy as heat, not fluorescence, as confirmed by the ratio of dissipation energy to the number of active RCs (Figures 2, 4). In the younger plants (DF and GDF), the mentioned minor increase in ABS/RC was accompanied by a similar rise in TR₀/RC. Simultaneously, the maximum quantum yield of PSII photochemistry underwent minor changes indicating that antenna size supplied excitation energy to active RCs, and that there was a probable increase in the young plants' functional antenna size (Figures 2–4).

The donor side of PSII is a place of the catalytic site of water cleavage and formation of the dioxygen molecule (Stirbet and Govindjee, 2011). Differential curves of Chl fluorescence transient, calculated between O-J steps representing K-band (Supplementary Figure 4), variable fluorescence at K step, and the fraction of O₂ evolving centers (OEC), were used to examine the PSII donor side's condition (Figure 3). K-bands were strongly expressed after 72 h of Cd exposure. The amplitude increased with increase in Cd concentration (Supplementary Figure 4), while the size of the effect increased with plant age (Figures 3A,D). This V_K increase on the normalized O-J curve signified that OEC activity was damaged, and that damage was more intense in the older fronds.

The maximal rate of closed reaction center fraction (M₀) accumulation, the probability by which the captured exciton moves the electron from the primary acceptor Q_A to Q_B (ψE₀) described by variable fluorescence at J step (V_J), and the quantum yield of electron transport from QA- to PQ PSII (ϕE₀) were used to examine the effect of Cd on the PSII acceptor side. The initial slope of the relative variable fluorescence curve (M₀) indicates that the net closing rate of the reaction centers was increased by about 200% in MFs exposed to 56 (C4) and 111 (C5) μM Cd after 72 h, as opposed to the about 37 and 26 % increase in younger fronds (DF₂, GDF) exposed to the same concentrations of Cd (Figure 3E). However, some slight differences in M₀ increase among the fronds (range was from 16 to 18% increase) were noted when exposed to the lowest Cd concentration. At the same time, the probability that a trapped exciton moves an electron into the electron transport chain beyond Q_A (ψE₀) was decreased from 20% in young plants up to 80% in mother plants when exposed to C4 and C5 Cd after 72 h (Figure 4B). This decrease was concentration-dependent in mother fronds, but this concentration gradient was not observed in younger fronds. Therefore, the electron transport on the acceptor side of PSII between Q_A and Q_B was inhibited by Cd concentrations above 28 (C3) μM. Cadmium effect increased with plant age and exposure time (Supplementary Figure 4, Figures 3, 4).

Considerable decline in the probability that an electron is transferred from the reduced intersystem electron acceptors to PSI end-electron acceptors (δR₀) indicated that intersystem electron transport was inhibited, and that the size of the effect was more extensive in younger fronds than mother fronds (Figure 4D). However, cadmium increased the number of non-reducing reaction centers (V_I) with almost an equal effect size (Figure 3C). Electron transfer efficiency from PSII to PSI end-electron acceptors (ΔV_IP) was reduced by 20 % in all the fronds and concentrations within the first 6 h of exposure (Supplementary Figure 5). After 24 h, a concentration gradient was formed in older fronds, while same as with all the other parameters, no gradient was found in younger fronds. With more prolonged exposure to Cd, electron transfer efficiency from PSII to PSI end-electron acceptors was inhibited by 20–50% in the MFs, around 50 % in the DF₁s, and 40–60% in the younger fronds (DF₂s and GDFs). This lower effect of higher cadmium concentrations on older fronds resulted from more significant inhibition in previous electron transport steps in these fronds. In fronds exposed to lower Cd concentrations, there was an increase in the reduction rate of PSI end-electron acceptors, followed by a subsequent decrease. This traffic jam caused by a blockage on the acceptor side slowed down the electron transfer through both photosystems, causing a reduction in PI_ABS and PI_TOT (Figures 4E,F).

The presented results of oxygen exchange rates refer to measurements after exposure for 72 h to C1 (7 μM) and C3 (28 μM) Cd concentrations and represent a mean difference between treatment and control and treatment effect sizes (Figure 6). In mother fronds, the net oxygen uptake during the dark after 72 h of exposure to C1 was significantly decreased by 28 % relative to the control MFs. However, the net oxygen evolution rate was increased, although not significantly (6%). In the youngest GDF fronds, and the dark oxygen uptake rate after 72 h of exposure to C1 significantly increased relative to the control GDF fronds by 114 %. At the same time, the net exchange of O₂ was significantly reduced by 53%. The oxygen uptake rate in fronds exposed to C3 increased considerably in all the fronds, from 75% in the oldest MFs to 124% in the younger DF₂s. On the other hand, the net O₂ evolution rate was significantly decreased from 183 % in MFs up to more than 300% in more immature fronds (DF₂s and GDFs), a six-fold decrease in contrast to C1.

Prasad et al. (2001) determined that Lemna trisulca exposed to low concentrations of Cd increases its respiration rates, mobilizing metabolism, and that under severe stress, respiration decreases, indicating metabolism damage. According to Amthor (2000), adverse growth conditions will increase respiration relative to photosynthesis and, thus, reduce the total carbon yield in the plant. Under stressful conditions, significantly increased respiration rates of S. polyrhiza indicated an improvement in physiological activities, whether the rate of respiration or photosynthesis. Improving photosynthetic activities may subsequently increase growth rates (Jager et al., 2013). However, this hormetic effect of Cd on respiration and photosynthesis may be an adaptive response of the plant organism to distribute the amount of energy available to suppress losses. Because low concentrations can cause partially repairable tissue injuries, the metabolic rate will increase by activating mechanisms involved in mitigating the harmful effects of Cd. Why? To repair stress-induced damage by, e.g., removing reactive oxygen species (Tkalec et al., 2008) or binding Cd to starch (Higuchi et al., 2015). Repair is a process that consumes energy and increases respiration rate (Jager et al., 2013). As the respiration rate increases, substrates are diverted from growth. Consequently, the growth rate of S. polyrhiza decreases, even at the lowest cadmium concentration (Supplementary Figure 1).

Transmission electron microscopy (TEM) was conducted to compare ultrastructural changes in S. polyrhiza chloroplasts upon exposure for 6, 24 and 72 h to C1 (7 μM) and C3 (28 μM) Cd concentrations in older and younger fronds (Figure 7). In the control older fronds, the observed chloroplasts with well-developed granum stacks connected by intergranal thylakoids, small starch grains (rarely), and several large plastoglobuli (Figures 8A,C) are consistent with their age. In the younger control fronds (GDFs), elongated intergranal thylakoids in one or two layers are embedded in the chloroplast stroma with rarely formed granum stacks (Figures 8B,D), small plastoglobuli, and regular shaped starch grain (one or two). Single or paired thylakoids without large granum stacks could be associated with a reduced risk of photoinhibition under stress (Krupinska et al., 2012). Cd-induced changes in the ultrastructure of the chloroplasts were visible even after 6 h of exposure to the lowest concentration used.

Gradual degradation of the thylakoid system was observed in all the fronds. For example, wavy and dilated thylakoids were marked in mother fronds (Figure 8G), similar to Lemna minor exposed to Cd (Tkalec et al., 2008). The size of plastoglobuli decreases after 24 h of exposure. Thylakoids are pushed toward the chloroplast periphery in younger fronds, and starch grains occupy chloroplast volume. With prolonged exposure to Cd, chloroplasts are inflated and contain larger starch grains of irregular shape and structure. Plastoglobuli are small but numerous. In younger fronds, starch content increased with cadmium concentration and exposure duration, while in older fronds, starch content was increased in low Cd and decreased in higher concentrations (Figures 7A–F, 9D,E). The accumulation of starch grains resulted from an imbalance between photosynthate production and export. Therefore, the energy produced by metabolic reactions either reinforced the younger frond's resistance to cadmium stress or compensated for the reduced enzyme activity caused by the same Cd stress.

Although not directly analyzed in this study, possible differences in Cd uptake and accumulation in plants of different ages could influence photosynthetic activity, since Cd is a cumulative toxicant (Haider et al., 2021). It has been determined that S. polyrhiza can tolerate and accumulate Cd for 1 week even when exposed to 7 μg Cd ml⁻¹(Sajwan and Ornes, 1994). The stage of plant development significantly affects physiological activity, especially its roots. It is known that Cd bonds to duckweed roots (Xue et al., 2018), affects the formation of turions by causing nutrient deficiency, and accumulates in mature fronds (Oláh et al., 2015). Cd accumulation depends on the number of binding sites in cells and the capacity to uptake Cd (Chen et al., 2021), and in S. polyrhiza, it is directly proportional to its exposure concentration and duration (Rolli et al., 2010). After several hours of exposure, we observed that S. polyrhiza rejected its roots to avoid further uptake. We have also observed the abscission phenomenon of colony disintegration upon exposure to Cd with almost immediate release of DFs from the maternal colony before reaching maturity (Figure 9 and Supplementary Figure 1). This avoidance strategy could also result in differences in Cd accumulation in young and mature fronds and needs to be taken into future consideration in explaining the sensitivity of the photosynthetic apparatus to Cd toxicity.

The oxygen-evolving complex was not affected by low Cd concentration in the mother fronds. Therefore, gross O₂ production rate was not affected, and the supply of electrons was similar to the control fronds. However, with a higher PI_TOT decrease, both performance indices decreased, meaning that electrons had to be redirected toward alternative electron sinks. A shortage in NADPH could have been, thus induced, affecting the Calvin Benson cycle (Krupa et al., 1999; Hald et al., 2008). The increased starch grains observed in chloroplasts of MFs treated with the lowest cadmium concentration could also mean that the Calvin cycle was in a latent state. Since the dark O₂ uptake rate was reduced, starch probably was not mobilized during the night, so decreased respiration rate could have possibly led to accumulation of reducing equivalents, changing the ratio of ATP/NADPH. The accumulation of reducing equivalents favored cyclic electron transport around PSI, which supplied additional ATP to chloroplasts by acidifying the thylakoid lumen (Huang et al., 2019, 2021).

On the other hand, acidification downregulates electron transport through the cytochrome b₆f complex (Krieger-Liszkay et al., 2000; Shikanai, 2014). In this way, the regulation of homeostasis was achieved with additional ATP needed to maintain system stability. This alternative cyclic electron flow could have been mediated by either the proton gradient regulation 5 (PGR5)/PGR5-like 1 (PGRL1) pathway or by a NAD(P)H plastoquinone oxidoreductase (NDH) complex (Munekage, 2002; Shikanai, 2007), since both can reduce plastoquinone using ferredoxin as an electron donor. However, lumen acidification typically decreases the rate of plastoquinol oxidation, suggesting, according to Cardol et al. (2011), that this step is the bottleneck of photosynthesis.

The respiration rate in MFs was increased by increasing Cd concentration to C3, but the oxygen-evolving rate was decreased, and starch was mobilized, as shown in Figures 6, 9. Since NADPH deficit was probably partially compensated because of increased respiration, e.g., with activation of NADPH producing reactions by malic enzyme and glucose-6-phosphate dehydrogenase (Van Assche and Clijsters, 1990), the cyclic electron flow around PSI was activated to recompense increased needs for ATP. Furthermore, the mother fronds have numerous plastoglobules inside chloroplasts (Figure 7A). Their number and size were observed to decrease during 72 h of exposure to low Cd concentration (C1, Figure 7C). However, their number and size were unchanged in higher cadmium concentration (C3, Figure 7F). According to Austin et al. (2006) and Besagni and Kessler (2012), plastoglobuli participate in the synthesis, storage, and regeneration of tocopherols plastochromanol and phylloquinone, which they release to the thylakoid membrane through the attachment site. Plastoglobules also contain carotenoids. Carotenoids in the C1 treatment were probably recruited from plastoglobules and contributed to the detoxification of reactive oxygen. Besides, plastoglobules on their lipid monolayer surface contain specific proteins, many of which are involved in lipid metabolism like NADPH dehydrogenase (NDH), a known player in prenylquinone metabolism that reduces the PQ pool to PQH₂ (Eugeni Piller et al., 2012; Rottet et al., 2016). Since enhanced synthesis of prenylquinones is implicated under oxidative stress, the reduced rate of plastoquinol oxidation by increased acidification in the C3 treatment could have been mitigated by the action of NADPH dehydrogenase. Thus, prenylquinone, namely plastoquinol, could have served as an electron carrier or more likely to reduce oxygen radicals O2- formed by electron redirection to O₂ in the water-water cycle (Munekage et al., 2004; Miyake, 2010). Therefore, in C1 treatments of the mother fronds, carotenoids were sufficient for detoxification of ROS that originated around PSI, but in C3 other mechanisms had to be involved.

As for the younger daughter fronds (DF₂s and GDFs), the faster respiration rate in both cadmium treatments did not result in starch mobilization, since starch accumulated with increase in Cd concentration (Figures 9F,G). Therefore, according to Vanlerberghe et al. (2020), faster respiration rate probably resulted in the activation of NADPH-producing reactions. However, the net exchange of oxygen in the light was 6-fold higher in C1 than in C3 because of the faster oxygen production rate in C1 and slower oxygen production rate in C3 (Figure 6). At the same time, the OEC complex was unaffected by both cadmium concentrations, meaning that, in both treatments, electrons were supplied as usual. However, electron transport from QA- to PQ was reduced in the C1 treatment and was unaffected in the C3 treatment, which was reflected in PI_ABS.

Similarly, based on PI_TOT, the whole electron transport was more affected by C1 than by C3. So, why is electron flow to the PQ pool reduced in the C1 treatment? The increased oxygen production in C1 treatment could be linked to increased specific energy fluxes per active PSII (ABS/RC, TR₀/RC, and DI₀/RC), activating cyclic electron flow around PSII (CEF-II) to enhance the electron flux through PSII and oxidize the PQ pool. CEF-II, in turn, could activate cyclic electron flow around PSI, accelerating the change in pH (or acidification), leading to accumulation of powerful reductant P700⁺ and contributing to the dissipation of excess energy as heat to protect PSI (Miyake and Okamura, 2003; Kadota et al., 2019). The decrease in electron transport from QA- to PQ is responsible for the subsequent reduction of PI_ABS, while electron transport from QA- to final electron acceptors was responsible for reducing PI_TOT. Unaffected OEC complex means that O₂ was produced as usual, confirmed with no net change in O₂ fluxes in the light. However, due to some RCs' inactivation, increased absorption, and trapping per active RCs, more O₂ was produced. Phenomenological energy fluxes per cross-section were no different from the control values, except for RE₀/CS₀, which was decreased significantly. On the other hand, in the C3 cadmium treatment, ABS, TR₀, and ET₀ per active RC were similar to the control values, while the density of active RCs per CS was increased. All phenomenological energy fluxes, except for RE₀, increased, indicating increased photosynthetic capacity per CS. These increased fluxes had a stimulating effect that stopped PI_ABS decrease. However, the energy was lost more (DI₀/CS₀ and DI₀/CS_m), reducing the available energy for photosynthesis.