Longleaf pondweed (Potamogeton nodosus, Potamogetonaceae) was grown at the University of Delhi, as described by Shabnam et al. (2015). For studying the impact of Ag⁺ on floating and submerged leaves of longleaf pondweed, fully expanded mature leaves were used. Leaves, after washing three times with double-distilled water, were acclimatized under laboratory conditions for 3 h. Silver nitrate (AgNO₃) was used to impose silver ion (Ag⁺)-toxicity. The leaves were exposed to different levels (0, 5, 10, 50, 100, 250, and 500 μM) of AgNO₃ in Borosil dishes (190 mm diameter × 100 mm height) under continuous white light (120 μmol photons m^-2 s^-1) for 24 h. Understandably, in this experimental setup floating leaves float and submerged leaves get submerged in test solution during experimental exposure.

Impact of Ag⁺ on floating and submerged leaves of longleaf pondweed was evaluated by measuring (i) photosystem (PS) II efficiency; (ii) carboxylase activity of ribulose bisphosphate carboxylase/oxygenase (Rubisco); and (iii) enzymatic and non-enzymatic antioxidants according to the protocols of Shabnam and Pardha-Saradhi (2016).

All the experiments were carried out independently six times. The data obtained were statistically tested with ANOVA using the general linear model. The variations between the means of treatments were compared using Duncan’s multiple range test (at P ≤ 0.05). All these statistical analyses were performed using IBM-SPSS statistical software, version 22.0 (IBM Corporation, Armonk, NY, United States).

We observed alteration of clear colorless AgNO₃ solutions to colloidal brown when incubated with floating and submerged leaves of longleaf pondweed within 24 h (Figures 1A,B). Clear colorless AgNO₃ solutions turned colloidal brown due to the formation of Ag-NPs (Shabnam et al., 2016). AgNO₃ solutions incubated in the absence of leaves did not show any alteration in color, thus confirming that leaves were responsible for the observed color change. Floating leaves turned AgNO₃ solutions colloidal brown more intensively compared to the submerged leaves, although only one side of floating leaves was in contact with test solution. Supplementary Figure 1 shows experimental setup revealing that the floating leaves possess superior potential to turn clear colorless AgNO₃ (500 μM) solutions colloidal brown compared to submerged leaves. For depicting the gradation in color with better clarity the test solutions (i.e., different concentrations of AgNO₃) incubated with floating and submerged leaves for 24 h were transferred to test tubes along with leaves (Figures 1A,B). However, absorption spectra of the brown colloidal solution did not show any Ag-NP specific absorption peak.

Transmission electron microscopy revealed the presence of distinct NPs of varying shapes and sizes (∼10–80 nm) in these colloidal brown solutions (Figures 1C–F). EDX of these NPs showed peaks specific to Ag (Figures 1I,J). SAED pattern revealed the crystalline nature of these Ag-NPs (Figures 1G,H). PXRD patterns showed Bragg reflections (111), (200), and (311), revealing crystalline nature and face centered cubic structure of Ag⁰-NPs (Figures 1K,L) (Pardha-Saradhi et al., 2014a). Additional peaks observed in the PXRD spectra might be due to Bragg reflections (111)^∗, (211)^∗, (220)^∗, (221)^∗ of cubic Ag₂O (Figures 1K,L).

In view of large differences in photosynthetic activities between floating and submerged leaves (Shabnam et al., 2015), we examined the effects of Ag⁺ on Photosystem II (PS II) efficiency of these leaves. Photosystem II efficiency (maximum quantum yield) is often determined as a ratio of variable Chl a fluorescence to maximum Chl a, i.e., F_v/F_m. Chl a fluorescence of oxygenic organisms shows a rise from a basal level (F_o) (i.e., minimum fluorescence) to the maximum (F_m) (Strasser et al., 1995; Stirbet and Govindjee, 2012; Shabnam et al., 2015, 2017). F_o and F_m values of both floating and submerged leaves declined significantly on exposure to Ag⁺; the decline was significantly higher in the submerged leaves than in the floating ones (Table 1). Ag⁺, like other heavy metal ions, brought about a significant decline in the quantum yield of PS II activity, as inferred from F_v/F_m values, in both floating and submerged leaves (Figure 2). However, at any given concentration, the decline in F_v/F_m was significantly higher in the submerged leaves.

Figure 3 shows the fast (up to a second) polyphasic Chl a fluorescence transients of floating and submerged leaves which were exposed to different Ag⁺ levels. All oxygen-evolving organisms show polyphasic Chl a fluorescence transients (also called the OJIP curves) with distinct O, J, I, and P steps (Figure 3). In these curves, “O” is the minimum fluorescence (F_o), “P” is the peak (F_m), and “J” and “I” are intermediate levels. The polyphasic rise of Chl a fluorescence transient was severely reduced in both floating and submerged leaves, exposed to Ag⁺ (Figure 3), which is in agreement with the decline in F_v/F_m. The decline in the amplitude of fluorescence was significantly higher in submerged leaves than in floating leaves, revealing superior potential of the latter to tolerate Ag⁺ than the former. Extreme sensitivity of submerged leaves to Ag⁺-toxicity was also evident from the loss in the polyphasic nature of Chl a fluorescence transients even at concentration as low as 5 μM of Ag⁺.

Chlorophyll a fluorescence kinetics is affected by such factors as Chl content. Therefore, we evaluated the impact of 24 h Ag⁺ treatment on both Chl a and Chl b levels. Decline of these Chls was recorded in both floating and submerged leaves, as a function of silver concentration (Table 2). However, the decline in the levels of both Chl a and Chl b levels were significantly lower in the floating leaves. Interestingly, decline in levels of Chl a was significantly higher than that of Chl b in both floating and submerged leaves.

Figure 4 depicts impact of 24 h Ag⁺ treatment on carboxylase activity of Rubisco in floating and submerged leaves. Upon exposure to 10 μM Ag⁺, the carboxylase activity of Rubisco declined by 50% in the submerged leaves, whereas it remained unaltered in the floating ones. However, 100 μM Ag⁺ caused ∼50 and ∼90% decline in Rubisco activity in the floating and submerged leaves, respectively (Figure 4).

Since there was a significant variation in the impact of Ag⁺ on photosynthetic efficiency between floating and submerged leaves, we measured Ag content in these leaves. Both floating and submerged leaves of longleaf pondweed exposed to Ag⁺ showed the presence of Ag. The level of Ag in these leaves increased as a function of Ag⁺ concentration to which they were exposed (Figure 5A). At any Ag⁺ concentration, silver content in submerged leaves was ∼3 times higher than that in floating ones. XPS analysis also confirmed the presence of Ag in both leaves. XPS spectra showed two peaks at binding energies of 368 and 374 eV (Figures 5B,C), which arise due to the emission of 3d_5/2 and 3d_3/2 photoelectrons, respectively (Adegboyega et al., 2013).

Like our earlier findings (Shabnam and Pardha-Saradhi, 2016), we did not observe any significant variation in the levels of ascorbate, phenolics, and thiols amongst floating and submerged leaves which were not exposed to Ag⁺. However, both floating and submerged leaves exposed to 10 and 100 μM Ag⁺ showed a decline in the levels of all these three non-enzymatic antioxidants (Figure 6). Irrespective of the Ag⁺ concentrations to which leaves were exposed, the decline in the level of phenolics was significantly higher in the submerged leaves (Figure 6A). However, the decline in the levels of ascorbate and thiols was almost similar for both leaves (Figures 6B,C).

Antioxidant enzymes, such as SOD, catalase (CAT), GPX, ascorbate peroxidase (APX), MDHAR, dehydroascorbate reductase (DHAR), and GR play an important role in scavenging ROS in plants exposed to heavy metals (Prasad et al., 1999; Dhir et al., 2009). Therefore, we evaluated the impact of Ag⁺ on activities of these enzymes in both floating and submerged leaves of longleaf pondweed. Ag⁺ treatment caused a significant decline in SOD activity in both floating and submerged leaves, although the decline was significantly higher in the latter compared to the former (Figure 7A). Both floating and submerged leaves, with the exception of floating leaves exposed to 10 μM Ag⁺, showed a significant decrease in the catalase activity compared to their respective controls (Figure 7B). However, the degree of loss in catalase activity was higher in submerged leaves. Contrary to decreased activity of SOD and catalase, activity of GPX increased by 2–2.5-fold in submerged leaves exposed to Ag⁺ (Figure 7C). However, floating leaves showed a decrease in GPX activity on exposure to Ag⁺.

Activities of enzymes of ascorbate-glutathione cycle, e.g., APX, MDHAR, DHAR, and GR, decreased significantly in both floating and submerged leaves as a function of given Ag⁺ level (Figure 8). In general, irrespective of the concentration of Ag⁺ used, the activities of ascorbate-glutathione cycle enzymes were significantly higher in floating leaves. Amongst enzymes of ascorbate-glutathione cycle, Ag⁺ induced highest decrease in the activity of GR, followed by those of APX, MDHAR, and DHAR.

Earlier, Pardha-Saradhi et al. (2014a,b,c) reported that plants reduce toxic ionic forms of heavy metals into non/less-toxic NPs as a defense mechanism. During our investigations, we found that both floating and submerged leaves of longleaf pondweed could turn clear colorless AgNO₃ solutions to colloidal brown (Figures 1A,B and Supplementary Figure 1). TEM coupled with SAED and EDX confirmed the presence of distinct crystalline NPs composed of Ag in these colloidal brown solutions (Figures 1C–F). However, similar to our earlier findings (Pardha-Saradhi et al., 2014a), these brown colloidal solutions did not show any Ag-NP specific peak in the absorption spectra. In addition to SAED, PXRD confirmed the crystalline nature of Ag-NPs; PXRD pattern showed peaks specific to face centered cubic structured Ag⁰ and cubic structured Ag₂O. These PXRD analyses clearly showed that Ag-NPs generated by floating and submerged leaves are composed of both Ag⁰ and Ag₂O. XPS analysis confirmed the presence of Ag in both leaves and that the accumulated Ag in these leaves existed predominantly as Ag₂O state. It is well-known that Ag⁰ and Ag⁰-NPs are prone to oxidation (Pardha-Saradhi et al., 2014a; Shabnam et al., 2016). Recently, Shabnam et al. (2016) demonstrated the potential of photosynthetic electron transport to reduce Ag⁺ to Ag⁰ and to generate Ag⁰-NPs. They also specifically showed that O₂ released as a byproduct during photosynthetic electron transport promotes oxidation of Ag⁰ and/or Ag⁰-NPs to generate Ag₂O-NPs. Therefore, we believe that O₂ released by light harvesting photosynthetic machinery of leaves promotes oxidation of Ag⁰ and/or Ag⁰-NPs to generate Ag₂O-NPs.

As evident from the Supplementary Figure 1, only one side of floating leaves is in immediate contact with test solution and further, overall size (i.e., surface area) of submerged leaves is nearly double of floating leaves (Shabnam and Pardha-Saradhi, 2016). In spite of such a vast variation in the surface area in contact with the test solution, the color intensity of the AgNO₃ solutions incubated with floating leaves was higher than the ones incubated with submerged leaves. This reveals superior potential of floating leaves to generate Ag-NPs compared to submerged leaves. Superior potential of floating leaves to generate Ag-NPs might be due to their superior photosynthetic photochemical reactions (Shabnam et al., 2015; Shabnam and Pardha-Saradhi, 2016).

Inductive coupled plasma analysis revealed that submerged leaves exposed to Ag⁺ possessed 3–4 fold higher levels of silver than floating leaves, which discloses that the uptake of Ag by the former leaves is significantly higher than the latter leaves. Higher levels of Ag in submerged leaves can be accounted to (i) larger surface area (as detailed above) available for uptake of Ag; and (ii) lower potential to reduce Ag⁺ and generate Ag-NPs, compared to floating leaves. We have recently demonstrated that the uptake of Ag by plants of Spirodela polyrhiza in the ionic state is 3–4 times higher than in the NP state (Shabnam et al., 2017). Therefore, we believe that superior potential to efficiently reduce Ag⁺ and generate Ag-NPs is one of the mechanisms acquired by floating leaves to curb the uptake of Ag.

Photosystem II plays a vital role in photochemical reactions. Accordingly, overall photosynthetic capacity of plants often relies on PS II activity (Shabnam et al., 2015). Therefore, during the present investigations, we evaluated the impact of Ag⁺ on PS II efficiency. F_v/F_m is a commonly used parameter to determine Photosystem II efficiency of plants (Strasser et al., 1995; Stirbet and Govindjee, 2012). PS II efficiency, measured in terms of F_v/F_m as well as through Chl a fluorescence kinetics, was significantly higher in floating leaves compared to submerged leaves, just as in our earlier findings (Shabnam et al., 2015).

In this paper, we found that Ag⁺ caused a significant decline in PS II efficiency of both floating and submerged leaves of longleaf pondweed. Ag⁺-induced decline in the quantum yield of PS II activity has also been reported in submerged P. crispus, as well as in free floating S. polyrhiza (Xu et al., 2010; Jiang et al., 2012; Shabnam et al., 2017). However, Ag⁺-induced suppression in PS II efficiency was significantly lower in the floating leaves compared to that in the submerged leaves. These findings unequivocally demonstrate the prevalence of superior mechanism(s) in floating leaves to counter Ag⁺.

As mentioned earlier, all oxygen-evolving organisms show polyphasic Chl a fluorescence transients with distinct O-J, J-I, and I-P photochemical phases (Figure 3). While O-J rise (0.05–2 ms) involves the reduction of Q_A to Q_A^-, the J-I rise (2–30 ms) denotes reduction of PQ pool and the I-P rise (30–300 ms) implies reduction of the acceptor side of PS I (Strasser et al., 1995; Stirbet and Govindjee, 2012; Hamdani et al., 2015; Shabnam et al., 2015, 2017). The OJIP transient kinetics are highly sensitive to various stresses including heavy metal stress (Appenroth et al., 2001; Oukarroum et al., 2012; Shabnam et al., 2015, 2017). During this study, we observed a drastic negative impact of Ag⁺ on the OJIP transients in longleaf pondweed leaves, even at a concentration of 5 μM. Negative impact of Ag⁺ on the OJIP transients was significantly higher in the submerged leaves. As shown in Figure 3, while floating leaves retain polyphasic nature of the OJIP transients, the submerged leaves showed a complete loss in the polyphasic nature of this transient on exposure to 5 μM Ag⁺. Severe loss in fluorescence intensity or polyphasic nature of OJIP transients has also been observed in several algae and plants exposed to heavy metals (Appenroth et al., 2001; Oukarroum et al., 2012; Wang et al., 2014; Shabnam et al., 2017). There are reports of inhibition of the oxygen evolving complex (OEC) by metal ions such as Cd and Cr (see e.g., Atal et al., 1991). Severe negative impact of Ag⁺ on the OJIP transients during this study suggests that Ag⁺ could be inhibiting the OEC of PSII as well as the flow of electrons from Q_A^- to the electron acceptor side of PS I (via the PQ-pool), in both floating and submerged leaves of longleaf pondweed. Higher Ag content in submerged leaves could be responsible for a significantly higher decline in their photosynthetic efficiency, compared to that of floating leaves.

As noted earlier, Chl a fluorescence kinetics can be affected by such factors like Chl content. A 24-h Ag⁺ treatment caused a decline in the levels of Chl a and Chl b in a concentration-dependent manner, in both floating and submerged leaves (Table 2). However, the decline in the levels of both Chl a and Chl b were significantly lower in the floating leaves. Our present findings clearly demonstrate that floating leaves are better equipped to protect their photosynthetic machinery against Ag⁺-toxicity than submerged leaves. Superior potential of the floating leaves to withstand Ag⁺ induced suppression of photosynthetic efficiency could be due to (i) higher potential to reduce Ag⁺ to Ag-NPs at the surface, and (ii) restricted uptake of Ag compared to submerged leaves.

In spite of having similar carboxylase activity of Rubisco, the control submerged leaves possess lesser carbon skeletons compared to the control floating leaves. Higher carbon skeletons in floating leaves is due to significantly higher and efficient light harvesting photochemical reactions (pivotal for the generation of assimilatory power essential for CO₂ fixation and the synthesis of various carbon skeletons). Lower decline in carboxylase activity of Rubisco in floating leaves revealed that floating leaves are better equipped to protect the carboxylase activity of Rubisco than submerged leaves.

Decline in the carboxylase activity of Rubisco has been reported earlier in terrestrial plants, such as Phaseolus vulgaris, Zea mays, Oryza sativa, and Citrus grandis, exposed to Zn, Cd, and Mn (Van Assche and Clijsters, 1986; Krantev et al., 2008; Li et al., 2010; Wang et al., 2014) and aquatic plants, such as Salvinia natans and Ceratopteris pteridoides, exposed to Cr and Cd (Dhir et al., 2008; Deng et al., 2014). Ag⁺ induced decline in PS II efficiency and carboxylase activity of Rubisco of these plants may be due to (i) ROS induced inactivation (Sharma and Dietz, 2009; Foyer and Noctor, 2016), (ii) enhanced proteolytic activity (Hajduch et al., 2001; Gajewska et al., 2013), (iii) interference with enzyme’s structure by substitution of native ions (Van Assche and Clijsters, 1986) and/or interaction with SH groups (e.g., by Cu and Cd) (Stiborova, 1998; Stiborova et al., 1998), and (iv) impaired protein biosynthesis (Kremer and Markham, 1982). A simple comparison of the impact of Ag⁺ on PSII efficiency with carboxylase activity of Rubisco reveals that the light harvesting photochemical reactions are more sensitive to Ag⁺ than carbon fixation reactions.

In general, heavy metals promote generation of ROS by interfering with electron transport and redox reactions (Prasad et al., 1999; Hall, 2002; Metwally et al., 2003; Sharma and Dietz, 2009; Pardha-Saradhi et al., 2014b). Accordingly, plants have evolved antioxidant systems, both non-enzymatic and enzymatic, to counter oxidative stress (Prasad et al., 1999; Shabnam and Pardha-Saradhi, 2016). Amongst the non-enzymatic antioxidants, ascorbate, phenolics and thiols play important roles in scavenging ROS and/or chelating heavy metals (Sakihama et al., 2002; Shabnam et al., 2014; Shabnam and Pardha-Saradhi, 2016). Plants exposed to heavy metal ions, such as Cd, Pb, and Zn, show enhanced levels of non-enzymatic antioxidants (Prasad et al., 1999; Oncel et al., 2000; Mishra et al., 2006). On the contrary, we observed a decline in the levels of all the non-enzymatic antioxidants in both floating and submerged leaves exposed to Ag⁺ (Figure 6). Significantly lower Ag⁺-induced decline in the content of phenolics in floating leaves could be one of the factors contributing to their superior potential to withstand Ag⁺-stress. Posmyk et al. (2009) also reported a decline in the phenolic content in red cabbage under Cu stress. A significant decline in the levels of ascorbate and/or thiols/GSH has been reported in studies with (i) submerged P. crispus plants exposed to Ag⁺ (Xu et al., 2010); (ii) pigeon pea seedlings exposed to Ni²⁺ and Zn²⁺ (Rao and Sresty, 2000); (iii) roots of soybean exposed to Cd²⁺ (Balestrasse et al., 2001); and (iv) roots and shoots of maize exposed to Cd²⁺ (Tukendorf and Rauser, 1990; Meuwly and Rauser, 1992). Ag⁺ has a strong affinity for thiols (Rao and Sresty, 2000; Blaske et al., 2013) like other heavy metals. A positive correlation has been established between the depletion of thiol content and the amount of metal ions (Cu²⁺, Zn²⁺) accumulated by plants (Tripathi et al., 2006). Thus, a decline in the levels of thiols, shown in this study, could be due to the binding of thiols to Ag⁺. In general, decrease in the levels of non-enzymatic antioxidants could be due to (i) enhanced catabolic degradation, (ii) alteration in their structure via chelation or reduction of metal ions, and/or (iii) decreased biosynthesis.

Significantly higher activities of antioxidant enzymes in floating leaves impart superior potential to counter oxidative damage compared to submerged leaves. Ag⁺ induced a significant decline in activity of SOD and catalase in both floating and submerged leaves; the decline was significantly higher in the latter (Figure 7A). Contrary to the decreased activity of SOD and catalase, the activity of GPX increased by 2–2.5-fold in the submerged leaves exposed to Ag⁺ (Figure 7C). However, the floating leaves showed a decrease in GPX activity on exposure to Ag⁺. In contrast to the decline in the activities of SOD and catalase, and an increase in GPX activity recorded in the submerged leaves of longleaf pondweed (present study), Xu et al. (2010) noted increase in activities of SOD and catalase, and a decline in the activity of GPX in submerged P. crispus, exposed to Ag⁺. Salvinia natans exposed to Cr-rich water, however, showed an increase in GPX activity, without any significant alteration in CAT activity (Dhir et al., 2009). Interestingly, Shah et al. (2001) also noted enhancement of GPX activity accompanied with a decline in CAT activity in rice exposed to cadmium. Peroxidases play a significant role in the synthesis of lignin, which is impermeable to metal ions (Hegedüs et al., 2001; Parrotta et al., 2015). Therefore, we believe that an increased GPX activity in submerged leaves might be a strategy to restrict the uptake of Ag⁺.

Silver ions also suppressed activities of enzymes of the ascorbate glutathione cycle in both types of the leaves. Ag⁺ induced decline in the activities of antioxidant enzymes in both floating and submerged leaves, except GPX in the submerged leaves, which is in agreement with those measured in the roots of soybean and poplar exposed to Cd²⁺ (Balestrasse et al., 2001; Schutzendubel et al., 2002). A similar decline in the activities of several antioxidant enzymes was observed in cotyledons and leaves of sunflower seedlings under Cd²⁺, Fe²⁺, and Cu²⁺ stress (Gallego et al., 1996a,b). In addition, the potential of Ag⁺ to displace native metal cations from their usual binding sites in enzymes has been reported (Ghandour et al., 1988). Ag⁺ induced decline in the activity of the antioxidant enzymes might be due to the effect of Ag⁺ on expression of the relevant genes at the transcriptional/translational level by binding with DNA/RNA. Further, this effect might be at the post-translational level. The binding of Ag⁺ to SH and other active groups might alter 3-D structure of these antioxidant enzymes affecting the catalytic/active site(s) vital for their activities (Ghandour et al., 1988).

A summary of the differential impacts of Ag⁺ on the floating and the submerged leaves of longleaf pondweed is presented in a hypothetical model (Figure 9). Superior photosynthesis in floating leaves leads to production of more carbon skeletons and energy resources compared to that in submerged leaves. Accordingly, floating leaves are better equipped to counter/tolerate stress imposed by heavy metals, such as Ag⁺. This includes (i) a superior capacity to biotransform toxic ionic state of heavy metals (such as Ag⁺) into less/non-toxic NPs (such as Ag⁰/Ag₂O-NPs); and (ii) a better capacity to counter oxidative stress through a superior antioxidant system. In addition, significantly higher levels of Ag accumulated in submerged leaves would also directly interfere with their cellular metabolism.