Photosystem II of Ligustrum lucidum in response to different levels of manganese exposure

The toxic effect of excessive manganese (Mn) on photosystem II (PSII) of woody species remains largely unexplored. In this study, five Mn concentrations (0, 12, 24, 36, and 48 mM) were used, and the toxicity of Mn on PSII behavior in leaves of Ligustrum lucidum was investigated using in vivo chlorophyll fluorescence transients. Results showed that excessive Mn levels induced positive L- and K- bands. Variable fluorescence at 2 ms (VJ) and 30 ms (VI), absorption flux (ABS/RC), trapped energy flux (TRo/RC), and dissipated energy flux (DIo/RC) increased in Mn-treated leaves, whereas the performance index (PIABS), electron transport flux (ETo/RC), maximum quantum yield (φPo), quantum yield of electron transport (φEo), and probability that an electron moves further than QA− (ψo) decreased. Also, excessive Mn significantly decreased the net photosynthesis rate and increased intercellular CO2 concentration. The results indicated that Mn blocked the electron transfer from the donor side to the acceptor side in PSII, which might be associated with the accumulation of QA−, hence limiting the net photosynthetic rate.


Materials and Methods
Plant culture and Mn treatments. Two-year-old L. lucidum seedlings (average diameter, ~9 mm; height, ~133 cm) were purchased from a local nursery. All plants were individually transplanted into plastic pots (diameter, 25.4 cm; height, 17.8 cm) filled with 7 kg of air-dried soil. The plants were grown under natural illumination (30/25 °C day/night temperature, 12/12 h day/night cycle and a maximum photosynthetically active radiation of about 1,000 μmol photons m −2 s −1 ) for 4 months to acclimatize them to the soil microclimate before initiating Mn treatment. Each pot was supplied with 400 mL of pure water every 2 to 3 days.
Samples of soil free from heavy metal pollution were collected from the CSUFT campus soil at a depth of 5-20 cm. The soil samples were taken back to the laboratory, and were sieved through 5 × 5 mm sieves to remove rocks, and were then air-dried at room temperature. The chemical properties of soil samples were as measured: pH 4.9, 0.227 g N/kg, 0.129 g P/kg, and 355.978 mg Mn/kg.
For the Mn-treated soil, distilled water containing 1.2 mM, 2.4 mM, 3.6 mM and 4.8 mM Mn from MnCl 2 ·5H 2 O was added to the pots every other day at a rate of 400 mL per day for 20 days. The four Mn treatments were designated as L1, L2, L3, and L4, respectively. For the control (CK), about 400 mL of distilled water without Mn was added into the pots. In total we have five treatments: CK, L1, L2, L3 and L4, and each treatment was replicated five times. Measurements were carried out on three fully expanded L. lucidum leaves of similar size on days 10, 25, and 40 after the Mn treatment.

Fast Chl a fluorescence kinetics and JIP-test. Fast Chl a fluorescence was measured by M-PEA
(Multifunctional Plant Efficiency Analyzer, Hansatech Instrument, UK). Leaves were exposed to a pulse of saturating red light (5,000 μmol m −2 s −1 , peak 625 nm, duration 50 μs-2 s, records of 128 points) and measured daily between 8:30-11:00 am after 1 h of dark adaptation using dark adaptation clips. The fluorescence transients (OJIP curves) were analyzed to determine energy distribution through PSII per RC (ABS/RC, TR o /RC, ET o /RC, DI o / RC, see Table 1), flux ratios (ϕ Po , ϕ Eo , and ψ o ) and performance index (PI ABS ) according to the JIP-test 19 . Relative variable fluorescence at time t, at the J-step, and at the I-step (i.e., V t , V J and V I , respectively) was calculated using the following equations 16,21 : where F J is fluorescence intensity at 2 ms and F I is the fluorescence intensity at 30 ms.
To further characterize the effect of Mn on L. lucidum PSII, some functional parameters were calculated from the JIP-test. The OJIP transients were double normalized between O (50 μs) and P steps to estimate relative variable fluorescence W OP = (F t − F o )/(F P − F o ). Normalization between O and K (300 μs) steps revealed L-band (150 μs), resulting in the variable fluorescence 24 : OK OK OK control Normalization between O and J (2 ms) steps revealed K-band (300 μs), resulting in the variable fluorescence 24 : OJ OJ OJ control The F I , F J , F K , F M , and F O represent fluorescence at I-step, J-step, and K-step, dark-adapted maximum fluorescence, and dark-adapted minimum fluorescence, respectively. ΔV J , ΔV I , ΔW OK , and ΔW OJ represent the J-band, I-band, L-band, and K-band, respectively, and are associated with the accumulation of Q A −21 , the proportion of Data analysis. Data were reported as means of each group based on at least six independent replicates.
Results are presented as means ± standard error (SE). Statistical differences between measurements were analyzed using one-way ANOVA, followed by a least significant difference (LSD) test at P < 0.05. Chl a fluorescence parameters (FPs) associated with Mn levels and stress time were assessed using two-way ANOVA with α = 0.05. All graphs were made using Sigmaplot 12.0.

Effects of Mn on Chl a fluorescence transient and related parameters in L. lucidum leaves. All
OJIP transients from both Mn-treated and control leaves showed a typical polyphasic rise with the basic steps of O-J-I-P. Mn-treated leaves displayed positive trends of K-, J-, and I-bands compared with the controls at 300 μs, 2 ms, and 30 ms, respectively (Fig. 1). K, J, or I step was positively correlated with the Mn level or the stress time in the Mn-treated leaves. Positive L-band and K-band increased with the Mn levels on days 10, 25, and 40 ( Fig. 2). In the L1-treated group, the L-band achieved the maximum value on day 10 and then decreased continuously to reach the control levels on day 40. In the L2-treated group, the maximum value of L-band appeared on day 25 and was maintained until day 40. The changes of K-band were similar to L-band except that the K-band in the L1-treated group on day 40 was higher than that of the controls.
Variable fluorescence at 2 ms (V J ) increased with the Mn levels ( Table 2). There was no significant difference between the L1-or the L2-treated groups and control group except for the L2-treated group on day 40. A significant difference was observed between the L3-and L4-treated groups and the control group. Significant differences were also observed between the L4-treated group and the L1-or L2-treated groups. V J was significantly higher on day 40 compared with day 10 in all Mn treatment levels. The variable fluorescence at 30 ms (V I ) increased with Mn levels ( Table 2). V I was significantly increased in L2-, L3-and L4-treated groups compared with the control group, while there was no significant difference between the L1-treated group and the control group. V I was significantly increased in the L4-treated group compared with the L2-treated group on days 25 and day 40. There were no significant differences among the different stress time points.
Effects of Mn on the performance index, energy distribution, and the quantum yield of excitation energy trapping of pSii in L. lucidum leaves. We analyzed several functional parameters from the JIP-test to further characterize the effect of Mn on PSII of L. lucidum. Performance index (PI ABS ) showed a declining trend during the test period (Fig. 3a). PI ABS in the L4-treated group was significantly decreased compared Relative variable fluorescence at the J-step (2 ms), reflects the activity of acceptor side of PSII Performance index (potential) for energy conservation from photons absorbed by PSII to the reduction of intersystem electron acceptors www.nature.com/scientificreports www.nature.com/scientificreports/ with the L1-treated group and the control group in a time-dependent manner. There was no significant difference between the L1-or L2-treated groups and the control group at any time point, except between the L2-treated group and the control group on day 40. In L3-and L4-treated groups, PI ABS was significantly decreased on day 40 compared with day 10.
Absorption ABS/RC (Fig. 3b), trapping TR o /RC (Fig. 3c), and dissipation DI o /RC (Fig. 3e) were increased in a Mn-concentration dependent manner during the test period, and these parameters were significantly increased in the L4-treated group compared with the control group during the test period. No significant difference at the same levels of Mn was observed among various time points (day 10, 25 and 40). Electron transport ET o /RC www.nature.com/scientificreports www.nature.com/scientificreports/ ( Fig. 3d) first increased and then decreased along with the increase of Mn levels, and the maximum value of ET o / RC was observed in the L2-treated group. There was no significant difference between the Mn-treated groups and control group at various time points, except between the L4-treated group and the control group on day 40.
Maximum quantum yield ϕ Po (Fig. 3f), the probability that an absorbed photon moves an electron further than Q A − (ϕ Eo ψ o ) (Fig. 3g), and the probability that a trapped exciton moves an electron further than Q A − (ψ o ) (Fig. 3h) at various time points showed a declining trend with increasing Mn levels. ϕ Po was significantly decreased in the L4-treated group compared with the control on days 10, 25, and 40, and ϕ Po was significantly decreased in L2, L3, and L4-treated groups compared with the control on day 40. ϕ Eo and ψ o were significantly decreased in the L2-, L3-and L4-treated groups compared with the control on day 40.

Discussion
In this study, the OJIP curve was observed to be O-L-K-J-I-P when the Mn levels increased (Figs 1 and 2). The OJIP curve is very sensitive to environmental stress 16,21,24 . In leaves that have been exposed to a disturbed environment for a short period of time, Chl a fluorescence shows a polyphasic rise before J step, and the O-J-I-P becomes O-K-J-I-P and even O-L-K-J-I-P 16,26 .
The L-band (~150 μs) is an indicator of energetic connectivity of the antennae to PSII units 24,27 , implying better excitation energy utilization and system stability of PSII units 21,27 . In our study, the presence of positive L-band in the Mn-treated leaves indicated an inferior performance of antennae connectivity compared to the control leaves and might be a sign of disturbed energy transfer 28 Table 3. Two-way ANOVA results for JIP-test parameters. www.nature.com/scientificreports www.nature.com/scientificreports/ positive L-band implies that the PSII units were less tightly grouped, or that less energy was exchanged between the independent PSII units. Therefore, PSII units of Mn-treated leaves had lower stability and became more fragile. However, an amplitude change in the L-band (from positive to negative) of the L1-treated group was observed www.nature.com/scientificreports www.nature.com/scientificreports/ from day 10 to day 40 (Fig. 2a,c,e) suggesting that the PSII units had better excitation energy utilization and system stability on day 40 without any irreversible damage. This may be associated with a lack of significant Mn accumulation in the leaves of the L1-treated groups (compared to controls, Table 5).
The K-band can be explained by the imbalance of electron flow from the donor side to the acceptor side in the PSII RCs 28 . When the electron transfer from the OEC to tyrosine Z (Y z ) is slower than the electron transfer from P680 to Q A and beyond, there is a high accumulation of Y z

+25
. Thus, this accumulation of Y z + causes the appearance of K-step, which is directly associated with an inactivation of the OEC 25 . In this study, the appearance of K step suggested that Mn inhibit the electron flow from the donor to the acceptor side of PSII even at low levels (L1) (Fig. 1ac). Meanwhile, the presence of positive K-band in the Mn-treated leaves indicates an inactivation of the OEC 24,25 ( Fig. 2d-f). Therefore, it may be inferred that the competition between Ca 2+ and Mn 2+29 in the OEC led to more sites held by Mn 2+ in the OEC, and this may depend on the similar ion radius and charge properties of Mn 2+ and Ca 2+ 30 .
OJIP transients can be used to examine the electron transport flux from PSII RCs to PSI through Q A and Q B . In this study, leaves in the L3-and L4-treated groups had significantly increased V J compared with the control leaves ( Table 2), indicating that high levels of Mn induced the accumulation of Q A − . This result is consistent with the previous findings 16,26,27 . The increased value of V I could be related to the blockade of electron transport downstream of Q A by Mn stress 31 . This finding is also supported by the decrease of ϕ Eo and ψ o (Fig. 3g,h), as Q B was unable to be reduced by Q B -non-reducing PSII RCs 27,32 . Correspondingly, the higher levels of Q B -non-reducing centers blocked electron transport towards PSI 32 . Lower redox state of Q B implies altered reduction potential of PSII at the acceptor side in Mn-stressed plants 17 . Since Q A is in quasi-equilibrium with Q B and the PQ pool, the lower redox potential of Q B will decrease the probability of forward electron transfer between the two quinone acceptors by shifting the redox equilibrium between Q A − Q B and Q A Q B − towards Q A − Q B 33,34 . The significant reduction of PI ABS , which is a very sensitive indicator of plant functionality 27 , indicates that excessive Mn may down-regulate PSII function, resulting in prolonged negative effect with irreversible damage. An increase in both ABS/RC and TR o /RC, and a decrease in ϕ Po indicates inactivation of a certain part of RCs, which was most likely due to inactivation of OEC as well as the transformation of active RCs to silent ones, because the functional antenna that supplies excitation energy to active RCs was increased in size 24,27 . However, an increase in ET o /RC under low levels of Mn (L1 and L2) implies that these inactive RCs 35 could prevent further damage to themselves and protect neighboring active RCs in response to the absorbed light energy in the active RCs 36 . Significantly increased DI o /RC and decreased ET o /RC in the highest Mn treatment group (L4) shows that the excess excitation energy was mostly dissipated 21,24 .
ANOVA results revealed that all JIP-test parameters used in this study were significantly affected by Mn stress (P < 0.05), but the interactive influences of Mn stress and stress time on the examined parameters were not significant (P > 0.05) ( Table 3). We also found that L. lucidum leaves were more sensitive to the Mn levels compared with the stress time. Additionally, ET o /RC, ϕ Eo, and ψ o were significantly influenced by Mn stress time, indicating that the blockage of PSII electron flow beyond Q A − was more severe in response to the increasing stress time. The blockage of PSII electron flow was also supported by the phenomena of the accumulation of Q A − and the increase in V J .
The Mn-induced changes in the shape of OJIP transient curves and other related parameters of L. lucidum as observed in this study were also found in the studies of Mn-treated Citrus grandis seedlings 16 , Al-treated    26 , and Cd-treated Solanum lycopersicum 37 . But different from our results here, Cr-treated Spirodela polyrhiza was found to have a decreasing trend of TR o /RC, indicating that the Cr damages LHCs 38 . Therefore, the sensitivity of different parts of the PSII units vary, and this response is the different for different heavy metals and is species-dependent.
This study found that Pn of the plants in L2, L3 and L4 treatments was significantly lower than that in the control (Table 4), and Pn and Ci were negatively correlated. Therefore the reduced Pn observed in our study was not caused by Ci limitation 39,40 . A negative correlation between Ci and Pn was suggested as an indicator to describe the decrease in carboxylation efficiency by Rouhi et al. 41 . A positive relationship between maximum quantum yield of PSII (Fv/Fm) and Pn was also found by Tezara et al. 42 . These results suggested that the reduction of Pn could be explained by the limitation in photochemical activity of PSII, which impeded the utilization of CO 2 in the assimilation process. The current study found that excessive Mn impaired the functional PSII, as supported by the observed positive L-band and the observed decrease in PI ABS . Thus, Mn toxicity contributed to the observed significant reduction of Pn through its effects on photosynthetic apparatus 43 . conclusions We conclude that an excess level of Mn affected the net photosynthesis rate, the OJIP transient, and other related parameters of L. lucidum seedlings. The imaging of JIP-Test parameters revealed Mn-induced photo-damage on the PSII RCs, including a decrease in energy absorption and excitation energy trapping, and an increase in energy dissipation. The disturbance of the PSII electron transport from the donor side to the acceptor side might be associated with inactivation of OEC. This, in turn, resulted in a decrease in the rate of electron transport beyond Q A and an accumulation of Q A − .