## Introduction

It is well-known that manganese (Mn) is an essential micronutrient element required for the growth and development of plants. Especially, Mn is involved in metabolic pathways of chlorophyll (Chl) synthesis and breakdown in the chloroplasts1,2. The oxygen-evolving complex (OEC) of photosystem II (PSII) contains a Mn-containing metalloenzyme core. This inorganic core, binding to the reaction center (RC) protein D1 in PSII, has the empirical formula Mn4CaO5 and is known as the tetra-nuclear Mn cluster3,4. However, Mn, in excess, is also considered as one of the most toxic trace metals to plants. Mn pollution often originates from industrial disposal5, manufacturing sewage6,7, as well as mineral exploitation8.

Toxic effects of Mn on plants is well documented. Excessive Mn level impedes plant growth and development by interfering with metabolic processes9,10. Moreover, a number of studies have shown that Mn mainly exerts its toxicity to plant leaves by inhibiting photosynthesis and chloroplast activity9,10,11,12 leading to reduced chloroplast content and suppressed CO2 assimilation13,14,15,16. PSII is highly sensitive to Mn level. Feng et al.13 have reported that excessive Mn inhibits the maximum photochemical efficiency (Fv/FM) and effective quantum yield of PSII (ΦPSII) in cucumber. Doncheva et al.17 have shown that excessive Mn significantly affects the quantum efficiency of PSII in Mn-sensitive maize (Zea mays L.) ‘Kneja 605’, but not in Mn-tolerant maize ‘Kneja 434’. Interestingly, some other studies have revealed that Fv/FM is not substantially affected by Mn accumulation in tobacco and rice bean seedlings11,18. In addition, Kitao et al.19 have suggested that excessive Mn affects the activity of CO2 reduction cycle rather than Fv/FM in white birch, while increased QA reduction and thermal energy dissipation, as well as decreased quantum yield of PSII, have been observed. Similar results have been found in Alnus hirsuta Turcz., Betula ermanii Cham., Ulmus davidiana Planch., and Acer mono Maxim15. Li et al.16 have reported that excessive Mn impairs the whole photosynthetic electron transport chain from the donor side of PSII up to the reduction of end acceptors of PSI in Citrus grandis seedlings, followed by the increase in ABS/RC, TRo/RC, and DIo/RC, as well as decrease in ETo/RC, φPo, and ψo. However, the mechanism of toxicity of Mn to PSII remains largely unexplored, and most previous studies have only focused on herbaceous plants11,13,17,18.

Chl a fluorescence, a non-invasive spectroscopic technique, has been widely used to detect and measure the in vivo behavior of PSII under different environmental stresses20,21. Analysis of the polyphasic fluorescence transient under physiological conditions shows that the fluorescence increases in the typical shape of OJIP kinetics21. The “JIP-test” analysis of the OJIP transients allows the calculation of structural, conformational, and functional parameters quantifying the PSII behavior under environmental stresses, including absorption flux, trapped energy flux, electron transport flux, and dissipated energy flux21.

Hunan Province in the southern area of China has a high density of Mn mines. Severe pollution in agricultural lands, stream water, sediments, and soils have been reported in this area, threatening human health8. As an important evergreen broad-leaved tree species in the southern regions of China, Ligustrum lucidum is highly tolerant to heavy metals22,23. In this study, we aimed to investigate changes in the OJIP transient and related parameters in the leaves of L. lucidum in the presence of Mn. In addition, we evaluated the toxicity of Mn to PSII behavior when L. lucidum was cultured under Mn stress for up to 40 days.

## 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 MnCl2·5H2O 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, TRo/RC, ETo/RC, DIo/RC, see Table 1), flux ratios (φPo, φEo, and ψo) and performance index (PIABS) according to the JIP-test19. Relative variable fluorescence at time t, at the J-step, and at the I-step (i.e., Vt, VJ and VI, respectively) was calculated using the following equations16,21:

$${{\rm{V}}}_{{\rm{t}}}=({{\rm{F}}}_{{\rm{t}}}-{{\rm{F}}}_{{\rm{o}}})/({{\rm{F}}}_{{\rm{M}}}-{{\rm{F}}}_{{\rm{o}}})$$
(1)
$${{\rm{V}}}_{{\rm{J}}}=({{\rm{F}}}_{{\rm{J}}}-{{\rm{F}}}_{{\rm{o}}})/({{\rm{F}}}_{{\rm{M}}}-{{\rm{F}}}_{{\rm{o}}})$$
(2)
$${{\rm{V}}}_{{\rm{I}}}=({{\rm{F}}}_{{\rm{I}}}-{{\rm{F}}}_{{\rm{o}}})/({{\rm{F}}}_{{\rm{M}}}-{{\rm{F}}}_{{\rm{o}}})$$
(3)
$${{\rm{\Delta }}V}_{{\rm{t}}}={{\rm{V}}}_{{\rm{t}}}-{{\rm{V}}}_{{\rm{t}}({\rm{control}})}$$
(4)

where FJ is fluorescence intensity at 2 ms and FI 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 WOP = (Ft − Fo)/(FP − Fo). Normalization between O and K (300 μs) steps revealed L-band (150 μs), resulting in the variable fluorescence24:

$${{\rm{W}}}_{{\rm{OK}}}=({{\rm{F}}}_{{\rm{t}}}-{{\rm{F}}}_{{\rm{o}}})/({{\rm{F}}}_{{\rm{K}}}-{{\rm{F}}}_{{\rm{o}}})$$
(5)
$${{\rm{W}}}_{{\rm{OK}}({\rm{control}})}=({{\rm{F}}}_{{\rm{t}}}-{{\rm{F}}}_{{\rm{o}}})/({{\rm{F}}}_{{\rm{K}}}-{{\rm{F}}}_{{\rm{o}}})$$
(6)
$${{\rm{\Delta }}{\rm{W}}}_{{\rm{OK}}}={{\rm{W}}}_{{\rm{OK}}}-{({{\rm{W}}}_{{\rm{OK}}})}_{{\rm{control}}}$$
(7)

Normalization between O and J (2 ms) steps revealed K-band (300 μs), resulting in the variable fluorescence24:

$${{\rm{W}}}_{{\rm{OJ}}}=({{\rm{F}}}_{{\rm{t}}}-{{\rm{F}}}_{{\rm{o}}})/({{\rm{F}}}_{{\rm{J}}}-{{\rm{F}}}_{{\rm{o}}})$$
(8)
$${{\rm{\Delta }}{\rm{W}}}_{{\rm{OJ}}}={{\rm{W}}}_{{\rm{OJ}}}-{({{\rm{W}}}_{{\rm{OJ}}})}_{{\rm{control}}}$$
(9)

The FI, FJ, FK, FM, and FO represent fluorescence at I-step, J-step, and K-step, dark-adapted maximum fluorescence, and dark-adapted minimum fluorescence, respectively. ΔVJ, ΔVI, ΔWOK, and ΔWOJ represent the J-band, I-band, L-band, and K-band, respectively, and are associated with the accumulation of QA21, the proportion of QB-non-reducing PSII RCs16, energetic connectivity of antennae to PSII RC units24, and the activity of OEC of PSII donor side25, respectively.

### Gas exchange

Net photosynthetic rate (Pn) and intercellular CO2 concentration (Ci) were measured by LI-COR 6400 portable photosynthesis system (LI-COR Bioscience, Lincoln, NE, USA). Measurements were carried out on three leaves per plant on day 40 with 1,000 μmol photon m−2 s−1, and a 2 min measurement duration per sample.

### Determination of total Mn content

The dried biomass of different organs (roots, stems, and leaves) was powdered and used to digested with 15 mL acid mixture (HClO4/HNO3 = 1/4). The concentration of Mn was determined by ICP-AES (Optima 8300, American platinum Elmer, USA).

### 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.

## Results

### 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 (VJ) 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. VJ was significantly higher on day 40 compared with day 10 in all Mn treatment levels. The variable fluorescence at 30 ms (VI) increased with Mn levels (Table 2). VI 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. VI 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 (PIABS) showed a declining trend during the test period (Fig. 3a). PIABS in the L4-treated group was significantly decreased compared 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, PIABS was significantly decreased on day 40 compared with day 10.

Absorption ABS/RC (Fig. 3b), trapping TRo/RC (Fig. 3c), and dissipation DIo/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 ETo/RC (Fig. 3d) first increased and then decreased along with the increase of Mn levels, and the maximum value of ETo/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 φPo (Fig. 3f), the probability that an absorbed photon moves an electron further than QAEoψo) (Fig. 3g), and the probability that a trapped exciton moves an electron further than QAo) (Fig. 3h) at various time points showed a declining trend with increasing Mn levels. φPo was significantly decreased in the L4-treated group compared with the control on days 10, 25, and 40, and φPo was significantly decreased in L2, L3, and L4-treated groups compared with the control on day 40. φEo and ψo were significantly decreased in the L2-, L3- and L4-treated groups compared with the control on day 40.

### Interaction of stress time and Mn levels for Chl a fluorescence parameters (FPs) in L. lucidum leaves

All JIP-test parameters significantly varied with the Mn levels, and all parameters, except VI, ABS/RC, TRo/RC, DIo/RC, and φPo, were significantly affected by the stress time. However, none of the parameters significantly responded to the interaction between Mn levels and stress time.

### Effects of Mn on net photosynthesis rate (Pn) and intercellular CO2 concentration (Ci) in L. lucidum leaves

As reported in Table 4, Pn was significantly decreased in L2-, L3-, and L4-treated groups compared with the control. Further, Pn in the L3- and L4-treated groups was significantly decreased compared with the L1-treated group. Ci increased with the Mn levels (Table 4), but no significant differences were observed between the control and the Mn-treated groups.

### Total contents of Mn in L. lucidum leaves, stems, and roots

The total Mn contents of plant organs were significantly higher in the Mn-treated L. lucidum than in the control except for the Mn levels in the leaves of the L1-treated group (Table 5). The Mn concentrations were highest in the roots, followed by the leaves, and the stems. The Mn content was significantly different among roots, stems, and leaves, except for between the stems and leaves in the L1-treated group.

## 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 stress16,21,24. 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-P16,26.

The L-band (~150 μs) is an indicator of energetic connectivity of the antennae to PSII units24,27, implying better excitation energy utilization and system stability of PSII units21,27. 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 transfer28. According to the Grouping Concept and JIP-test21,26, the 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 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 RCs28. When the electron transfer from the OEC to tyrosine Z (Yz) is slower than the electron transfer from P680 to QA and beyond, there is a high accumulation of Yz+25. Thus, this accumulation of Yz+ causes the appearance of K-step, which is directly associated with an inactivation of the OEC25. 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. 1a–c). Meanwhile, the presence of positive K-band in the Mn-treated leaves indicates an inactivation of the OEC24,25 (Fig. 2d–f). Therefore, it may be inferred that the competition between Ca2+ and Mn2+29 in the OEC led to more sites held by Mn2+ in the OEC, and this may depend on the similar ion radius and charge properties of Mn2+ and Ca2+30.

OJIP transients can be used to examine the electron transport flux from PSII RCs to PSI through QA and QB. In this study, leaves in the L3- and L4-treated groups had significantly increased VJ compared with the control leaves (Table 2), indicating that high levels of Mn induced the accumulation of QA. This result is consistent with the previous findings16,26,27. The increased value of VI could be related to the blockade of electron transport downstream of QA by Mn stress31. This finding is also supported by the decrease of φEo and ψo (Fig. 3g,h), as QB was unable to be reduced by QB-non-reducing PSII RCs27,32. Correspondingly, the higher levels of QB-non-reducing centers blocked electron transport towards PSI32. Lower redox state of QB implies altered reduction potential of PSII at the acceptor side in Mn-stressed plants17. Since QA is in quasi-equilibrium with QB and the PQ pool, the lower redox potential of QB will decrease the probability of forward electron transfer between the two quinone acceptors by shifting the redox equilibrium between QAQB and QAQB towards QAQB33,34.

The significant reduction of PIABS, which is a very sensitive indicator of plant functionality27, indicates that excessive Mn may down-regulate PSII function, resulting in prolonged negative effect with irreversible damage. An increase in both ABS/RC and TRo/RC, and a decrease in φPo 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 size24,27. However, an increase in ETo/RC under low levels of Mn (L1 and L2) implies that these inactive RCs35 could prevent further damage to themselves and protect neighboring active RCs in response to the absorbed light energy in the active RCs36. Significantly increased DIo/RC and decreased ETo/RC in the highest Mn treatment group (L4) shows that the excess excitation energy was mostly dissipated21,24.

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, ETo/RC, φEo, and ψo were significantly influenced by Mn stress time, indicating that the blockage of PSII electron flow beyond QA 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 QA and the increase in VJ.

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 seedlings16, Al-treated Citrus grandis26, and Cd-treated Solanum lycopersicum37. But different from our results here, Cr-treated Spirodela polyrhiza was found to have a decreasing trend of TRo/RC, indicating that the Cr damages LHCs38. 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 limitation39,40. 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 CO2 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 PIABS. Thus, Mn toxicity contributed to the observed significant reduction of Pn through its effects on photosynthetic apparatus43.

## 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 QA and an accumulation of QA.