Healthy specimens of P. iwatensis, characterized by intact rhizome systems and fresh leaves that lacked mechanical injury (Figure 1), were collected from the rocky intertidal zone of Rongcheng (37° 16′N, 122° 41′E), Weihai, Shandong Province, China, throughout May 2021. The samples were precultured in aquaria with filtered seawater for 3 days under conditions of 15°C and a 10:14 h light: dark cycle with a minimum saturating irradiance of 40 μmol photons/m² s.

Precultured P. iwatensis samples were acclimatized in the dark overnight prior to experimentation. To evaluate the dynamics of the OEC activity, dark-adapted samples were exposed to a light intensity of 400 μmol photons/m² s, which was the usual underwater light conditions measured in P. iwatensis habitats during midday. The samples were exposed for 6 h, with measurements taken every 1 h during this period. Furthermore, to investigate the ability of OEC to recover, two light environments were simulated to treat the samples. Pretreated samples were exposed to 400 μmol photons/m² s for 3 h to simulate the high light environment at midday under usual conditions. Following 1, 2, and 3 h of light exposure, a portion of the samples was taken for recovery under dark conditions in 15°C seawater. Measurements were taken every 1 and 3 h during exposure to light and recovery, respectively. In addition, based on the light environments of cloudless sunny neap tide days and continual sunny days, the times of exposure were extended to 6 h per day for 3 continuous days, which was established as the harsh light stress. Following light exposure, samples were recovered in the dark at 15°C. Measurements were taken at the beginning and end of each light exposure. All the experiments were conducted in aquaria. The light source was provided by an LED lamp with a color temperature range of 6,000 K (LI-COR Inc., Lincoln, NE, United States). The leaf segments were randomly collected from 2 cm above the leaf sheath before the parameters were measured, with a portion of the samples used for fluorescence measurements after 15 min of dark adaptation, while the other portions were immediately frozen in liquid nitrogen for subsequent Western blot analyses. The subscript “control” indicated that the parameter was derived from dark-adapted samples under controlled laboratory conditions. The subscript “treatment” indicated that the parameter was derived from samples treated with light stress in the laboratory. Each measurement was conducted in triplicate.

To examine the in situ inactivation of the OEC, in situ measurements were conducted on a cloudless sunny neap tide day in May 2021 at the intertidal zone of a rocky shore with high seawater transparency in North Rongcheng (37°16’N, 122°41’E). Apparently healthy P. iwatensis plants were collected by diving every 2 h from 6:00 to 18:00 and at 6:00 and 8:00 on the following morning. Leaf segments that had been collected from ∼2 cm above the leaf sheath were partially used for chlorophyll fluorescence measurements, while the remainder were immediately frozen in liquid nitrogen for subsequent Western blot analyses. The light intensity of P. iwatensis habitats was measured using QSPL2100 (Biospherical Instruments Inc., San Diego, CA, United States). The subscript “control” indicated that the parameter was derived from samples collected at 6:00 on the first day of measurement under natural conditions in the field. The subscript “treatment” indicated that the parameter was derived from samples at different time points under natural conditions. Each measurement was conducted in triplicate.

To analyze the photosynthetic physiological parameters, M-PEA-2 (Hansatech, Norfolk, United Kingdom) was used to monitor the rapid chlorophyll fluorescence induction kinetic curve (OJIP curve). The OJIP curve was induced by 5,000 μmol photons/m² s red light at a measurement time of 2 s. Chlorophyll fluorescence parameters were calculated as previously described (Strasser et al., 2010; Guo et al., 2020). The normalized OJIP fluorescence rise kinetics could be calculated using the formula ΔVt = Δ[(F_t – F_O)/(F_m – F_O)]; F_v/F_m = (F_m – F_O)/F_m, which represented the maximal quantum yield of the PSII; W_K = (F_K – F_O)/(F_J – F_O) and ΔW_K = [(F_K – F_O)/(F_J – F_O)]_treatment – [(F_K – F_O)/(F_J – F_O)]_control, which reflected the extent of damage on the donor side of PSII; the active fraction of OEC centers, characterized by OEC_centers, could be calculated as OEC_centers = [1 – (V_K/V_J)]_treatment/[1 – (V_K/V_J)]_control. A G-band observed between the I-step and P-step was supposed to relate to the redox state of the end PSI acceptor pool (Zagorchev et al., 2021).

The levels of expression of the OEC peripheral proteins PsbO, PsbP, PsbQ, and PSII core protein D1 were used to verify the activities of the OEC and PSII reaction center. The chloroplasts from leaves were separated using a Plants Leaf Chloroplast Rude Divide Kit (GenMED Scientifics Inc., Arlington, MA, United States). The chlorophyll content of the separated chloroplasts was measured as described earlier (Porra et al., 1989). To compare quantitative differences, serial dilutions (1.25, 2.5, and 5 μg of chlorophyll corresponding to 25, 50, and 100% of the control samples) of the control samples were loaded onto the first three lanes of a 12% SDS-triglycine-PAGE gel, followed by loading equal contents of chlorophyll (5 μg) of the solubilized materials of treated samples onto the same gel for separation. They were then transferred to PVDF membranes (0.22 μm; Sartorius Stedim, Göttingen, Germany). Western blot assays with antibodies against PsbO, PsbP, PsbQ, and D1 (Agrisera, Vännäa, Sweden) were performed as described earlier (Fristedt et al., 2009). RuBisCo large subunits (RbcL) with the same loading volume (5 μg of chlorophyll) as the proteins above were used as equal loading controls. The chemiluminescent bands were quantified on a Gel Doc XR + system (Bio-Rad, Hercules, CA, United States) using Image Lab software (Bio-Rad). The total sample density in each case was normalized based on the RbcL density. Each measurement was conducted in triplicate.

The rate of photosynthetic O₂ evolution was measured to evaluate the overall photosynthetic performance. This rate was determined using a liquid-phase oxygen electrode system (Chlorolab2 + ; Hansatech, Norfolk, United Kingdom) at 15°C. Leaf fragments (∼25 mg) were placed in the reaction chamber with 2 ml of seawater. A white light of 40 μmol photons/m² s was used to measure the evolution of O₂. The net photosynthetic rate (Pn) of the leaves was measured within 3 min of light irradiation, and the respiration rate (R) was measured within 2 min of the dark state. The rate of O₂ evolution (P) was calculated as P = R + Pn and expressed as nmol O₂/min g fresh mass. Each measurement was conducted in triplicate.

A statistical analysis of the collected parameters was performed using a one-way ANOVA in SPSS 22.0 (IBM Inc., Armonk, NY, United States). Post hoc comparisons were made using Tukey’s trend test. Statistical significance was assessed at a threshold of p < 0.05.

The ΔVt curves of fluorescence rise kinetics normalized by OJIP changed with an increase in the time of exposure, suggesting that the performance of PSII was significantly affected by the duration of exposure (Figure 2A). The intensity of K-band at 0.3 ms in the curve of ΔW_k increased gradually with the duration of exposure, indicating that the OEC was continuously inactivated (Figure 2B). Furthermore, the activity of OEC, characterized by the peripheral protective protein contents of PsbO, PsbP, and PsbQ, decreased significantly during light exposure (Figure 2C and Supplementary Figure 1). Indeed, high light (HL) induced the partial inactivation of active OEC, as demonstrated by the variation in the physiological parameter OEC_centers (Figure 2D). The percentage of OEC inactivation continuously increased with the duration of exposure, with 6 h of exposure resulting in about 10% of inactivation (Figure 2D).

Following a single HL exposure for 3 h, both the F_v/F_m and W_k gradually recovered during darkness (Figures 3A,B). Furthermore, the Western blot showed that the protein contents of PsbO, PsbP, and PsbQ increased during the recovery period (Figure 3C and Supplementary Figure 1). All these parameters reached their initial levels after 6 h of recovery, which was also confirmed by the full recovery of OEC_centers (Tukey’s test, p = 0.171, p = 0.13, p = 0.055, p = 0.073, p = 0.068, and p = 0.54, respectively; Figures 3A–D). No net loss of the D1 protein was observed during illumination (Tukey’s test, p = 0.86; Figure 3C and Supplementary Figure 1), indicating that the PSII reaction centers did not suffer net damage.

To further verify the effect of light exposure on the photosynthetic performance of P. iwatensis, the rate of O₂ evolution was measured. Within a single 3-h period of exposure to HL, the rate of O₂ evolution gradually increased, exhibiting a typical photoinduction process (Figure 3E), which represents a normal photosynthetic performance.

Following a single 6-h exposure, F_v/F_m and W_k failed to reach their initial levels until the following day (Tukey’s test, p < 0.05, p < 0.05, respectively; Figure 4A). This was also true for the changes in both the OEC peripheral protein contents and OEC_centers (Tukey’s test, p < 0.05, for both OEC peripheral proteins and OEC_centers; Figures 4B,C and Supplementary Figure 1). These observations indicated the occurrence of irreversible photoinactivation of OEC. Furthermore, irreversible oxidative damage to the PSII reaction centers was observed, as indicated by the D1 protein, which suffered a net loss during illumination and did not return to its original state by the morning of the next day (Tukey’s test, p < 0.05; Figure 4D and Supplementary Figure 1).

During 6 h of HL exposure per day for 3 continuous days, F_v/F_m and W_k exhibited rhythmic changes of the light-dark cycle, with gradual decreases and increases, respectively, during the day and incomplete recovery at night, which made F_v/F_m and W_k keep decreasing and increasing as the days progressed (Figure 4A). Moreover, aside from the decrease in OEC_centers with the extended days of exposure, the proportion of OEC that did not regain activity after dark adaptation overnight also increased daily (Figure 4C). These results indicated the occurrence of continuous irreversible photoinactivation of OEC.

The complete measurement process of the O₂ evolution rate is shown in Figure 5A. When light exposure exceeded 3 h, the rate of O₂ evolution decreased gradually, indicating that the photosynthetic performance was inhibited following long light exposure (Figure 5B). Additionally, under daily light stress, both the net photosynthetic rate in the light and the respiration rate in the dark decreased daily (Figure 5C). The O₂ evolution rate calculated from them exhibited a similar trend (Figure 5B).

To gain insight into the OEC photoinactivation of P. iwatensis in its natural environment, the chlorophyll fluorescence and protein contents were measured in situ on seagrass beds. As shown in Figure 6A, the ΔVt curves significantly fluctuated in a diurnal manner. In the morning, the decrease in F_v/F_m was accompanied by an increase in W_k, reaching their extreme values at 14:00. In the afternoon, F_v/F_m and W_k gradually recovered as the light intensity diminished, but their full recoveries were not observed by the next morning (Tukey’s test, p < 0.05, p < 0.05, respectively; Figure 6B). Similarly, an incomplete recovery was also demonstrated in the OEC peripheral proteins and PSII core D1 protein contents (Tukey’s test, p < 0.05, for both OEC and D1 proteins; Figure 6C and Supplementary Figure 2). The OEC_centers, as shown in Figure 6D, changed with the process of diurnal light fluctuations, with the highest rate of inactivation of approximately 15% at 14:00, which remained at 6% inactivation following an overnight recovery (Tukey’s test, p < 0.05). Those results confirmed that P. iwatensis suffers irreversible OEC photoinactivation in a natural harsh light environment.