Photosystem II photoinhibition and photoprotection in a lycophyte, Selaginella martensii

Abstract The Lycophyte Selaginella martensii efficiently acclimates to diverse light environments, from deep shade to full sunlight. The plant does not modulate the abundance of the Light Harvesting Complex II, mostly found as a free trimer, and does not alter the maximum capacity of thermal dissipation (NPQ). Nevertheless, the photoprotection is expected to be modulatable upon long‐term light acclimation to preserve the photosystems (PSII, PSI). The effects of long‐term light acclimation on PSII photoprotection were investigated using the chlorophyll fluorometric method known as “photochemical quenching measured in the dark” (qPd). Singularly high‐qPd values at relatively low irradiance suggest a heterogeneous antenna system (PSII antenna uncoupling). The extent of antenna uncoupling largely depends on the light regime, reaching the highest value in sun‐acclimated plants. In parallel, the photoprotective NPQ (pNPQ) increased from deep‐shade to high‐light grown plants. It is proposed that the differences in the long‐term modulation in the photoprotective capacity are proportional to the amount of uncoupled LHCII. In deep‐shade plants, the inconsistency between invariable maximum NPQ and lower pNPQ is attributed to the thermal dissipation occurring in the PSII core.


| INTRODUCTION
The evolution of the photosynthetic apparatus allowed land plants to adapt to a broad range of light conditions, from extreme shade to full sunlight. However, any change in light regime during the plants' lifetime represents a major threat to their survival and requires structural and functional adjustments of their photosynthetic machinery (developmental acclimation) (Lichtenthaler et al., 2007;Pribil et al., 2014;. Selaginella martensii Spring is a shade plant typical of the understory of tropical and equatorial rainforests. However, this ancient tracheophyte is sufficiently flexible to acclimate to extreme light regimes, such as deep shade or full sunlight (Ferroni et al., 2016;Ferroni, Brestič, et al., 2021). Its long-term acclimation to different light regimes produces major rearrangements in the thylakoid organization and photosystem I (PSI) and II (PSII) relative abundance, whereas, unlike most angiosperms, it does not modulate the lightharvesting antenna complex II (LHCII) content and the total thermal dissipation capacity of absorbed excess energy (Ferroni et al., 2016).
Deep-shade (L) acclimated thylakoids of S. martensii are characterized by a peculiar pseudo-lamellar organization, while both midshade (M) and full-sunlight (H) plants display a predominant granal structure. The PSI/PSII ratio increases from L to H plants because the PSI content rises in parallel to the increasing light availability, while PSII is more abundant in L and M plants than H. In contrast, the relative amount of LHCII does not change in response to light acclimation (Ferroni et al., 2016). This characteristic seems typical of seedless plants (Gerotto et al., 2011), while angiosperms generally cope with increasing light availability by decreasing the LHCII content (Albanese et al., 2019;Ballottari et al., 2007;Flannery et al., 2021;Schumann et al., 2017). However, despite the invariable LHCII content, the longterm light acclimation in S. martensii strongly influences the LHCII association with PSII. PSII-LHCII supercomplexes are not high in abundance in native gels of S. martensii thylakoids, but the amount is clearly higher in L and M than in H plants (Ferroni et al., 2014(Ferroni et al., , 2016. Higher abundance of PSII-LHCII supercomplexes in L plants responds to the need for a larger PSII antenna to enhance the harvesting process under limiting light conditions. In contrast, H plants conceivably need a smaller PSII antenna because the light availability is not limiting, and the safe management of excess light is instead the priority. In fact, in S. martensii the great majority of LHCII antennae do not form stable complexes with PSII but are found in the form of free trimers (Ferroni et al., 2016). Free LHCII trimers are common in Viridiplantae, and their function is a hot topic in photosynthesis research, being possibly involved in thermal dissipation of excess absorbed energy (Holzwarth et al., 2009;Horton et al., 2005;Johnson et al., 2011;Nicol et al., 2019;Shukla et al., 2020), PSII connectivity (Haferkamp et al., 2010;Zivcak et al., 2014), PSI-PSII interconnectivity (Grieco et al., 2015;Wientjes et al., 2013;Wood & Johnson, 2020). Moreover, the thylakoid membrane of S. martensii is characterized by permanent megacomplexes comprised of PSII, PSI, and LHCII, which presence increases from L to H plants (Ferroni et al., 2016). The abundance of these megacomplexes is regulated in response to a short-term highlight exposure; in particular, their increase suggests a facilitating role for the energy repartition between PSI and PSII through a mechanism of energy spillover (Ferroni et al., 2016;Yokono et al., 2015).
Non-photochemical quenching (NPQ) is an operative parameter in fluorescence analysis quantifying the decrease in maximum fluorescence of PSII (F m ) in the dark-acclimated state to a lower value F m 0 in the light-acclimated state (Bilger & Björkman, 1990). NPQ is due to a series of light-induced dissipative processes in competition with PSII photochemistry, and, in general, NPQ can be divided into photoprotective and photoinhibitory quenching components. The main photoprotective component is qE, the high energy-dependent quenching caused by the onset of the transthylakoid ΔpH and upregulated by PsbS activity and zeaxanthin formation (see for review Ruban, 2016). The other minor NPQ components are related to a sustained violaxanthin de-epoxidation to zeaxanthin (qZ), state transitions linked to phosphorylated LHCII movement from PSII to PSI (qT), light avoidance chloroplast movements (qM) and plastid lipocalindependent antenna quenching qH (see for review Malnoë, 2018;Roach & Krieger-Liszkay, 2014). The photoinhibitory component is qI, which depends on the thermal dissipation occurring at the photoinactivated PSII (Aro et al., 1993;Demmig-Adams et al., 2012).
Upon exposure to intense light, PSII photoinactivation can be quantified destructively by monitoring the degradation rate of the D1 PSII core protein (Aro et al., 1993;Kato et al., 2012;Keren et al., 1995) or by the light-saturated oxygen evolution of PSII in the presence of an artificial electron acceptor (Delieu & Walker, 1983;Mattila et al., 2020;Öquist et al., 1992;Schansker & van Rensen, 1999); however, it is more easily and precisely analyzed in vivo as the decline of PSII photochemical quantum yield (Campbell & Tyystjärvi, 2012;Chow et al., 1991;Mattila et al., 2020;Schansker & van Rensen, 1999) and/or the persistence of a sustained NPQ fraction in darkness (Demmig-Adams et al., 2012;Nilkens et al., 2010). Ruban and Murchie (2012) proposed an alternative, fast and non-invasive method to monitor the PSII photoinactivation. Their chlorophyll fluorescence approach is based on the calculation of the parameter qP d , "photochemical quenching measured in the dark." qP d assesses the onset of PSII photoinactivation by comparing two values of minimum fluorescence (F 0 0 ): (a) the actual minimum fluorescence measured after a short far-red stimulation (F 0 0 act ) and (b) the value of F 0 0 calculated according to Oxborough and Baker (1997), which is an estimate of F 0 0 as a function of NPQ (F 0 0 calc ). qP d varies theoretically between 0 and 1; in the absence of photoinactivation, F 0 0 calc matches F 0 0 act , and correspondingly qP d = 1. The occurrence of photoinactivation affects only F 0 0 act , whereas F 0 0 calc does not account for it; hence, F 0 0 calc underestimates F 0 0 (i.e., F 0 0 calc < F 0 0 act ), and qP d drops consequently below 1. The theoretical lower limit qP d = 0 could be only reached when all the PSII reaction centers are closed and photoinactivated. qP d values are monitored during experiments in which a plant sample is exposed to subsequent steps with increasing irradiance (light curves). Ruban and Murchie (2012) empirically fixed qP d ≤ 0.98 as the threshold to assess the onset of PSII photoinactivation during a light curve. Accordingly, the effectiveness of photoprotection provided by NPQ to PSII corresponds to the last value of NPQ that allows a qP d value above 0.98.

This method was developed and broadly validated in Alexander
Ruban's Laboratory in the model angiosperm Arabidopsis thaliana, including mutants, chemical treatments, and acclimation to contrasting light regimes (Giovagnetti & Ruban, 2015;Ruban & Belgio, 2014;Tian et al., 2017;Townsend et al., 2018;Ware et al., 2015Ware et al., , 2016Wilson & Ruban, 2019, 2020b. More recently, the qP d method was also applied to other photosynthetic organisms, such as the spring ephemeral Bertereoa incana, Prunus cerasifera, Oryza sativa, and the algal reef builder Neogoniolithon sp. (Gefen-Treves et al., 2020;Lo Piccolo et al., 2020;Okegawa et al., 2020;Wilson & Ruban, 2020a). However, the phototropin 2 mutant of A. thaliana, which is unable to produce light-avoidance chloroplast movements, was found to be completely insensitive to photoinhibition according to the qP d method (Wilson & Ruban, 2020b), while it is instead known to be extremely prone to photobleaching (Cazzaniga et al., 2013). Consequently, Bassi and Dall'Osto (2021) consider the qP d method insufficiently validated, that is, not always leading to results consistent with independent methods.
In S. martensii, the variability in PSII photoprotection is expected from an ecophysiological point of view, but it seems hardly compatible with the constancy of NPQ and qE amplitudes across L, M, and H plants. The use of the qP d method in the lycophyte S. martensii may potentially unveil whether the long-term acclimation to contrasting light regimes influences the PSII photoprotection capacity. Given the recent introduction of the method and the non-angiosperm plant species, the qP d method was checked for consistency with a direct assessment of PSII quantum yield loss upon light exposure. The present study shows that the photoprotective capacity of NPQ well matches the growth light regime in S. martensii. Moreover, the qP d method indicates the relevance of antenna uncoupling in PSII photoprotection, suggesting a physiological role for the abundant and invariable amount of LHCII in ancient vascular plants. (PPFR <80 μmol m À2 s À1 ), that is, the typical light environment experienced by S. martensii in its natural habitat. Finally, high-light grown plants (H) were exposed to direct sunlight, which provided typically a maximal PPFR higher than 800 μmol m À2 s À1 . Subsequent biochemical and fluorometric analyses were performed on the terminal branches after at least 3 weeks of acclimation to each light regime.

| Thylakoid isolation and blue-native gel electrophoresis
Branches of S. martensii plants were dark acclimated for 1 h. Terminal branches were harvested and grinded in an ice-cold (À20 C) mortar in the presence of a grinding buffer (Järvi et al., 2011). The whole thylakoid isolation was performed according to Järvi et al., 2011. Extracted thylakoids were promptly frozen and stored in liquid nitrogen until use. For quantification of pigments, thylakoids were solubilized in 90% (v/v) acetone buffered with HEPES-KOH (pH 7.8) and analyzed using a spectrophotometer Ultrospec 2000 (Pharmacia Biotech). Chlorophyll a and b content were determined according to Ritchie (2006), while Wellburn's equation (Wellburn, 1994) was used to determine the carotenoid content. For electrophoresis, thylakoid solubilization was performed according to Järvi et al. (2011) using 1.5% β-dodecylmaltoside. Bluenative gel electrophoresis (BN-PAGE) was performed according to Järvi et al. (2011), with modifications as in Giovanardi et al. (2018), maintaining the electrophoretic chamber at 0 C.

| Chlorophyll fluorescence measurements
Modulated chlorophyll fluorescence was measured using a Walz Junior PAM (Walz) on independent samples previously dark-acclimated for 30 min. All the measurements were performed in the morning to avoid the presence of photoinhibition, especially in H plants. Light curves were recorded applying a simplified version of the method described by Ruban and Murchie (2012). Before light exposure, minimum (F 0 ) and maximum (F m ) fluorescence levels in the dark were measured with the saturating pulse (SP, 0.6 s) method, and the variable fluorescence was  Hendrickson et al. (2004). The 1-qP parameter was calculated as an indicator of the excitation pressure inside PSII according to Schreiber et al. (1986). F m quenching to F m 0 following the onset of light-induced thermal dissipation was quantified using the Stern-Volmer NPQ parameter (Bilger & Björkman, 1990). NPQ equals the Y(NPQ)/Y (NO) ratio (Ferroni et al., 2014;Lazár, 2015).
In addition to the light curves, induction curves were also recorded at fixed independent irradiances. After the 30-min darkacclimation, the samples were exposed to 19 min of continuous actinic light illumination (24,45,65,90,125,190,285,420,625, and 820 μmol m À2 s À1 ), each followed by 38 min and 30 s of dark relaxation. F m 0 and F 0 0 fluorescence levels were measured every minute during the light induction and at intervals with increasing length during the dark relaxation (30 s, 1 min, 2 min, 5 min, 10 min, and 20 min).
The PSII quantum yield loss because of photoinhibition, Y(qI), was calculated as the difference between the PSII quantum yields in the dark-acclimated sample before the induction curve (F v /F m ) and at the end of the dark-relaxation period.
2.4 | qP d parameter and photoprotective NPQ quantification qP d parameter was calculated at the end of each actinic light illumination step according to Ruban and Murchie (2012). Briefly, qP d compares the actual and calculated values of minimal fluorescence of the light-adapted state samples as it follows: act is the measured minimum fluorescence level, and F 0 0 calc is the theoretical minimum fluorescence level according to Oxborough and Baker (1997). However, because at low-medium irradiances qP d was generally found >1, F 0 0 calc values were corrected as described by Ware et al. (2015) accounting for uncoupled and loosely coupled PSII antenna, thus obtaining the new estimate of F 0 0 calc in the case of a heterogeneous antenna, F 0 0 het : where F 0 and F m are the entry constants and NPQ is the indepen- pNPQ was determined according to Ruban and Murchie (2012) as the last value of NPQ corresponding to qP d het > 0.98. pNPQ values relative to the independent samples were used to obtain the average pNPQ values for each plant group.

| Light-tolerance curves
Light tolerance curves were determined for each plant group plotting the fraction of photoinactivated samples at a given irradiance (those with qP d het < 0.98) with the light intensity as described by Ware et al. (2015). Instead of the Hill equation used by Ware et al. (2015), regression curves were produced fitting the data with the following logistic equation using Origin software: where I 50 is the irradiance responsible for the photoinactivation of half the samples and I X is the irradiance corresponding to a given fraction of photoinactivated samples. The fitting parameter p can be related to the intrinsic propensity of the specific plant group to PSII photoinactivation.

| Data treatment
Statistical analyses and graphical representations were performed using Origin software. Statistical differences were tested by ANOVA followed by a post-hoc Tukey test, using a threshold of P < 0.05.

| RESULTS
Thermal dissipation capacity in S. martensii acclimated to different light regimes improved ability to control the plastoquinone pool reduction state compared with L and M plants . This contributed to a lower excitation pressure inside PSII (1-qP) in H plants ( Figure 1E).
At higher irradiances Y(NO) stabilized to a plateau value both in M and H plants, while in L plants, it decreased continuously, suggesting, in the latter, the occurrence of an additional mechanism responsible for a decrease in the electron inflow into the chain ( Figure 1C). Finally, the differences in Y(NPQ) trend were relatively minor, indicating that the thermal dissipation mechanisms had a similar amplitude in the three plant groups. To the scope of the qP d method, the thermal dissipation was quantified using NPQ.
The steep rise in NPQ at the initial irradiances was very similar in all three groups, while the curves diverged at 125 μmol m À2 s À1 when  Figure 2B).  Ware et al. (2015). For instance, in an artificial system, the quenching capacity by uncoupled antennae was found to be similar or even smaller than that of the coupled antennae (Tian et al., 2015). However, in our study, fitting tests using different combinations of F and m, for example, closer to the value of 1, were unproductive. Conversely, the Ware's heterogeneous antenna model is internally consistent and indicated that the apparent differences among plant groups were almost exclusively due to the parameter U. This is the fraction of uncoupled antenna and can vary between 0 (all antenna is coupled with PSII) and 1 (all antenna is uncoupled from PSII). NPQ that maintains qP d het above the 0.98 threshold (Ware et al., 2015). qP d het was maintained above 0.98 for higher NPQ values in H plants than in M and L, showing a higher photoprotective capacity in the former, although to a well-reduced extent as compared to the estimates from uncorrected qP d (see Figure 3A-C and 4C-E for comparison). H plants benefitted from 29% and 38% more pNPQ than M and L, respectively ( Figure 5A). Photoinhibitory irradiances were re-determined as 70, 90, and 120 μmol m À2 s À1 for L, M, and H, respectively. Although pNPQ estimation can be strongly dependent on the light treatment (length, number, and intensity of the light intervals), pNPQ was strongly consistent with the growth light regime, in contrast to what observed for NPQ MAX ( Figure 2B).

| In sun plants PSII photoprotection is higher and accompanied by PSII antenna uncoupling
The pNPQ/NPQ MAX ratio reported in Figure 5B indicated that ca. 40% of NPQ MAX was photoprotective in H and M plants, while in L plants the photoprotective fraction was reduced to 29%.

| Validation of qP d sensitivity to the onset of PSII photoinhibition
The light curves account for cumulative light-related effects occurring on the same sample exposed to increasing light intensities, but cannot allow a direct comparison of qP d and PSII photoinhibition. To this aim, the qP d method was also applied on the data obtained after independent light inductions to stable irradiances, followed by dark relaxation (see induction curves of NPQ, Figure S4A Figure S3). In particular, the quenching of F 0 0 act can be assigned to the marked NPQ increase occurring during the late stages of the light curve, characterizing specifically the L plants (Figure 2A). This result shows that enhanced thermal dissipation processes could effectively contribute to mitigate PSII photoinhibition rate in L plants at irradiance levels > 400 μmol m À2 s À1 (Figure 4C-E).

| Light tolerance curves offer an alternative and consistent quantification of phototolerance
To further substantiate the results of comparative photoprotection in S. martensii plants, light tolerance curves were built on the same datasets and used as an approach independent from the pNPQ quantification. Plots of light intensity against the respective fraction of photoinactivated samples (those yielding qP d het < 0.98)were fitted with a logistic function: an increased steepness of the curve indicates a higher propension of plants to PSII photoinactivation.
Phototolerance was estimated by I 50 parameter, that is, the light intensity causing the PSII inactivation in half the analyzed samples.
The sensitivity of PSII to photoinactivation decreased from shade to sun acclimation ( Figure 7A-C). However, despite the strongly contrasting light regimes, the difference in I 50 between the two extremes was of only 74 μmol m À2 s À1 (72 vs. 146 μmol m À2 s À1 in L and H plants, respectively; Figure 7D).
Phototolerance trends revealed by I 50 resembled the gradient previously observed for pNPQ ( Figures 5A and 7D). Because there was no obvious relationship between pNPQ and NPQ MAX , it was interesting to find out how the I 50 and pNPQ positioned on the NPQ-light curves. For each type of long-term acclimation, the position of pNPQ marked the end of the linear growth of NPQ in response to increasing irradiance; for NPQ > pNPQ (or irradiance>I 50 ) the linear response with light intensity was lost, that is, NPQ increased more slowly ( Figure 8A-C). This scenario was uniform in all the analyzed samples and indicated that PSII photoprotection was efficient until NPQ increased linearly as a function of light intensity.

| DISCUSSION
The present study demonstrates that in the ancient vascular plant S. martensii the pNPQ is not proportional to the total NPQ amplitude (NPQ MAX ) inducible in plants acclimated to strongly contrasting light regimes. Instead, the PSII photoprotection effectiveness is strongly dependent on the light regime, with a remarkable increase in pNPQ from L to H plants ( Figure 5A). Developmental acclimation to higher light availability results indeed in a higher phototolerance to increasing irradiance (Figure 7). The inconsistency between pNPQ and NPQ MAX finds its major explanation in the special regulation of excitation energy management in deep-shade plants when exposed to exceedingly high light.
F I G U R E 6 Comparison between photochemical quenching measured in the dark in context of a heterogeneous antenna system (qP d het ) and quantum yield of PSII photoinhibition (Y(qI)) as functions of the light intensity in Selaginella martensii acclimated to three natural light regimes-deep shade (A), intermediate shade (B) and high light (C) (see the text for details). In all plants, stable and low values of Y(qI) correspond to stable qP d het around 1. The increment in Y(qI) corresponds to a drop in qP d het . Average values ± standard error for n = 3-5 According to Ruban (2016), pNPQ is mainly due to qE. Because in angiosperms qE is more induced in sun-grown plants, it can be satisfying to explain the variations in photoprotective capacity upon longterm light-acclimation (Anderson & Aro, 1994;Demmig-Adams et al., 2015;Mathur et al., 2018;Mishra et al., 2012;Park II et al., 1997;Schumann et al., 2017). Differently, in S. martensii, qE is only slightly variable between L, M, and H plants (Ferroni et al., 2016).
A different view about the PSII photoprotection offered by NPQ was presented by Lambrev et al. (2012), based on ultrafast time-resolved fluorescence measurement in A. thaliana. Although qE contributes largely to the total NPQ amplitude, it was not considered the main component of photoprotective NPQ, but qZ was instead proposed as the prevailing mechanism that brings photoprotection to PSII (Lambrev et al., 2012). However, the interpretation of the same kinetic NPQ component as qZ in S. martensii is quite questionable (Ferroni, Colpo, et al., 2021). In fact, rather than depending on zeaxanthin, this component, termed qX, seems to be triggered by a reduced electron transport chain and to exploit PSI as a thermal quencher to  Figure 5). NPQ loses its linear response to light at NPQ > pNPQ. Each point represents average value ± standard error for n = 12 (L), 16 (M), and 18 (H) prevent PSII photodamage Ferroni, Colpo, et al., 2021). qX activity is deemed related to PSII interactions with PSI as mediated by LHCII (Ferroni et al., 2014), not only the formation of the state transition complex (Galka et al., 2012;Pesaresi et al., 2009;Wood & Johnson, 2020), but also the assembly of PSI-LHCII-PSII megacomplexes responsible for an extensive connectivity between photosystems, including the chance for energy spillover of excitation energy from PSII to PSI (Barber, 1980;Grieco et al., 2015;Jajoo et al., 2014;Järvi et al., 2011;Tiwari et al., 2016;Yokono et al., 2015).
Currently, energy spillover in megacomplexes is considered relevant to effective PSII photoprotection (Yokono et al., 2019). (2014) and Ware et al. (2015), the role of additional F 0 0 quenchers could be played by the antennae uncoupled from PSII. These hypothetical quenchers would be characterized by an enhanced NPQ capacity and by a lower fluorescence emission than the coupled population. At present, this is the only well-modeled interpretation of qPd > 1 and, as such, it was used in our work. Accordingly, S. martensii would be characterized by a larger population of uncoupled/loosely coupled antenna than the angiosperms, in particular A. thaliana (Ware et al., 2015). In the latter, the antenna uncoupling distorting the qP d trends is specific to the low light-grown plants and related to the acclimative accumulation of LHCII (Ware et al., 2015). It is not surprising that the shade-tolerant lycophyte S. martensii is affected by similar distortions, because of the great amount of LHCII as compared to PSII (Ferroni et al., 2014(Ferroni et al., , 2016 (Ferroni et al., 2016). Therefore, in S. martensii the involvement of uncoupled quenched antennae in qP d determination seems well grounded from a biochemical point of view. However, considering that the excitation quenching capacity by uncoupled LHCII is a debated issue, other explanations are possible (Tian et al., 2015). Another reason for a too low F 0 0 act at non-photoinhibitory irradiances could be the reduction in PSII absorption cross-section due to state-transition-like antenna detachment. Interestingly, in M plants the maximum divergence between F 0 0 act and F 0 0 calc -that is, the peak in qP d -is in very good agreement with the peak of LHCII phosphorylation previously reported . Both events occur approximately at the irradiance of growth (50-100 μmol m À2 s À1 ). If the qP d increase is a reflection of state-transition-like processes, the antenna uncoupling plays again a pivotal role. This inference allows the interpretation of qP d in the more general frame of the multiple roles assigned to the free LHCII in the thylakoid membrane, including the regulation of PSI-PSII interaction at the grana margins (Grieco et al., 2015;Wientjes et al., 2013;Wood & Johnson, 2020;Zivcak et al., 2014). It is very probable that a more complete interpretation of qP d should also take into account the photoprotective contribution by PSI, together with mixed populations of uncoupled LHCII, which could be "functionally isolated" from PSII (quenched or unquenched) and/or connected to PSI.
Among PSII uncoupled antennae, a consistent fraction probably serves as qE quenching site (Holzwarth et al., 2009;Miloslavina et al., 2011;Ruban, 2016). Because in S. martensii the qE amplitude is almost invariable irrespective of the light regime (Ferroni et al., 2016), the remaining, non-qE-related fraction of uncoupled antenna must be responsible for the increased photoprotection from L to H plants, for example, via interactions involving PSI as a photochemical or nonphotochemical quencher (Brestic et al., 2015;Tiwari et al., 2016;Wood & Johnson, 2020;Yokono et al., 2019). In S. martensii the amount of PSI and PSI-LHCII-PSII megacomplexes increases under high light (Ferroni et al., 2016) and the assembly of the latter requires the recruitment of free LHCII trimers to mediate labile interactions between PSII and PSI (Grieco et al., 2015). Terashima et al. (2021) suggested that the energy spillover process could be particularly important in shade-tolerant plants to confer them resistance against strong sunflecks. In a lycophyte with invariable LHCII amount and low carbon fixation capacity (Ferroni et al., 2016;Ferroni, Brestič, et al., 2021), the extensive PSII antenna uncoupling can allow an emphasized exploitation of PSI-linked photoprotection also upon long-term acclimation to high light. Conversely, in the complete absence of sunflecks, the photoprotective role of uncoupled antennae and PSI seems diminished, potentially exposing the small PSI pool of L plants to photodamage upon short-term exposure to even moderate light (Brestic et al., 2015). Because PSI is particularly sensitive to donor-side over-reduction (Takagi et al., 2017), its photoprotection primarily depends on a reduced inflow of electrons from PSII into the membrane (Yamamoto & Shikanai, 2019). The qP d het results suggest that in L plants of S. martensii the safe accumulation of a stable population of photoinactivated PSII under moderate/high light may serve to the scope of downregulating the electron flow and preserve PSI . Beside photoprotective thermal dissipation mechanisms, PSII photoinactivation is also counteracted by the repair cycle of PSII based on the D1 core protein turnover (Keren et al., 1995, Baena-González & Aro, 2002, Kato & Sakamoto, 2009, Nath et al., 2013. The PSII repair cycle requires the migration of photodamaged PSII to the non-appressed grana margins, where the turnover takes place (Anderson & Aro, 1994;Järvi et al., 2015;Li et al., 2018;Kirchhoff, 2014). D1 turnover is more active in sun plants, whose thylakoid membranes are enriched in grana margins (Anderson & Aro, 1994). Differently, shade plants are characterized by a higher grana stacking, further increased when exposed to high irradiance; the extensive thylakoid appression hinders the PSII turnover, so that the grana contain a kind of reservoir of inactive PSII (Anderson & Aro, 1994;Mathur et al., 2018;Matsubara & Chow, 2004;Tietz et al., 2015). The accumulation of photoinactivated PSII upon increasing irradiances also occurs in S. martensii, starting from relatively low light intensities (see I 50 values, Figure 7D). However, in L plants-the richest in PSII-qP d surprisingly slows its drop at the highest irradiances, indicating the achievement of a constant ratio between intact and photoinactivated PSII ( Figure 4C). A stable reservoir of photoinactivated PSII in long appressed pseudo-lamellar thylakoids may have an important photoprotective role, because they safely dissipate the excess of absorbed energy, preventing the photoinactivation of the remaining, active PSII, but also restricting the electron inflow directed to PSI (Mathur et al., 2018;Matsubara & Chow, 2004). According to Ruban (2016), qI does not contribute to pNPQ. However, the qP d method indirectly evidences the physiological function of qI in mitigating the PSII photoinactivation, although the small qI extent (5%-10% of total NPQ amplitude, Ferroni et al., 2016) could not explain per se the constant increase in NPQ at high irradiances observed in L plants (Figure 2A). A possible interpretation of this phenomenon can be related to an additional thermal dissipation mechanism produced by PSII cores (Nicol et al., 2019), more relevant in L plants because of their higher content in PSII.
In conclusion, although qE might still represent the main component of pNPQ as postulated by Ruban (2016), in S. martensii the pNPQ could also strongly depend on the PSII antenna uncoupling and the relative amount of PSII and PSI. After the correction for the antenna heterogeneity, qP d het is confirmed as a very precise indicator of incipient PSII photoinhibition. Furthermore, the example of S. martensii suggests that the qP d method can be sensitive to PSI-related mechanisms and to the PSII core-related thermal dissipation. A sustained PSII photoinhibition can have a photoprotective function to increase physiologically a low PSI/PSII ratio (Shimakawa & Miyake, 2019). Evidence for the importance of such processes is quite sparse in the literature regarding angiosperms.
The results obtained in S. martensii may indicate that processes collateral to qE, and often considered as minor, can have had a special relevance for thylakoid membrane photoprotection in ancient land plants, which do not modulate extensively the LHCII total content (Gerotto et al., 2019;Lei et al., 2021). However, any evolutionary

DATA AVAILABILITY STATEMENT
The data supporting the findings of this study are available from the corresponding author Lorenzo Ferroni, upon request.