Coral reefs are one of the world's most productive and diverse ecosystems, sustained through a symbiosis between reef-building corals and their endosymbiont eukaryotic microalgal partner (Symbiodinium sp.) and a complex bacterial consortia (Ainsworth et al., 2010). Nutrient exchange between partners is fuelled primarily by Symbiodinium's photosynthetic productivity and hence light utilization is a key factor driving coral metabolism from the organismal (Roth, 2014) to the entire ecological (Muir et al., 2015) reef scale. As the highly orchestrated symbiotic metabolic exchange can be threatened by several environmental stress factors that may impact coral metabolism, it is crucial to understand stress impacts at the holobiont level through integrated bio-optical/bio-physical, metabolomics and molecular approaches (Pernice and Levy, 2014). The importance of understanding the photosynthetic processes is particularly critical to revealing the light utilization efficiency and thus niche specialization of corals across a range of biogeographical habitats. Such knowledge also characterizes a range of regulatory mechanisms that respond to environmental stress that define the resilience of the coral holobiont (Roth, 2014; Warner and Suggett, 2016). Photosynthetic productivity of Symbiodinium sp. is highly regulated via the coupled inter-system electron transfer processes between photosystem II (P680) and photosystem I (P700), but the regulation mechanisms of this process in Symbiodinium are still poorly understood.

Under steady state environmental conditions, PSII and PSI work in series to ensure the linear flow of electrons from the donor side of PSII to the acceptor side of PSI, and in turn drive CO₂ fixation via the Calvin-Benson Cycle. This linear electron flow is coupled with translocation of protons from the stroma to the lumen to build up a proton motive force that drives ATP synthase activity, producing a defined ratio of ATP and NADPH (e.g., Kramer and Evans, 2011). Over-reduction of the plastoquinone pool that could naturally occur under midday high irradiance levels e.g., in shallow reefs (Gorbunov et al., 2001) or as a result of impaired CO₂ fixation may initiate alternative pathways of electron flow to dissipate excessive excitation energy to avoid photo-oxidative stress while maintaining coordinated level of ATP production and NADPH synthesis (Cardol et al., 2011). For Symbiodinium, these processes are much less characterized than in higher plants or green algae (Warner and Suggett, 2016), but evidence suggests that alternative electron transport can manifest itself in the form of cyclic electron flow (CEF) operating between PSI and the cytochrome b₆/f complex possibly via the ferredoxin-dependent pathway (Aihara et al., 2016), or pseudo-cyclic electron flow of Mehler reaction, that plays a significant role in excess energy dissipation (Roberty et al., 2014). Expression of these pathways, however, may differ significantly between different Symbiodinium genotypes (Aihara et al., 2016; Warner and Suggett, 2016).

To characterize photosynthetic efficiency on multiple levels of organization of the symbiont algae, e.g., from free living state to single nubbins and in situ colonies, one of the most frequently applied methods is the detection of variable fluorescence of chlorophyll a (Chl a) (Warner et al., 2010). Such methods have been broadly applied to investigate light use efficiency (Gorbunov et al., 2001; Ralph et al., 2005; Szabó et al., 2014), photoacclimation (Hill and Ralph, 2005; Hennige et al., 2008; Lichtenberg et al., 2016), and photodamage (Ragni et al., 2010; Hill et al., 2011; Hill and Takahashi, 2014; Schrameyer et al., 2016) of intact corals under various environmental stress conditions. However, chlorophyll a fluorescence measurements of whole coral tissues are not always straightforward due to a strong light gradient and other complex optical properties specific to coral tissues (Enríquez et al., 2005; Wangpraseurt et al., 2012, 2014; Schrameyer et al., 2014; Lichtenberg et al., 2016). Light attenuation within coral tissue is strongly wavelength dependent (Wangpraseurt et al., 2012; Szabó et al., 2014), which particularly influences both the penetration depth of the excitation light beam projected onto the coral surface and the re-absorption of the fluorescence. Attempts to overcome this fundamental problem, based on attenuation of the functional absorption-cross section of PSII (σ) as a measure of effective irradiance within the tissue, are limited by the fact that these measurements are still performed at the coral surface thereby giving rise to potential artifacts of light attenuation of the probing and actinic light from the surface (Szabó et al., 2014). More recently, combined microfiber PAM and dissolved oxygen measurements were performed throughout the entire depth of the tissue, revealing stratification of chlorophyll a fluorescence signals from different symbiont layers with potentially different photoacclimation states (Lichtenberg et al., 2016). In addition, such measurements are still fundamentally invasive and less convenient for rapid monitoring of photosynthetic performance of whole corals, and do not provide information about PSI operation and/or electron transfer, an important regulatory process of alternative electron flow processes.

Whilst past studies have applied a combination of biophysical/photobiological methods (e.g., oxygen evolution and uptake measurements, variable chlorophyll a fluorescence and PSI reaction center (P700) redox kinetics) to evaluate the mechanisms underpinning photobiological operation (e.g., Suggett et al., 2008; Roberty et al., 2014; Aihara et al., 2016), most of these measurements require invasive treatment that is impractical on intact corals. Simultaneous measurements of Photosystems I and II electron transport rates [ETR(I) and ETR(II), respectively] in intact corals have been reported (Hoogenboom et al., 2012). However, their approach did not enable meaningful information to be derived about the kinetics of the intersystem electron transfer and the plastoquinone (PQ) pool size, which are the most significant descriptors of “active” PSII. Moreover, no assessment of the active pool of PSII based on P700 redox kinetics signal has been undertaken. P700 kinetics based on near-infrared (NIR) reflectance detection is a known whole-tissue measure for whole chain PSII and PSI electron transport e.g., in plant leaves (Jia et al., 2014), but its applicability for concerted PSII-PSI activity has not yet been evaluated in corals.

Our objective here was therefore to determine the applicability of simple P700 redox kinetic measurements to (i) assay both PSII and PSI activity, (ii) evaluate the applicability of PSI redox kinetics measurements in comparison with PSII chlorophyll a fluorescence kinetics measurements and (iii) reveal the behavior of coupled PSII-PSI electron transport under acute heat stress in the common Great Barrier Reef coral Pavona decussata, which is considered a relatively heat tolerant species in terms of photosynthetic activity. Due to such high thermal tolerance, this model species allows the investigation of fine changes of photosynthetic activity under short, acute stress conditions without initiating major alterations in photosynthetic pigment content, permanent physiological damage or symbiont expulsion e.g., during coral bleaching (McClanahan, 2004; Hill et al., 2012; Krämer et al., 2013; Schrameyer et al., 2014, 2016).

We show that (i) P700 redox kinetics as applied here provide accurate estimate of both the active PSII electron pool and the steady-state and maximal levels of P700⁺ and (ii) sub-lethal heat stress impacts the redox state of P700⁺ and PSII electron transfer processes, maintaining an enhanced electron turnover via PSI, even when linear electron flow from PSII is transiently impaired.

Photosynthetic performance of reef-building corals plays a key role in determining their ecological success under normal growth conditions as well as in response to transient stressors (Warner et al., 2002; Anthony et al., 2007). Photobiological performance and photosynthetic energy budgets of Symbiodinium in hospite have been characterized and reviewed in several studies (Brodersen et al., 2014; Roth, 2014; Warner and Suggett, 2016). However, surprisingly, the available data regarding the fundamental intersystem PSII-PSI electron transport properties in corals is scarce. While changes in PSII activity represent one of the best known indicators of heat stress sensitivity (Warner et al., 1999; Hill et al., 2004b), the loss of PSII quantum efficiency, deduced from variable Chl fluorescence measurements, usually manifests itself under severe prolonged heat impacts which lead to extensive loss of photosynthetic performance (e.g., Hill et al., 2012). Intuitively, severe inhibition of PSII activity should be preceded by more subtle temperature perturbations of the electron transport flow through the whole photosynthetic machinery, lowering photosynthetic performance. Such decline in photosynthetic activity, if persistent, may lead to a gradual deterioration of the photosynthetic performance of the endosymbiont algae without impacting the most characteristic indicator of the stress, the decline in PSII quantum efficiency. Unfortunately, the PSII quantum efficiency is generally used as the most universal (and in most cases the only) indicator of the stress presence in corals. Earlier studies (Hoogenboom et al., 2012) indicated that PSII is a more vulnerable component to heat stress than PSI, which is in agreement with our finding that forward electron transfer kinetics slow down (Figure 7). However, the main aim of our approach was to ascertain whether using a single measurement of P700 redox kinetics yields information about both active PSII and PSI, possibly complementing the performance indicators obtained by more commonly applied chlorophyll fluorescence measurements.

The fact that acute heat stress caused remarkable changes in P700 kinetics, the steady-state level of P700⁺ and the P700 kinetics area suggests that electron flow from PSII has a strong impact on the redox state of P700⁺, similarly to the situation in higher plants (Oguchi et al., 2011; Jia et al., 2014). This is corroborated with the observations that the P700 kinetics area completely disappeared in the presence of DCMU (Figure 3) and the steady state P700⁺ increased, as the linear electron flow from PSII reducing P700⁺ has completely stopped. When heat stress was applied in the presence of DCMU, the steady-state P700⁺ decreased, although to a much lesser extent as compared to the heat treatment in the absence of inhibitor (cf. Figures 2, 4). The fact that the steady-state P700⁺ level drops even in the presence of DCMU (when linear electron flow is blocked) indicates that alternative electron transport pathways might have been initiated under heat stress, the existence of which has been shown in Symbiodinium (Reynolds et al., 2008; Roberty et al., 2014; Aihara et al., 2016). The diminished response of steady-state P700⁺ under heat stress, when DCMU was applied might be due to the disturbance of the redox poise of P700 by DCMU (Fan et al., 2016a). Our chlorophyll fluorescence measurements indicated that under moderate heat stress conditions, maximum PSII quantum efficiency and the functional cross-section of PSII were marginally affected by the heat stress. Instead, kinetic parameters of the photosynthetic electron transport responded to a higher degree. In plants, τ₁ reflects electron transport from Q_A to PQ, whereas τ₂ reflects a slower electron transfer component from PQ to PSI (Osmond et al., 2017). However, these electron transfer steps are uncharacterised in detail for corals; as such, here we refer to τ₁ as the fast component of electron transfer, an inherent property of PSII, which is largely unchanged under acute heat stress. In contrast, τ₂ is most likely closely related to the reduction level of the PQ pool (and possibly the electron transfer steps beyond PQ pool, indicated by the large time constant values), which is related to the phenomenon that the Q_B site is occupied by an electron that is unable to proceed to the PQ pool, because an oxidized PQ molecule from the PQ pool is unavailable. This is corroborated by the calculated oxidized portion of the PQ pool, which strongly declined upon heating. The concentration of oxidized PQ decreases when the PQ pool is more reduced due to limitation of electron transport at the acceptor side of PSI. This is in agreement with earlier findings that persistent reduction of the PQ pool was more prevalent under heat stress or bleaching conditions (Jones and Hoegh-Guldberg, 2001; Hill et al., 2004a).

This interpretation is further corroborated by the finding that when active electron flow from PSII to PSI is impaired under heat stress, maximal fluorescence of the fast induction (OJIP) curves occurs already at the J level, ~2 ms, with only a small thermal phase (JIP) present. Together these measurements indicate that the maximal photochemical capacity of PSII reaction centers is not affected (unchanged F_v/F_m and presence of photochemical OJ phase) under moderate heat stress, whereas the subsequent electron transfer steps became significantly impaired probably due to the heat-induced reduction of the PQ pool, as has been shown for higher plants (Tóth et al., 2007). Taken together, our observations suggest that when referring to the “active PSII electron transfer” involved in reducing P700⁺, the critical steps appear to be the efficiency of electron transfer after the primary acceptor, QA-, rather than the actual photochemical activity of PSII reaction centers (represented by the presence of OJ phase). While F_v/F_m measurements indicated little stress, the simultaneous measurements of PSII to PSI electron transfer rates clearly indicated severe impacts on the PSII-PSI intersystem electron flow, i.e. the forward electron transfer from Q_A to Q_B and the PQ pool. Steady-state P700⁺ levels recorded at 820 nm, in the presence of weak FR light (740 nm) were very sensitive to heat-induced changes, indicating that under acute heat stress there is less P700⁺ under continuous weak FR light, possibly due to enhanced electron pressure, which may originate from non-linear electron flow pathways.

The P700 kinetics signal underwent similar changes to heat stress when chemical inhibition of the Calvin-Benson cycle was applied. Glycolaldehyde (GA) is a known inhibitor of Calvin-Benson cycle in corals (Bhagooli, 2013; Hill et al., 2014) and earlier applications have demonstrated that under conditions when CO₂ fixation is impaired by chemical inhibition (Hill et al., 2014) or by decreased concentrations of dissolved inorganic carbon (Oakley et al., 2014), linear electron flow of photosynthesis is downregulated even at ambient temperatures in the absence of heat stress (Hill et al., 2014; Oakley et al., 2014). Such a response is in agreement with the current study, where P700 kinetics area (bounded by the horizontal line corresponding to steady-state illumination with far-red light and the dipping curve of P700⁺ signal upon application of a saturating flash, representing the delivery of PSII electrons to PSI), completely disappears in the presence of GA, but in the absence of heat stress (Figure 5), in agreement with earlier findings on decreased oxygen evolution in corals as a result of GA treatment (Hill et al., 2014). In parallel with the loss of P700 kinetics area, the initial steady-state P700 level decreases, possibly because inhibition of Calvin-Benson cycle leads to elevated NADPH and ferredoxin levels that cause over-reduction of PQ pool through cyclic electron transport pathway(s) and blockage of the PSI acceptor side. This in fact leads to more reduced P700 (lower steady-state P700⁺) and slower electron transfer rates (smaller P700 kinetics area). Therefore, GA treatment alone mimics the heat-induced changes in photosynthetic electron flow, but the acute heat stress applied in the current study may cause only partial inactivation of Calvin cycle (P700 kinetics area not completely lost), which can be recovered during the applied recovery period at 26°C. As for all dinoflagellates, Symbiodinium sp. carry a type II Rubisco (Whitney et al., 1995) that is considered generally more heat sensitive than type I Rubisco (Leggat et al., 2004). The same heat stress assay employed here was applied in earlier studies to cause partial inactivation of Rubisco in Symbiodinium (Lilley et al., 2010), which may lead to a decrease in relative oxidation level of PQ and thus electron flow back-pressure on PSII (Hill et al., 2014).

In summary, we suggest that under conditions when the chlorophyll fluorescence signals may be complex to interpret i.e., due to the vertical light heterogeneity in coral tissues (Wangpraseurt et al., 2012), P700 kinetics measurements performed in the NIR region may serve as a whole tissue marker for operation of both PSII and PSI and a non-invasive stress indicator of photosynthetic efficiency of coral endosymbionts (the combination of P700 and chlorophyll fluorescence methods to understand intersystem PSII-PSI electron transfer properties are shown in Figure S2). How these current findings under transient and rapid (acute) heat stress ultimately apply to more prolonged heat stress events that drive coral bleaching in nature will be an important next question to investigate, as is, for example, whether the transient perturbation of intersystem electron flow, represented by the P700 kinetics signal, provides a sensitive marker of photosynthetic activity under other sub-lethal stress conditions as well. It has to be noted that during prolonged stress exposure, the P700 signal might also be influenced by bio-optical/physiological alterations of coral tissues (e.g., symbiont expulsion, changes in pigment concentrations across the tissue) that need to be investigated further. Physiological phenomena of the coral holobiont such as tentacle movement and/or contraction could have occurred in the experimental timeframe applied in the current study, and it may have influenced the cross-section and thus light availability within the coral tissues. However, we suggest that such impacts are minimal, because the functional absorption cross-section of PSII was found unchanged (Figure 7B). It also remains to be investigated whether P700 redox signal can be assigned as a functional trait, as it has been shown that chlorophyll fluorescence parameters can be associated with the different Symbiodinium genetic types as photobiological traits (Suggett et al., 2015; Goyen et al., 2017).

Finally, our results are in agreement with earlier observations (Jones et al., 1998; Lilley et al., 2010; Bhagooli, 2013; Hill et al., 2014) that a general weakness in corals (potentially leading to coral bleaching) could be the heat sensitivity of the Type II Rubisco, which causes partial inhibition of the Calvin-Benson cycle and CO₂ fixation, although the direct effect of acute heat stress on the Rubisco and on the whole photosynthetic productivity remains to be investigated. Under these circumstances, as indicated here, there may be side effects on cyclic electron transport and the Mehler-Ascorbate-Peroxidase (MAP) pathway. Recent works (Reynolds et al., 2008; Roberty et al., 2014; Aihara et al., 2016) have indicated the possibility that singlet oxygen production is generated when PQ pool is reduced, i.e., the relative size of oxidized PQ pool is smaller (and thus charge recombination in PSII reaction centers is promoted), which may be a primary factor in coral bleaching (Vass, 2012; Rehman et al., 2016). Nevertheless, these effects have to be documented individually in corals, where it is known that there are varied responses to similar acute thermal stress scenarios (Downs et al., 2013; Gardner et al., 2017). It is entirely possible that despite the “Achilles Heel” of a heat sensitive Rubisco, different Symbiodinium types have evolved different strategies to cope with this problem, and that the diversity of these strategies account for the different heat sensitivities of corals to coral bleaching.

MS, AL, IV, and WC planned the study. MS and AZ collected the data. MS, AL, AZ, IV, LS, DS, BO, and WC interpreted and analyzed the data. PR and WC provided consumables, reagents and tools. All authors contributed to drafting, critical commenting the manuscript and approved it for publication.