Photoprotective Switching Reveals a Thermal Achilles’ Heel in Breviolum minutum at 41 °C

Abstract

Non-photochemical quenching (NPQ) is a key photoprotective mechanism in Symbiodiniaceae, enabling photosystem II (PSII) to dissipate excess excitation energy under stress. The balance between regulated (Φ_NPQ) and unregulated (Φ_NO) energy dissipation influences thermal tolerance, yet the temperature thresholds at which this balance shifts remain poorly defined. Here, we used the Phenoplate, a high-throughput fluorometric platform integrating rapid light curves with controlled temperature ramping, to examine short-term thermal responses in Breviolum minutum across 6–71 °C. We identified a sharp transition at 41 °C where Φ_NPQ collapsed and was replaced by Φ_NO, indicating loss of regulated photoprotection. This switch coincided with a pronounced drop in PSII effective quantum yield (Φ_II) and substantial reductions in cell density, marking a thermal Achilles’ heel in the photoprotective capacity of this species. Despite this regulatory breakdown, a fraction of cells persisted for at least three days post-exposure. These results demonstrate that B. minutum maintains regulated photoprotection up to a discrete threshold, beyond which unregulated becomes the dominant pathway and survival is compromised. Identifying such thermal inflection points in coral symbionts provides mechanistic insight into their vulnerability under acute heat stress and may inform early-warning indicators for coral bleaching susceptibility.

1. Introduction

Reef-building corals rely on dinoflagellates from the family Symbiodiniaceae for most of their energy requirements. Within this symbiosis, the photophysiological performance of the dinoflagellate partner is a primary determinant of coral resilience to thermal stress [1,2]. Among Symbiodiniaceae, Breviolum minutum is a common and ecologically important species whose thermal tolerance influences bleaching susceptibility in its coral host [3]. Elevated temperatures impair photosynthesis, trigger oxidative stress, and ultimately lead to symbiont loss [4].

A central component of photoprotection in Symbiodiniaceae is non-photochemical quenching (NPQ), which dissipates excess excitation energy from photosystem II (PSII) as heat [5]. Energy quenching at the level of PSII can be divided into regulated non-photochemical quenching (Φ_NPQ) and unregulated components (Φ_NO) [6]. Regulated dissipation (Φ_NPQ) is generally protective, preventing the over-reduction of the electron transport chain and limiting oxidative damage. In contrast, unregulated dissipation (Φ_NO) reflects energy loss via uncontrolled pathways, often associated with photodamage [7]. The balance between Φ_NPQ and Φ_NO provides useful context on photosynthetic status and complements maximum quantum yield of PSII (Fv/Fm) which is widely used as a key indicator of photosynthetic health under stress.

While the temperature dependence of NPQ has been examined [8], the precise temperature at which the balance shifts from regulated dissipation (Φ_NPQ) to unregulated dissipation (Φ_NO)—that is, when Φ_NPQ fails and Φ_NO becomes the predominant pathway—remains poorly defined. Most studies have focused on prolonged stress exposures (hours to days) to simulate natural bleaching conditions in corals [9], but such approaches can obscure short-term physiological tipping points [10]. Identifying these photosynthetic thresholds where Φ_NPQ gives way to Φ_NO clarifies how PSII energy is managed during acute heat stress. While such thresholds can inform early indicators of bleaching risk under rapid heat events, a full mechanistic account of acute thermal damage will require additional physiological measurements beyond chlorophyll a fluorescence.

Symbiodiniaceae mitigate excess excitation through (I) rapid, energy-dependent non-photochemical quenching (superquenching, often xanthophyll-cycle associated) [11,12,13], (II) alternative electron-transport routes that divert/recycle electrons and relieve pressure on the photosynthetic chain [3], and (III) enzymatic and non-enzymatic antioxidant defenses [14]. When these regulated processes saturate or fail, residual energy is increasingly lost via Φ_NO, which remains comparatively poorly resolved under short-term heat stress. Because light and temperature interact, we examine responses across low, moderate, and high irradiance to distinguish temperature-driven thresholds from purely light-driven changes. In the energy partitioning framework, absorbed energy is divided among photochemistry, regulated heat dissipation, and other losses (Φ_II + Φ_NPQ + Φ_NO = 1), while qL estimates the fraction of open PSII reaction centres [6]: open centres favour photochemistry; as centres close under stress, Φ_NPQ and then Φ_NO pathways increasingly dissipate excess energy.

In this study, we used the Phenoplate, a high-throughput fluorometric platform integrating rapid light curves (RLC) with precise temperature control [15], to map short-term thermal responses in B. minutum. We reveal a discrete temperature threshold at 41 °C where Φ_NPQ collapses and is abruptly replaced by Φ_NO. This photoprotective switch represents a thermal Achilles’ heel in B. minutum, coinciding with a sharp decline in PSII effective quantum yield (Φ_II) and substantial cell loss. We discuss the implications of this threshold for the thermal resilience of B. minutum and its potential role in modulating host coral susceptibility to bleaching.

2. Materials and Methods

The algae were sourced from the Climate Change Culture collection, and were grown for up to two weeks at 28 °C in a 12:12 light dark cycle in a temperature-controlled incubator (Labec, 26-30 Farr Street, Marrickville, NSW, Australia) fitted with a fluorescent white light (TLD18W/840 Cool White, Phillips, Amsterdam, The Netherlands) illumination set at 20 µmol photons m⁻² s⁻¹ (Figure 1A). Cultures were maintained in F/2 media in seawater, in 25 cm² cell culture flasks (430,639, Corning, One Riverfront Plaza, Corning, NY 14831, USA). Cell density before the experiment was at optical density of 0.1 measured at 680 nm. Culture density in the survival assay was inferred by measuring relative fluorescence (RFU) emission at 685 nm with excitation at 485 nm, with a fixed gain in a plate reader spectrophotometer (Tecan Spark, Tecan, Männedorf, Switzerland). Plates were sealed with breathable membrane to minimize evaporation (Breath-Easy, Merck KGaA, Darmstadt, Germany) and fluorescence reading was performed from bottom of the plate at day 3 and day 7 after the Phenoplate measurement. Recovery plates were kept in standard growth conditions (28 °C in a 12:12 light dark cycle, at 20 µmol photons m⁻² s⁻¹) for up to 7 days. Percent relative survival was computed as (RFU_Temp/RFU_Control) × 100, where RFU_Temp is the fluorescence value of the culture exposed to temperature during the Phenoplate assay and RFU_Control is a culture that was never exposed to the Phenoplate assay.

Phenoplate measurements were carried out using the system previously described [15], with minor modifications to the pulse amplitude modulated (PAM) fluorometer protocol. Briefly, 200 µL of algae culture was transferred into a 96-well plate (HSP9601, Bio-Rad Laboratories, Inc., Hercules, CA, USA) (Figure 1B), which was positioned on to a thermocycler (ABI Veriti, Applied Biosystems, Waltham, MA, USA). The PAM fluorometer (Open FluorCam FC 800-O/1010, Photon Systems Instruments, Brno, Czech Republic) was positioned above the plate (Figure 1C) and programmed to carry out a sequence of measurements: (1) Fv/Fm after 5 min of dark adaptation at 28 °C (Supplementary Figure S1); (2) Fv/Fm after another 5 min of dark adaptation at various temperatures (Supplementary Figure S1); (3) 10 step Rapid Light Curve (RLC) with 8 steps of subsequent darkness (Figure 1D). The RLC illumination range was from 10 to 1085 µmol photons m⁻² s⁻¹ white actinic light; each RLC and dark recovery step was of 30 s. During the RLC, Φ_II was calculated as Φ_II = (F_m^′ − F_s)/F_m^′ [16]. Energy partitioning parameters Φ_NPQ and Φ_NO were computed following [6] such that Φ_II + Φ_NPQ + Φ_NO = 1. The fraction of open PSII reaction centres (qL) was calculated as qL = (F_m^′ − F_s)/(F_m^′ − F_o^′) × (F_o^′/F_s) [6]. Here, F_s is the steady-state (light-adapted) fluorescence during the actinic step, F_m^′ is the maximum light-adapted fluorescence elicited by a saturating pulse, and F_o^′ is the minimal light-adapted fluorescence. We interpret Φ_II, Φ_NPQ, Φ_NO, and qL as instantaneous values under the given step, not steady state. During plate preparation, cultures were kept at room temperature (24 °C) for 30–60 min before re-equilibration to growth conditions. The thermocycler was programmed to bring the samples to 28 °C for 5 min (growth temperature), followed by temperature gradient for another 14 min. The temperature range covered was from 5 to 71 °C; this was achieved by distributing the experiment across 5 separate runs (plates), each covering a different subset of target temperatures due to instrument capacity. Temperatures were tested at 1–2 °C increments with four biological replicates per temperature per run. Temperature was validated using a thermal camera (FLIR C2, Teledyne FLIR, LLC, Wilsonville, OR, USA). All experiments were replicated 3 times over the course of 1 year.

3. Results

3.1. Phenoplate Measurements

To characterize the temperature-dependent photophysiological response of B. minutum, we employed the Phenoplate system, which integrates a programmable thermocycler with an imaging PAM fluorometer. The experimental configuration is shown in Figure 1. This configuration enabled high-throughput assessment of thermal effects on regulated (Φ_NPQ) and unregulated (Φ_NO) energy dissipation in PSII in B. minutum.

3.2. Energetic Pathways in PSII

The distribution of absorbed light energy between photochemistry (Φ_II), regulated non-photochemical quenching (Φ_NPQ), and unregulated dissipation (Φ_NO) revealed distinct temperature-dependent patterns (Figure 2).

Φ_II (Figure 2A) increased with light intensity up to 20 °C at low light (40 µmol photons m⁻² s⁻¹) and up to 30 °C at moderate light (100 µmol photons m⁻² s⁻¹). From those temperatures onwards, Φ_II declined sharply and dropped close to zero at 41 °C, remaining mostly inactive at higher temperatures. Φ_NPQ (Figure 2B) increased with both light and temperature, with two distinct maxima: the first at 35 °C under high light (1200 µmol photons m⁻² s⁻¹), and a second above 47 °C, peaking at 65 °C. Between 41 and 47 °C, Φ_NPQ was completely absent.

This gap in Φ_NPQ corresponded to a rise in Φ_NO (Figure 2C). Φ_NO exhibited two main components: a low-temperature one with a peak at 5 °C, present under moderate to high light but absent at low light, and a high-temperature component spanning 41–47 °C at all light levels. The latter extended to 70 °C under low light but contracted under high light. The dotted white line marks the transition at 41 °C, where regulated dissipation collapses and unregulated pathways dominate. This transition temperature point is also evident in the Fv/Fm data measured after the RLC (Supplementary Figure S1).

3.3. PSII Open Reaction Centres and Post-Stress Survival

The fraction of open PSII reaction centres (qL) across all temperatures and light intensities is shown in Figure 3A. At elevated light intensities and temperatures below 40 °C PSII was predominantly closed, as shown by low qL values. Above 41 °C, qL values became highly variable at both high and low light, suggesting a loss of regulatory control over PSII closure.

At the lowest light intensity (Figure 3B), qL indicated that PSII was mostly open around 15 °C and gradually closed as temperature approached 40 °C. At 41 °C, PSII centres opened sharply, followed by a marked closure up to approximately 55 °C. Above this point, values became more scattered but showed a tendency toward reopening of reaction centres. The grey dotted line indicates the 41 °C transition, marking the shift from regulated to unregulated quenching and coinciding with a brief maximum in qL.

In this study, the acute Phenoplate exposure (5 min at 28 °C followed by 14 min at the target temperature) was considered a heat shock. After exposure cultures were returned to 28 °C. Survival was measured on Day 3 and Day 7 as chlorophyll a fluorescence at 685 nm, expressed as a percent of same-day unexposed controls. Cell survival data (Figure 3C) showed that, relative to control cultures which were never exposed to the Phenoplate assay, survival declined sharply with increasing temperature exposure. The steepest decline occurred at the 41 °C transition temperature. After 3 days, survival (red line) dropped substantially; after 7 days (black line), recovery was minimal, indicating lasting damage associated with the photoprotective collapse. Interestingly, more cells survived heat shocks between 41–55 °C than at exactly 41 °C. Survival at 35 °C and 55 °C was similar after 3 days, whereas survival at 41 °C was markedly lower than at temperatures both above and below this point. The survival pattern around the 41 °C transition was consistent between the 3-day and 7-day measurements, indicating that this temperature represents a persistent vulnerability in B. minutum’s thermal response.

4. Discussion

This study identified a discrete thermal threshold in B. minutum at 41 °C, where regulated non-photochemical quenching of energy in PSII (Φ_NPQ) collapsed and was abruptly replaced by unregulated energy dissipation (Φ_NO). This transition marked a clear photoprotective failure point, coinciding with the complete loss of Φ_II, relatively higher qL, and a sharp reduction in cell survival. Above this temperature, qL indicated a loss of coordinated PSII regulation, with open and closed states distributed unpredictably across light intensities. By contrast, under non-stress conditions qL is expected to decline progressively with increasing actinic irradiance during a rapid light curve, reflecting greater PSII reaction-centre closure as Q_A becomes reduced [6]. Notably, survival analysis revealed that fewer cells persisted after exposure to 41 °C than at temperatures both above and below this point, including the range from 41–55 °C where Φ_NO was the quenching process dominating. The survival pattern was consistent at both 3 and 7 days post-exposure, showing the lasting impact of this photoprotective collapse. Together, these findings define 41 °C as a thermal Achilles’ heel for B. minutum, representing a narrow but critical vulnerability in its short-term heat stress response.

The sharp transition from Φ_NPQ to Φ_NO at 41 °C indicates a discrete temperature at which regulated dissipation ceases and unregulated pathways predominate under acute heat. Previous studies in Symbiodiniaceae have documented gradual declines in Fv/Fm under elevated temperatures [17,18], but few have reported the Φ_NPQ to Φ_NO transition over an expanded temperature range [19]. The present data suggests that this threshold is not simply a product of accumulated photodamage, but rather a discrete shift in the balance of energy pathways (Figure 2B,C), potentially linked to structural or functional destabilisation of the light-harvesting complexes or PSII reaction centres (Figure 3A,B).

The concomitant variability in qL above 41 °C suggests that the mechanisms governing PSII openness become unpredictable under these conditions. This agrees with earlier work showing that antenna complex dissociation and PSI–PSII spillover can occur rapidly under extreme stress in dinoflagellates [11], but the temperature at which this occurs in B. minutum now appears precisely constrained.

Survival was lowest exactly at 41 °C, indicating a mismatch between protective responses. At this temperature Φ_NPQ has collapsed, while Φ_NO—and other high-temperature safety valves—has not yet fully engaged, creating a short, critical vulnerability in which PSII experiences sustained excitation pressure without effective dissipation. Beyond 47–55 °C, Φ_NO becomes established and, although unregulated, can transiently reduce damaging charge separation, blunting mortality relative to the 41 °C window. While our abrupt thermal ramping is unlikely in nature, it isolates a temperature-specific switching threshold—rather than a purely cumulative heat-load effect—highlighting where photoprotective pathways fail and where vulnerabilities arise.

The 41 °C threshold we report should be interpreted in the context of our acute exposure profile (5 min at 28 °C followed by 14 min at the target temperature) and prior acclimation at 28 °C. Thermal limits determined in corals and their symbionts will vary with heating rate and exposure duration [20] in addition to light intensity, fragment/colony traits, and acclimation history; therefore, thresholds derived from different protocols are not directly comparable. We frame 41 °C as an assay-specific value for B. minutum under our conditions and encourage transparent reporting and use of common response metrics to aid synthesis across studies. This aligns with community recommendations for improving cross-study comparability and with evaluations of acute heat-stress assays that document protocol-sensitivity and reproducibility considerations [21,22,23,24].

Future work should aim to resolve the molecular basis of the protection gap observed at 41 °C in B. minutum. Controlled experiments varying both peak temperature and ramping rate could distinguish whether this threshold is driven by absolute temperature, cumulative heat load, or the kinetics of photoprotective pathway engagement. High-resolution measurements of qE, qI, and PSI spillover during the transition could clarify which components fail first and how quickly alternative dissipation pathways are recruited. Structural studies of antenna complexes and PSII–PSI connectivity under rapid thermal stress may reveal specific protein or membrane domains vulnerable to this temperature. Extending this approach to other Symbiodiniaceae species and comparing in vitro and in hospite responses will determine whether the 41 °C protection gap is a universal feature or clade-specific. Ultimately, incorporating such mechanistic thresholds into predictive bleaching models could improve our ability to forecast coral resilience under short-duration, high-intensity marine heatwaves and rapidly fluctuating light conditions.

5. Conclusions

Using the Phenoplate high-throughput thermal–light assay, we mapped the activity of multiple photoprotective mechanisms in the coral symbiont Breviolum minutum across a broad temperature range. This approach revealed distinct temperature optima for regulated and unregulated energy dissipation in PSII, with a sharp transition at 41 °C marking a collapse in photoprotective regulation. This “protection gap” coincided with altered PSII reaction centre dynamics and the lowest cell survival observed, identifying 41 °C as a thermal weak point in this species’ short-term stress response. While such abrupt thermal ramping is unlikely to occur in the natural environment of corals, this finding provides valuable mechanistic insight into how photoprotective pathways switch under extreme stress. Extending this approach to other symbiont types will reveal whether the protection gap is a universal feature, offering a new metric for assessing thermal vulnerability in coral–algal partnerships.

6. Limitations

The 41 °C threshold is assay-specific to our acute protocol (5 min at 28 °C + 14 min at target temperature; 30-s RLC steps). Reported Φ_II, Φ_NPQ, Φ_NO and qL are instantaneous, not steady-state; regulated NPQ may have a greater influence for longer assays. Survival was quantified as chlorophyll fluorescence at 685 nm, normalized to same-day unexposed controls, and should be interpreted as a relative proxy, not absolute counts. At very high temperature/light, signal amplitudes fall, modestly increasing scatter despite fixed-gain averaging.