Photosynthetic organisms living in the intertidal zone need to be able to withstand sudden changes in their environmental factors (such as temperature, salinity, light and desiccation) associated with alternating periods of emergence and submergence. In fact, on rocky shores, seaweed species grow in differentiated horizontal belts characterised by specific levels of light and temperature stresses . Light, influenced not only by the daily course of solar irradiance but also by the tidal range, is one of the most variable and limiting factors for intertidal species. Consequently, seaweeds have developed a set of photoprotective mechanisms that allow them to adjust the amount of light absorbed by their photosynthetic apparatus, thus optimizing its use when light limits photosynthesis, and developing fast photoprotective responses under the effect of photoinhibitory light intensities. Algae possess different photoprotective mechanisms, such as the adjustment of chloroplast orientation ; repair mechanisms ; the accumulation of UV-absorbing phenolic compounds ; or the xanthophyll cycles-related dissipation of the excess of absorbed light energy. The last of these mechanisms is the more flexible and rapid, being crucial for the prevention of photoinhibitory damage. In P. canaliculata, the V-cycle pool size is considerably higher than in other brown algae, as corresponds to its position in the uppermost intertidal belt . In addition, it has been demonstrated that this species develops high, non-photochemical quenching (NPQ) when exposed to high irradiance . As shown in Figure 1, light-induced de-epoxidation reverted after the onset of darkness, confirming the complete operation of the V-cycle in this species.
García-Mendoza and Colombo-Pallota , have recently described that the brown alga Macrocystis pyrifera lacks the initial quenching of fluorescence shown for vascular plants , which is associated with the transthylakoidal-ΔpH. As a result, this species shows a direct and linear relationship between the V-cycle de-epoxidation ratio and the NPQ, which does not occur in vascular plants in which the initial NPQ component (qE) is developed due to the ΔpH, independently of V-cycle de-epoxidation. Similarly, a linear negative correlation was found between AZ/VAZ and Fv/Fm (Figure 1, inset), which implies a decrease in photochemical efficiency, mainly dependent on the V-cycle operation.
As previously demonstrated for other phototrophs, such as ferns and chlorolichens [11, 12], in P. canaliculata the V-cycle operation can be induced solely by dehydration, independently of light (Figure 2). Furthermore, dehydration triggered the de-epoxidation of V and the down-regulation of Fv/Fm to the same extent as illumination did (Figures 1 and 2). To clarify the similarities between light- and dehydration-induced V-cycle activities, a separate experiment was performed in which each branch of the same thalli were either exposed to light or kept in darkness, and subsequently dehydrated in darkness (thereby generating a branch in which A + Z formation was triggered by light and another branch in which this formation was induced by dehydration). As shown in Figure 3, Fv/Fm decreased to the same extent irrespective of the origin of V de-epoxidation, suggesting that A + Z play the same regulatory role when their formation is induced either by light or by dehydration.
Emersion frequently favours overheating, which damages temperate algae . When Pelvetia thalli were exposed to 32°C, the heat treatment induced an increase in A + Z but the AZ/VAZ reached was lower than that reached after any other treatment, i.e., dehydration (Figure 2), immersion (Figure 5), or anoxia (Figure 6). The decrease in Fv/Fm was notably larger than that observed for the same level of de-epoxidation in other treatments (note the outlayer point in Figure 7). Besides, the decrease in Fv/Fm and the increase in AZ/VAZ were not reversible after treatment had ceased, indicating that the stress was probably beyond the lethal threshold for a cool water macroalga. Indeed, for other species it has been recently shown that moderate heat stress can affect thylakoid reactions greatly .
Besides desiccation and high temperature, periodic hypoxic episodes in intertidal pools may be detrimental for the photosynthetic organisms of those habitats [34, 35], especially at night (when no photosynthetic production of oxygen can counteract respiration). Astonishingly, immersion (Figure 5) or anoxia followed by re-oxygenation in an absence of light (Figure 6), induced the same effects on the V-cycle and Fv/Fm as did dehydration-rehydration or light-darkness cycles. The activation of VDE by immersion may be due to the cellular acidification induced by the release of respiratory CO2, or by fermentative metabolism , but this hypothesis seems unlikely because of the intrathylakoidal location of this enzyme.
Considering the unlikelihood of that hypothesis, the acidification of lumen due to chlororespiration would become a mechanism to take into account. Essentially, during chlororespiration, the plastid terminal oxidase (PTOX) may oxidize plastoquinol (PQH2) by transferring electrons to oxygen  and at the same time, the pumping of protons towards the lumen would take place , providing the lumenal acidic pH required for the VDE activation. Several authors have described the activation of the chlororespiratory pathway by anoxia [39, 40]. Although the accumulation of de-epoxidized xanthophylls under chlororespiratory conditions was never observed in green algae , chlororespiration has been recently proposed as the mechanism responsible for the dark activation of the diadinoxanthin cycle in diatoms under anoxic conditions . Apart from anoxia, Brüggemann and co-workers have recently proposed chlororespiration as the mechanism responsible for the AZ/VAZ increase observed during the dark incubation of some winter-acclimated oaks at room temperature . Both these examples suggest that chlororespiration may represent a plausible explanation for the activation of V de-epoxidation showed by P. canaliculata in darkness. This may be the case of desiccating or overheated tissues, but under anoxia, it is unlikely that PTOX would be able to oxidize PQH2 due to the absence of the electron acceptor (oxygen), even when it is considered that under these conditions the PQ pool should be over-reduced . Furthermore, the inhibition of PTOX by n-PG did not block the de-epoxidation of V-cycle pigments that occurred in darkness during desiccation (Figure 8). This result indicates that another mechanism, other than the PTOX-mediated oxidation of PQ, may be responsible for the generation of the transthylakoidal-ΔpH needed for the VDE activation. Nevertheless, the fact that Z accumulation during desiccation of samples in darkness was unaltered by the application of the uncoupler NH4Cl (Figure 8) led us to consider an alternative explanation for the de-epoxidation of V in darkness.
It is sometimes thought that under strong light conditions only VDE is activated whereas only ZE works at night or under low light conditions. Nevertheless, ZE activity seems to be constitutive . The complete de-epoxidation of V induced in darkness by SA (Figure 8), together with the inhibition of V de-epoxidation induced by DTT, suggests that also VDE activity is constitutive, even in darkness. In the absence of light, Z accumulation is not observed under non-stressful conditions because ZE prevents its accumulation. However, the inhibition of ZE induced by stress would lead to the de-epoxidation of most of V, and to the consequent increase in the protective Z. Since ZE requires molecular oxygen as second substrate an NADPH as cofactor, both molecules may limit the ZE activity . The underlying mechanism responsible for such inhibition is unknown, but it may be associated with the availability of NADPH, as has been shown in Arabidopsis mutants lacking a chloroplastic NAD Kinase . In these mutants, ZE reduces its activity due to the reduced availability of its cofactor NADPH. Consequently, these plants accumulate high amounts of Z even in low light .