Abstract
Photoinhibition is the popular topic in plant photosynthesis. However, restricted to experimental systems ofin vitro membranes, knowledge of photosystem II (PSII) donor-side photoinhibition remains limited. Here, we report the first in vivo study of the mechanism in the marine higher plantZostera marina . Preferential oxygen-evolving complex photoinactivation decreased the light-harvesting capacity and enhanced photosystem I cyclic electron flow (CEF). Non-photochemical quenching was inefficient and alternative electron flows, e.g. chlororespiration, Mehler reaction, malic acid synthesis, and photorespiration, remained unactivated, thereby reducing the unnecessary consumption of limited electron resources and maintaining a well carbon assimilation level. At variance with the PSII acceptor-side photoinhibition, the PSII photodamage of Z. marina was not attributed to1O2but was associated with the long-lived P680+ resulted from the photoinactivated OEC. Furthermore, we provided the novel insights into the PSII donor-side photoinhibition that rare PSII-CEF and ascorbate assumed photoprotective roles in Z. marina , which could donate electrons to the PSII reaction center to prevent the oxidative damage by P680+. This study addressed an important knowledge gap in PSII donor-side photoinhibition, providing a novel understanding of photosynthetic regulation mechanism responding to light stress.
Keywords: alternative electron donor; oxygen-evolving complex; P680+; PSII donor-side photoinhibition; Zostera marina
Introduction
Photosystem II (PSII) photoinhibition is commonly expected to occur when photosystems cannot sufficiently utilize the energy absorbed by their antenna system, its occurrence depends on the redox state of PSII acceptor components (Gururani et al., 2015). Strong illumination creates excessive excitation, which in turns leads to over-reduction of plastoquinone (PQ) acceptors, thus rendering the PSII reaction center (RC) inactive (Barber and Andersson, 1992; Melis, 1999). In contrast to acceptor-side photoinhibition, PSII donor-side photoinhibition is caused by water-splitting dysfunction, e.g., chemical damage like the removal of the Mn cluster by NH2OH, Tris or high-salt washing (Callahan and Cheniae, 1985; Yadav and Pospíšil, 2012), and light damage like the photoinactivated oxygen-evolving complex (OEC) caused by direct UV illumination absorption (Yadav and Pospíšil, 2012; Havurinne and Tyystjärvi, 2017). These can be observed early in the photosynthetic membrane systemin vitro . Recently, vulnerable OEC under visible wavelengths have also been reported in vivoin specific unicelluar algae including the diatom Phaeodactylum tricornutum and the cyanobacteriaSynechocystis , which are likely the result of a lack of photoprotective sunscreen compounds such as non-photosynthetic pigments or divinyl chlorophyll (Havurinne and Tyystjärvi, 2017; Soitamo et al., 2017). So far,in vivo PSII photoinhibition, derived from photoinactivated OEC, has not been demonstrated with direct experimental evidence in higher plants under visible light.
Several explicit characteristics of the mechanism can be summarized as follows: (1) the mechanism even occurs under low light conditions (Keren and Krieger-Liszkay, 2011); (2) non-photochemical quenching (NPQ) has low protective efficiency (Tyystjärvi, 2013); (3) the photoinhibition rate constant (KPI) and light intensity are directly proportional (Tyystjärvi and Aro, 1996); (4) light leads to primary inactivation of OEC and secondary damage of PSII RC (Hakala et al., 2005; Tyystjärvi, 2008). The in vitro occurrence of donor-side photoinhibition, although mimicking, might not completely reflect in vivo phenomena (Dall’Osto et al., 2017) because of the incomplete photosynthetic apparatus. Restricted to imperfect experimental systems of photosynthetic membranesin vitro , knowledge of the mechanism remains limited and fragmented (Zavafer et al., 2015, 2017).
During PSII donor-side photoinhibition, the PSII RC may be susceptible to damage by both P680+ and 1O2 (Aro et al., 1993; Vass, 2011). When the electron donation of OEC is inferior to the rate of electron withdrawal by P680+• (Vass, 2012), the long-lived P680+, a strong oxidant damaging component of PSII RC, is assumed to be a source of damage in this mechanism, which appears to be uncontroversial. However, whether the highly reactive 1O2 is generated in the mechanism remains unclear, since the relevant results are mainly based on speculation and in vitro experiments (Nishiyama et al., 2011; Yadav and Pospíšil, 2012; Pospíšil, 2016). It has been considered that peroxidation of lipids by the highly oxidizing P680+ and TyrZ+could cause 1O2 formation (Yadav and Pospíšil, 2012; Pospíšil, 2016). The impaired OEC might damage specific oxygen channels that block oxygen molecules to P680 and conduct formative oxygen molecules outward, thus resulting in an accumulation of 1O2(Nishiyama et al., 2011). However, it has also been proposed that1O2formation only occurs in preparations that contain functional OEC (Hideg et al., 1994; Johnson et al., 1995). When OEC is damaged, the redox potential of the QA/QA pair shifts to a higher value, thus decelerating the conversion from P680+QA to3[P680+Phe] (Ivanov et al., 2008). Even though the inactive OEC first promotes1O2 generation, conformational changes induced by the persistent OEC inactivity protects against1O2 formation (Tyystjärvi, 2008).
Ascorbate (AsA), as an alternative PSII electron donor, exerts a photoprotective role by continually supporting electron transport through PSII (Tóth et al., 2011). Although the amount of AsA and its affinity to PSII varies with species, this alternative electron transport appears to be ubiquitous in both plants and green alga, and serves a more vital protection in heat-stressed plants (Tóth et al., 2009). When the OEC in thylakoids is impaired, either by acidic pH or by UV-B exposure, AsA is also photooxidized at the donor side of PSII (Mano et al., 2004). During normal OEC function, no electrons are donated from AsA to PSII. It has thus been considered that the AsA acts as emergency electron donor when water oxidation is impaired (Mano et al., 2004). As another electron donation event, PSII cyclic electron flow (PSII-CEF), a pathway by which electrons on the QB site of PSII are returned to P680+ via cytb559, has been reported to exert a species-dependent role in photoprotection (Ananyev et al., 2017). Typically, strong light would lead to the over-reduction of QA through linear electron transfer, thereby enhancing the activity of PSII-CEF to consume the excess energy (Feikema et al., 2006; Lavaud, 2007). Other factors, such as nitrogen limitation which caused over-reduction of PQ pool, can stimulate the electron flow, thus preventing PSII RC photodestruction (Wagner et al., 2016). PSII-CEF also plays a vital role in drought-tolerant species via proton gradient formation as a contribution to ATP production and photoprotection without consuming the limited water supply (Ananyev et al., 2016, 2017). Electron transport from PSII-CEF to PSII RC occurs when suppressed transfer of electrons from OEC to P680 extend the life time of P680+ (Thompson and Brudvig, 1988; Miyake, 2002). Hence, it is reasonable to assume that both AsA and PSII-CEF can donate electrons to prevent the accumulation of the long-lived P680+, exerting photoprotective roles during the PSII donor-side photoinhibition.
Zostera marina (Zosteraceae), a widespread seagrass species throughout the temperate northern hemisphere, playing ecological service function, evolved from a freshwater ancestor of terrestrial monocots and successfully adapted to a fully submerged marine environment (Wissler et al., 2011) where it must deal with shifted spectral composition, characterized by a high penetration of blue-green light (Olsen et al., 2016). The lack of cryptochrome blue-light receptors demonstrated by genome-based research inZ. marina (Olsen et al., 2016) would lead to insufficient anthocyanin levels (Li et al., 2013), thus weakening the screening of high-energy blue-green wavelengths (Hughes et al., 2010). Accordingly, Z. marina would possess more susceptible OEC with absorption peaks ranging between UV and blue-green light wavelengths (Tyystjärvi, 2008), providing the condition for the occurrence of PSII donor-side photoinhibition. Our recent study (Yang et al., 2017) showed that: (1) photoinhibition of Z. marinaalso occurred in low light conditions; (2) photoinhibition is closely relevant to the inactivation of OEC described by delayed fluorescence (DF); (3) the time-course changes ofF v/F m exhibited the deficiency of the P fast component of the Hanelt photoinhibition model, suggesting the slow NPQ development. These results agreed with the characteristics of PSII donor-side photoinhibition. Therefore,Z. marina , a marine angiosperm with complete photosynthetic apparatus and functional differentiation, may be a valid model species to study PSII donor-side photoinhibition.
In the present study, in vivoPSII photoinhibition derived from photoinactivated OEC under visible light was first determined in Z. marina , by identifying the primary target of the light-induced impairment. The integrated characteristics of the PSII photoinhibition associated with light absorption, electron transfer, and energy conversion were explored. Furthermore, damaging and photoprotective mechanisms were verified, based on the assumption that the alternative PSII electron donation pathways were activated to promote depletion of the long-lived P680+.