In Vivo Identification of Photosystem II Light Harvesting Complexes Interacting with PHOTOSYSTEM II SUBUNIT S

Light is the primary energy source for photosynthetic organisms, but in excess, it can generate reactive oxygen species and lead to cell damage. Plants evolved multiple mechanisms to modulate light use efficiency depending on illumination intensity to thrive in a highly dynamic natural environment. One of the main mechanisms for protection from intense illumination is the dissipation of excess excitation energy as heat, a process called nonphotochemical quenching. In plants, nonphotochemical quenching induction depends on the generation of a pH gradient across thylakoid membranes and on the presence of a protein called PHOTOSYSTEM II SUBUNIT S (PSBS). Here, we generated Physcomitrella patens lines expressing histidine-tagged PSBS that were exploited to purify the native protein by affinity chromatography. The mild conditions used in the purification allowed copurifying PSBS with its interactors, which were identified by mass spectrometry analysis to be mainly photosystem II antenna proteins, such as LIGHT-HARVESTING COMPLEX B (LHCB). PSBS interaction with other proteins appears to be promiscuous and not exclusive, although the major proteins copurified with PSBS were components of the LHCII trimers (LHCB3 and LHCBM). These results provide evidence of a physical interaction between specific photosystem II light-harvesting complexes and PSBS in the thylakoids, suggesting that these subunits are major players in heat dissipation of excess energy.Photosynthetic organisms exploit sunlight energy to support their metabolism. However, if absorbed in excess, light can produce harmful reactive oxygen species (Li et al., 2009; Murchie and Niyogi, 2011). In a natural environment, light intensity is highly variable and can rapidly change from being limited to being in excess. To survive and thrive in such a variable habitat, plants evolved multiple strategies to modulate their light use efficiency to limit reactive oxygen species formation when exposed to excess illumination while maintaining the ability to harvest light efficiently when required (Li et al., 2009; Murchie and Niyogi, 2011; Ruban, 2015). Among these different protection processes, the fastest, called nonphotochemical quenching (NPQ), is activated in a few seconds after a change in illumination, and it leads to the thermal dissipation of excess absorbed energy. NPQ is a complex phenomenon with different components that are distinguished according to their activation/relaxation time scale (Demmig-Adams et al., 1996; Szabó et al., 2005; Niyogi and Truong, 2013). The primary and fastest NPQ component, called qE (for energy-quenching component) or feedback deexcitation, depends on the generation of a pH gradient across the thylakoid membranes (Niyogi and Truong, 2013). In land plants, qE activation requires the presence of a thylakoid protein called PHOTOSYSTEM II SUBUNIT S (PSBS; Li et al., 2000, 2004). The Arabidopsis (Arabidopsis thaliana) PSBS-depleted mutant psbs KO (Li et al., 2000) is unable to activate qE and also showed reduced fitness when exposed to natural light variations in the field, supporting a major role for this protein in responding to illumination intensity fluctuations (Li et al., 2000; Külheim et al., 2002). Mutational analyses showed that the PSBS role in qE strictly depends on the presence of two protonable Glu residues, which are most likely involved in sensing the pH decrease in the lumen (Li et al., 2004). Despite several studies, however, the precise molecular mechanism by which PSBS controls NPQ induction remains debatable, and contrasting hypotheses have been presented (for review, see Ruban et al., 2012). PSBS has been hypothesized to bind pigments and to be directly responsible for energy dissipation based on its sequence similarity with LIGHT HARVESTING COMPLEX (LHC) proteins (Li et al., 2000; Aspinall-O’Dea et al., 2002). An alternative hypothesis instead suggested that PSBS is unable to bind pigments (Funk et al., 1995; Crouchman et al., 2006; Bonente et al., 2008a) and that it plays an indirect role in NPQ by modulating the PSII antenna protein transition from light harvesting to an energy dissipative state (Betterle et al., 2009; Johnson et al., 2011). This transition has been suggested to depend on the control of the macroorganization of the PSII-LHCII supercomplexes that are present in the grana membranes (Kiss et al., 2008; Betterle et al., 2009; Kereïche et al., 2010; Johnson et al., 2011). Consistent with this hypothesis, it was recently demonstrated that PSBS is able to induce a dissipative state in isolated LHCII proteins in liposomes (Wilk et al., 2013), suggesting that its interactions with antenna proteins play a key role in its biological activity. However, the precise identity of PSBS interactors (Teardo et al., 2007; Betterle et al., 2009), the PSBS oligomerization state (Bergantino et al., 2003), and its localization within PSII supercomplexes (Nield et al., 2000; Haniewicz et al., 2013) remain unclear or at least controversial, limiting the current understanding of PSBS molecular mechanisms.The moss Physcomitrella patens has recently emerged as a valuable model organism in which to study NPQ. As in the model angiosperm Arabidopsis, PSBS accumulation modulates NPQ amplitude and protects plants from photoinhibition under strong light in P. patens (Li et al., 2000; Alboresi et al., 2010; Zia et al., 2011; Gerotto et al., 2012). PSBS-mediated NPQ in P. patens also showed zeaxanthin dependence as in other plants (Niyogi et al., 1998; Pinnola et al., 2013). The moss P. patens has another protein involved in NPQ, LHCSR, which is typically found in algae and is different from proteins found in vascular plants (Peers et al., 2009; Bailleul et al., 2010; Gerotto and Morosinotto, 2013). Even if LHCSR is present in P. patens, LHCSR- and PSBS-dependent NPQ mechanisms were shown to be independent and to have an additive effect without any significant functional synergy (Gerotto et al., 2012).Previous data also demonstrated the possibility of achieving strong overexpression of PSBS in P. patens (Gerotto et al., 2012), which, however, was never observed in Arabidopsis (Li et al., 2002). This property was exploited in this work to overexpress a His-tagged PSBS isoform, which was afterward purified in its native state from dark-adapted thylakoid membranes. Several PSII antenna proteins were copurified with PSBS and identified by mass spectrometry analyses, demonstrating that they interact physically in dark-adapted thylakoid membranes. Components of LHCII trimers (LHCB3 and LHCBM) appear to be major, but not exclusive, components of PSBS interactors.