Plants Actively Avoid State Transitions upon Changes in Light Intensity: Role of Light-Harvesting Complex II Protein Dephosphorylation in High Light

Photosystem II (PSII) core and light-harvesting complex II (LHCII) proteins in plant chloroplasts undergo reversible phosphorylation upon changes in light intensity (being under control of redox-regulated STN7 and STN8 kinases and TAP38/PPH1 and PSII core phosphatases). Shift of plants from growth light to high light results in an increase of PSII core phosphorylation, whereas LHCII phosphorylation concomitantly decreases. Exactly the opposite takes place when plants are shifted to lower light intensity. Despite distinct changes occurring in thylakoid protein phosphorylation upon light intensity changes, the excitation balance between PSII and photosystem I remains unchanged. This differs drastically from the canonical-state transition model induced by artificial states 1 and 2 lights that concomitantly either dephosphorylate or phosphorylate, respectively, both the PSII core and LHCII phosphoproteins. Analysis of the kinase and phosphatase mutants revealed that TAP38/PPH1 phosphatase is crucial in preventing state transition upon increase in light intensity. Indeed, tap38/pph1 mutant revealed strong concomitant phosphorylation of both the PSII core and LHCII proteins upon transfer to high light, thus resembling the wild type under state 2 light. Coordinated function of thylakoid protein kinases and phosphatases is shown to secure balanced excitation energy for both photosystems by preventing state transitions upon changes in light intensity. Moreover, PROTON GRADIENT REGULATION5 (PGR5) is required for proper regulation of thylakoid protein kinases and phosphatases, and the pgr5 mutant mimics phenotypes of tap38/pph1. This shows that there is a close cooperation between the redox- and proton gradient-dependent regulatory mechanisms for proper function of the photosynthetic machinery.Photosynthetic light reactions take place in the chloroplast thylakoid membrane. Primary energy conversion reactions are performed by synchronized function of the two light energy-driven enzymes PSII and PSI. PSII uses excitation energy to split water into electrons and protons. PSII feeds electrons to the intersystem electron transfer chain (ETC) consisting of plastoquinone, cytochrome b₆f, and plastocyanin. PSI oxidizes the ETC in a light-driven reduction of NADP to NADPH. Light energy is collected by the light-harvesting antenna systems in the thylakoid membrane composed of specific pigment-protein complexes (light-harvesting complex I [LHCI] and LHCII). The majority of the light-absorbing pigments are bound to LHCII trimers that can serve the light harvesting of both photosystems (Galka et al., 2012; Kouřil et al., 2013; Wientjes et al., 2013b). Energy distribution from LHCII is regulated by protein phosphorylation (Bennett, 1979; Bennett et al., 1980; Allen et al., 1981) under control of the STN7 and STN8 kinases (Depège et al., 2003; Bellafiore et al., 2005; Bonardi et al., 2005; Vainonen et al., 2005) and the TAP38/PPH1 and Photosystem II Core Phosphatase (PBCP) phosphatases (Pribil et al., 2010; Shapiguzov et al., 2010; Samol et al., 2012). LHCII trimers are composed of LHCB1, LHCB2, and LHCB3 proteins, and in addition to reversible phosphorylation of LHCB1 and LHCB2, the protein composition of the LHCII trimers also affects the energy distribution from the light-harvesting system to photosystems (Damkjaer et al., 2009; Pietrzykowska et al., 2014). Most of the LHCII trimers are located in the PSII-rich grana membranes and PSII- and PSI-rich grana margins of the thylakoid membrane, and only a minor fraction resides in PSI- and ATP synthase-rich stroma lamellae (Tikkanen et al., 2008b; Suorsa et al., 2014). Both photosystems bind a small amount of LHCII trimers in biochemically isolatable PSII-LHCII and PSI-LHCII complexes (Pesaresi et al., 2009; Järvi et al., 2011; Caffarri et al., 2014). The large portion of the LHCII, however, does not form isolatable complexes with PSII or PSI, and therefore, it separates as free LHCII trimers upon biochemical fractionation of the thylakoid membrane by Suc gradient centrifugation or in native gel analyses (Caffarri et al., 2009; Järvi et al., 2011), the amount being dependent on the thylakoid isolation method. Nonetheless, in vivo, this major LHCII antenna fraction serves the light-harvesting function. This is based on the fact that fluorescence from free LHCII, peaking at 680 nm in 77-K fluorescence emission spectra, can only be detected when the energy transfer properties of the thylakoid membrane are disturbed by detergents (Grieco et al., 2015).Regulation of excitation energy distribution from LHCII to PSII and PSI has, for decades, been linked to LHCII phosphorylation and state transitions (Bennett, 1979; Bennett et al., 1980; Allen et al., 1981). It has been explained that a fraction of LHCII gets phosphorylated and migrates from PSII to PSI, which can be evidenced as increase in PSI cross section and was assigned as transition to state 2 (for review, see Allen, 2003; Rochaix et al., 2012). The LHCII proteins are, however, phosphorylated all over the thylakoid membrane (i.e. in the PSII- and LHCII-rich grana core) in grana margins containing PSII, LHCII, and PSI as well as in PSI-rich stroma lamellae also harboring PSII-LHCII, LHCII, and PSI-LHCII complexes in minor amounts (Tikkanen et al., 2008b; Grieco et al., 2012; Leoni et al., 2013; Wientjes et al., 2013a)—making the canonical-state transition theory inadequate to explain the physiological role of reversible LHCII phosphorylation (Tikkanen and Aro, 2014). Moreover, the traditional-state transition model is based on lateral segregation of PSII-LHCII and PSI-LHCI to different thylakoid domains. It, however, seems likely that PSII and PSI are energetically connected through a shared light-harvesting system composed of LHCII trimers (Grieco et al., 2015), and there is efficient excitation energy transfer between the two photosystems (Yokono et al., 2015). Nevertheless, it is clear that LHCII phosphorylation is a prerequisite to form an isolatable PSI-LHCII complex called the state transition complex (Pesaresi et al., 2009; Järvi et al., 2011). Existence of a minor state transition complex, however, does not explain why LHCII is phosphorylated all over the thylakoid membrane and how the energy transfer is regulated from the majority of LHCII antenna that is shared between PSII and PSI but does not form isolatable complexes with them (Grieco et al., 2015).Plants grown under any steady-state white light condition show the following characteristics of the thylakoid membrane: PSII core and LHCII phosphoproteins are moderately phosphorylated, phosphorylation takes place all over the thylakoid membrane, and the PSI-LHCII state transition complex is present (Järvi et al., 2011; Grieco et al., 2012; Wientjes et al., 2013b). Upon changes in the light intensity, the relative phosphorylation level between PSII core and LHCII phosphoproteins drastically changes (Rintamäki et al., 1997, 2000) in the timescale of 5 to 30 min. When light intensity increases, the PSII core protein phosphorylation increases, whereas the level of LHCII phosphorylation decreases. On the contrary, a decrease in light intensity decreases the phosphorylation level of PSII core proteins but strongly increases the phosphorylation of the LHCII proteins (Rintamäki et al., 1997, 2000). The presence and absence of the PSI-LHCII state transition complex correlate with LHCII phosphorylation (similar to the state transitions; Pesaresi et al., 2009; Wientjes et al., 2013b). Despite all of these changes in thylakoid protein phosphorylation, the relative excitation of PSII and PSI (i.e. the absorption cross section of PSII and PSI measured by 77-K fluorescence) remains nearly unchanged upon changes in white-light intensity (i.e. no state transitions can be observed despite massive differences in LHCII protein phosphorylation; Tikkanen et al., 2010).The existence of the opposing behaviors of PSII core and LHCII protein phosphorylation, as described above, has been known for more than 15 years (Rintamäki et al., 1997, 2000), but the physiological significance of this phenomenon has remained elusive. It is known that PSII core protein phosphorylation in high light (HL) facilitates the unpacking of PSII-LHCII complexes required for proper processing of the damaged PSII centers and thus, prevents oxidative damage of the photosynthetic machinery (Tikkanen et al., 2008a; Fristedt et al., 2009; Goral et al., 2010; Kirchhoff et al., 2011). It is also known that the damaged PSII core protein D1 needs to be dephosphorylated before its proteolytic degradation upon PSII turnover (Koivuniemi et al., 1995). There is, however, no coherent understanding available to explain why LHCII proteins are dephosphorylated upon exposure of plants to HL and PSII core proteins are dephosphorylated upon exposure to low light (LL).The above-described light quantity-dependent control of thylakoid protein phosphorylation drastically differs from the light quality-dependent protein phosphorylation (Tikkanen et al., 2010). State transitions are generally investigated by using different light qualities, preferentially exciting either PSI or PSII. State 1 light favors PSI excitation, leading to oxidation of the ETC and dephosphorylation of both the PSII core and LHCII proteins. State 2 light, in turn, preferentially excites PSII, leading to reduction of ETC and strong concomitant phosphorylation of both the PSII core and LHCII proteins (Haldrup et al., 2001). Shifts between states 1 and 2 lights induce state transitions, mechanisms that change the excitation between PSII and PSI (Murata and Sugahara, 1969; Murata, 2009). Similar to shifts between state lights, the shifts between LL and HL intensity also change the phosphorylation of the PSII core and LHCII proteins (Rintamäki et al., 1997, 2000). Importantly, the white-light intensity-induced changes in thylakoid protein phosphorylation do not change the excitation energy distribution between the two photosystems (Tikkanen et al., 2010). Despite this fundamental difference between the light quantity- and light quality-induced thylakoid protein phosphorylations, a common feature for both mechanisms is a strict requirement of LHCII phosphorylation for formation of the PSI-LHCII complex. However, it is worth noting that LHCII phosphorylation under state 2 light is not enough to induce the state 2 transition but that the P-LHCII docking proteins in the PSI complex are required (Lunde et al., 2000; Jensen et al., 2004; Zhang and Scheller, 2004; Leoni et al., 2013).Thylakoid protein phosphorylation is a dynamic redox-regulated process dependent on the interplay between two kinases (STN7 and STN8; Depège et al., 2003; Bellafiore et al., 2005; Bonardi et al., 2005; Vainonen et al., 2005) and two phosphatases (TAP38/PPH1 and PBCP; Pribil et al., 2010; Shapiguzov et al., 2010; Samol et al., 2012). Concerning the redox regulation mechanisms in vivo, only the LHCII kinase (STN7) has so far been thoroughly studied (Vener et al., 1997; Rintamäki et al., 2000; Lemeille et al., 2009). The STN7 kinase is considered as the LHCII kinase, and indeed, it phosphorylates the LHCB1 and LHCB2 proteins (Bellafiore et al., 2005; Bonardi et al., 2005; Tikkanen et al., 2006). In addition to this, STN7 takes part in the phosphorylation of PSII core proteins (Vainonen et al., 2005), especially in LL (Tikkanen et al., 2008b, 2010). The STN8 kinase is required for phosphorylation of PSII core proteins in HL but does not significantly participate in phosphorylation of LHCII (Bellafiore et al., 2005; Bonardi et al., 2005; Vainonen et al., 2005; Tikkanen et al., 2010). It has been shown that, in traditional state 1 condition, which oxidizes the ETC, the dephosphorylation of LHCII is dependent on TAP38/PPH1 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010), whereas the PSII core protein dephosphorylation is dependent on the PBCP phosphatase (Samol et al., 2012). However, it remains unresolved whether and how the TAP38/PPH1 and PBCP phosphatases are involved in the light intensity-dependent regulation of thylakoid protein phosphorylation typical for natural environments.Here, we have used the two kinase (stn7 and stn8) and the two phosphatase (tap38/pph1and pbcp) mutants of Arabidopsis (Arabidopsis thaliana) to elucidate the individual roles of these enzymes in reversible thylakoid protein phosphorylation and distribution of excitation energy between PSII and PSI upon changes in light intensity. It is shown that the TAP38/PPH1-dependent, redox-regulated LHCII dephosphorylation is the key component to maintain excitation balance between PSII and PSI upon increase in light intensity, which at the same time, induces strong phosphorylation of the PSII core proteins. Collectively, reversible but opposite phosphorylation and dephosphorylation of the PSII core and LHCII proteins upon increase or decrease in light intensity are shown to be crucial for maintenance of even distribution of excitation energy to both photosystems, thus preventing state transitions. Moreover, evidence is provided indicating that the pH gradient across the thylakoid membrane is yet another important component in regulation of the distribution of excitation energy to PSII and PSI, possibly by affecting the regulation of thylakoid kinases and phosphatases.     

Phosphorylation of the Light-Harvesting Complex II Isoform Lhcb2 Is Central to State Transitions

Light-harvesting complex II (LHCII) is a crucial component of the photosynthetic machinery, with central roles in light capture and acclimation to changing light. The association of an LHCII trimer with PSI in the PSI-LHCII supercomplex is strictly dependent on LHCII phosphorylation mediated by the kinase STATE TRANSITION7, and is directly related to the light acclimation process called state transitions. In Arabidopsis (Arabidopsis thaliana), the LHCII trimers contain isoforms that belong to three classes: Lhcb1, Lhcb2, and Lhcb3. Only Lhcb1 and Lhcb2 can be phosphorylated in the N-terminal region. Here, we present an improved Phos-tag-based method to determine the absolute extent of phosphorylation of Lhcb1 and Lhcb2. Both classes show very similar phosphorylation kinetics during state transition. Nevertheless, only Lhcb2 is extensively phosphorylated (>98%) in PSI-LHCII, whereas phosphorylated Lhcb1 is largely excluded from this supercomplex. Both isoforms are phosphorylated to different extents in other photosystem supercomplexes and in different domains of the thylakoid membranes. The data imply that, despite their high sequence similarity, differential phosphorylation of Lhcb1 and Lhcb2 plays contrasting roles in light acclimation of photosynthesis.Light capture and its conversion to chemical energy occur in a set of transmembrane protein complexes of the thylakoid membrane. PSII, the cytochrome b₆f complex, and PSI drive photosynthetic electron flow and the creation of a proton gradient across the thylakoid membrane. ATP synthase couples the dissipation of this gradient to the synthesis of ATP. The light-harvesting antennae play an important role in collecting light and transferring energy to the photosystems. Light-Harvesting Complex I (LHCI) exclusively transfers light energy to PSI, with which it is tightly associated (Croce and van Amerongen, 2014). In contrast, LHCII, which is the most abundant complex of the thylakoid membrane, can transfer energy to PSI or PSII (Grieco et al., 2015). Light is highly variable in natural environments, and plants experience continuous changes in both the spectrum and intensity of light on timescales as short as seconds. Changes in light quality may unbalance the activity of the two photosystems since their absorption spectra differ, whereas high light intensity can lead to overexcitation and induce photodamage. At low or moderate light intensities, the LHCII complex differentially associates with PSII or PSI, in a phosphorylation-dependent process known as state transitions, to rapidly respond to changes in the spectrum of light. In brief, under light quality that activates PSII more than PSI (e.g. blue light), LHCII is phosphorylated, and as a consequence, its binding to PSI is favored (state 2). Conversely, under light that preferentially excites PSI (enriched in far-red), this association can be reverted by dephosphorylation of the LHCII antenna, which favors its binding to PSII (state 1; Goldschmidt-Clermont and Bassi, 2015; Kim et al., 2015). A protein kinase, STATE TRANSITION7 (STN7), and a protein phosphatase, PROTEIN PHOSPHATASE1 (PPH1)/THYLAKOID-ASSOCIATED PHOSPHATASE38 (TAP38), are essential for the rapid phosphorylation and dephosphorylation of the LHCII antenna that regulates its differential association to PSI or PSII (Bellafiore et al., 2005; Pribil et al., 2010; Shapiguzov et al., 2010). Only a relatively small fraction of the LHCII antenna (<20%) is estimated to participate in state transitions in Arabidopsis (Arabidopsis thaliana; Allen, 1992). However, the process is conserved across the green eukaryotes and is relevant to plant fitness (Frenkel et al., 2007). Under high light, energy-dependent quenching of LHCII predominates, and furthermore, this antenna can uncouple from PSII (Wientjes et al., 2013b).The differential association of photosystems, LHCII, and other components of the thylakoid membrane gives rise to a set of supercomplexes that are central in ensuring photosynthetic efficiency and a rapid response to environmental cues (Caffarri et al., 2009; Duffy et al., 2013; Pietrzykowska et al., 2014; Fristedt et al., 2015). Fine tuning the dynamic assembly of these supercomplexes involves the association of antennae containing specific sets of Lhcb proteins. The major LHCII antenna comprises homo- and heterotrimers of Lhcb1 to Lhcb3 (Jackowski et al., 2001), whereas the minor LHCII isoforms (Lhcb4–Lhcb6) are monomeric (de Bianchi et al., 2008). Lhcb1 and Lhcb2 share a very similar primary structure and associated pigments (Formaggio et al., 2001; Zhang et al., 2008), whereas Lhcb3 appears to have slightly different features (Standfuss and Kühlbrandt, 2004). In Arabidopsis, five genes encode Lhcb1 isoforms, three genes encode Lhcb2 isoforms, and a single gene encodes Lhcb3. The principal discriminant between these classes is a short stretch of residues at the N-terminal end, which is of particular importance since it contains the Thr that is reversibly phosphorylated during light-acclimation processes (Goldschmidt-Clermont and Bassi, 2015). During evolution, land plants have maintained a major LHCII composed of different classes of Lhcb subunits. The phosphorylated N terminus of Lhcb2 was particularly well conserved (Alboresi et al., 2008; Zhang et al., 2008).PSII-LHCII supercomplexes have been isolated from Arabidopsis with up to four LHCII trimers bound to a PSII dimer, as well as the three minor monomeric antennae (Lhcb4–Lhcb6; Caffarri et al., 2009; Kouřil et al., 2012). In the LHCII trimers of these supercomplexes, different classes of Lhcb subunits are distributed differently, suggesting a specific role in light acclimation for each of them (Damkjaer et al., 2009; Pietrzykowska et al., 2014). In the stably bound S trimer, Lhcb1 and Lhcb2 are more abundant, whereas the moderately bound M trimer contains mostly Lhcb1 and Lhcb3 (Galka et al., 2012). PSII supercomplexes isolated from spinach (Spinacia oleracea) showed the presence of an extra LHCII trimer (L trimer); therefore, it is possible that, in Arabidopsis, other trimers are associated with the PSII dimer in a more labile supercomplex that cannot be isolated (Boekema et al., 1999). A single LHCII trimer, containing Lhcb1 and Lhcb2, stably associates with PSI to constitute the PSI-LHCII supercomplex, whose formation is dependent on LHCII phosphorylation by STN7 in state 2 (Kouřil et al., 2005; Galka et al., 2012).Previous reports have shown that the relative phosphorylation of Lhcb1 and Lhcb2 isoforms differs among thylakoid supercomplexes (Galka et al., 2012; Leoni et al., 2013). Here, we address the specific roles of Lhcb1 and Lhcb2 phosphorylation in photosynthetic acclimation. The improved protocol for SDS-PAGE in the presence of Phos-tag (Wako Chemicals) that we present allows quantification of the extent of phosphorylation for each class of antenna isoforms. We report that, in the PSI-LHCII supercomplex that is assembled in state 2, only the phosphorylated form of Lhcb2 is present, whereas the phosphorylated form of Lhcb1 is excluded. In contrast, both Lhcb1 and Lhcb2 are phosphorylated to different levels in other supercomplexes. This quantitative information on the level of phosphorylation of Lhcb1 and Lhcb2 offers new insights into the specific roles of the two classes of LHCII isoforms in light acclimation and supercomplex formation.     

STATE TRANSITION7-Dependent Phosphorylation Is Modulated by Changing Environmental Conditions,and Its Absence Triggers Remodeling of Photosynthetic Protein Complexes

In plants and algae, the serine/threonine kinase STN7/STT7, orthologous protein kinases in Chlamydomonas reinhardtii and Arabidopsis (Arabidopsis thaliana), respectively, is an important regulator in acclimation to changing light environments. In this work, we assessed STT7-dependent protein phosphorylation under high light in C. reinhardtii, known to fully induce the expression of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) and a nonphotochemical quenching mechanism, in relationship to anoxia where the activity of cyclic electron flow is stimulated. Our quantitative proteomics data revealed numerous unique STT7 protein substrates and STT7-dependent protein phosphorylation variations that were reliant on the environmental condition. These results indicate that STT7-dependent phosphorylation is modulated by the environment and point to an intricate chloroplast phosphorylation network responding in a highly sensitive and dynamic manner to environmental cues and alterations in kinase function. Functionally, the absence of the STT7 kinase triggered changes in protein expression and photoinhibition of photosystem I (PSI) and resulted in the remodeling of photosynthetic complexes. This remodeling initiated a pronounced association of LHCSR3 with PSI-LIGHT HARVESTING COMPLEX I (LHCI)-ferredoxin-NADPH oxidoreductase supercomplexes. Lack of STT7 kinase strongly diminished PSII-LHCII supercomplexes, while PSII core complex phosphorylation and accumulation were significantly enhanced. In conclusion, our study provides strong evidence that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments and gives new insights into acclimation strategies to these environmental conditions.Oxygenic photosynthesis converts solar energy into chemical energy. This energy is utilized for carbon dioxide assimilation, allowing the formation of complex organic material. Plant photosynthesis is performed by a series of reactions in and at the thylakoid membrane, resulting in light-dependent water oxidation, NADP reduction, and ATP formation (Whatley et al., 1963). These light reactions are catalyzed by two photosystems (PSI and PSII). A third multiprotein complex, also embedded in the thylakoid membrane, is the cytochrome b₆f (cyt b₆f) complex that links photosynthetic electron transfer processes between the two photosystems and functions in proton translocation. The ATP synthase takes advantage of the proton-motive force that is generated by the light reactions (Mitchell, 1961) to produce ATP. ATP and NADPH, generated through linear electron flow from PSII to PSI, drive the Calvin-Benson-Bassham cycle (Bassham et al., 1950) to fix CO₂. Alternatively, cyclic electron flow (CEF) between PSI and the cyt b₆f complex solely produces ATP (Arnon, 1959).Under normal growth conditions, CEF provides additionally required ATP for CO₂ fixation (Lucker and Kramer, 2013), counteracts overreduction of the PSI acceptor side under stressful environmental cues, and readjusts the ATP poise, leading to increased lumen acidification important for photoprotection (Alric, 2010; Peltier et al., 2010; Leister and Shikanai, 2013; Shikanai, 2014). In microalgae and vascular plants, CEF relies on the NAD(P)H dehydrogenase-dependent and/or PROTON GRADIENT REGULATION5 (PGR5)-related pathways (Munekage et al., 2002, 2004; Petroutsos et al., 2009; Tolleter et al., 2011; Johnson et al., 2014). For both pathways, supercomplexes consisting of PSI-LIGHT HARVESTING COMPLEX I (LHCI) and components of the respective electron transfer routes have been identified. In Arabidopsis (Arabidopsis thaliana), a unique NAD(P)H dehydrogenase-PSI supercomplex with a molecular mass of more than 1,000 kD was discovered (Peng et al., 2008). From Chlamydomonas reinhardtii, Iwai et al. (2010) isolated a protein supercomplex composed of PSI-LHCI, LHCII, the cyt b₆/f complex, ferredoxin-NADPH oxidoreductase (FNR), and PROTON GRADIENT REGULATION-LIKE1 (PGRL1).PGRL1 and PGR5 interact physically in Arabidopsis and associate with PSI to allow the operation of CEF (DalCorso et al., 2008). Functional data suggest that PGRL1 might operate as a ferredoxin-plastoquinone reductase (Hertle et al., 2013). The PGRL1-containing CEF supercomplex isolated from C. reinhardtii is capable of CEF under in vitro conditions in the presence of exogenously added soluble plastocyanin and ferredoxin (Iwai et al., 2010). Terashima et al. (2012) isolated a CEF supercomplex of similar composition from anaerobic growth conditions that was active in vitro and contained proteins such as the chloroplast-localized Ca²⁺ sensor CAS and ANAEROBIC RESPONSE1 (ANR1), which were also shown to be functionally important for efficient CEF in the alga. Notably, it was suggested that the onset of CEF in C. reinhardtii is redox controlled (Takahashi et al., 2013).It has been demonstrated that efficient CEF is crucial for successful acclimation to excess light (Munekage et al., 2004; Dang et al., 2014; Johnson et al., 2014; Kukuczka et al., 2014). The most rapid response to excess light, however, relies on a mechanism called nonphotochemical quenching (NPQ). The fastest constituent of NPQ is energy-dependent (qE) quenching, which operates at a time scale of seconds to minutes and regulates the thermal dissipation of excess absorbed light energy, thereby providing effective photoprotection. In vascular plants, the PSII protein PSII SUBUNIT S is essential for qE (Li et al., 2000), whereas qE induction in the green alga C. reinhardtii is mediated by LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3), an ancient light-harvesting protein that is missing in vascular plants (Peers et al., 2009). CEF and qE are complementary for acclimation to excess light, as double mutants deficient in both mechanisms possess additive phenotypes and are highly sensitive to light (Kukuczka et al., 2014). Another constituent of NPQ is the quenching by state transitions. State transitions are important to balance the excitation energy between PSI and PSII (Bonaventura and Myers, 1969; Murata, 1969). Under light conditions where PSII is preferentially excited, both PSII core and LHCII proteins become phosphorylated (Lemeille and Rochaix, 2010). As a consequence, phosphorylated LHCII proteins detach from PSII and partly connect to PSI (state 2). Under conditions where PSI excitation is predominant, this process is reversed. LHCII proteins are dephosphorylated and associate with PSII (state 1). The extent of state transition between vascular plants such as Arabidopsis and C. reinhardtii differs significantly. The proportion of mobile LHCII antenna is about 80% in the alga, whereas in Arabidopsis, only 15% to 20% of LHCII is transferred to PSI under state 2 conditions (Lemeille and Rochaix, 2010). However, the large increase in PSI antenna size in C. reinhardtii has recently been challenged (Nagy et al., 2014; Ünlü et al., 2014): while 70% to 80% of mobile LHCII detached from PSII in response to transition to state 2 conditions, only a fraction of about 20% functionally attached to PSI.Phosphorylation of LHC proteins requires the function of the STT7 kinase or its ortholog STN7 in C. reinhardtii or Arabidopsis, respectively. In the absence of the STT7/STN7 kinase, the initiation of state transitions is blocked (Depège et al., 2003; Bellafiore et al., 2005). The mobile LHCII fraction of C. reinhardtii includes the two monomeric minor LHCII antenna proteins, CP26 and CP29 (encoded by lhcb5 and lhcb4 genes), and the major chlorophyll a/b binding protein of LHCII, LHCBM5 (Takahashi et al., 2006), but also the LHCSR3 protein was suggested to migrate during state transitions (Allorent et al., 2013). Takahashi et al. (2014) suggested that only CP29 and LHCBM5 directly associate with PSI to form the PSI-LHCI-LHCII supercomplex, while the binding of CP26 could occur indirectly or via the other two proteins. However, it is not yet known whether STT7 directly phosphorylates the LHCII proteins or if this takes place as part of a kinase cascade (Rochaix, 2007). Nevertheless, the direct interaction between STT7 and the LHCII proteins is quite likely, since none of the other chloroplast kinases was found to be specifically required for LHCII phosphorylation (Rochaix, 2014). The activity of the STT7 kinase is mainly determined by the redox status of the plastoquinone pool (Vener et al., 1997; Zito et al., 1999). The identification of a PROTEIN PHOSPHATASE 2C (PP2C)-type phosphatase responsible for the dephosphorylation of the LHCII proteins in Arabidopsis has been described by two studies in parallel pointing to the fact that this enzyme, called PROTEIN PHOSPHATASE1/THYLAKOID-ASSOCIATED PHOSPHATASE38, acts directly on phosphorylated LHCII proteins, in particular when they are associated with the PSI-LHCI supercomplex (Pribil et al., 2010; Shapiguzov et al., 2010). Moreover, it is not known whether these phosphatases are constitutively active or if they are regulated by other means, for example through the redox state of the plastoquinone pool. Nonetheless, both enzymes are conserved in land plants and exhibit orthologous proteins in C. reinhardtii (Rochaix et al., 2012).Another kinase related to STN7/STT7 is encoded in the Arabidopsis and C. reinhardtii genomes and named STN8 and STATE TRANSITION-LIKE1 (STL1), respectively. STN8 is involved in PSII core subunit phosphorylation and influences the repair of PSII after photodamage (Bonardi et al., 2005; Vainonen et al., 2005). Remarkably, the disassembly of the PSII holocomplex is inhibited in STN7/STN8 double mutants (Tikkanen et al., 2008; Fristedt et al., 2009; Dietzel et al., 2011; Nath et al., 2013), suggesting that the phosphorylation of core subunits is required for PSII disassembly. It was further suggested that STN8 controls the transition between linear electron flow and CEF by the phosphorylation of PGRL1 in Arabidopsis (Reiland et al., 2011). As described for STN7, the activity of STN8 is probably regulated via the redox state of the plastoquinone pool (Bennett, 1991; Fristedt et al., 2009). Notably, the action of STN8 is counteracted by a chloroplast PP2C phosphatase (Samol et al., 2012), allowing for the fast reversibility of STN8-mediated acclimation responses. Thus, it appears that an intricate regulatory network of chloroplast protein kinases and phosphatases evolved in vascular plants and algae that drives the acclimation response to various environmental cues, including excess and changing light settings (Rochaix et al., 2012). As STN7/STT7 and STN8/STL1 kinase activities appear to be controlled by the redox poise of the plastoquinone pool, the plastoquinone pool would be a central player in these acclimation responses. On the other hand, the kinases themselves are subjected to phosphorylation (Reiland et al., 2009, 2011; Lemeille et al., 2010; Wang et al., 2013). However, the functional consequences of this phosphorylation are unknown.Recent comparative analyses revealed the presence of at least 15 distinct chloroplast protein kinases, suggesting an intricate kinase phosphorylation network in the chloroplast (Bayer et al., 2012). Generally, the phosphorylation of proteins is one of the most abundant posttranslational modifications. In complex eukaryotic systems, protein phosphorylation occurs most frequently on Ser followed by Thr residues, whereas protein phosphorylation of Tyr residues (1,800:200:1) is comparatively rare (Hunter, 1998; Mann et al., 2002). Protein phosphorylation is a general phenomenon in vivo; it is assumed that about one-third of all proteins are phosphorylated at a given time (Cohen, 2000; Ahn and Resing, 2001; Venter et al., 2001; Manning et al., 2002; Knight et al., 2003). A recent large-scale quantitative evaluation of human proteomic data strengthened the importance of protein phosphorylation for cellular function and human biology (Wilhelm et al., 2014). The C. reinhardtii and Arabidopsis genomes encode large kinase families (Arabidopsis Genome Initiative, 2000; Kerk et al., 2002; Merchant et al., 2007), supporting the view that protein phosphorylation also plays an important role in a plant’s life cycle. It is thus evident that the understanding of protein phosphorylation, including the specificity of residues phosphorylated or dephosphorylated in response to cellular as well as environmental factors, is one key to understanding the complex functional biological networks at the whole-system level. Likewise, it is crucial to design experimental setups allowing the linkage between phosphorylation events and particular physiological consequences to be elucidated.In this regard, we designed experiments to investigate STT7 kinase-dependent phosphorylation dynamics in C. reinhardtii in response to high light and anoxia, employing quantitative proteomics in conjunction with in-depth physiological characterization. These conditions are particularly interesting, as high light conditions are known to fully induce LHCSR3 protein expression and qE, while anoxia promotes CEF activity. Recently, it was demonstrated that qE and CEF are complementary and crucial in acclimation to these environmental cues (Kukuczka et al., 2014). Notably, LHCSR3 phosphorylation was suggested to depend on STT7 function (Bonente et al., 2011), while CEF supercomplex formation was found to be independent of STT7 kinase function (Takahashi et al., 2013), indicating that STT7 function might impact the acclimation to high light and anoxia in different ways. However, our quantitative proteomics and physiological data reveal that STT7-dependent variations in protein phosphorylation profiles have similar dramatic phenotypic consequences in both conditions, strongly suggesting that the regulation of protein phosphorylation is critical for driving successful acclimation to high light and anoxic growth environments.     

Comparative Analysis of Light-Harvesting Antennae and State Transition in chlorina and cpSRP Mutants

State transitions in photosynthesis provide for the dynamic allocation of a mobile fraction of light-harvesting complex II (LHCII) to photosystem II (PSII) in state I and to photosystem I (PSI) in state II. In the state I-to-state II transition, LHCII is phosphorylated by STN7 and associates with PSI to favor absorption cross-section of PSI. Here, we used Arabidopsis (Arabidopsis thaliana) mutants with defects in chlorophyll (Chl) b biosynthesis or in the chloroplast signal recognition particle (cpSRP) machinery to study the flexible formation of PS-LHC supercomplexes. Intriguingly, we found that impaired Chl b biosynthesis in chlorina1-2 (ch1-2) led to preferentially stabilized LHCI rather than LHCII, while the contents of both LHCI and LHCII were equally depressed in the cpSRP43-deficient mutant (chaos). In view of recent findings on the modified state transitions in LHCI-deficient mutants (Benson et al., 2015), the ch1-2 and chaos mutants were used to assess the influence of varying LHCI/LHCII antenna size on state transitions. Under state II conditions, LHCII-PSI supercomplexes were not formed in both ch1-2 and chaos plants. LHCII phosphorylation was drastically reduced in ch1-2, and the inactivation of STN7 correlates with the lack of state transitions. In contrast, phosphorylated LHCII in chaos was observed to be exclusively associated with PSII complexes, indicating a lack of mobile LHCII in chaos. Thus, the comparative analysis of ch1-2 and chaos mutants provides new evidence for the flexible organization of LHCs and enhances our understanding of the reversible allocation of LHCII to the two photosystems.In oxygenic photosynthesis, PSII and PSI function in series to convert light energy into the chemical energy that fuels multiple metabolic processes. Most of this light energy is captured by the chlorophyll (Chl) and carotenoid pigments in the light-harvesting antenna complexes (LHCs) that are peripherally associated with the core complexes of both photosystems (Wobbe et al., 2016). However, since the two photosystems exhibit different absorption spectra (Nelson and Yocum, 2006; Nield and Barber, 2006; Qin et al., 2015), PSI or PSII is preferentially excited under naturally fluctuating light intensities and qualities. To optimize photosynthetic electron transfer, the excitation state of the two photosystems must be rebalanced in response to changes in lighting conditions. To achieve this, higher plants and green algae require rapid and precise acclimatory mechanisms to adjust the relative absorption cross-sections of the two photosystems.To date, the phenomenon of state transitions is one of the well-documented short-term acclimatory mechanisms. It allows a mobile portion of the light-harvesting antenna complex II (LHCII) to be allocated to either photosystem, depending on the spectral composition and intensity of the ambient light (Allen and Forsberg, 2001; Rochaix, 2011; Goldschmidt-Clermont and Bassi, 2015; Gollan et al., 2015). State transitions are driven by the redox state of the plastoquinone (PQ) pool (Vener et al., 1997; Zito et al., 1999). When PSI is preferentially excited (by far-red light), the PQ pool is oxidized and all the LHCII is associated with PSII. This allocation of antenna complexes is defined as state I. When light conditions (blue/red light or low light) favor exciton trapping of PSII, the transition from state I to state II occurs. The over-reduced PQ pool triggers the activation of the membrane-localized Ser-Thr kinase STN7, which phosphorylates an N-terminal Thr on each of two major LHCII proteins, LHCB1 and LHCB2 (Allen, 1992; Bellafiore et al., 2005; Shapiguzov et al., 2016). Phosphorylation of LHCII results in the dissociation of LHCII from PSII and triggers its reversible relocation to PSI (Allen, 1992; Rochaix, 2011). Conversely, when the PQ pool is reoxidized, STN7 is inactivated and the constitutively active, thylakoid-associated phosphatase TAP38/PPH1 dephosphorylates LHCII, which then reassociates with PSII (Pribil et al., 2010; Shapiguzov et al., 2010). The physiological significance of state transitions has been demonstrated by the reduction in growth rate seen in the stn7 knock-out mutant under fluctuating light conditions (Bellafiore et al., 2005; Tikkanen et al., 2010).The canonical state transitions model implies spatial and temporal regulation of the allocation of LHC between the two spatially segregated photosystems (Dekker and Boekema, 2005). PSII-LHCII supercomplexes are organized in a tightly packed form in the stacked grana regions of thylakoid membranes, while PSI-LHCI supercomplexes are mainly localized in the nonstacked stromal lamellae and grana margin regions (Dekker and Boekema, 2005; Haferkamp et al., 2010). It has been proposed that, in the grana margin regions, which harbor LHCII and both photosystems, LHCII can migrate rapidly between them (Albertsson et al., 1990; Albertsson, 2001). This idea is supported by the recent discovery of mega complexes containing both photosystems in the grana margin regions (Yokono et al., 2015). Furthermore, phosphorylation of LHCII was found to increase not only the amount of PSI found in the grana margin region of thylakoid membranes (Tikkanen et al., 2008a), but also to modulate the pattern of PSI-PSII megacomplexes under changing light conditions (Suorsa et al., 2015). Nonetheless, open questions remain in relation to the physiological significance of the detection of phosphorylated LHCII in all thylakoid regions, even under the constant light conditions (Grieco et al., 2012; Leoni et al., 2013; Wientjes et al., 2013), although LHCII phosphorylation has been shown to modify the stacking of thylakoid membranes (Chuartzman et al., 2008; Pietrzykowska et al., 2014).State I-to-state II transition is featured by the formation of LHCII-PSI-LHCI supercomplexes, in which LHCII favors the light-harvesting capacity of PSI. Recently, LHCII-PSI-LHCI supercomplexes have been successfully isolated and purified using various detergents (Galka et al., 2012; Drop et al., 2014; Crepin and Caffarri, 2015) or a styrene-maleic acid copolymer (Bell et al., 2015). These findings yielded further insights into the reorganization of supercomplexes associated with state transitions, and it was suggested that phosphorylation of LHCB2 rather than LHCB1 is the essential trigger for the formation of state transition supercomplexes (Leoni et al., 2013; Pietrzykowska et al., 2014; Crepin and Caffarri, 2015; Longoni et al., 2015). Furthermore, characterization of mutants deficient in individual PSI core subunits indicates that PsaH, L, and I are required for docking of LHCII at PSI (Lunde et al., 2000; Zhang and Scheller, 2004; Kouril et al., 2005; Plöchinger et al., 2016).Recently, the state transition capacity has been characterized in the Arabidopsis (Arabidopsis thaliana) mutants with missing LHCI components. Although the Arabidopsis knock-out mutants lacking one of the four LHCI proteins (LHCA1-4) showed enhanced accumulation of LHCII-PSI complexes, the absorption cross-section of PSI under state II conditions was still compromised in the lhca1-4 mutants, and it is suggested that LHCI mediates the detergent-sensitive interaction between ‘extra LHCII’ and PSI (Benson et al., 2015; Grieco et al., 2015). Furthermore, the Arabidopsis mutant ΔLhca lacking all LHCA1-4 proteins was shown to be compensated for the deficiency of LHCI by binding LHCII under state II conditions (Bressan et al., 2016). In spite of this finding, the significant reduction in the absorption cross-section of PSI was still observed in the ΔLhca mutant, suggesting a substantial role of LHCI in light absorption under canopy conditions (Bressan et al., 2016). However, these findings emphasize the acclimatory function of state transitions in balancing light absorption capacity between the two photosystems by modifying their relative antenna size and imply the dynamic and variable organization of PS-LHC supercomplexes.LHC proteins are encoded by the nuclear Lhc superfamily (Jansson, 1994). The biogenesis of LHCs includes the cytoplasmic synthesis of the LHC precursor proteins, their translocation into chloroplasts via the TOC/TIC complex, and their posttranslational targeting and integration into the thylakoid membranes by means of the chloroplast signal recognition particle (cpSRP) machinery (Jarvis and Lopez-Juez, 2013). The posttranslational cpSRP-dependent pathway for the final translocation of LHC proteins into the thylakoid membrane includes interaction of cpSRP43 with LHC apo-proteins and recruitment of cpSRP54 to form a transit complex. Then binding of this tripartite cpSRP transit complex to the SRP receptor cpFtsY follows, which supports docking of the transit complex to thylakoid membranes and its association with the LHC translocase ALB3. Ultimately, ALB3 inserts LHC apo-proteins into the thylakoid membrane (Richter et al., 2010). Importantly, stoichiometric amounts of newly synthesized Chl a and Chl b as well as carotenoid are inserted into the LHC apo-proteins by unknown mechanisms to form the functional LHCs that associate with the core complexes of both photosystems in the thylakoid membranes (Dall’Osto et al., 2015; Wang and Grimm, 2015).The first committed steps in Chl synthesis occur in the Mg branch of the tetrapyrrole biosynthesis pathway. 5-Aminolevulinic acid synthesis provides the precursor for the formation of protoporphyrin IX, which is directed into the Mg branch (Tanaka and Tanaka, 2007; Brzezowski et al., 2015). Chl synthesis ends with the conversion of Chl a to Chl b catalyzed by Chl a oxygenase (CAO; Tanaka et al., 1998; Tomitani et al., 1999). It has been hypothesized that coordination between Chl synthesis and the posttranslational cpSRP pathway is a prerequisite for the efficient integration of Chls into LHC apo-proteins.In this study, we intend to characterize the assembly of LHCs when the availability of Chl molecules or the integration of LHC apo-proteins into thylakoid membranes is limiting. To this end, we compared the assembly of LHCs and the organization of PS-LHC complexes in two different sets of Arabidopsis mutants. Firstly, we used the chlorina1-2 (ch1-2) mutant, which is defective in the CAO gene. The members of the second set of mutants carry knock-out mutations in genes involved in the chloroplast SRP pathway (Richter et al., 2010).Our studies revealed distinct accumulation of PS-LHC supercomplexes between the two sets of mutant relative to wild-type plants. In spite of the defect in synthesis of Chl b, ch1-2 retains predominantly intact PSI-LHCI supercomplexes but has strongly reduced amounts of LHCII. In contrast, the chaos (cpSRP43) mutant exhibits synchronously reduced contents of both LHCI and LHCII, which results in the accumulation of PS core complexes without accompanying LHCs. Thus, the distribution of LHCs in the thylakoid membranes of the two mutants, ch1-2 and chaos, were explored under varying light conditions with the aim of elucidating the influence of modified LHCI/LHCII antenna size on state transitions. Our results contribute to an expanding view on the variety of photosynthetic complexes, which can be observed in Arabidopsis plants with specified mutations in LHC biogenesis.     

The Antarctic Psychrophile Chlamydomonas sp. UWO 241 Preferentially Phosphorylates a Photosystem I-Cytochrome b6/f Supercomplex

Chlamydomonas sp. UWO 241 (UWO 241) is a psychrophilic green alga isolated from Antarctica. A unique characteristic of this algal strain is its inability to undergo state transitions coupled with the absence of photosystem II (PSII) light-harvesting complex protein phosphorylation. We show that UWO 241 preferentially phosphorylates specific polypeptides associated with an approximately 1,000-kD pigment-protein supercomplex that contains components of both photosystem I (PSI) and the cytochrome b₆/f (Cyt b₆/f) complex. Liquid chromatography nano-tandem mass spectrometry was used to identify three major phosphorylated proteins associated with this PSI-Cyt b₆/f supercomplex, two 17-kD PSII subunit P-like proteins and a 70-kD ATP-dependent zinc metalloprotease, FtsH. The PSII subunit P-like protein sequence exhibited 70.6% similarity to the authentic PSII subunit P protein associated with the oxygen-evolving complex of PSII in Chlamydomonas reinhardtii. Tyrosine-146 was identified as a unique phosphorylation site on the UWO 241 PSII subunit P-like polypeptide. Assessment of PSI cyclic electron transport by in vivo P700 photooxidation and the dark relaxation kinetics of P700⁺ indicated that UWO 241 exhibited PSI cyclic electron transport rates that were 3 times faster and more sensitive to antimycin A than the mesophile control, Chlamydomonas raudensis SAG 49.72. The stability of the PSI-Cyt b₆/f supercomplex was dependent upon the phosphorylation status of the PsbP-like protein and the zinc metalloprotease FtsH as well as the presence of high salt. We suggest that adaptation of UWO 241 to its unique low-temperature and high-salt environment favors the phosphorylation of a PSI-Cyt b₆/f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of PSII light-harvesting complex proteins.The Antarctic psychrophilic green alga Chlamydomonas sp. UWO 241 (UWO 241) originates from the lowest trophic zone of Lake Bonney, which is characterized by an extremely stable environment of low temperatures (4°C–6°C), low irradiance (less than 50 µmol photons m⁻² s⁻¹), high salt concentrations (700 mm), and a narrow spectral distribution enriched in the blue-green region (Lizotte and Priscu, 1992; Morgan-Kiss et al., 2006). Adaptation of UWO 241 to this unique natural aquatic environment has resulted in the evolution of a structurally and functionally distinct photosynthetic apparatus relative to the mesophilic strains Chlamydomonas raudensis SAG 49.72 (SAG 49.72; Pocock et al., 2004) and the model green alga Chlamydomonas reinhardtii (Morgan et al., 1998; Morgan-Kiss et al., 2006). UWO 241 is a halotolerant psychrophile (Morgan-Kiss et al., 2006; Takizawa et al., 2009) that dies at temperatures of 20°C or higher (Possmayer et al., 2011). This is consistent with the fact that temperature-response curves for light-saturated rates of CO₂-saturated oxygen evolution indicate that UWO 241 photosynthesizes maximally at 8°C at rates that are comparable to rates of the mesophile, C. reinhardtii, grown and measured at 29°C (Pocock et al., 2007). Although UWO 241 exhibits a low quantum requirement for photoinhibition and the degradation of the PSII reaction center polypeptide D1 (PsbA), this is complemented by a rapid, light-dependent recovery of PSII photochemistry associated with the de novo biosynthesis of D1 at low temperature (Pocock et al., 2007). Thus, this psychrophile appears to be photosynthetically adapted to growth at low temperature (Pocock et al., 2007).UWO 241 exhibits significantly enhanced fatty acid unsaturation associated with all of the major thylakoid lipid classes (monogalactosyldiacylglyceride, digalactosyldiacylglyceride, sulfoquinovosyldiacylglyceride, and phosphatidyldiacylglyceride) as well as a 2- to 10-fold increase in the unique, unsaturated fatty acid 16:4, depending on the specific thylakoid lipid species (Morgan-Kiss et al., 2002a). Consequently, the biophysical determination of the critical temperature for thylakoid membrane destabilization for UWO 241 (40°C) was significantly lower than that for C. reinhardtii (50°C), which is consistent with the adaptation of UWO 241 to low temperature (Morgan-Kiss et al., 2002a).Biochemical analyses of the chlorophyll-protein complexes coupled with immunoblots of their constituent polypeptides indicate that UWO 241 exhibits abundant PSII light-harvesting complex (LHCII) associated with a low chlorophyll a/b (Chl a/b) ratio (1.8–2) relative to the mesophiles, SAG 49.72 and C. reinhardtii (Chl a/b ratio = 3). In addition, UWO 241 exhibits an unusually low level of PSI such that the stoichiometry of PSI/PSII was estimated to be about 0.5 in UWO 241, whereas the mesophiles, SAG 49.72 and C. reinhardtii, grown under optimal growth conditions, exhibited a PSI/PSII of about 1. These biochemical data were confirmed by measurements of P700 photooxidation (Morgan-Kiss et al., 2002b; Szyszka et al., 2007), which indicated that UWO 241 exhibits high rates of PSI cyclic electron flow (CEF; Morgan-Kiss et al., 2002b).Recently, we reported that acclimation of UWO 241 to low temperature and low growth irradiance results in alterations in the partitioning of excess excitation energy to maintain cellular energy balance compared with the mesophile, SAG 49.72 (Szyszka et al., 2007). While SAG 49.72 favors energy partitioning for photoprotection through the induction of the xanthophyll cycle, the psychrophilic strain, UWO 241, favors energy partitioning for photoprotection through constitutive quenching processes involved in energy dissipation, even though UWO 241 exhibits an active xanthophyll cycle (Pocock et al., 2007; Szyszka et al., 2007). Although the molecular basis of the constitutive quenching process for photoprotection has not been elucidated unequivocally, this may reflect the differences in the predisposition for energy dissipation through either the Q2 or the Q1 site in PSII-LHCII supercomplexes (Jahns and Holzwarth 2012; Derks et al., 2015) or, alternatively, it may indicate quenching through PSII reaction centers, as suggested previously (Hüner et al., 2006; Sane et al., 2012). Regardless of the mechanism, one consequence of this enhanced energy-quenching capacity of UWO 241 is that the psychrophile does not exhibit any pigment change in response to photoacclimation (Morgan-Kiss et al., 2006), typically observed for other mesophilic green algae such as C. reinhardtii, Dunaliella tertiolecta (Escoubas et al., 1995), Dunaliella salina (Smith et al., 1990; Maxwell et al., 1995), and Chlorella vulgaris (Maxwell et al., 1995; Wilson et al., 2003). In addition, maximum growth rates of UWO 241 are sensitive to light quality, since rates of growth and photosynthesis are inhibited under red light, which results in increased excitation pressure in the psychrophile (Morgan-Kiss et al., 2005).However, the most unusual feature of UWO 241 is that it represents a natural variant that is deficient in state transitions (Morgan-Kiss et al., 2002b; Takizawa et al., 2009). State transitions have been well documented as a short-term mechanism for photoacclimation employed by algae and plants to balance light excitation between PSII and PSI (Allen et al., 1981; Allen, 2003; Eberhard et al., 2008; Rochaix, 2011, 2014). Overexcitation of PSII relative to PSI results in the phosphorylation of several peripheral Chl a/b-binding LHCII proteins, which causes their dissociation from the PSII core and subsequent association with PSI (Eberhard et al., 2008; Rochaix, 2011). As a result, excitation energy is redistributed in favor of PSI at the expense of PSII. Phosphorylation of LHCII polypeptides is essential in the regulation of state transitions and energy distribution between the two photosystems (Allen, 2003; Eberhard et al., 2008; Kargul and Barber, 2008; Rochaix, 2011, 2014). LHCII phosphorylation is initiated by modulation of the redox state of the plastoquinone (PQ) pool, which is sensed through the preferential binding of plastoquinol to the quinone-binding site of the cytochrome b₆/f (Cyt b₆/f) complex. As a consequence, the thylakoid protein kinases STT7 in C. reinhardtii and its ortholog, STN7, in Arabidopsis (Arabidopsis thaliana) are activated and LHCII is phosphorylated (Rochaix, 2011, 2014; Wunder et al., 2013). Similar to all other photosynthetic organisms, the LHCII polypeptides represent the major phosphorylated polypeptides detected in thylakoids of the mesophile, SAG 49.72 (Szyszka et al., 2007). Consistent with a deficiency in state transitions, UWO 241 does not phosphorylate the major LHCII polypeptides in response to changes in either growth irradiance or growth temperature (Morgan-Kiss et al., 2002b; Szyszka et al., 2007; Takizawa et al., 2009). In fact, UWO 241 exhibits a unique thylakoid membrane phosphorylation profile compared with either SAG 49.72 or C. reinhardtii (Morgan-Kiss et al., 2005; Szyszka et al., 2007; Takizawa et al., 2009). Rather than phosphorylation of LHCII polypeptides, UWO 241 preferentially phosphorylates several novel high-molecular-mass polypeptides (greater than 70 kD; Morgan-Kiss et al., 2002b; Szyszka et al., 2007).The Cyt b₆/f complex of the photosynthetic intersystem electron transport chain is essential in the regulation of state transitions and the activation of the STT7 kinase (Rochaix, 2011, 2014). The Cyt b₆/f complex of UWO 241 exhibits a unique cytochrome f (Cyt f) that is 7 kD smaller than the expected molecular mass of 41 kD exhibited by C. reinhardtii based on SDS-PAGE (Morgan-Kiss et al., 2006; Gudynaite-Savitch et al., 2006, 2007). No other differences in the structure and composition of the Cyt b₆/f complex are apparent. Sequencing of the entire Cytochrome f gene (petA) from UWO 241 indicated that the amino acid sequence of Cyt f from UWO 241 exhibited 79% identity to that of C. reinhardtii. Through domain swapping between petA of UWO 241 and that of C. reinhardtii and subsequent transformation of a ΔpetA mutant of C. reinhardtii with the chimeric gene constructs, we reported that the apparent differences in molecular masses observed for petA in UWO 241 are due to differences in the amino acid sequences of the small domain of Cyt f. However, complementation of the ΔpetA mutant of C. reinhardtii with the entire petA from either UWO 241 or C. reinhardtii completely restored the capacity for state transitions in the ΔpetA mutant. Thus, we concluded that the changes in the amino acid sequence of the small domain of Cyt f of UWO 241 cannot account for the inability of UWO 241 to undergo state transitions (Gudynaite-Savitch et al., 2006, 2007).Since state transitions are inhibited in UWO 241, we hypothesized that the unique protein phosphorylation pattern observed in UWO 241 reflects an alternative mechanism to regulate energy flow within the photosynthetic apparatus of this Antarctic psychrophile. Thus, the objective of this research was to identify and characterize the high-molecular-mass polypeptides phosphorylated in the psychrophile, UWO 241. We report that UWO 241 preferentially phosphorylates specific polypeptides associated with a PSI-Cyt b₆/f supercomplex. The role of the PSI-Cyt b₆/f supercomplex and its phosphorylation status in the regulation of PSI cyclic electron transport in UWO 241 are discussed. We suggest that adaptation of UWO 241 to its unique low-temperature and low-light environment favors the phosphorylation of a PSI-Cyt b₆/f supercomplex to regulate PSI cyclic electron transport rather than the regulation of state transitions through the phosphorylation of LHCII proteins.     

PHOTOSYSTEM II SUBUNIT R Is Required for Efficient Binding of LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 to Photosystem II-Light-Harvesting Supercomplexes in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii, the LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3) protein is crucial for efficient energy-dependent thermal dissipation of excess absorbed light energy and functionally associates with photosystem II-light-harvesting complex II (PSII-LHCII) supercomplexes. Currently, it is unknown how LHCSR3 binds to the PSII-LHCII supercomplex. In this study, we investigated the role of PHOTOSYSTEM II SUBUNIT R (PSBR) an intrinsic membrane-spanning PSII subunit, in the binding of LHCSR3 to PSII-LHCII supercomplexes. Down-regulation of PSBR expression diminished the efficiency of oxygen evolution and the extent of nonphotochemical quenching and had an impact on the stability of the oxygen-evolving complex as well as on PSII-LHCII-LHCSR3 supercomplex formation. Its down-regulation destabilized the PSII-LHCII supercomplex and strongly reduced the binding of LHCSR3 to PSII-LHCII supercomplexes, as revealed by quantitative proteomics. PHOTOSYSTEM II SUBUNIT P deletion, on the contrary, destabilized PHOTOSYSTEM II SUBUNIT Q binding but did not affect PSBR and LHCSR3 association with PSII-LHCII. In summary, these data provide clear evidence that PSBR is required for the stable binding of LHCSR3 to PSII-LHCII supercomplexes and is essential for efficient energy-dependent quenching and the integrity of the PSII-LHCII-LHCSR3 supercomplex under continuous high light.Plant photosynthetic electron transfer is conducted by a series of reactions at the chloroplast thylakoid membrane, resulting in light-dependent water oxidation, NADP reduction, and ATP formation (Whatley et al., 1963). Two separate photosystems (PSI and PSII) and an ATP synthase catalyze these reactions. PSI and PSII are multiprotein complexes that are mainly embedded in unstacked and stacked regions of the thylakoid membrane, respectively. PSI consists of more than 10 subunits and a number of cofactors such as chlorophyll a, β-carotene, phylloquinone, and three iron-sulfur (4Fe-4S) clusters (Busch and Hippler, 2011). PSI catalyzes light-driven electron transfer from luminal plastocyanin to stromal ferredoxin. The latter reduces the ferredoxin-NADP reductase that, in turn, leads to the formation of NADPH. PSII catalyzes light-induced electron transfer from water to the plastoquinone pool by using chlorophyll a, carotenoids, as well as redox-active cofactors, causing the release of oxygen and protons (Pagliano et al., 2013). The core complex is organized as a dimer. Monomers are composed of the reaction center subunits PSBA (D1) and PSBD (D2), the inner antenna proteins PSBB (CP47) and PSBC (CP43), the α- and β-subunits (PSBE and PSBF) of cytochrome b₅₅₉, as well as a number of intrinsic low-molecular-mass subunits. The core monomer is further associated with an inorganic Mn₄O₅Ca cluster and a few chloride ions (Rivalta et al., 2011; Umena et al., 2011) required for photosynthetic water oxidation. To optimize this process, the oxygen-evolving complex is formed at the luminal side by the extrinsic polypeptides PSBO, PSBP, PSBQ, and PSBR (for review, see Pagliano et al., 2013).To enhance the light-harvesting capacity of PSII, various light-harvesting proteins bind to dimeric PSII core complexes (Dekker and Boekema, 2005). A common structure found for vascular plants and green algae is the C₂S₂ supercomplex, where two copies of monomeric Lhcb4 and Lhcb5 and two LHCII trimers (S-trimer; Boekema et al., 1995) bind to the dimeric PSII core. In vascular plants, larger but less stable PSII supercomplexes, known as C₂S₂M₂, are composed of two extra copies of the monomeric Lhcb6 with two additional LHCII trimers (M-trimer) bound through Lhcb4 and Lhcb5 (Dekker and Boekema, 2005; Caffarri et al., 2009). Even larger complexes containing two additional LHCII trimers (L-trimer), bound via Lhcb6, are found and are known as C₂S₂M₂L_1–2 (Boekema et al., 1999). A recent study in Chlamydomonas reinhardtii identified PSII-LHCII supercomplexes with three LHCII trimers attached to each side of the core (C₂S₂M₂L₂; Tokutsu et al., 2012). Interestingly, such PSII-LHCII supercomplexes associate with LIGHT-HARVESTING COMPLEX STRESS-RELATED PROTEIN3 (LHCSR3; Tokutsu and Minagawa, 2013), an ancient light-harvesting protein required for efficient energy-dependent (qE) quenching in the alga (Peers et al., 2009). The qE component of nonphotochemical quenching (NPQ) is an energy-dependent constituent of NPQ and regulates the thermal dissipation of excess absorbed light energy (Li et al., 2000; Peers et al., 2009). The qE capacity in C. reinhardtii increases proportionally to the light-dependent accumulation of the LHCSR3 protein (Peers et al., 2009). In contrast, in vascular plants, qE is constitutively active and dependent on PSBS, a PSII polypeptide (Li et al., 2000). Mass spectrometric analyses of isolated C₂S₂M₂ PSII supercomplexes revealed the presence of extrinsic subunits PSBP, PSBQ, and PSBR, while PSBS was not identified, suggesting that PSBS does not influence the association of the PSII core with the outer light-harvesting complex system (Pagliano et al., 2014). In line with the proteomic findings, recent data suggest that subunits PSBP, PSBQ, and PSBR contribute to the stability of PSII-LHCII supercomplexes in vascular plants (Caffarri et al., 2009; Ifuku et al., 2011; Allahverdiyeva et al., 2013). A recent quantitative proteomic study performed with C. reinhardtii identified PSBR as the only PSII subunit to be induced upon the shift from photoheterotrophic to photoautotrophic growth conditions similar to LHCSR3 (Höhner et al., 2013).In vascular plants and green algae, PSBR is nucleus encoded and has a mass of about 10 kD. The mature protein has a predicted 70-amino acid luminal N-terminal part and a C-terminal transmembrane span (Ljungberg et al., 1986; Lautner et al., 1988; Webber et al., 1989). An association of PSBR with the oxygen-evolving complex has been suggested, as its presence is required for the stable assembly of PSBP with the PSII core and its absence also impacts the binding of PSBQ to the core (Suorsa et al., 2006; Liu et al., 2009). For stable association with the PSII core complex, PSBR needs the presence of PSBJ (Suorsa et al., 2006). Functionally, the depletion of PSBR protein expression decreased rates of oxygen evolution (Allahverdiyeva et al., 2007, 2013) and quinone reoxidation (Allahverdiyeva et al., 2007). PSBR phosphorylation is known for Arabidopsis (Arabidopsis thaliana; Reiland et al., 2009, 2011; Nakagami et al., 2010) and in the green alga C. reinhardtii (Turkina et al., 2006), although phosphorylation sites are not conserved between the alga and the vascular plant.In this work, we addressed the question of whether down-regulation of PSBR expression would affect LHCSR3 binding to the PSII-LHCII supercomplex in C. reinhardtii. To this end, we took advantage of artificial microRNA (amiRNA) technology to down-regulate PSBR expression and investigated the impact of PSBR down-regulation on photosynthetic performance as well as on PSII-LHCII-LHCSR3 supercomplex formation. Our data provide evidence that PSBR is required for the stable binding of LHCSR3 to PSII-LHCII supercomplexes.     

TEF30 Interacts with Photosystem II Monomers and Is Involved in the Repair of Photodamaged Photosystem II in Chlamydomonas reinhardtii

The remarkable capability of photosystem II (PSII) to oxidize water comes along with its vulnerability to oxidative damage. Accordingly, organisms harboring PSII have developed strategies to protect PSII from oxidative damage and to repair damaged PSII. Here, we report on the characterization of the THYLAKOID ENRICHED FRACTION30 (TEF30) protein in Chlamydomonas reinhardtii, which is conserved in the green lineage and induced by high light. Fractionation studies revealed that TEF30 is associated with the stromal side of thylakoid membranes. By using blue native/Deriphat-polyacrylamide gel electrophoresis, sucrose density gradients, and isolated PSII particles, we found TEF30 to quantitatively interact with monomeric PSII complexes. Electron microscopy images revealed significantly reduced thylakoid membrane stacking in TEF30-underexpressing cells when compared with control cells. Biophysical and immunological data point to an impaired PSII repair cycle in TEF30-underexpressing cells and a reduced ability to form PSII supercomplexes after high-light exposure. Taken together, our data suggest potential roles for TEF30 in facilitating the incorporation of a new D1 protein and/or the reintegration of CP43 into repaired PSII monomers, protecting repaired PSII monomers from undergoing repeated repair cycles or facilitating the migration of repaired PSII monomers back to stacked regions for supercomplex reassembly.Oxygenic photosynthesis is essential for almost all life on Earth, as it provides the reduced carbon and the oxygen required for respiration. A key enzyme in oxygenic photosynthesis is PSII, which catalyzes the light-driven oxidation of water. The core of PSII in algae and land plants contains D1 (PsbA), D2 (PsbD), CP43 (PsbC), CP47 (PsbB), the α-subunit (PsbE) and β-subunit (PsbF) of cytochrome b₅₅₉, as well as several intrinsic low-molecular-mass subunits. The core monomer is associated with the extrinsic oxygen-evolving complex (OEC) consisting of OEE1 (PSBO), OEE2 (PSBP), and OEE3 (PSBQ), which stabilize the inorganic Mn₄O₅Ca cluster required for water oxidation (for review, see Pagliano et al., 2013). PSII core monomers assemble into dimers to which, at both sides, light-harvesting proteins (LHCII) bind to form PSII supercomplexes. In land plants, each PSII dimer binds two each of the monomeric minor LHCII proteins CP24, CP26, and CP29 in addition to up to four major LHCII trimers (Caffarri et al., 2009; Kouřil et al., 2011). Biochemical evidence suggests that, in the thylakoid membrane, up to eight LHCII trimers can be present per PSII core dimer, presumably because of the existence of a pool of extra LHCII (Kouřil et al., 2013). In Chlamydomonas reinhardtii, lacking CP24, each PSII dimer binds two each of the CP26 and CP29 monomers as well as up to six major LHCII trimers (Tokutsu et al., 2012). The reaction center proteins D1 and D2 bind all the redox-active cofactors required for PSII electron transport (Umena et al., 2011). Light captured by the internal antenna proteins CP43 and CP47 and the outer antenna induces charge separation in PSII, which in turn enables the OEC to oxidize water and provide electrons to the electron transfer chain. In land plants and green algae, PSII supercomplexes are localized to stacked regions of the thylakoid membranes, while the synthesis of PSII cores is considered to take place in stroma lamellae.A particular feature of PSII is its vulnerability to light, with the D1 protein being a target of light-induced damage and the damage being proportional to the photon flux density (PFD) applied (Tyystjärvi and Aro, 1996). To cope with this damage, an elaborate, highly conserved repair mechanism has evolved termed the PSII repair cycle, during which damaged PSII complexes are partially disassembled and the defective D1 protein is replaced by a de novo synthesized copy (for review, see Nixon et al., 2010; Komenda et al., 2012; Mulo et al., 2012; Nath et al., 2013a; Nickelsen and Rengstl, 2013; Tyystjärvi, 2013; Järvi et al., 2015). Photodamage occurs at all light intensities, but when the rate of damage exceeds the capacity for repair, photoinhibition is manifested as a decrease in the proportion of active PSII reaction centers (Aro et al., 1993). While PSII photodamage occurs in the supercomplexes in the stacked membrane regions, the replacement of damaged D1 takes place in stroma lamellae (Aro et al., 2005). Thus, the PSII repair cycle requires the lateral migration of PSII complexes, which is impaired by the macromolecular crowding in stacked thylakoid membranes (Kirchhoff, 2014). Lateral migration of damaged PSII complexes is facilitated by thylakoid membrane unfolding and PSII supercomplex disassembly. Both processes are enhanced by the phosphorylation of the PSII core subunits D1, D2, CP43, and PsbH, which is mainly mediated by the protein kinase STATE TRANSITION8 (STN8; Tikkanen et al., 2008; Fristedt et al., 2009; Herbstová et al., 2012; Nath et al., 2013b; Wunder et al., 2013). Efficient PSII supercomplex disassembly also requires the THYLAKOID FORMATION1 (THF1)/NON-YELLOW COLORING4 (NYC4)/Psb29 protein (Huang et al., 2013; Yamatani et al., 2013). After the migration of PSII monomers to unstacked thylakoid regions, PSII core subunits are dephosphorylated by the PSII core phosphatase PBCP (Samol et al., 2012), which is required for the efficient degradation of D1 (Koivuniemi et al., 1995; Rintamäki et al., 1996; Kato and Sakamoto, 2014). Degradation of D1 is subsequently realized by the membrane-integral FtsH protease (Lindahl et al., 2000; Silva et al., 2003) and by lumenal and stromal Deg proteases (Haussühl et al., 2001; Kapri-Pardes et al., 2007; Sun et al., 2010). Degradation is assisted by the THYLAKOID LUMEN PROTEIN18.3 (TLP18.3), presumably by its phosphatase activity and ability to interact with lumenal Deg1 (Sirpiö et al., 2007; Wu et al., 2011; Zienkiewicz et al., 2012). D1 proteolysis follows the partial disassembly of the PSII complex, during which CP43 and low-molecular-mass subunits are released to generate a CP43-free PSII monomer (Aro et al., 2005). Thereafter, a newly synthesized D1 copy is cotranslationally inserted from a plastidial 70S ribosome into the thylakoid membrane and processed by the CARBOXYL TERMINAL PEPTIDASE A (CTPA; Zhang et al., 1999, 2000; Che et al., 2013). In Arabidopsis (Arabidopsis thaliana), the D1 synthesis rate appears to be negatively regulated by the PROTEIN DISULFIDE ISOMERASE6 (PDI6; Wittenberg et al., 2014). Moreover, yet unknown steps during PSII repair require the stromal cyclophilin ROTAMASE CYP4 and stromal HEAT SHOCK PROTEIN70 (Schroda et al., 1999; Yokthongwattana et al., 2001; Cai et al., 2008). The PSII repair cycle is completed by the reassembly of the CP43 protein, ligation of the OEC, back migration of PSII to stacked membrane regions, and supercomplex formation. Except for CtpA, all mentioned factors appear to be specific for PSII repair, while many more auxiliary factors play roles in PSII de novo synthesis and repair (for review, see Järvi et al., 2015).In this study, we report on the functional characterization of the THYLAKOID ENRICHED FRACTION30 (TEF30) protein in C. reinhardtii. In this organism, TEF30 was first identified in a proteomics study on isolated thylakoid membranes (Allmer et al., 2006). TEF30 attracted our attention because its abundance increased 1.7-fold in membrane-enriched fractions of C. reinhardtii cells that had been shifted from 41 to 145 µmol photons m⁻² s⁻¹ for 8 h (Mettler et al., 2014; Supplemental Fig. S1). The TEF30 ortholog in Arabidopsis M-ENRICHED THYLAKOID PROTEIN1 (MET1; where M stands for mesophyll cells) was functionally characterized only recently (Bhuiyan et al., 2015). Both MET1 and TEF30 interact quantitatively with monomeric PSII core particles at the stroma side of the thylakoid membranes and play a role in the assembly of PSII monomers and/or their migration to stacked membrane regions for supercomplex assembly. While MET1 appears to exert this function during PSII de novo biogenesis and during the PSII repair cycle in Arabidopsis, TEF30 appears to function exclusively during PSII repair in C. reinhardtii.     

The Solar Action Spectrum of Photosystem II Damage

The production of oxygen and the supply of energy for life on earth rely on the process of photosynthesis using sunlight. Paradoxically, sunlight damages the photosynthetic machinery, primarily photosystem II (PSII), leading to photoinhibition and loss of plant performance. However, there is uncertainty about which wavelengths are most damaging to PSII under sunlight. In this work we examined this in a simple experiment where Arabidopsis (Arabidopsis thaliana) leaves were exposed to different wavelengths of sunlight by dispersing the solar radiation across the surface of the leaf via a prism. To isolate only the process of photodamage, the repair of photodamaged PSII was inhibited by infiltration of chloramphenicol into the exposed leaves. The extent of photodamage was then measured as the decrease in the maximum quantum yield of PSII using an imaging pulse amplitude modulation fluorometer. Under the experimental light conditions, photodamage to PSII occurred most strongly in regions exposed to ultraviolet (UV) or yellow light. The extent of UV photodamage under incident sunlight would be greater than we observed when one corrects for the optical efficiency of our system. Our results suggest that photodamage to PSII under sunlight is primarily associated with UV rather than photosynthetically active light wavelengths.Plants absorb sunlight to power the productive photochemical reactions of photosynthesis. Absorption of sunlight may also lead to deleterious photochemistry that damages the photosynthetic machinery. The PSII protein complex is important in this regard as it seems to be most susceptible to photodamage that results in photoinhibition and ultimately suppresses photosynthetic CO₂ assimilation, growth, and productivity (Long et al., 1994; Takahashi and Murata, 2008). Although plants have photoprotection mechanisms (Niyogi, 1999) and can effectively repair photodamaged PSII through the PSII repair cycle (Aro et al., 1993), photoinhibition still occurs under stressful environmental conditions (Nishiyama et al., 2006; Murata et al., 2007; Takahashi and Murata, 2008).The onset of photoinhibition is strongly correlated with the absorption of excessive excitation energy for photosynthesis. Therefore, photodamage to PSII was most readily assumed to be attributed to the excess light absorbed by photosynthetic pigments (Melis, 1999). However, the extent of photodamage that is measured under conditions where the repair of photodamaged PSII is prevented by inhibiting chloroplast protein synthesis (i.e. lincomycin or chloramphenicol) is directly proportional to the intensity of light (Mattoo et al., 1984; Tyystjärvi and Aro, 1996; Nishiyama et al., 2001, 2004; Allakhverdiev and Murata, 2004; Chow et al., 2005). Furthermore, recent studies have demonstrated that interruption of the Calvin cycle (Hakala et al., 2005; Takahashi and Murata, 2005; Takahashi et al., 2007) and inhibition of electron transfer between Q_A and Q_B (Jegerschöld et al., 1990; Kirilovsky et al., 1994; Allakhverdiev et al., 2005) have no effect on the rate of photodamage to PSII, but in fact cause inhibition of the repair of photodamaged PSII due to suppression of the de novo synthesis of PSII proteins (Allakhverdiev et al., 2005; Takahashi and Murata, 2005, 2006). Thus, photodamage to PSII is paradoxically not associated with the excess light absorbed by photosynthetic pigments (Nishiyama et al., 2006; Murata et al., 2007; Takahashi and Murata, 2008).Studies of the effect of monochromatic light on the photodamage process have suggested that photodamage to PSII primarily occurs at the manganese cluster of the oxygen-evolving complex (OEC) through a direct photoexcitation of manganese (Hakala et al., 2005; Ohnishi et al., 2005). Release of manganese ions (Mn²⁺) from thylakoid membranes is accompanied by photodamage to PSII (Hakala et al., 2005; Zsiros et al., 2006), suggesting that disruption of the manganese cluster upon absorption of light might be a primary event in photodamage. It is likely that the reaction center of PSII is secondarily damaged by light absorbed by photosynthetic pigments after inactivation of the OEC (Hakala et al., 2005; Ohnishi et al., 2005), if an alternative electron transfer donor from lumenal ascorbate is not available (Mano et al., 2004; Tóth et al., 2009). These findings have lead to a recent photodamage model called the manganese (or two-step; Ohnishi et al., 2005) mechanism of photoinhibition (Tyystjärvi, 2008).Studies of the action spectrum of photodamage to PSII have shown that UV damages PSII more effectively than visible light (Jones and Kok, 1966; Jung and Kim, 1990; Hakala et al., 2005; Ohnishi et al., 2005). Thus, under identical light intensity, UV is the most damaging wavelength to PSII. However, inferring damage under natural sunlight is not straight forward as there is a need to account for the spectral distribution and intensity of sunlight. It is unclear which wavelengths of sunlight are most damaging to PSII and we cannot discount the premise that significant primary photodamage to PSII is caused by light absorbed by photosynthetic pigments (Vass and Cser, 2009). To identify which wavelengths of sunlight are most damaging to PSII, sunlight was spectrally dispersed via a prism onto an Arabidopsis (Arabidopsis thaliana) leaf infiltrated with chloramphenicol and decrease in the maximum quantum yield of PSII (F_v/F_m) was measured using an imaging pulse amplitude modulation (PAM) fluorometer. This simple but powerful approach revealed the in vivo spectral dependence of photodamage that had two peaks at UV and yellow wavelengths. Since the spectral efficiency of our optical system decreased below 400 nm, we calculated photodamage to PSII under incident sunlight. Our results show that photodamage to PSII was primarily associated with UV wavelengths and secondarily with yellow light wavelengths. This finding indicates that photodamage to PSII is less associated with light absorbed by photosynthetic pigments under sunlight and suggest that most of photodamage to PSII is potentially avoidable during photosynthesis.     

Steady-State Phosphorylation of Light-Harvesting Complex II Proteins Preserves Photosystem I under Fluctuating White Light

According to the “state transitions” theory, the light-harvesting complex II (LHCII) phosphorylation in plant chloroplasts is essential to adjust the relative absorption cross section of photosystem II (PSII) and PSI upon changes in light quality. The role of LHCII phosphorylation upon changes in light intensity is less thoroughly investigated, particularly when changes in light intensity are too fast to allow the phosphorylation/dephosphorylation processes to occur. Here, we demonstrate that the Arabidopsis (Arabidopsis thaliana) stn7 (for state transition7) mutant, devoid of the STN7 kinase and LHCII phosphorylation, shows a growth penalty only under fluctuating white light due to a low amount of PSI. Under constant growth light conditions, stn7 acquires chloroplast redox homeostasis by increasing the relative amount of PSI centers. Thus, in plant chloroplasts, the steady-state LHCII phosphorylation plays a major role in preserving PSI upon rapid fluctuations in white light intensity. Such protection of PSI results from LHCII phosphorylation-dependent equal distribution of excitation energy to both PSII and PSI from the shared LHCII antenna and occurs in cooperation with nonphotochemical quenching and the proton gradient regulation5-dependent control of electron flow, which are likewise strictly regulated by white light intensity. LHCII phosphorylation is concluded to function both as a stabilizer (in time scales of seconds to minutes) and a dynamic regulator (in time scales from tens of minutes to hours and days) of redox homeostasis in chloroplasts, subject to modifications by both environmental and metabolic cues. Exceeding the capacity of LHCII phosphorylation/dephosphorylation to balance the distribution of excitation energy between PSII and PSI results in readjustment of photosystem stoichiometry.Plant acclimation to different quantities and qualities of light has been extensively investigated. The light quality experiments have usually concerned the red/blue and far-red light acclimation strategies, which have been closely related to the state transitions and the phosphorylation of the light-harvesting complex II (LHCII) proteins, Lhcb1 and Lhcb2, by the state transition7 (STN7) kinase (Allen, 2003; Bellafiore et al., 2005; Bonardi et al., 2005; Tikkanen et al., 2006; Rochaix, 2007). Such studies on acclimation to different qualities of light have uncovered key mechanisms required for the maintenance of photosynthetic efficiency in dense populations and canopies (Dietzel et al., 2008). However, the role of LHCII phosphorylation under fluctuations in the quantity of white light has been scarcely investigated. Light conditions in natural environments may be very complex with respect to the quantity of white light, which constantly fluctuates both in short- and long-term durations (Smith, 1982; Külheim et al., 2002). Thus, the acclimation strategies to natural environments must concomitantly meet the challenges of both high- and low-light acclimation. Changing cloudiness, for example, would initiate both the high-light and low-light acclimation signals in the time scale of minutes and hours, whereas the movements of leaves in the wind or the rapid movement of clouds would initiate even more frequent light acclimation signals. The kinetics of reversible LHCII phosphorylation is far too slow to cope with rapid environmental changes.The phosphorylation level of LHCII proteins in the thylakoid membrane is regulated by both the STN7 kinase and the counteracting PPH1/TAP38 phosphatase (Pribil et al., 2010; Shapiguzov et al., 2010). No definite results are available about regulation of the PPH1/TAP38 phosphatase, but the STN7 kinase is strongly under redox regulation (Lemeille et al., 2009) and controls the phosphorylation level of LHCII proteins under varying white light intensities as well as according to chloroplast metabolic cues, as described already decades ago (Fernyhough et al., 1983; Rintamäki et al., 2000; Hou et al., 2003). So far, research on the role of the STN7 kinase and LHCII phosphorylation in the light acclimation of higher plants has heavily focused on reversible LHCII phosphorylation and concomitant state transitions. The state 1-to-state 2 transition, by definition, means the phosphorylation of LHCII proteins, their detachment from PSII in grana membranes, and migration to the stroma membranes to serve in the collection of excitation energy to PSI (Fork and Satoh, 1986; Williams and Allen, 1987; Wollman, 2001; Rochaix, 2007; Kargul and Barber, 2008; Murata, 2009; Lemeille et al., 2010; Minagawa, 2011). Concomitantly, the absorption cross section of PSII decreases and that of PSI increases (Canaani and Malkin, 1984; Malkin et al., 1986; Ruban and Johnson, 2009). Indeed, state transitions have been well documented when different qualities (blue/red and far red) of light, preferentially exciting either PSII or PSI, have been applied.Different from state transitions, the white light intensity-dependent reversible LHCII phosphorylation does not result in differential excitation of the two photosystems (Tikkanen et al., 2010). Instead, both photosystems remain nearly equally excited independently whether the LHCII proteins are heavily phosphorylated or strongly dephosphorylated. Moreover, it is worth noting that the different qualities of light generally used to induce reversible LHCII phosphorylation and state transitions (blue/red and far-red lights) have usually been of very low intensity (for review, see Haldrup et al., 2001), and apparently, minimal protonation of the lumen takes place under such illumination conditions. Yet another difference between induction of LHCII protein phosphorylation by different qualities of light or different quantities of white light concerns the concomitant induction of PSII core protein phosphorylation. In the former case, the level of PSII core protein phosphorylation follows the phosphorylation pattern of LHCII proteins, whereas under different quantities of white light, the phosphorylation behavior of PSII core and LHCII proteins is the opposite (Tikkanen et al., 2008b).To gain a more comprehensive understanding of the physiological role of white light-induced changes in LHCII protein phosphorylation, we have integrated Arabidopsis (Arabidopsis thaliana) LHCII phosphorylation with other light-dependent regulatory modifications of light harvesting and electron transfer in the thylakoid membrane, which include the nonphotochemical quenching of excitation energy (for review, see Niyogi, 1999; Horton and Ruban, 2005; Barros and Kühlbrandt, 2009; de Bianchi et al., 2010; Jahns and Holzwarth, 2012; Ruban et al., 2012) and the photosynthetic control of electron transfer by the cytochrome b₆f (Cytb6f) complex (Rumberg and Siggel, 1969; Witt, 1979; Tikhonov et al., 1981; Bendall, 1982; Nishio and Whitmarsh, 1993; Joliot and Johnson, 2011; Suorsa et al., 2012; for review, see Foyer et al., 1990, 2012), both strongly dependent on lumenal protonation.It is demonstrated that the steady-state LHCII phosphorylation is particularly important under rapidly fluctuating light (FL) conditions. This ensures equal energy distribution to both photosystems, prevents the accumulation of electrons in the intersystem electron transfer chain (ETC), eliminates perturbations in chloroplast redox balance, and maintains PSI functionality upon rapid fluctuations in white light intensity.     

Kinetic and Spectral Resolution of Multiple Nonphotochemical Quenching Components in Arabidopsis Leaves

Using novel specially designed instrumentation, fluorescence emission spectra were recorded from Arabidopsis (Arabidopsis thaliana) leaves during the induction period of dark to high-light adaptation in order to follow the spectral changes associated with the formation of nonphotochemical quenching. In addition to an overall decrease of photosystem II fluorescence (quenching) across the entire spectrum, high light induced two specific relative changes in the spectra: (1) a decrease of the main emission band at 682 nm relative to the far-red (750–760 nm) part of the spectrum (Δ F₆₈₂); and (2) an increase at 720 to 730 nm (Δ F₇₂₀) relative to 750 to 760 nm. The kinetics of the two relative spectral changes and their dependence on various mutants revealed that they do not originate from the same process but rather from at least two independent processes. The Δ F₇₂₀ change is specifically associated with the rapidly reversible energy-dependent quenching. Comparison of the wild-type Arabidopsis with mutants unable to produce or overexpressing the PsbS subunit of photosystem II showed that PsbS was a necessary component for Δ F₇₂₀. The spectral change Δ F₆₈₂ is induced both by energy-dependent quenching and by PsbS-independent mechanism(s). A third novel quenching process, independent from both PsbS and zeaxanthin, is activated by a high turnover rate of photosystem II. Its induction and relaxation occur on a time scale of a few minutes. Analysis of the spectral inhomogeneity of nonphotochemical quenching allows extraction of mechanistically valuable information from the fluorescence induction kinetics when registered in a spectrally resolved fashion.One of the most important photoprotective mechanisms against high-light (HL) stress in photosynthetic organisms is the nonphotochemical quenching (NPQ) of excitation energy, which is mostly due to thermal deactivation of pigment excited states in the antenna of PSII. There exist a number of literature reviews on the subject (Demmig-Adams and Adams, 1992; Horton et al., 1996; Horton and Ruban, 1999, 2005; Niyogi, 1999, 2000; Müller et al., 2001; Golan et al., 2004; Krause and Jahns, 2004). Chlorophyll (Chl) fluorescence, and in particular pulse amplitude-modulated (PAM) fluorometry as introduced by Schreiber et al. (1986), has become by far the dominant technique to measure NPQ in leaves, chloroplasts, and intact microorganisms (Krause and Weis, 1991; Govindjee, 1995; Maxwell and Johnson, 2000; Krause and Jahns, 2003; Schreiber, 2004), more recently often combined with specific NPQ mutant studies (Golan et al., 2004; Kalituho et al., 2006, 2007; Dall''Osto et al., 2007). In this technique, periodic saturating light pulses are applied, superimposed on the continuous actinic irradiation applied to induce NPQ, in order to transiently close the PSII reaction centers (RCs). Since the photochemistry contribution (photochemical quenching) is thus brought to zero, the method allows us to follow the dynamics of the NPQ development and relaxation by fluorescence in a relatively simple manner (Krause and Jahns, 2003, 2004).Mostly based on its relaxation kinetics, NPQ has been divided technically into the three kinetic components qE, qT, and qI, for the rapid, middle, and slow phases of relaxation (Horton and Hague, 1988), initially attributed to energy-dependent quenching, state transitions, and photoinhibitory quenching (Quick and Stitt, 1989). The rapidly forming and reversible part of NPQ, qE, is the most thoroughly studied. It is well established that this type of quenching is a finely regulated process in which the main governing factors are the proton gradient across the chloroplast thylakoid membrane, Δ pH (Wraight and Crofts, 1970; Briantais et al., 1979), the xanthophyll cycle (i.e. conversion of violaxanthin to antheraxanthin and zeaxanthin [Zx]; Demmig et al., 1987; Demmig-Adams, 1990; Demmig-Adams and Adams, 1992), and the action of the PsbS protein (Funk et al., 1995; Li et al., 2000, 2004; Niyogi et al., 2005). The actual molecular mechanism is still unknown, although there is no shortage of hypotheses and proposed quencher candidates: energy transfer from Chl to Zx in the major light-harvesting complex (LHCII; Frank et al., 2000); electron transfer from a carotenoid to Chl forming a Zx-Chl or lutein-Chl charge-transfer state (Holt et al., 2005; Avenson et al., 2009); direct or indirect quenching by the PsbS protein (Li et al., 2000; Niyogi et al., 2005); energy transfer from Chl to lutein in LHCII (Horton et al., 1991; Ruban et al., 2007) linked to the aggregation of or a conformational change in LHCII; and last but not least, a far-red (FR) light-emitting quenched Chl-Chl charge-transfer state formed by the aggregation of LHCII (Miloslavina et al., 2008). Quenching in the PSII RC has also been proposed (Weis and Berry, 1987; Finazzi et al., 2004; Huner et al., 2005; Ivanov et al., 2008) as an additional type of Zx-independent quenching. Alternatively, it has been suggested that quenching by lutein can complement the Zx-dependent quenching (Niyogi et al., 2001; Li et al. 2009). Johnson et al. (2009) have recently given support to the notion that both Zx-dependent and Zx-independent quenching originate from the same PsbS-dependent mechanism, which is modulated by Zx (Crouchman et al., 2006).While the rapidly relaxing phase qE is now well characterized in its dependence on the various factors, the much slower qT and qI phases are still controversial, and each of them may have contributions from more than one mechanism. The qI component has been traditionally attributed to photoinhibition of PSII (Somersalo and Krause, 1988), associated with coordinated degradation and repair of the photosystem (Powles and Björkman, 1982; Kyle, 1987; Krause, 1988; Aro et al., 1993; Long et al., 1994; Murata et al., 2007). Lately, though, it is more widely accepted that under most conditions the photoinhibition is low and qI, like qE, is a result of thermal deactivation of excited states. Different hypotheses have been put forward to account for its seeming irreversibility: persistent transmembrane Δ pH (Gilmore and Yamamoto, 1992), stable protonation of proteins (Horton et al., 1994), accumulation of inactive PSII reaction centers (Briantais et al., 1992; Schansker and van Rensen, 1999), or stable binding of Zx to CP29 (Färber et al., 1997). The connection of the qT phase with state transitions has been doubted as well, and in fact it is now thought that the fraction of energy redistributed from PSI to PSII under high-light conditions is negligible (Walters and Horton, 1991, 1993) and that the qT must have a different origin or that it has erroneously been ascribed as NPQ (Schansker et al., 2006).Along with the large amount of contradictory evidence on the nature and location of the NPQ quenching site(s), the question of whether the light-induced reversible NPQ represents one single mechanism of deexcitation located in a single site brought about by the combined action of PsbS and Zx (Johnson et al., 2009) or whether it comprises several parallel and largely independent mechanisms acting on different parts of the PSII antenna has not been finally answered. One way to answer this question might be to carefully examine the spectral properties of NPQ-related fluorescence changes. Quenching in different locations of the PSII antenna or with different mechanisms might give rise to a differential quenching in various parts of the PSII antenna that might affect the PSII fluorescence spectra in different ways. This appears possible, since the various pigment-protein complexes of the photosynthetic apparatus have slightly different absorption and emission spectra (Holzwarth, 1991; Holzwarth and Roelofs, 1992). However, in the vast majority of modulated Chl fluorescence instrumentation, including the most widely used PAM fluorometer (Schreiber et al., 1986), the signal is integrated over a broad wavelength range, usually covering the whole range of 710 nm or greater. This integration over the long-wave part of the spectrum has several undesirable consequences and is associated with the unnecessary loss of available information. For example, the fluorescence of PSII peaks in the region of 680 to 685 nm, whereas beyond 700 nm, the PSII fluorescence intensity drops to less than 20% of its peak intensity. In contrast, the fluorescence of intact PSI complexes is dominant in the region above 710 nm (Haehnel et al., 1982; Karukstis and Sauer, 1983; Holzwarth et al., 1985; Holzwarth, 1986; Slavov et al., 2008). Thus, the widely used instrumentation measures the NPQ parameters in a region with reduced PSII contribution and relatively high PSI contribution to total fluorescence, despite the fact that NPQ is generally considered to be primarily a PSII phenomenon. Only in a few studies has the fluorescence in the red and the FR region been separated in order to evaluate the contribution of PSI and its influence on the NPQ parameters (Genty et al., 1990; Peterson et al., 2001). NPQ might also shift the fluorescence properties of the PSII antenna complexes or give rise to entirely new fluorescing components (Miloslavina et al., 2008). This would remain undetected if the NPQ fluorescence changes are not resolved in the spectral domain. It follows from these considerations that a great deal of insight into the NPQ mechanisms and locations may be gained if the spectral dimension is added to the NPQ fluorescence characterization. Among the many advantages of such an approach, one would then be able to distinguish whether NPQ simply leads to a uniform decrease of PSII fluorescence across the emission range or whether this decrease is nonuniform, localized in specific pigment protein complexes, and/or whether new fluorescing species are actually being produced in the NPQ process.The HL-induced NPQ effects on the leaf fluorescence spectra have often been studied also at low temperature, where the differentiation between pigment sites is better (Krause et al., 1983; Demmig and Björkman, 1987; Ruban and Horton, 1994). However, the possibility to resolve the kinetics of NPQ development and relaxation is largely lost when performing the measurements at low temperatures. The 77 K spectra of leaves and thylakoid membranes are characterized by three main peaks, F₆₈₅, F₆₉₅, and F₇₃₀, believed to originate predominantly from Chl a in CP47 of PSII, a specific Chl in CP43 of PSII, and PSI, respectively (Satoh and Butler, 1978; van Dorssen et al., 1987; Andrizhiyevskaya et al., 2005; Komura et al., 2007). Fluorescence from the major LHCII peaks at 680 nm (Rijgersberg and Amesz, 1978) and from the PSII reaction center Chls at 683 nm (Roelofs et al., 1993; Andrizhiyevskaya et al., 2005). Low-temperature studies on the effects of HL irradiation are confined to the changes in the FR-to-red fluorescence ratio, which are the result of the quenching of PSII fluorescence or energy redistribution between the photosystems (state transitions). Ruban and Horton (1994) have shown that photochemical quenching in Guzmania is maximal at 688 nm, whereas nonphotochemical processes quench preferentially at 683 and 698 nm.In this study, we undertook a detailed investigation of the NPQ-associated spectral changes in the fluorescence spectra of Arabidopsis (Arabidopsis thaliana) measured at room temperature (RT) and at 77 K. It follows from the above discussion that deeper insight into the mechanisms of NPQ processes may be gained by combining the kinetic and the spectral information of the fluorescence changes occurring in NPQ. For this purpose, we developed a multiwavelength spectrometer with parallel detection, allowing us to follow the entire time-dependent fluorescence spectra of leaves during the induction and relaxation phases of NPQ with high sensitivity.Specific questions to be addressed in this study are the following. Are there more than one NPQ processes and NPQ sites? Are these processes occurring in a linked fashion or are they independent? How do they depend on the various cofactors known to affect NPQ, in particular regarding the roles of PsbS and Zx? Using this novel approach of adding the spectral information to the NPQ fluorescence changes, we discovered specific spectral changes associated with different NPQ components. By comparing the effects measured on various NPQ mutants of Arabidopsis, it is possible to assign these NPQ components to specific quenching processes. The results provide evidence that the total NPQ is a combination of several parallel and largely independent processes, likely occurring at different locations in the photosynthetic apparatus.     

The Zeaxanthin-Independent and Zeaxanthin-Dependent qE Components of Nonphotochemical Quenching Involve Common Conformational Changes within the Photosystem II Antenna in Arabidopsis

The light-harvesting antenna of higher plant photosystem II (LHCII) has the intrinsic capacity to dissipate excess light energy as heat in a process termed nonphotochemical quenching (NPQ). Recent studies suggest that zeaxanthin and lutein both contribute to the rapidly relaxing component of NPQ, qE, possibly acting in the minor monomeric antenna complexes and the major trimeric LHCII, respectively. To distinguish whether zeaxanthin and lutein act independently as quenchers at separate sites, or alternatively whether zeaxanthin fulfills an allosteric role regulating lutein-mediated quenching, the kinetics of qE and the qE-related conformational changes (ΔA₅₃₅) were compared in Arabidopsis (Arabidopsis thaliana) mutant/antisense plants with altered contents of minor antenna (kolhcb6, aslhcb4), trimeric LHCII (aslhcb2), lutein (lut2, lut2npq1, lut2npq2), and zeaxanthin (npq1, npq2). The kinetics of the two components of NPQ induction arising from zeaxanthin-independent and zeaxanthin-dependent qE were both sensitive to changes in the protein composition of the photosystem II antenna. The replacement of lutein by zeaxanthin or violaxanthin in the internal Lhcb protein-binding sites affected the kinetics and relative amplitude of each component as well as the absolute chlorophyll fluorescence lifetime. Both components of qE were characterized by a conformational change leading to nearly identical absorption changes in the Soret region that indicated the involvement of the LHCII lutein 1 domain. Based on these observations, we suggest that both components of qE arise from a common quenching mechanism based upon a conformational change within the photosystem II antenna, optimized by Lhcb subunit-subunit interactions and tuned by the synergistic effects of external and internally bound xanthophylls.The chlorophyll a/b-binding light-harvesting antenna of photosystem II (PSII of higher plants is responsible for the efficient collection and transfer of excitation energy to the reaction center. The PSII antenna comprises the main trimeric light-harvesting complex, LHCII, which is composed of the Lhcb1 to -3 polypeptides, and the minor light-harvesting complexes, CP29, CP26, and CP24, composed of Lhcb4, -5, and -6, respectively. In Arabidopsis (Arabidopsis thaliana), four LHCII trimers associate with two copies each of CP24, CP26, and CP29 and a core dimer of PSII (CP43/D1/D2/CP47) to form the C₂S₂M₂ LHCII-PSII supercomplex (Dekker and Boekema, 2005). In addition, depending upon the growth conditions, two or three extra LHCII trimers per PSII may be present in LHCII-only regions of the grana, providing additional light-harvesting capacity.The PSII antenna is a highly dynamic system that is able to tune the amount of excitation delivered to the PSII reaction center to match physiological need (Horton et al., 1996). The regulation of energy flow occurs by control of the thermal dissipation of excess excitation within the PSII antenna, a process termed nonphotochemical quenching (NPQ). NPQ is heterogeneous, comprising a slowly reversible qI component and a rapidly reversible qE component (Horton et al., 1996). The trigger for qE is the buildup of the transmembrane proton gradient or ΔpH (Briantais et al., 1979). The ΔpH is sensed by the PsbS protein (Li et al., 2004), without which the rapidly reversible behavior of NPQ is lost (Li et al., 2000). Full expression of qE in vivo is associated with the enzymatic deepoxidation of the epoxy-xanthophyll violaxanthin to zeaxanthin, via the action of the xanthophyll cycle (Demmig-Adams, 1990). The majority of the photoconvertible xanthophyll cycle pool is associated with trimeric LHCII, bound at the external V1 binding site (Ruban et al., 1999, 2002a; Caffarri et al., 2001; Liu et al., 2004). Trimeric LHCII binds two other types of xanthophylls internally: two all-trans-luteins at the L1 and L2 sites associated with the central membrane-spanning α-helices; and a 9-cis-neoxanthin at the N1 site associated with the C-helix chlorophyll b domain (Liu et al., 2004). The minor monomeric complexes CP24, CP26, and CP29 all bind lutein at the L1 site. In addition, CP29 binds two xanthophyll cycle carotenoids and one-half to one neoxanthin, CP24 binds two xanthophyll cycle carotenoids, while CP26 binds one xanthophyll cycle carotenoid and one neoxanthin (Peter and Thornber, 1991; Bassi et al., 1993; Ruban et al., 1994, 1999; Morosinotto et al., 2002).Although there is strong evidence that qE occurs in the PSII antenna light-harvesting proteins and that xanthophylls are involved, the mechanism of energy dissipation remains unclear. There is evidence for two distinct quenching mechanisms, one involving zeaxanthin (type I) and the other lutein (type II). In the type I mechanism, it is proposed that qE obligatorily depends upon zeaxanthin acting as a quencher of excited chlorophyll via the formation of a charge transfer state. Evidence for type I is the formation of a carotenoid radical cation absorbing at approximately 1,000 nm that correlates with the extent of qE (Holt et al., 2005). Recently, evidence was obtained that formation of the zeaxanthin radical cation occurs exclusively at the L2 binding site of the minor antenna complexes (Ahn et al., 2008; Avenson et al., 2008), quenching therefore requiring reversible insertion of zeaxanthin into this internal site. Because the effect of this cation on the excited-state lifetime of the minor antenna complexes was found to be very small, it was suggested that in vivo, under the influence of the ΔpH, a large population of complexes would adopt a conformation in which this species could form (Avenson et al., 2008). Evidence was also obtained that a zeaxanthin radical cation may form in trimeric LHCII (Amarie et al., 2007). Again, the effect on the chlorophyll excited-state lifetime was very small, leading these authors to conclude that the type I mechanism could not be responsible for qE (Amarie et al., 2007; Dreuw and Wormit, 2008).In the type II mechanism, qE is an inbuilt property of LHCII proteins; a protein conformational change alters the configuration of bound pigments and results in the xanthophyll bound at the L1 site (normally lutein) becoming an effective quencher of chlorophyll excited states (Ruban et al., 2007; Ilioaia et al., 2008). Evidence for a type II mechanism came from studies of trimeric LHCII aggregates (Ruban et al., 2007). Here, it was concluded that energy dissipation occurs by energy transfer from chlorophyll a to the S₁ state (2Ag¹) of lutein bound at the L1 site. Notably, this quenching mechanism decreases the chlorophyll excited-state lifetime by a magnitude sufficient to fully account for qE in vivo. A change in the conformation of another LHCII-bound xanthophyll (neoxanthin) correlates with the extent of quenching. This conformational change takes place in vivo with an amplitude that correlates with the amount of qE. In the model for type II quenching proposed by Horton and coworkers (1991, 2005), zeaxanthin acts not as a quencher but as an allosteric modulator of the ΔpH sensitivity of this intrinsic LHCII quenching process.Although the type I and type II mechanisms involve different xanthophylls operating at different sites, there are similarities: in particular, both are proposed to involve a ΔpH-triggered, PsbS-mediated conformational change (Ruban et al., 2007; Ahn et al., 2008). Indeed, it is possible that both mechanisms contribute to in vivo qE, since the process occurs in both the presence and absence of zeaxanthin (Adams et al., 1990; Crouchman et al., 2006). The crucial question is whether zeaxanthin-dependent and zeaxanthin-independent qE arise from the same mechanism (type II) or from two different ones (types I and II, respectively). The kinetics of NPQ formation upon the illumination of dark-adapted leaves comprise two components: the first forms rapidly and is zeaxanthin independent; the second, slower component correlates with violaxanthin deepoxidation and therefore is described as zeaxanthin dependent (Adams et al., 1990; Ruban and Horton, 1999). The two components of NPQ formation are of the qE type: both relax rapidly upon darkening (Adams et al., 1990); both are dependent upon PsbS (Li et al., 2000); and both are enhanced by PsbS overexpression (Li et al., 2002; Crouchman et al., 2006). Investigation of these kinetics provides an opportunity to determine whether a single mechanism can account for qE and to give clues to which type of mechanism is involved. Here, we test the hypothesis that the two components arise from different mechanisms: the zeaxanthin-dependent component arising in the minor monomeric antenna by a type I mechanism (Gilmore et al., 1998; Ahn et al., 2008; Avenson et al., 2008), and the zeaxanthin-independent component arising in the major trimeric LHCII by the type II mechanism. An alternative explanation for zeaxanthin-independent qE, at least under low-light conditions, when qE forms transiently, is that it is caused by quenching in the PSII reaction center (Finazzi et al., 2004). Several predictions emerge from this hypothesis. First, the removal of certain Lhcb proteins by mutation would differentially affect the two components of qE. Second, because the two components would be additive and could not compensate for the loss of one another (Niyogi et al., 1998; Pogson et al., 1998), they should each contribute a discrete component to the kinetics of qE formation and relaxation. Third, in mutants lacking lutein, the capacity of the type II mechanism would be reduced, while the zeaxanthin-dependent component would be unaffected. Finally, the two components may be expected to be characterized by different absorption changes in the Soret region, which reflect changes in the absorption spectra of bound pigments brought about by conformational changes within the PSII antenna upon qE formation (Ruban et al., 1993a, 1993b, 2002b; Bilger and Björkman, 1994). We tested this hypothesis by analysis of qE kinetics, fluorescence lifetimes, and qE-related absorption difference spectra. Contrary to the above predictions, the data indicated that both steady-state and transient qE arise from a common mechanism within the PSII antenna, in both the presence and absence of zeaxanthin.     

Phosphorylation of Photosystem II Controls Functional Macroscopic Folding of Photosynthetic Membranes in Arabidopsis

Photosynthetic thylakoid membranes in plants contain highly folded membrane layers enriched in photosystem II, which uses light energy to oxidize water and produce oxygen. The sunlight also causes quantitative phosphorylation of major photosystem II proteins. Analysis of the Arabidopsis thaliana stn7xstn8 double mutant deficient in thylakoid protein kinases STN7 and STN8 revealed light-independent phosphorylation of PsbH protein and greatly reduced N-terminal phosphorylation of D2 protein. The stn7xstn8 and stn8 mutants deficient in light-induced phosphorylation of photosystem II had increased thylakoid membrane folding compared with wild-type and stn7 plants. Significant enhancement in the size of stacked thylakoid membranes in stn7xstn8 and stn8 accelerated gravity-driven sedimentation of isolated thylakoids and was observed directly in plant leaves by transmission electron microscopy. Increased membrane folding, caused by the loss of light-induced protein phosphorylation, obstructed lateral migration of the photosystem II reaction center protein D1 and of processing protease FtsH between the stacked and unstacked membrane domains, suppressing turnover of damaged D1 in the leaves exposed to high light. These findings show that the high level of photosystem II phosphorylation in plants is required for adjustment of macroscopic folding of large photosynthetic membranes modulating lateral mobility of membrane proteins and sustained photosynthetic activity.The use of captured sunlight energy to split water and drive oxygenic photosynthesis by photosystem II (PSII) (Barber, 2006) inevitably generates reactive oxygen species and causes oxidative damage to the PSII protein pigment complex. The light-induced damage to PSII, in particular to the D1 reaction center protein, requires PSII repair to sustain its photosynthetic function (Takahashi and Murata, 2008). Impairment and degradation of D1 increase with rising light intensities, and this protein has the fastest turnover rate among the photosynthetic proteins of plants, algae, and cyanobacteria (Yokthongwattana and Melis, 2006). However, in plants, the PSII is segregated in highly stacked membrane layers of very large thylakoid membranes (Andersson and Anderson, 1980; Kirchhoff et al., 2008), which are densely folded to fit inside chloroplasts (Mullineaux, 2005; Shimoni et al., 2005). As a consequence, the PSII repair cycle in plants is slower than in cyanobacteria (Yokthongwattana and Melis, 2006), and it includes migration of the PSII complex from the stacked membrane domains (grana) to the unstacked membranes (stroma lamellae), where proteolysis and insertion of a newly synthesized D1 protein occurs (Baena-Gonzalez and Aro, 2002; Yokthongwattana and Melis, 2006). High light also causes quantitative phosphorylation of the membrane surface–exposed regions of D1, D2, CP43, and PsbH proteins of PSII in plants (Rintamäki et al., 1997; Vener et al., 2001), but the function of this phosphorylation is largely unknown and reports on its importance for the D1 protein turnover are conflicting (Bonardi et al., 2005; Tikkanen et al., 2008).Phosphorylation of the PSII proteins in Arabidopsis thaliana depends mostly on the light-activated protein kinase STN8 (Vainonen et al., 2005), while the STN7 kinase is essential for phosphorylation of the light-harvesting proteins of PSII (Bellafiore et al., 2005; Bonardi et al., 2005; Tikkanen et al., 2006). An earlier study on Arabidopsis mutants lacking both STN7 and STN8 (stn7xstn8), as well as only STN8, concluded that protein phosphorylation was not essential for PSII repair (Bonardi et al., 2005), while more recent work revealed a dramatic retardation in D1 degradation under high light in the stn8 and stn7xstn8 mutants (Tikkanen et al., 2008). Moreover, it was shown that the lack of PSII phosphorylation resulted in accumulation of photodamaged PSII complexes and in general oxidative damage of photosynthetic proteins in the thylakoid membranes under high light (Tikkanen et al., 2008). The other study revealed that the stn7xstn8 double mutant grown under natural field conditions produced 41% less seeds than wild-type plants (Frenkel et al., 2007), which also indicated physiological importance of thylakoid protein phosphorylation in maintenance of plant fitness.To uncover the function of light-dependent protein phosphorylation in plant photosynthetic membranes, we performed a detailed analysis of the Arabidopsis mutants deficient in the protein kinases STN7 and STN8. The earlier published results on protein phosphorylation analyses in the stn7xstn8 mutant of Arabidopsis were restricted to antiphosphothreonine antibody-based immunodetection and did not reveal any phosphorylation of PSII core proteins (Bonardi et al., 2005; Tikkanen et al., 2008). Using a mass spectrometry (MS) approach and immunoblot analyses with two complementary antiphosphothreonine antibodies, we find remaining light-independent phosphorylation of PsbH and D2 proteins of PSII in stn7xstn8. We demonstrate that degradation and aggregation patterns of the D1 protein in stn7xstn8 differ from those in wild-type, stn7, and stn8 plants. We also observe a reproducible delay in the degradation of D1 in high light–treated leaves of stn7xstn8 and stn8 compared with the wild-type and stn7 plants. Finally, we show that phosphorylation of PSII proteins modulates macroscopic rearrangements of the entire membrane network of plant thylakoids, which facilitates lateral mobility of membrane proteins, required for repair and sustained activity of PSII.     

Thylakoid Membrane Reduction Affects the Photosystem Stoichiometry in the Cyanobacterium Synechocystis sp. PCC 6803

Biogenesis of thylakoid membranes in both chloroplasts and cyanobacteria is largely not understood today. The vesicle-inducing protein in plastids 1 (Vipp1) has been suggested to be essential for thylakoid membrane formation in Arabidopsis (Arabidopsis thaliana), as well as in the cyanobacterium Synechocystis sp. PCC 6803, although its exact physiological function remains elusive so far. Here, we report that, upon depletion of Vipp1 in Synechocystis cells, the number of thylakoid layers in individual Synechocystis cells decreased, and that, in particular, the content of photosystem I (PSI) complexes was highly diminished in thylakoids. Furthermore, separation of native photosynthetic complexes indicated that PSI trimers are destabilized and the monomeric species is enriched. Therefore, depletion of thylakoid membranes specifically affects biogenesis and/or stabilization of PSI in cyanobacteria.In chloroplasts and cyanobacteria the energy transfer between PSI and PSII is regulated in a light-dependent manner (for a recent review, see Kramer et al., 2004). The two photosystems are connected by the cytochrome b₆f complex, and electron transfer from PSII via the cytochrome b₆f complex to PSI is believed to be regulated by the redox state of the plastoquinol pool potentially also involving the cytochrome b₆f complex (Fujita et al., 1987; Murakami and Fujita, 1993; Schneider et al., 2001, 2004; Pfannschmidt, 2003; Volkmer et al., 2007). Transfer of light energy to the two photosystems is mediated by light-harvesting complexes, and in cyanobacteria light is harvested by the soluble extramembranous phycobilisomes. The efficient energy transfer to PSI and PSII has to be balanced to synchronize the function of the two photosystems. In response to changing light intensities and qualities, energy coupling between the phycobilisomes and the photosystems changes, which allows a rapid adjustment of light absorbance by the individual photosystems. Furthermore, besides this short-term adaptation mechanism, it has been shown in many studies that on a longer term in cyanobacteria the ratio of the two photosystems changes depending on the light conditions (Manodori and Melis, 1986; Murakami and Fujita, 1993; Murakami et al., 1997). Upon shifting cyanobacterial cells from low-light to high-light growth conditions, the PSI-to-PSII ratio decreases due to selective suppression of the amount of functional PSI. In recent years, some genes have already been identified that are involved in this regulation of the photosystem stoichiometry (Hihara et al., 1998; Sonoike et al., 2001; Fujimori et al., 2005; Ozaki et al., 2007).Whereas in chloroplasts of higher plants and green algae the amounts of the two photosystems change in response to changing light conditions (Melis, 1984; Chow et al., 1990; Smith et al., 1990; Kim et al., 1993), it has already been noted a long time ago that the chloroplast ultrastructure also adapts to high-light and low-light conditions (Melis, 1984). Chloroplasts of plants grown under low light or far-red light have more thylakoid membranes than chloroplasts of plants grown under high light or blue light (Anderson et al., 1973; Lichtenthaler et al., 1981; Melis and Harvey, 1981). There appears to be a direct correlation between the chlorophyll content and the amount of thylakoids per chloroplast because light harvesting is increased by enhanced chlorophyll and thylakoid membrane content per chloroplast. Thus, chloroplasts adapt to high light both by a reduction of thylakoid membranes and by a decrease in the PSI-to-PSII ratio.Thylakoid membranes are exclusive features of both cyanobacteria and chloroplasts, and it still remains mysterious how formation of thylakoid membranes is organized. Many cellular processes, like lipid biosynthesis, membrane formation, protein synthesis in the cytoplasm and/or at a membrane, protein transport, protein translocation, and protein folding have to be organized and aligned for formation of internal thylakoid membranes. The recent observation that deletion of the vipp1 gene in Arabidopsis (Arabidopsis thaliana) results in complete loss of thylakoid membranes has indicated that Vipp1 is involved in biogenesis of thylakoid membranes. Further analysis has suggested that Vipp1 could be involved in vesicle trafficking between the inner envelope and the thylakoid membrane of chloroplasts (Kroll et al., 2001). Because of this, the protein was named Vipp1, for vesicle-inducing protein in plastids 1. Depletion of Vipp1 strongly affected the ability of cyanobacterial cells to form proper thylakoid membranes (Westphal et al., 2001) and, consequently, also in cyanobacteria Vipp1 appears to be involved in formation of thylakoid membranes. A Vipp1 depletion strain of Arabidopsis is deficient in photosynthesis, although the defect could not be assigned to a deficiency of a single photosynthetic complex, but appeared to be caused by dysfunction of the entire photosynthetic electron transfer chain (Kroll et al., 2001). Therefore, depletion of Vipp1 in Arabidopsis seems to affect thylakoid membrane formation rather than the assembly of thylakoid membrane protein complexes (Aseeva et al., 2007). However, for cyanobacteria, it is not clear yet how diminishing the amount of thylakoid membrane layers would affect the amount and stoichiometry of the two photosystems.Here, we present the generation and characterization of a Vipp1 depletion strain of the cyanobacterium Synechocystis sp. PCC 6803. Upon depletion of Vipp1, a decrease in thylakoid membrane pairs in the generated mutant strain and, furthermore, a significant decrease in active PSI centers was observed. Moreover, trimerization of PSI also appeared to be impaired in the mutant strain. These results suggest that thylakoid membrane perturbations caused by the Vipp1 depletion directly affects PSI assembly and stability in cyanobacterial thylakoid membranes.     

The Variegated Mutants Lacking Chloroplastic FtsHs Are Defective in D1 Degradation and Accumulate Reactive Oxygen Species

In the photosynthetic apparatus, a major target of photodamage is the D1 reaction center protein of photosystem II (PSII). Photosynthetic organisms have developed a PSII repair cycle in which photodamaged D1 is selectively degraded. A thylakoid membrane-bound metalloprotease, FtsH, was shown to play a critical role in this process. Here, the effect of FtsHs in D1 degradation was investigated in Arabidopsis (Arabidopsis thaliana) mutants lacking FtsH2 (yellow variegated2 [var2]) or FtsH5 (var1). Because these mutants are characterized by variegated leaves that sometimes complicate biochemical studies, we employed another mutation, fu-gaeri1 (fug1), that suppresses leaf variegation in var1 and var2 to examine D1 degradation. Two-dimensional blue native PAGE showed that var2 has less PSII supercomplex and more PSII intermediate lacking CP43, termed RC47, than the wild type under normal growth light. Moreover, our histochemical and quantitative analyses revealed that chloroplasts in var2 accumulate significant levels of reactive oxygen species, such as superoxide radical and hydrogen peroxide. These results indicate that the lack of FtsH2 leads to impaired D1 degradation at the step of RC47 formation in PSII repair and to photooxidative stress even under nonphotoinhibitory conditions. Our in vivo D1 degradation assays, carried out by nonvariegated var2 fug1 and var1 fug1 leaves, demonstrated that D1 degradation was impaired in different light conditions. Taken together, our results suggest the important role of chloroplastic FtsHs, which was not precisely examined in vivo. Attenuated D1 degradation in the nonvariegated mutants also suggests that leaf variegation seems to be independent of the PSII repair.Excessive light often limits the growth of photosynthetic organisms by irreversibly inactivating the photosynthetic apparatus, a process called photoinhibition (for review, see Barber and Andersson, 1992; Aro et al., 1993). A major target of photodamage is PSII (for review, see Barber and Andersson, 1992; Aro et al., 1993; Murata et al., 2007), a large pigment-protein complex in the thylakoid membrane. In particular, the reaction center D1 protein, which binds cooperatively to D2 and carries cofactors required for electron flow from the manganese cluster of the water-oxidizing complex to the plastoquinone pool (Zouni et al., 2001; Loll et al., 2005), is the primary target of light-induced irreversible oxidative damage (Mattoo et al., 1981; Ohad et al., 1990). Because D1 can be damaged by even low light intensities, photosynthetic organisms cannot avoid photodamage (Tyystjärvi and Aro, 1996; for review, see Barber and Andersson, 1992). To overcome this, photosynthetic organisms have evolved an efficient PSII repair cycle, which involves disassembling PSII, degrading photodamaged D1, and replacing newly synthesized D1 (for review, see Baena-Gonzalez and Aro, 2002). The rate of photodamage is proportional to light energy. When the light intensity exceeds the repair capacity, damaged D1 accumulates, resulting in photoinhibition.In the PSII repair, recent studies in Synechocystis species PCC 6803 and Arabidopsis (Arabidopsis thaliana) suggest important roles of prokaryotic proteases (Lindahl et al., 1996, 2000; Bailey et al., 2002; Sakamoto et al., 2003; Silva et al., 2003; Komenda et al., 2006; Sun et al., 2007; Kapri-Pardes et al., 2007). Among them, FtsH appears to be a major protease. It is a membrane-anchored ATP-dependent zinc metalloprotease that belongs to the ATPases associated with a variety of cellular activities (AAA)+ protein family (for review, see Patel and Latterich, 1998; Ogura and Wilkinson, 2001). The ATPase and protease domain of FtsH was shown to form a hexameric-ring structure (Suno et al., 2006). In Synechocystis species PCC 6803, deletion of one of the thylakoidal FtsHs, slr0228, results in light-sensitive growth, impairment of the PSII repair cycle, and slower D1 degradation under high-light conditions (Silva et al., 2003). In Arabidopsis, 12 FtsH homologues were identified, nine of which are targeted to chloroplasts (Sakamoto et al., 2003). FtsH2 and FtsH5 are the most abundant among all chloroplastic FtsHs and are located in thylakoid membranes (Sakamoto et al., 2003; Yu et al., 2004, 2005). Chloroplastic FtsHs predominantly exist as a heterocomplex consisting of at least two types of isomers, A and B, represented by FtsH1/5 and FtsH2/8, respectively. These two types are functionally distinguishable from each other (Sakamoto et al., 2003; Yu et al., 2004, 2005; Zaltsman et al., 2005b).We have extensively studied Arabidopsis mutants lacking chloroplast FtsHs. A mutant lacking FtsH5 (called yellow variegated1 [var1]) or lacking FtsH2 (var2) is highly vulnerable to PSII photodamage under high light (Chen et al., 2000; Lindahl et al., 2000; Takechi et al., 2000; Sakamoto et al., 2002, 2004). One notable feature in these mutants, in addition to the defective PSII repair, is the leaf-variegated phenotype that displays two sectors in the same leaf (green sectors containing normal chloroplasts and white sectors containing abnormal plastids lacking thylakoid membranes; Supplemental Fig. S1). White sectors are made by living cells and appear to be comparable with green sectors, except for lacking photosynthetic proteins (Kato et al., 2007). These results demonstrated that white sectors in var2 are arrested in chloroplast development. It is thus proposed that FtsH is not only involved in PSII repair but also in the formation of thylakoid membranes. Moreover, a series of genetic studies enabled us to identify trans-acting mutations that suppressed leaf variegation in var2 (Park and Rodermel, 2004; Miura et al., 2007; Yu et al., 2008). Many suppressors appeared to be associated with chloroplast translation, suggesting that the formation of variegated sectors is not simply governed by a specific factor but rather by factors related to chloroplast development (Miura et al., 2007; Yu et al., 2008). Since the variegated phenotype complicates our biochemical study in Arabidopsis, unlike cyanobacteria, a combined usage of var and its suppressors is necessary to further investigate the role of FtsH in the PSII repair cycle.In this study, we show that chloroplasts in green sectors accumulate less PSII supercomplex and more PSII partial complexes than wild-type chloroplasts, likely due to the compromised PSII repair. Interestingly, we found that chloroplasts in green sectors accumulated significantly high levels of reactive oxygen species (ROS), suggesting that var2 indeed suffers from photooxidative stress. Given the essential role of FtsH, we evaluated impaired D1 degradation by the lack of FtsH2 and FtsH5 under different light conditions. Although a defect in degrading PSII reaction center proteins in var2 has been reported to occur under a photoinhibitory light condition (Bailey et al., 2002), D1 degradation examined in this study was very limited. This is because in vivo degradation of D1 is very difficult to measure in variegated leaf tissues (e.g. the presence of green and white leaf sectors interferes with protein normalization). To overcome the difficulty of handling variegated sectors, nonvariegated suppressor lines were subjected to these experiments. Our D1 degradation assays demonstrated that the lack of FtsH2 or FtsH5 significantly impairs D1 degradation. Collectively, our data corroborate important roles of FtsH2 and FtsH5 in avoiding photooxidative stress in chloroplasts.     

High Light-Dependent Phosphorylation of Photosystem II Inner Antenna CP29 in Monocots Is STN7 Independent and Enhances Nonphotochemical Quenching

Phosphorylation of the photosystem II antenna protein CP29 has been reported to be induced by excess light and further enhanced by low temperature, increasing resistance to these stressing factors. Moreover, high light-induced CP29 phosphorylation was specifically found in monocots, both C3 and C4, which include the large majority of food crops. Recently, knockout collections have become available in rice (Oryza sativa), a model organism for monocots. In this work, we have used reverse genetics coupled to biochemical and physiological analysis to elucidate the molecular basis of high light-induced phosphorylation of CP29 and the mechanisms by which it exerts a photoprotective effect. We found that kinases and phosphatases involved in CP29 phosphorylation are distinct from those reported to act in State 1-State 2 transitions. In addition, we elucidated the photoprotective role of CP29 phosphorylation in reducing singlet oxygen production and enhancing excess energy dissipation. We thus established, in monocots, a mechanistic connection between phosphorylation of CP29 and nonphotochemical quenching, two processes so far considered independent from one another.In eukaryotic photosynthesis, light-dependent reactions are performed by two supramolecular complexes, PSII and PSI, which catalyze light harvesting and electron transport from water to NADP⁺. To this aim, water is oxidized by PSII, which, in turn, is oxidized by PSI, which becomes a reductant for ferredoxin-NADP(+) oxidoreductase and NADP⁺ (Nelson and Ben-Shem, 2004). The two photosystems are functionally connected by the plastoquinone (PQ) and cytochrome (cyt) b₆/f, which catalyze the building of the transthylakoid proton gradient, which is dissipated by ATP synthase (ATPase) activity for ATP synthesis from ADP and inorganic phosphate (P_i). PSII and PSI have clearly distinct absorption spectra, with PSI-light-harvesting complex I (LHCI) complexes being enriched in red-shifted absorption forms (Gobets and van Grondelle, 2001). Within canopies, this leads to differential excitation depending on available light quality. This effect needs to be compensated to avoid imbalance of electron transport rates, yielding into either photoinhibition or decrease of photon use efficiency. Two major regulatory mechanisms counteract these effects. (1) State 1-State 2 transitions are active in limiting light conditions (Rintamäki et al., 2000) and inhibited by reduction of a disulfide bridge in high light (HL; Lemeille et al., 2009). This mechanism is activated by overreduction of PQ to plastoquinol (PQH₂) through activation of a thylakoid bound kinase, STN7, acting on LHCII (Depège et al., 2003; Bellafiore et al., 2005). This causes a fraction of PSII antenna system, mainly Lhcb2 (Leoni et al., 2013), to be transferred to PSI in stroma-exposed membranes. The consequent increase in PSI antenna size (Galka et al., 2012) bursts the electron transfer rate and reequilibrates PQ/PQH₂ redox poise, thus causing feedback inactivation of kinase activity. A phosphatase, PPH1-TAP38 (Pribil et al., 2010; Shapiguzov et al., 2010), dephosphorylates LHCII, allowing for its return to PSII in grana partitions. (2) The process of nonphotochemical quenching (NPQ) is rather aimed to photoprotection from excess light (Horton and Ruban, 2005; de Bianchi et al., 2010). In this condition, saturation of downstream metabolic reactions causes depletion of ADP and P_i and inhibition of ATPase activity, which normally brings back protons from lumen to the stromal compartment. This causes accumulation of protons in the lumen, triggering dissipation into heat of the energy absorbed in excess by PSII (de Bianchi et al., 2010; Niyogi and Truong, 2013). Thus, the synergic, although independent, activities of State-1-State 2 transitions and NPQ cover the needs for regulation of photosynthesis over the wide dynamic range of light intensity experienced by plants when shaded or by daily light changes. However, a number of experimental results do not fit within this scheme: phosphorylation of PSII antenna proteins, namely CP29 (LHCB4) has been reported to be induced in very HL and further enhanced by low temperature (Bergantino et al., 1995), implying inhibition of LHC protein phosphorylation by HL is not a general feature of plant photosynthesis. Moreover, CP29 phosphorylation has been shown to be protective in condition of HL combined with low temperature (Mauro et al., 1997), a major stressing factor limiting crop productivity. HL-induced CP29 phosphorylation has been specifically found in monocots, either C3 or C4 (Bergantino et al., 1998), which include the large majority of food crops, thus making the study of this process of interest for both basic and applied research. In dicots, CP29 phosphorylation has been detected at very low level (Fristedt and Vener, 2011) and targeted to different sites within the N-terminal domain with respect to monocots (Testi et al., 1996). Although early research focused on biochemical and physiological characterization of CP29 phosphorylation (Croce et al., 1996; Mauro et al., 1997; Hwang et al., 2003), genetic dissection of this regulation process has been hampered by lack of genetic resources. More recently, rice (Oryza sativa) has become a model organism for monocots, and knockout collections have become available. In this study, we have used reverse genetics coupled to biochemical and physiological analysis to elucidate the molecular basis of HL-induced phosphorylation of CP29 and the mechanisms by which it exerts a photoprotective effect. We found that a different set of kinases and phosphatases is involved in CP29 reversible phosphorylation with respect to that reported to act in State 1-State 2 transitions and that the photoprotection effect is mediated by an enhancement of excess energy dissipation. These results establish, for the first time, a mechanistic connection between thylakoid protein phosphorylation and NPQ, so far believed to be independent processes.     

Large-Scale Phosphoprotein Analysis in Medicago truncatula Roots Provides Insight into in Vivo Kinase Activity in Legumes

Nitrogen fixation in legumes requires the development of root organs called nodules and their infection by symbiotic rhizobia. Over the last decade, Medicago truncatula has emerged as a major model plant for the analysis of plant-microbe symbioses and for addressing questions pertaining to legume biology. While the initiation of symbiosis and the development of nitrogen-fixing root nodules depend on the activation of a protein phosphorylation-mediated signal transduction cascade in response to symbiotic signals produced by the rhizobia, few sites of in vivo phosphorylation have previously been identified in M. truncatula. We have characterized sites of phosphorylation on proteins from M. truncatula roots, from both whole cell lysates and membrane-enriched fractions, using immobilized metal affinity chromatography and tandem mass spectrometry. Here, we report 3,457 unique phosphopeptides spanning 3,404 nonredundant sites of in vivo phosphorylation on 829 proteins in M. truncatula Jemalong A17 roots, identified using the complementary tandem mass spectrometry fragmentation methods electron transfer dissociation and collision-activated dissociation. With this being, to our knowledge, the first large-scale plant phosphoproteomic study to utilize electron transfer dissociation, analysis of the identified phosphorylation sites revealed phosphorylation motifs not previously observed in plants. Furthermore, several of the phosphorylation motifs, including LxKxxs and RxxSxxxs, have yet to be reported as kinase specificities for in vivo substrates in any species, to our knowledge. Multiple sites of phosphorylation were identified on several key proteins involved in initiating rhizobial symbiosis, including SICKLE, NUCLEOPORIN133, and INTERACTING PROTEIN OF DMI3. Finally, we used these data to create an open-access online database for M. truncatula phosphoproteomic data.Medicago truncatula has become a model for studying the biology of leguminous plants such as soybean (Glycine max), alfalfa (Medicago sativa), and clover (Trifolium spp.; Singh et al., 2007). Most members of this vast family have the ability to fix atmospheric nitrogen by virtue of an endosymbiotic association with rhizobial bacteria, through which legumes undergo nodulation, the process of forming root nodules (Jones et al., 2007). Legumes are central to modern agriculture and civilization because of their ability to grow in nitrogen-depleted soils and replenish nitrogen through crop rotation. Consequently, there is great interest in understanding the molecular events that allow legumes to recognize their symbionts, develop root nodules, and fix nitrogen. Nod factors are lipochitooligosaccharidic signals secreted by the rhizobia and are required, in most legumes, for intracellular infection and nodule development. In recent decades, an elegant combination of genetics, biochemistry, and cell biology has shown that Nod factors activate intricate signaling events within cells of legume roots, including protein phosphorylation cascades and intracellular ion fluxes (Oldroyd and Downie, 2008).Protein phosphorylation is a central mechanism of signal transfer in cells (Laugesen et al., 2006; Peck, 2006; Huber, 2007). Several characterized protein kinases are required for symbiosis signal transduction in M. truncatula roots (Lévy et al., 2004; Yoshida and Parniske, 2005; Smit et al., 2007). A recent antibody-based study of cultured M. truncatula cells observed protein phosphorylation changes at the proteomic level in response to fungal infection (Trapphoff et al., 2009); however, the target residues of the phosphorylation events were not determined. A variety of studies have determined in vitro phosphorylation sites on legume proteins and demonstrated the biological importance of the target residues by mutagenesis (Yoshida and Parniske, 2005; Arrighi et al., 2006; Lima et al., 2006; Miyahara et al., 2008; Yano et al., 2008). To our knowledge, only six sites of in vivo protein phosphorylation have been detected for M. truncatula (Laugesen et al., 2006; Lima et al., 2006; Wienkoop et al., 2008), demonstrating the need for the identification of endogenous protein phosphorylation sites in legume model organisms on a proteome-wide scale.While considerable advancements have been made in the global analysis of protein phosphorylation (Nita-Lazar et al., 2008; Macek et al., 2009; Piggee, 2009; Thingholm et al., 2009), phosphoproteomics in plants has lagged years behind that of the mammalian systems (Kersten et al., 2006, 2009; Peck, 2006), which have more fully sequenced genomes and better annotated protein predictions. Arabidopsis (Arabidopsis thaliana), the first plant genome sequenced (Arabidopsis Genome Initiative, 2000), is now predicted to have over 1,000 protein kinases (Finn et al., 2008), approximately twice as many as in human (Manning et al., 2002). Because many of the kinases in the commonly studied mammalian systems are not conserved in the plant kingdom, there is significant need for large-scale phosphoproteomic technologies to discern the intricacies of phosphorylation-mediated cell signaling in plants. With the high mass accuracy afforded by the linear ion trap-orbitrap hybrid mass spectrometer (Makarov et al., 2006; Yates et al., 2006), recent studies in Arabidopsis have reported 2,597 phosphopeptides from suspension cell culture (Sugiyama et al., 2008) and 3,029 phosphopeptides from seedlings (Reiland et al., 2009).All previous large-scale plant phosphoproteomic studies have relied solely on collision-activated dissociation (CAD) during tandem mass spectrometry (MS/MS) and have not taken advantage of the more recently developed methods (Kersten et al., 2009) electron capture dissociation (Kelleher et al., 1999) or electron transfer dissociation (ETD; Coon et al., 2004; Syka et al., 2004). Mapping sites of posttranslational modifications, such as phosphorylation, is often more straightforward using electron-based fragmentation methods, as they frequently produce a full spectrum of sequence-informative ions without causing neutral loss of the modifying functional groups (Meng et al., 2005; Chi et al., 2007; Khidekel et al., 2007; Molina et al., 2007; Wiesner et al., 2008; Chalkley et al., 2009; Swaney et al., 2009). With an ETD-enabled hybrid orbitrap mass spectrometer (McAlister et al., 2007, 2008), we previously compared the performance of CAD and ETD tandem MS for large-scale identification of phosphopeptides (Swaney et al., 2009). ETD identified a greater percentage of unique phosphopeptides and more frequently localized phosphorylation sites. Still, the low overlap of identified phosphopeptides indicates that the two methods are highly complementary. With this in mind, we recently developed a decision tree-driven tandem MS algorithm to select the optimal fragmentation method for each precursor (Swaney et al., 2008).Here, we utilize this technology to map sites of in vivo protein phosphorylation in roots of M. truncatula Jemalong A17 plants. Phosphoproteins, from both whole-cell lysate and membrane-enriched fractions, were analyzed after digestion with a variety of different enzymes individually. Utilizing the complementary fragmentation methods of ETD and CAD, we report 3,404 nonredundant phosphorylation sites at an estimated false discovery rate (FDR) of 1%. Analysis of these data revealed several phosphorylation motifs not previously observed in plants. The phosphorylation sites identified provide insight into the potential regulation of key proteins involved in rhizobial symbiosis, potential consensus sequences by which kinases recognize their substrates, and critical phosphorylation events that are conserved between plant species.