Protein Phosphorylation and Disassembly of the Ps Ii Holocomplex

Photosystem II reaction centers occur as dimer complexes, which collectively contain 50, or more, transmembrane and peripheral proteins (Hankamer et al., 1997; Zouni et al., 2001; Ferreira et al., 2004). The repair process, which entails the selective removal and replacement of the affected D1 protein, requires a partial disassembly of the PS II holocomplex prior to D1 degradation. A prompt disassembly of the PS II holo-complex has been observed upon photodamage (Aro et al., 1993), however, the driving force for this disassembly is not well understood. It is possible that reversible PS II protein phosphorylation is responsible for the disassembly of photodamaged PS II holocom-plexes.

In green plants and algae, several major PS II proteins including D1, D2, PsbH, CP43, and sub-units of the LHC-II become reversibly phosphorylated upon exposure to strong illumination (Michel et al., 1988; Bennett, 1991). Although phosphorylation of the LHC-II is reported to serve in balancing the distribution of excitation energy between PS II and PS I (Bennett, 1979; Allen and Nilsson, 1997; Allen, 2003), the possibility cannot be excluded that such phosphorylation simply contributes to a negative charge density increase in the stroma-exposed regions of the NH2-termini of these PS II proteins. Such a negative charge field on the PS II proteins might increase repulsive forces between them, leading to their electrostatic separation (unfolding of the PS II holocomplex).

Reversible phosphorylation of PS II proteins has been linked directly to the regulation of D1 protein turnover (Aro et al., 1992; Elich et al., 1992; Koivuniemi et al., 1995; Kruse et al., 1997). Under ambient physiological conditions, phosphorylation of the D1 reaction center protein appears to be controlled by an endogenous circadian rhythm (Booij-James etal., 2002). Phosphorylation of D1 does not alter its sensitivity to photodamage but rather prevents its degradation (Koivuniemi et al., 1995; Kruse et al., 1997). As a result, dephosphorylation of P ~ D1 is required for the D1 protein degradation to occur (Rintamaki et al., 1996). In the presence of NaF, a protein phosphatase inhibitor, photoinhibited PS II was found to disassemble into monomers, while still in the phosphorylated state, and the monomer PS II complex was found to migrate from the appressed thylakoids of the grana to the stroma-exposed thylakoid membranes (Baena-Gonzalez and Aro, 2002). It was proposed that phosphorylation of the PS II core proteins does not prevent monomerization of the PS II holocomplex but rather functions as a protective mechanism via inhibition of premature degradation of the damaged D1, i.e., before the latter reaches the stroma-exposed thylakoid region. Subsequent dephosphorylation of the PS II proteins in the stroma lamellae allows a coordinated D1 degradation and de novo D1 biosynthesis to take place. Phosphorylation of the D1 protein, however, has not been observed in cyanobacteria or red and green algae (Pursiheimo et al., 1998; A.K. Mattoo, personal communication). This suggests that D1 phosphorylation is not essential for D1 turnover in every photosynthetic organism. It also raises the prospect of other mechanisms employed by these organisms in the regulation ofD1 degradation.

In a unicellular green alga, Dunaliella salina, pho-todamaged and disassembled PS II reaction centers have been identified and isolated as distinct 160 kD complexes on SDS-PAGE (Kim et al., 1993; Melis and Nemson, 1995). Kinetics of the 160 kD accumulation and decay matched those of photodamage and PS II repair (Kim et al., 1993; Baroli and Melis, 1996). The 160 kD complex was found to be a cross-linked derivative of D1, D2, CP47, and Hsp70B, the latter being a chloroplast-localized heat-shock protein (Yokthongwattana et al., 2001). Other investigators have also reported cross-linked products between D1 and proximal PS II proteins on SDS-PAGE during photoinhibition (Barbato et al., 1992; Ishikawaetal., 1999; Yamamoto, 2001). It was postulated that cross-linking of these proteins occurs specifically in photoinhibited thylakoids and is an artifact of the solubilization process. Such cross-linking is unlikely to occur in vivo as it is generally accepted that cross-linked proteins are subject to prompt degradation. In this case, other components of the cross-linked complex would also have a high turnover rate similar to that of D1. However, only the D1 protein is selectively degraded and replaced in the course of the PS II damage and repair process (Vasilikiotis and Melis, 1994). A frequent turnover of D2 and of other PS II subunits has not been observed under normal physiological conditions (Jansen et al., 1999).

E. Involvement of a Chloroplast-Localized Hsp70 in the PS II Repair Process

The PS II repair process is induced by irradiance and operates only in the light. As such, no photodamage or repair occurs in the dark (Polle and Melis, 1999). The repair involves a coordinated and light-regulated expression of several genes and their respective proteins. A clear example of this 'induction' was provided by the prompt (less than 1 h) and specific 70-fold increase in Hsp70B gene transcripts following a LL ^ HL transition of a green alga culture (Drzymalla et al., 1996; Schroda et al., 1999; Yokthongwattana et al., 2001).

Evidence suggested that a molecular chaperone, the Hsp70B protein, might play a critical role in the PS II damage and repair process. A full-length cDNA of the D. salina Hsp70B gene was cloned and sequenced (GenBank Accession No. AF420430/AJ271605). Expression patterns of the Hsp70B gene were investigated upon shifting a D. salina culture from low-light to high-light-growth conditions, designed to significantly accelerate the rate of PS II photodamage. Northern blot analyses and nuclear run-on transcription assays revealed a prompt and substantial irradiance-dependent induction of Hsp70B gene transcription, followed by a subsequent increase in Hsp70B protein synthesis and accumulation. Mild detergent solubilization of pho-toinhibited thylakoid membranes, in which photodam-aged PS II centers had accumulated, followed by non-denaturing gel electrophoresis revealed formation of a 320 kD native protein complex that contained, in addition to the Hsp70B, the photodamaged but as yet undegraded D1 protein as well as D2 and CP47. Evidence suggested that the 320 kD complex is a transiently forming PS II repair intermediate. Denaturing solubilization of the 320 kD PS II repair intermediate by SDS-urea resulted in cross-linking of its constituent polypeptides, yielding a 160 kD protein complex. It was postulated that the Hsp70B protein plays a pivotal role in the repair process, e.g. in stabilizing the disassembled PS II-core complex and in facilitating the D1 removal and replacement (Fig. 5; see also Yokthongwattana et al., 2001).

Thus, the PS II damage and repair cycle lends itself to studies on the regulation of gene expression. Relevant and valid questions in this area range from signal (photodamage) perception to organelle-nucleus communication and coordination of gene expression for the repair.

F. Role of Zeaxanthin and of the Cbr Protein in the PS II Damage and Repair Process

The Cbr protein is a green alga homologue to the higher plant ELIP proteins (Banet et al., 2000), which are related to light stress (Adamska et al., 1992). In the

Fig. 5. A schematic model depicting the interaction of the HSP70B with the disassembled PS II-core complex prior to the degradation and replacement of the photodamaged D1 reaction center protein. This association results in the formation of a PS II repair intermediate.

Photosystem II Repair Intermediate

Fig. 5. A schematic model depicting the interaction of the HSP70B with the disassembled PS II-core complex prior to the degradation and replacement of the photodamaged D1 reaction center protein. This association results in the formation of a PS II repair intermediate.

model green alga Dunaliella salina, synthesis of Cbr is induced by light stress and occurs in parallel with the accumulation of zeaxanthin (Levy et al., 1993; Andreasson and Melis, 1995; Jin et al., 2001, 2003). Both Cbr and zeaxanthin appear to be reversibly associated with PS II, in which zeaxanthin acts as a quencher of excited Chl*molecules (Frank et al., 2000; Baroli and Niyogi, 2000; Maetal., 2003), thereby contributing to photoprotection. Recent work suggested a role for Cbr-zeaxanthin in the PS II repair process. This insight came as a result of irradiance-dependent studies, first with a photoinhibition-sensitive mutant of Dunaliella salina (denoted as dcd1; Jin et al., 2001). Photoinhibition in the dcd1 was manifested by a lowering of the Fv/Fm ratio, inhibition in QA photoreduction, and accumulation of zeaxanthin and of the 320 kD protein complex in the thylakoid membranes, onset of which occurred at a lower threshold of irradiance than in the wild type. In addition to these accepted markers of photoinhibition, de-epoxidation of the xanthophyll cycle carotenoids, accumulation of zeaxanthin and enhanced levels of the Cbr protein were observed. Although the onset of these changes occurred at different levels of irradiance for the wild type and for the dcd1 mutant, there appeared to be a strict correlation between xanthophyll de-epoxidation, amount of Cbr protein, and amount of photodamaged PS II centers. The notion of a relationship between PS II repair and Cbr-zeaxanthin was further strengthened in kinetic studies. These showed that zeaxanthin and the Cbr protein accumulate in parallel with the accumulation of photodamaged PS II centers following a LL ^ HL shift, and decay in tandem with a chloroplast recovery from photoinhibition (Jin et al., 2001, 2003).

Experimental evidence for the accumulation of zea-xanthin during photodamage, and possibly due to photodamage, was first presented by Trebst and cowork-ers (Depka et al., 1998). This body of evidence has been steadily growing in the literature (Smith et al., 1990; Baroli and Melis, 1996; Jahns and Miehe, 1996; Demmig-Adams et al., 1998; Xu et al., 1999; Jahns et al., 2000; Jin et al., 2001, 2003). Diverse observations, which cover both higher plants and green algae, raised the possibility that zeaxanthin and the Cbr protein accumulate not in response to irradiance per se but in proportion to photoinhibition. It was hypothesized that zeaxanthin and the Cbr protein might play a role in the protection of photodamaged and disassembled PS II reaction centers, apparently needed while PS II is in the process of degradation and replacement of the D1/32 kD reaction center protein (Jin et al., 2001, 2003). This notion is consistent with the recovery of pea chloroplasts from photoinhibition in which the kinetics of zeaxanthin epoxidation to vio-laxanthin resembled those of D1 degradation and replacement (Jahns and Miehe, 1996). The notion is also consistent with a study of an obligate shade species in which the de-epoxidation state of the xanthophyll-cycle carotenoids remained directly proportional to the level of photoinhibition in the leaves and independent of the light-intensity seen by the plant (Demmig-Adams et al., 1998). Further in this direction, of interest is the developing story of the underlying biochemistry in overwintering plant species, in which there appears to be interplay between photoprotection, zeaxanthin accumulation, and status of the PS II damage and repair cycle (Adams et al., 2002, 2004).

G. Sulfur-Deprivation Arrests the PS II Repair Process

In the absence of a sufficient supply of sulfur to the chloroplast, which is an essential component of cys-teine and methionine (Hell, 1997), D1 protein biosynthesis is impeded and the repair cycle is arrested in the PS II QB-nonreducing configuration (Wykoff et al., 1998). In consequence, the rate of photosynthesis declines quasi-exponentially in the light as a function of time in S-deprivation with a half time of about 18 h (Wykoff et al., 1998; Melis et al., 2000; Cao et al., 2001). This effect is specific to PS II in the thylakoid membrane. Thus, the supply of inorganic sulfur to the chloroplast may determine the rate of D1 turnover and may thus represent a significant regulatory step in the PS II repair process.

H. A Novel Nuclear-Encoded and Chloroplast-targeted Sulfate Permease Regulates the PS II Repair Process in Chlamydomonas reinhardtii

Genomic, proteomic, phylogenetic, and evolutionary aspects of a novel gene encoding a putative chloroplast-targeted sulfate permease of prokaryotic origin in the greenalga Chlamydomonas reinhardtii were described. This nuclear-encoded sulfate permease gene (SulP) contained four introns and five exons, whereas all other known chloroplast sulfate permease genes lack in-trons and are encoded by the chloroplast genome. The deduced amino acid sequence of the protein showed an extended N-terminus, which includes a putative chloroplast transit peptide. The mature protein contained 7 transmembrane domains and two large hy-drophilic loops (Fig. 6). This novel prokaryotic-origin

CpTP

Cp Stroma

Fig. 6. Folding-model of the nuclear-encoded and chloroplast-targeted Chlamydomonas reinhardtii SulP protein. CpTP refers to the chloroplast transit peptide prior to cleavage by a stroma-localized peptidase. TM1 represents the first N-terminal transmembrane domain of the SulP protein, which is exclusive to C. reinhardtii. The other six conserved transmembrane domains of green alga chloroplast sulfate permease are shown as A through F. Note the two extended hydrophilic loops, occurring between transmembrane helices TM1-A and D-E, facing toward the exterior of the chloroplast. (From Chen et al., 2003.)

CpTP

Cp Stroma

Fig. 6. Folding-model of the nuclear-encoded and chloroplast-targeted Chlamydomonas reinhardtii SulP protein. CpTP refers to the chloroplast transit peptide prior to cleavage by a stroma-localized peptidase. TM1 represents the first N-terminal transmembrane domain of the SulP protein, which is exclusive to C. reinhardtii. The other six conserved transmembrane domains of green alga chloroplast sulfate permease are shown as A through F. Note the two extended hydrophilic loops, occurring between transmembrane helices TM1-A and D-E, facing toward the exterior of the chloroplast. (From Chen et al., 2003.)

Fig. 7. Southern blot analysis to visualize the number of independent plasmid insertions in a group of plasmid-transformed Chlamydomonas reinhardtii. The arrow shows the position of the endogenous inactive Arg7 gene. The other hybridization bands originate from the insertion and non-homologous recombination of plasmid DNA within the Chlamydomonas reinhardtii nuclear genome. An overall 2:1 single/double plasmid insertion ratio was found. Lane 1: rep53; Lane 2: rep55; Lane 3: rep27; Lane 4: rep66; Lane 5: rep16; Lane 6: rep18.

Fig. 7. Southern blot analysis to visualize the number of independent plasmid insertions in a group of plasmid-transformed Chlamydomonas reinhardtii. The arrow shows the position of the endogenous inactive Arg7 gene. The other hybridization bands originate from the insertion and non-homologous recombination of plasmid DNA within the Chlamydomonas reinhardtii nuclear genome. An overall 2:1 single/double plasmid insertion ratio was found. Lane 1: rep53; Lane 2: rep55; Lane 3: rep27; Lane 4: rep66; Lane 5: rep16; Lane 6: rep18.

gene probably migrated from the chloroplast to the nuclear genome during evolution of C. reinhardtii. The SulP gene, or any of its homologues, has not been retained in vascular plants, e.g. Arabidopsis thaliana, although it is encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). A comparative structural analysis and phylogenetic origin of chloro-plast sulfate permeases in a variety of species was presented (Chen and Melis, 2002; Chen et al., 2002, 2003). Preliminary evidence suggested a dependence of the D1/32 kD protein turnover rate on the rate of sulfate uptake by the chloroplast. Thus, the SulP gene may directly or indirectly regulate the PS II repair process.

V. DNA Insertional Mutagenesis for the Isolation and Functional Characterization of PS II Repair Aberrant Mutants

Currently, DNA insertional mutagenesis appears to be the method of choice in efforts to unlock the "black box" of the PS II repair process. The successful isolation of many repair mutants will permit the identification and study of the respective genes and proteins, ultimately opening the way to a full elucidation of the repair process. A review of this technology and its application in this research is given below:

Mutagenesis and Screening Procedures: Chlamydomonas reinhardtii mutants are generated by transformation of an arginine auxotroph strain (CC-425) with plasmid DNA containing the complementing argini-nosuccinate lyase (arg+) gene (Gumpel and Purton, 1994; Davies et al., 1994, 1996). The integration of the transformant DNA occurs almost exclusively by nonhomologous recombination (Kindle, 1990; Tam and Lefebvre, 1993). Thus, transformants carrying integrated DNA at random locations in the Chlamy-domonas nuclear genome are generated (Fig. 7). The following screening procedure is suitable and has been successfully employed to isolate PS II repair mutants:

• Chlamydomonas reinhardtii Arg7 transformants are grown on TAP plates (medium lacking argi-nine).

• Replica plating on media lacking acetate is performed to identify and isolate acetate-requiring transformants. The latter are expected to include PS II repair mutants since they cannot grow photo-autotrophically. This is an important initial screening step, as it eliminates about 90% of the Arg7 transformants.

• Each acetate-requiring transformant is grown (in the presence of acetate) separately under low-light (LL: 10 ^mol photons m-2 s-1) or mediumlight (ML: 150 ^mol photons m-2 s-1) conditions. The steady-state amount of functional PS II is measured in such replica colonies. In repair-aberrant C. reinhardtii, the steady-state amount of functional PS II depends solely on the relationship between the rate of PS II biogenesis and photodamage. At 10 ^mol photons m-2 s-1, the rate of photodamage (about once every 48 h) is slower than the rate of de novo PS II biosynthesis (cell duplication time —24 h), thus permitting accumulation and detection of functional PS II centers in the chloroplast thylakoids. At 150 ^mol photons m-2 s-1, the rate ofphotodamage (once every — 5 h) is faster than the rate of de novo PS II biosynthesis (cell duplication time of — 18 h), thus causing a nearly quantitative accumulation ofphotodamaged PS II centers in the chloroplast thylakoids.

• Chlorophyll fluorescence transient analysis of the LL and ML-grown mutants is applied to measure the activity of PS II from the yield of the nonvariable (Fo), variable (Fv), and maximum (Fm) fluorescence emission (Guenther et al., 1990). Repair mutants are selected on the basis of differential fluorescence phenotype under LL versus ML conditions, as follows. LL-grown repair mutants display both Fo and Fv (Zhang et al., 1997), indicating the presence and functional integrity of PS II in the chloroplast thylakoids. ML-grown repair mutants display Fo but are mostly or entirely devoid of Fv. This irradiance-induced difference in the fluorescence induction characteristics indicates that chloroplasts synthesize, assemble, and retain functional D1 under low-light conditions (when the rate of photodamage is very slow) but could not repair photodamaged D1 under moderate light intensities.

• Transformants that meet the above criteria are isolated. Their functional and repair properties are investigated further by absorbance difference spectrophotometry, SDS-PAGE, Western blot analysis, and (35S) sulfate pulse-chase labeling (Melis, 1989; Vasilikiotis and Melis, 1994; Zhang et al., 1997; Melis, 1999), leading to the isolation of repair-aberrant mutants.

In summary, DNA insertional mutagenesis efforts for the generation, isolation, and characterization of PS II repair aberrant mutants is a useful tool in the discovery of additional genes and proteins that are involved in the PS II repair process. Identification of such genes will permit a subsequent study of their functional and regulatory properties and will thus advance knowledge about the PS II repair process. This experimental approach, when carefully and persistently implemented, may thus contribute to the complete elucidation of the PS II repair process.

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