Seiko Ishihara

PsbP Protein, But Not PsbQ Protein, Is Essential for the Regulation and Stabilization of Photosystem II in Higher Plants

PsbP and PsbQ proteins are extrinsic subunits of photosystem II (PSII) and participate in the normal function of photosynthetic water oxidation. Both proteins exist in a broad range of the oxygenic photosynthetic organisms; however, their physiological roles in vivo have not been well defined in higher plants. In this study, we established and analyzed transgenic tobacco (Nicotiana tabacum) plants in which the levels of PsbP or PsbQ were severely down-regulated by the RNA interference technique. A plant that lacked PsbQ showed no specific phenotype compared to a wild-type plant. This suggests that PsbQ in higher plants is dispensable under the normal growth condition. On the other hand, a plant that lacked PsbP showed prominent phenotypes: drastic retardation of growth, pale-green-colored leaves, and a marked decrease in the quantum yield of PSII evaluated by chlorophyll fluorescence. In PsbP-deficient plant, most PSII core subunits were accumulated in thylakoids, whereas PsbQ, which requires PsbP to bind PSII in vitro, was dramatically decreased. PSII without PsbP was hypersensitive to light and rapidly inactivated when the repair process of the damaged PSII was inhibited by chloramphenicol. Furthermore, thermoluminescence studies showed that the catalytic manganese cluster in PsbP-deficient leaves was markedly unstable and readily disassembled in the dark. The present results demonstrated that PsbP, but not PsbQ, is indispensable for the normal PSII function in higher plants in vivo.

Distinct Functions for the Two PsbP-Like Proteins PPL1 and PPL2 in the Chloroplast Thylakoid Lumen of Arabidopsis

PsbP, an extrinsic subunit of photosystem II (PSII), is a nuclear-encoded protein that optimizes the water-splitting reaction in vivo. In addition to PsbP, higher plants have two nuclear-encoded genes for PsbP homologs (PsbP-like proteins [PPLs]) that show significant sequence similarity to a cyanobacterial PsbP homolog (cyanoP); however, the function of PPLs in higher plants has not yet been elucidated. In this study, we characterized Arabidopsis (Arabidopsis thaliana) mutants lacking either of two PPLs, PPL1 and PPL2. Phylogenetic analysis suggests that PPL1 would be an ortholog of cyanoP, and PPL2 and PsbP may have a paralogous relationship with PPL1. Analysis on mRNA expression profiles showed that PPL1 expressed under stress conditions and PPL2 coexpressed with the subunits of chloroplast NAD(P)H dehydrogenase (NDH) complex. Consistent with these suggestions, PSII activity in a ppl1 mutant was more sensitive to high-intensity light than wild type, and the recovery of photoinhibited PSII activity was delayed in ppl1 plants. Therefore, PPL1 is required for efficient repair of photodamaged PSII. Furthermore, the stoichiometric level and activity of the chloroplast NDH complex in thylakoids were severely decreased in a ppl2 mutant, demonstrating that PPL2 is a novel thylakoid lumenal factor required for accumulation of the chloroplast NDH complex. These results suggest that during endosymbiosis and subsequent gene transfer to the host nucleus, cyanoP from ancient cyanobacteria evolved into PPL1, PPL2, and PsbP, and each of them has a distinct role in photosynthetic electron transfer in Arabidopsis.

Molecular Functions of Oxygen-Evolving Complex Family Proteins in Photosynthetic Electron Flow

Oxygen-evolving complex (OEC) protein is the original name for membrane-peripheral subunits of photosystem (PS) II. Recently, multiple isoforms and homologs for OEC proteins have been identified in the chloroplast thylakoid lumen, indicating that functional diversification has occurred in the OEC family. Gene expression profiles suggest that the Arabidopsis OEC proteins are roughly categorized into three groups: the authentic OEC group, the stress-responsive group, and the group including proteins related to the chloroplast NAD(P)H dehydrogenase (NDH) complex involved in cyclic electron transport around PSI. Based on the above gene expression profiles, molecular functions of the OEC family proteins are discussed together with our current knowledge about their functions.

Knockdown of the PsbP protein does not prevent assembly of the dimeric PSII core complex but impairs accumulation of photosystem II supercomplexes in tobacco

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically found in land plants and green algae. Using PsbP-RNAi tobacco, we have investigated effects of PsbP knockdown on protein supercomplex organization within the thylakoid membranes and photosynthetic properties of PSII. In PsbP-RNAi leaves, PSII dimers binding the extrinsic PsbO protein could be formed, while the light-harvesting complex II (LHCII)-PSII supercomplexes were severely decreased. Furthermore, LHCII and major PSII subunits were significantly dephosphorylated. Electron microscopic analysis showed that thylakoid grana stacking in PsbP-RNAi chloroplast was largely disordered and appeared similar to the stromally-exposed or marginal regions of wild-type thylakoids. Knockdown of PsbP modified both the donor and acceptor sides of PSII; In addition to the lower water-splitting activity, the primary quinone Q(A) in PSII was significantly reduced even when the photosystem I reaction center (P700) was noticeably oxidized, and thermoluminescence studies suggested the stabilization of the charged pair, S(2)/Q(A)(-). These data indicate that assembly and/or maintenance of the functional MnCa cluster is perturbed in absence of PsbP, which impairs accumulation of final active forms of PSII supercomplexes.

Three PsbQ-Like Proteins are Required for the Function of the Chloroplast NAD(P)H Dehydrogenase Complex in Arabidopsis

Arabidopsis has three PsbQ-like (PQL) proteins in addition to the PsbQ subunit of the oxygen-evolving complex of PSII. Recent bioinformatic and proteomic studies suggested that the two PQL proteins, PQL1 (At1g14150) and PQL2 (At3g01440), might function in the chloroplast NAD(P)H dehydrogenase (NDH) complex; however, their molecular function has not been characterized. In this study, we examined the function of the chloroplast NDH in the Arabidopsis pql1 and pql2 mutants. Post-illumination increases in Chl fluorescence, which are caused by an NDH-dependent cyclic electron flow, were absent in both mutants, indicating that PQL1 and PQL2 are required for NDH activity. In the thylakoid membranes of wild-type plants, PQL1 and PQL2 were tightly associated with the NDH-PSI supercomplex and protected from protease treatments, while unassembled PQLs were not stably accumulated in mutants lacking known NDH subunits. Subunit stability of the NDH complex was affected differently in the thylakoid membranes of the pql1 and pql2 mutants. These data indicate that PQL1 and PQL2 are novel NDH subunits and differ in their functional roles and in their binding sites in the NDH complex. Furthermore, functional analysis on PQL3 (At2g01918) using the pql3 mutant suggests that PQL3 is also required for NDH activity. Proteins homologous to each PQL protein are found in various plant species, but not in cyanobacteria, algae, mosses or ferns. These results suggest that seed plants that have NDH activity in chloroplasts specifically developed three PQL proteins for the function of the chloroplast NDH complex.

Structure and function of the PsbP protein of Photosystem II from higher plants

PsbP is a membrane extrinsic subunit of Photosystem II (PS II), which is involved in retaining Ca2+ and Cl−, two inorganic cofactors for the water-splitting reaction. In this study, we re-investigated the role of N-terminal region of PsbP on the basis of its three-dimensional structure. In previous paper [Ifuku and Sato (2002) Plant Cell Physiol 43: 1244–1249], a truncated PsbP lacking 19 N-terminal residues (Δ19) was found to bind to NaCl-washed PS II lacking PsbP and PsbQ without activation of oxygen evolution at all. Three-dimensional (3D) structure of PsbP suggests that deletion of 19 N-terminal residues would destabilize its protein structure, as indicated by the high sensitivity of Δ19 to trypsin digestion. Thus, a truncated PsbP lacking 15 N-terminal residues (Δ15), which retained core PsbP structure, was produced. Whereas Δ15 was resistant to trypsin digestion and bound to NaCl-washed PS II membranes, it did not show the activation of oxygen evolution. This result indicated that the interaction of 15-residue N-terminal flexible region of PsbP with PS II was important for Ca2+ and Cl− retention in PS II, although the 15 N-terminal residues were not essential for the binding of PsbP to PS II. The possible N-terminal residues of PsbP that would be involved in this interaction are discussed.

Functional Analysis of Two PsbP-Like (PPL) Proteins in Arabidopsis thaliana

PsbP is an extrinsic subunit of photosystem II (PSII) and optimizes the water- splitting reaction in higher plants in vivo. In addition to PsbP, recent genomic and proteomic studies have demonstrated the existence of many PsbP homologs in the chloroplast of higher plants; however, their molecular functions have not been investigated at all. In order to establish a unified view about the PsbP family in higher plants, PsbP homologs in a model plant Arabidopsis thaliana were classified into two PsbP-like (PPL) proteins and six PsbP domain (PPD) proteins by amino-acid sequence similarity. Their molecular functions, especially those of two PPLs, are discussed in terms of mRNA expression profiles in the public microarray database.

Functional Analysis of PsbP-Like Protein 1 (PPL1) in Arabidopsis

Higher plants have a number of PsbP homologs (PsbP-like proteins: PPLs, PsbP-domain proteins: PPDs) in addition to the authentic PsbP in the oxygen-evolving complex of photosystem II (PSII). Among the PsbP homologs, the PPL1 protein is most homologous to a cyanobacterial PsbP (cyanoP), and we previously reported that PPL1 is required for the efficient repair of photo-damaged PSII under high light conditions [Ishihara et al. (2007) Plant Physiol. 145: 668-679]. However, functional role of PPL1 in the PSII repair cycle has not been clarified yet. In this study, we further investigated molecular function of PPL1 by characterizing the phenotypes of the PPL1 knockdown plants (ppl1i) in which PPL1 expressions were differently suppressed. Although growth of the ppl1i mutants under low intensity light was comparable with that of wild type plants, PSII activity of the ppl1i mutants was more sensitive to high intensity light and the extent of photoinhibition was correlated with the levels of the knocked-down PPL1. The possible functional role of PPL1 in PSII repair is discussed.

Effects of PsbP Knockdown on the Photosynthetic Electron Transfer in Nicotiana tabacum

Transgenic tobacco plants deficient in PsbP (ΔPsbP), one of the extrinsic proteins of photosystem II (PSII), were characterized with respect to thylakoid functional properties. The ΔPsbP plants showed low PSII activity and the inhibition was located at the Mn-cluster in PSII. In addition, chlorophyll fluorescence and spectroscopic measurements indicated that the forward electron flow from QA - to plastoquinone was hindered in ΔPsbP leaves; significant accumulation of QA - was observed, while P700 in photosystem I (PSI) was noticeably oxidized. Consistent with this observation, thermoluminescence studies suggested the stabilization of the redox pair of S2QA - in ΔPsbP leaves. We speculate that these properties of PSII in ΔPsbP leaves resemble an intermediate state of PSII biogenesis, which is almost inactive but relatively stable in thylakoid membranes. The function of PsbP is discussed in terms of the dynamic life cycle of PSII.