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.     

Specific RNA interference in psbP genes encoded by a multigene family in Nicotiana tabacum with a short 3'-untranslated sequence

RNA interference with double-stranded RNA is a new method for the study of gene function in various organisms. In this report, we show that an inverted repeat of a short (103-bp) 3'-untranslated sequence of an isogene, 1A, of psbP genes, encoded by a small multigene family of four genes (1A, 2AF, 3F, and 5B) in Nicotiana tabacum, can specifically suppress the expression of psbP isogenes 1A and 5B with a 3'-untranslated sequence similar to a transcribed double-stranded RNA. The expression of other psbP isogenes, 2AF and 3F, was not affected, although the coding sequences of the psbP family genes are highly conserved. Consistent with this observation, small interfering RNAs were detected for the 3'-untranslated sequence used for the inverted-repeat transgene, and not for the coding sequence. These results suggest that double-stranded RNA having a 3'-untranslated sequence could be useful for an isogene-specific RNA interference of the family genes in Nicotiana tabacum.     

Molecular evolutionary analysis of the psbP gene family of the photosystem II oxygen-evolving complex in Nicotiana.

Photosystem II psbP protein of the oxygen-evolving complex is involved in the photosynthetic oxygen evolution in plants. Four psbP polypeptides were detected in Nicotiana tabacum on a two-dimensional gel by immunostaining the proteins with antiserum against the pea psbP Comparison of the protein patterns of psbP from N. tabacum and its ancestral parents, N. sylvestris and N. tomentosiformis, indicated that each of the ancestral parents has contributed a pair of psbP proteins. This was supported by Southern hybridization results, which suggested that psbP in Nicotiana is encoded by a gene family consisting of four members in N. tabacum and two members each in N. glauca, N. langsdorffii, N. sylvestris, and N. tomentosiformis. A scheme of molecular evolution of the psbP genes in Nicotiana is also proposed.     

Homologs of plant PsbP and PsbQ proteins are necessary for regulation of photosystem ii activity in the cyanobacterium Synechocystis 6803

The mechanism of oxygen evolution by photosystem II (PSII) has remained highly conserved during the course of evolution from ancestral cyanobacteria to green plants. A cluster of manganese, calcium, and chloride ions, whose binding environment is optimized by PSII extrinsic proteins, catalyzes this water-splitting reaction. The accepted view is that in plants and green algae, the three extrinsic proteins are PsbO, PsbP, and PsbQ, whereas in cyanobacteria, they are PsbO, PsbV, and PsbU. Our previous proteomic analysis established the presence of a PsbQ homolog in the cyanobacterium Synechocystis 6803. The current study additionally demonstrates the presence of a PsbP homolog in cyanobacterial PSII. Both psbP and psbQ inactivation mutants exhibited reduced photoautotrophic growth as well as decreased water oxidation activity under CaCl(2)-depleted conditions. Moreover, purified PSII complexes from each mutant had significantly reduced activity. In cyanobacteria, one PsbQ is present per PSII complex, whereas PsbP is significantly substoichiometric. These findings indicate that both PsbP and PsbQ proteins are regulators that are necessary for the biogenesis of optimally active PSII in Synechocystis 6803. The new picture emerging from these data is that five extrinsic PSII proteins, PsbO, PsbP, PsbQ, PsbU, and PsbV, are present in cyanobacteria, two of which, PsbU and PsbV, have been lost during the evolution of green plants.     

Structure, function, and evolution of the PsbP protein family in higher plants

The PsbP is a thylakoid lumenal subunit of photosystem II (PSII), which has developed specifically in higher plants and green algae. In higher plants, the molecular function of PsbP has been intensively investigated by release-reconstitution experiments in vitro. Recently, solution of a high-resolution structure of PsbP has enabled investigation of structure-function relationships, and efficient gene-silencing techniques have demonstrated the crucial role of PsbP in PSII activity in vivo. Furthermore, genomic and proteomic studies have shown that PsbP belongs to the divergent PsbP protein family, which consists of about 10 members in model plants such as Arabidopsis and rice. Characterization of the molecular function of PsbP homologs using Arabidopsis mutants suggests that each plays a distinct and important function in maintaining photosynthetic electron transfer. In this review, recent findings regarding the molecular functions of PsbP and other PsbP homologs in higher plants are summarized, and the molecular evolution of these proteins is discussed.     

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.     

The Tobacco mosaic virus 126-kDa protein associated with virus replication and movement suppresses RNA silencing

Systemic symptoms induced on Nicotiana tabacum cv. Xanthi by Tobacco mosaic virus (TMV) are modulated by one or both amino-coterminal viral 126- and 183-kDa proteins: proteins involved in virus replication and cell-to-cell movement. Here we compare the systemic accumulation and gene silencing characteristics of TMV strains and mutants that express altered 126- and 183-kDa proteins and induce varying intensities of systemic symptoms on N. tabacum. Through grafting experiments, it was determined that M(IC)1,3, a mutant of the masked strain of TMV that accumulated locally and induced no systemic symptoms, moved through vascular tissue but failed to accumulate to high levels in systemic leaves. The lack of M(IC)1,3 accumulation in systemic leaves was correlated with RNA silencing activity in this tissue through the appearance of virus-specific, approximately 25-nucleotide RNAs and the loss of fluorescence from leaves of transgenic plants expressing the 126-kDa protein fused with green fluorescent protein (GFP). The ability of TMV strains and mutants altered in the 126-kDa protein open reading frame to cause systemic symptoms was positively correlated with their ability to transiently extend expression of the 126-kDa protein:GFP fusion and transiently suppress the silencing of free GFP in transgenic N. tabacum and transgenic N. benthamiana, respectively. Suppression of GFP silencing in N. benthamiana occurred only where virus accumulated to high levels. Using agroinfiltration assays, it was determined that the 126-kDa protein alone could delay GFP silencing. Based on these results and the known synergies between TMV and other viruses, the mechanism of suppression by the 126-kDa protein is compared with those utilized by other originally characterized suppressors of RNA silencing.     

In vivo system for analyzing the function of the PsbP protein using <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The PsbP protein is an extrinsic subunit of photosystem II (PSII) specifically developed in green-plant species including land plants and green algae. The protein–protein interactions involving PsbP and its effect on oxygen evolution have been investigated in vitro using isolated PSII membranes. However, the importance of those interactions needs to be examined at the cellular level. To this end, we developed a system expressing exogenous PsbP in the background of the Chlamydomonas BF25 mutant lacking native PsbP. Expression of His-tagged PsbP successfully restored the oxygen-evolving activity and photoautotrophic growth of the mutant, while PsbP-?15 lacking the N-terminal 15 residues, which are crucial for the oxygen-evolving activity of spinach PSII in vitro, only partially did. This demonstrated the importance of N-terminal sequence of PsbP for the photosynthetic activity in vivo. Furthermore, the PSII–LHCII supercomplex can be specifically purified from the Chlamydomonas cells having His-tagged PsbP using a metal affinity chromatography. This study provides a platform not only for the functional analysis of PsbP in vivo but also for structural analysis of the PSII–LHCII supercomplex from green algae.     

Protein assembly of photosystem II and accumulation of subcomplexes in the absence of low molecular mass subunits PsbL and PsbJ.

The protein assembly and stability of photosystem II (PSII) (sub)complexes were studied in mature leaves of four plastid mutants of tobacco (Nicotiana tabacum L), each having one of the psbEFLJ operon genes inactivated. In the absence of psbL, no PSII core dimers or PSII-light harvesting complex (LHCII) supercomplexes were formed, and the assembly of CP43 into PSII core monomers was extremely labile. The assembly of CP43 into PSII core monomers was found to be necessary for the assembly of PsbO on the lumenal side of PSII. The two other oxygen-evolving complex (OEC) proteins, PsbP and PsbQ, were completely lacking in Delta psbL. In the absence of psbJ, both intact PSII core monomers and PSII core dimers harboring the PsbO protein were formed, whereas the LHCII antenna remained detached from the PSII dimers, as demonstrated by 77 K fluorescence measurements and by the lack of PSII-LHCII supercomplexes. The Delta psbJ mutant was characterized by a deficiency of PsbQ and a complete lack of PsbP. Thus, both the PsbL and PsbJ subunits of PSII are essential for proper assembly of the OEC. The absence of psbE and psbF resulted in a complete absence of all central PSII core and OEC proteins. In contrast, very young, vigorously expanding leaves of all psbEFLJ operon mutants accumulated at least traces of D2, CP43 and the OEC proteins PsbO and PsbQ, implying developmental control of the expression of the PSII core and OEC proteins. Despite severe problems in PSII assembly, the thylakoid membrane complexes other than PSII were present and correctly assembled in all psbEFLJ operon mutants.     

Manganese binding to the 23 kDa extrinsic protein of Photosystem II

The recombinant form of the extrinsic 23 kDa protein (psbP) of Photosystem II (PSII) was studied with respect to its capability to bind Mn. The stoichiometry was determined to be one manganese bound per protein. A very high binding constant, K(A)=10(-17) M(-1), was determined by dialysis of the Mn containing protein against increasing EDTA concentration. High Field EPR spectroscopy was used to distinguish between specific symmetrically ligated Mn(II) from those non-specifically Mn(II) attached to the protein surface. Upon Mn binding PsbP exhibited fluorescence emission with maxima at 415 and 435 nm when tryptophan residues were excited. The yield of this blue fluorescence was variable from sample to sample. It was likely that different conformational states of the protein were responsible for this variability. The importance of Mn binding to PsbP in the context of photoactivation of PSII is discussed.     

Post-transcriptional gene silencing in the root system of the actinorhizal tree Allocasuarina verticillata

In recent years, RNA interference has been exploited as a tool for investigating gene function in plants. We tested the potential of double-stranded RNA interference technology for silencing a transgene in the actinorhizal tree Allocasuarina verticillata. The approach was undertaken using stably transformed shoots expressing the beta-glucuronidase (GUS) gene under the control of the constitutive promoter 35S; the shoots were further transformed with the Agrobacterium rhizogenes A4RS containing hairpin RNA (hpRNA) directed toward the GUS gene, and driven by the 35S promoter. The silencing and control vectors contained the reporter gene of the green fluorescent protein (GFP), thus allowing a screening of GUS-silenced composite plantlets for autofluorescence. With this rapid procedure, histochemical data established that the reporter gene was strongly silenced in both fluorescent roots and actinorhizal nodules. Fluorometric data further established that the level of GUS silencing was usually greater than 90% in the hairy roots containing the hairpin GUS sequences. We found that the silencing process of the reporter gene did not spread to the aerial part of the composite A. verticillata plants. Real-time quantitative polymerase chain reaction showed that GUS mRNAs were substantially reduced in roots and, thereby, confirmed the knock-down of the GUS transgene in the GFP(+) hairy roots. The approach described here will provide a versatile tool for the rapid assessment of symbiotically related host genes in actinorhizal plants of the Casuarinaceae family.     

Cross-linking Evidence for Multiple Interactions of the PsbP and PsbQ Proteins in a Higher Plant Photosystem II Supercomplex

The extrinsic subunits of membrane-bound photosystem II (PSII) maintain an essential role in optimizing the water-splitting reaction of the oxygen-evolving complex (OEC), even though they have undergone drastic change during the evolution of oxyphototrophs from symbiotic cyanobacteria to chloroplasts. Two specific extrinsic proteins, PsbP and PsbQ, bind to the lumenal surface of PSII in green plants and maintain OEC conformation and stabilize overall enzymatic function; however, their precise location has not been fully resolved. In this study, PSII-enriched membranes, isolated from spinach, were subjected to chemical cross-linking combined with release-reconstitution experiments. We observed direct interactions between PsbP and PsbE, as well as with PsbR. Intriguingly, PsbP and PsbQ were further linked to the CP26 and CP43 light-harvesting proteins. In addition, two cross-linked sites, between PsbP and PsbR, and that of PsbP and CP26, were identified by tandem mass spectrometry. These data were used to estimate the binding topology and location of PsbP, and the putative positioning of PsbQ and PsbR on the lumenal surface of the PSII. Our model gives new insights into the organization of PSII extrinsic subunits in higher plants and their function in stabilizing the OEC of the PSII supercomplex.     

RNA干涉在纤毛虫中的研究进展

RNA干涉是dsRNA介导的基因沉默现象,本文简要介绍了其作用的机制和生物学意义,重点阐述了RNA干涉在原生动物纤毛虫中的发现与应用,比较了RNA干涉与纤毛虫大核基因组重排机理的异同,并对RNA干涉在纤毛虫中传输的技术途径-RNAi喂饲法的原理也做了详细的介绍。