The mechanism of the degradation of psaB gene product,one of the photosynthetic reaction center subunits of Photosystem I,upon photoinhibition

The psaB gene product (PsaB protein), one of the reaction center subunits of Photosystem I (PS I), was specifically degraded by light illumination of spinach thylakoid membranes. The degradation of the protein yielded N-terminal fragments of molecular mass 51 kDa and 45 kDa. The formation of the 51 kDa fragment was i) partially suppressed by the addition of phenylmethylsulfonyl fluoride or 3,4-dichloroisocoumarin, which are inhibitors of serine proteases, and ii) enhanced in the presence of hydrogen peroxide during photoinhibitory treatment, but iii) not detected following hydrogen peroxide treatment in the dark. These results suggest that the hydroxyl radical produced at the reduced iron-sulfur centers in PS I triggers the conformational change of the PS I complex, which allows access of a serine-type protease to PsaB. This results in the formation of the 51 kDa N-terminal fragment, presumably by cleavage on the loop exposed to the stromal side, between putative helices 8 and 9. On the other hand, the formation of the 45 kDa fragment, which was enhanced in the presence of methyl viologen but did not accompany the photoinhibition of PS I, was not affected by the addition of hydrogen peroxide or protease inhibitors. Another fragment of 18 kDa was identified as a C-terminal counterpart of the 45 kDa fragment. N-terminal sequence analysis of the 18 kDa fragment revealed that the cleavage occurred between Ala⁵⁰⁰ and Val⁵⁰¹ on the loop exposed to the lumenal side, between putative helices 7 and 8 of the PsaB protein.     

Presence of the CO2-concentrating mechanism in some species of the pyrenoid-less free-living algal genus Chloromonas (Volvocales, Chlorophyta)

Physiological and morphological characteristics related to the CO₂-concentrating mechanism (CCM) were examined in several species of the free-living, unicellular volvocalean genus Chloromonas (Chlorophyta), which differs morphologically from the genus Chlamydomonas only by lacking pyrenoids. The absence of pyrenoids in the chloroplasts of Chloromonas (Cr.) rosae UTEX 1337, Cr. serbinowii UTEX 492, Cr.␣clatharata UTEX 1970, Cr. rosae SAG 26.90, and Cr. palmelloides SAG 32.86 was confirmed by light and electron microscopy. In addition, immunogold electron microscopy demonstrated that ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC 4.1.1.39) molecules were distributed almost evenly throughout the chloroplasts in all five Chloromonas strains. However, Chloromonas exhibited two types of physiological characteristics related to the CCM depending on the species or strains examined. Chloromonas rosae UTEX 1337 and Cr. serbinowii had high photosynthetic affinities for CO₂ in cells grown in culture medium bubbled with air (low-CO₂ cells), compared with those grown in medium bubbled with 5% CO₂ (high-CO₂ cells), indicating the presence of the low-CO₂-inducible CCM. In addition, these two Chloromonas strains exhibited low-CO₂-inducible carbonic anhydrase (CA; EC 4.2.1.1) activity and seemed to have small intracellular inorganic carbon pools. Therefore, it appears that Cr. rosae UTEX 1337 and Cr. serbinowii possess the CCM as in pyrenoid-containing microalgae such as Chlamydomonas reinhardtii. By contrast, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides showed low photosynthetic affinities for CO₂ when grown under both CO₂ conditions. Moreover, these three strains exhibited an apparent absence of intracellular inorganic carbon pools and lacked low-CO₂-inducible CA activity. Thus, Cr. clatharata, Cr. rosae SAG 26.90 and Cr. palmelloides, like other pyrenoid-less algae (lichen photobionts) reported previously, seem to lack the CCM. The present study is the first demonstration of the CCM in pyrenoid-less algae, indicating that pyrenoids or accumulation of Rubisco in the chloroplasts are not always essential for the CCM in algae. Focusing on this type of CCM in pyrenoid-less algae, the physiological and evolutionary significance of pyrenoid absence is discussed. Received: 1 May 1997 / Accepted: 11 September 1997     

Selective Photoinhibition of Photosystem I in Isolated Thylakoid Membranes from Cucumber and Spinach

The site of photoinhibition at low temperatures in leaves ofa chilling-sensitive plant, cucumber, is photosystem I [Terashimaet al. (1994) Planta 193: 300]. As described herein, selectivephotoinhibition of PSI can also be induced in isolated thylakoidmembranes in vitro. Inhibition was observed both at chillingtemperatures and at 25°C, and not only in the thylakoidmembranes isolated from cucumber, but also in those isolatedfrom a chilling-tolerant plant, spinach. Comparison of theseobservations in vitro to the earlier results in vivo indicatesthat (1) photoinhibition of PSI is a universal phenomenon; (2)a mechanism exists to protect PSI in vivo; and (3) the protectivemechanism is chilling-sensitive in cucumber. The chilling-sensitivecomponent seems to be lost during the isolation of thylakoidmembranes. Very weak light (10–20µmol m^-2 s^-1) wassufficient to cause the inhibition of PSI. About 80% of theoxygen-evolving activity by PSII was maintained even after theactivity of PSI had decreased by more than 70%. This is thefirst report of the selective photoinhibition of PSI in vitro. (Received March 1, 1995; Accepted April 26, 1995)     

Large-scale analysis of chlorophyll fluorescence kinetics in Synechocystis sp. PCC 6803: identification of the factors involved in the modulation of photosystem stoichiometry

Since chlorophyll fluorescence reflects the redox state of photosynthetic electron transport chain, monitoring of chlorophyll fluorescence has been successfully applied for the screening of photosynthesis-related genes. Here we report that the mutants having a defect in the regulation of photosystem stoichiometry could be identified through the simple comparison of the induction kinetics of chlorophyll fluorescence. We made a library containing 500 mutants in the cyanobacterium Synechocystis sp. PCC 6803 with transposon-mediated gene disruption, and the mutants were used for the measurement of chlorophyll fluorescence kinetics for 45 s. We picked up two genes, pmgA and sll1961, which are involved in the modulation of photosystem stoichiometry. The disruptants of the two genes share common characteristics in their fluorescence kinetics, and we searched for mutants that showed such characteristics. Out of six mutants identified so far, five showed a different photosystem stoichiometry under high-light conditions. Thus, categorization based on the similarity of fluorescence kinetics is an excellent way to identify the function of genes.     

Chloroplast NAD kinase is essential for energy transduction through the xanthophyll cycle in photosynthesis

Photosynthetic parameters of the nadk2 mutant of Arabidopsis thaliana, which is defective in chloroplast NAD kinase, were investigated. In this plant, the effective efficiency of photosynthetic electron transport (PhiII) and the quantum yield of open reaction centers of photosystem II (Fv'/Fm') were decreased. Furthermore, an increase in non-photochemical quenching attributed to energy dissipation from the xanthophyll cycle was observed. The mutant showed an aberrant de-epoxidation state of xanthophyll cycle carotenoids and had a high level of zeaxanthin even under low light conditions. These results indicate that chloroplast NAD kinase, catalyzing phosphorylation of NAD, is essential for the proper photosynthetic machinery of PSII and the xanthophyll cycle.     

Mechanism of photosystem-I photoinhibition in leaves ofCucumis sativus L.

It was recently shown that the site of photoinhibition in leaves of Cucumis sativus L. at low temperatures is Photosystem I (PSI), not PSII (I. Terashima et al. 1994, Planta 193, 300–306). In the present study, the mechanisms of this PSI photoinhibition in vivo were examined. By lowering the photon flux density during the photoinhibitory treatment of leaves at 4°C for 5 h to less than 100 mol·m^–2s^–1, we were able to separate the steps of the destruction of the electron-transfer components. Although P-700, the reaction-center chlorophyll, was almost intact in this low-light treatment, the quantum yield of the electron transfer through PSI and photochemically induced absorption change at 701 nm were markedly inhibited. This, along with the results from the measurements of the light-induced absorption changes in the presence of various concentrations of methyl viologen, an artificial electron acceptor, indicates that the component on the acceptor side of the PSI, A₁ or F_x, is the first site of inactivation. When the photon flux density during the treatment was increased to 220 mol·m^–2s^–1, the destruction of P-700 itself was also observed. Furthermore, the partial degradation of the chlorophyll-binding large subunits was observed in photoinhibited leaves. This degradation of the subunits was not detected when the treatment was carried out under nitrogen atmosphere, the condition in which the electron transfer is not inhibited. Thus, the photoinhibitory processes in the reaction center of PSI go through three steps, the inactivation of the acceptor side, the destruction of the reaction-center chlorophyll and the degradation of the reaction center subunit(s). The similarities and the differences between the mechanisms of PSI photoinhibition and those of PSII photoinhibition are discussed.Abbreviations DAD 2,3,5,6-tetramethyl-p-phenylenediamine - LHCI, LHCII light-harvesting chlorophyll-a/b proteins associating with photosystems I and II, respectively - PFD photon flux density We are grateful to Dr. I. Enami (Department of Biology, Faculty of Science, Science University of Tokyo) and Drs. H. Matsubara and H. Oh-oka (Department of Biology, Faculty of Science, Osaka University) for generous gifts of antisera used in the present work. We also thank A. Aoyama for technical assistance. This work was partly supported by the grants from the Ministry of Education, Science and Culture, Japan.     

The Inhibition of Photosynthesis after Exposure of Bean Leaves to Various Low Levels of CO2

Mature first leaves of Phaseolus vulgaris L. were exposed tolow partial pressures of CO₂ (7, 6 and 0 Pa CO₂) for 24 h. Afterexposure of leaves to 6 Pa CO₂ for 24 h, there was a reductionin the carbon exchange rate (CER) at all partial pressures ofCO₂ at which measurements were made. After exposure to 7 PaCO₂, the CER decreased only at high partial pressures of CO₂.The rates of electron transport from water to methyl viologen,through the whole chain, decreased in parallel with the decreasein CER measured at 90 Pa CO₂. One site of inhibition in leavesexposed for 24 h to 6 Pa CO₂ appeared to be the intersystemelectron-transport chain since there were no significant changesin the activities of PSI and PSII, as determined from the levelof P-700 and measurement of fluorescence, respectively. Anotherinhibitory phenomenon appeared to be a negative change in theactivation state of Rubisco, while the level of Rubisco wasunaffected by the exposure to 6 Pa CO₂. These decreases in photosyntheticactivity caused by depletion of CO₂ explains at least in part,the inhibition of photosynthesis that is caused by rain treatment[Ishibashi and Terashima (1995) Plant Cell Environ. 18: 431]. (Received September 19, 1996; Accepted March 10, 1997)     

Presence of an N-terminal presequence in the PsaI protein of the Photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413

The psaI gene encoding the 5.2 kDa protein component (PsaI) of the photosystem I complex was cloned from the cyanobacterium Anabaena 29413. The gene is present in single copy in this cyanobacterial genome. The nucleotide sequence of a 500 bp region of the cloned DNA revealed the presence of an open reading frame encoding a 46 amino acid long polypeptide. The N-terminal 11 residues are absent in the mature polypeptide and thus represents the first identified cleavable presequence on the PsaI protein. We suggest that this presequence directs the N-terminus of the protein to the thylakoid lumen.     

A Novel 3.5 kDa Protein Component of Cyanobacterial Photosystem I Complexes

A novel protein component of 3.5 kDa was detected in photosystemI complexes prepared from several cyanobacteria, viz. Synechococcusvulcanus, Synechococcus elongotus BP-1, Synechococcus sp. FCC7002 and Synechocystis sp. FCC 6803. The complete amino acidsequence of this component was determined by direct proteinsequencing. The sequences of the 3.5 kDa proteins from thesefour organisms were highly homologous to each other, featuringa hydrophobic domain in the middle. The cyanobacterial consensussequence exhibits significant homology to the presumed productof ORF32 in the chloroplast DNA of liverwort (Marchantia polymorpha),but no homologous ORF is present in the chloroplast DNA of tobaccoor rice. Since this protein appears to interact strongly withthe PS I reaction center complex, it may play some role in thefunction and maintenance of the structure of PS I. (Received May 25, 1992; Accepted August 18, 1992)