Synthesis Of D1 Protein Research Articles

Mechanisms by which the infection of Sclerotinia sclerotiorum (Lib.) de Bary affects the photosynthetic performance in tobacco leaves.

BackgroundSclerotinia sclerotiorum (Lib.) de Bary is a necrotrophic fungal pathogen which causes disease in a wide range of plants. An observed decrease in photosynthetic performance is the primary reason for the reduction of crop yield induced by S. sclerotiorum. The H2C2O4 is the main pathogenic material secreted by S. sclerotiorum, but the effects of H2C2O4 acidity and the C2O42− ion on photosynthetic performance remain unknown.ResultsS. sclerotiorum infection significantly decreased photosynthetic O2 evolution and the maximum quantum yield of photosystem II (Fv/Fm) in tobacco leaves under high-light. H2C2O4 (the main pathogenic material secreted by S. sclerotiorum) with pH 4.0 also significantly decreased photosynthetic performance. However, treatment with H3PO4 and HCl at the same pH as H2C2O4 caused much less decrease in photosynthetic performance than H2C2O4 did. These results verify that the acidity of the H2C2O4 secreted by S. sclerotiorum was only partially responsible for the observed decreases in photosynthesis. Treatment with 40 mM K2C2O4 decreased Fv/Fm by about 70% of the levels observed under 40 mM H2C2O4, which further demonstrates that C2O42− was the primary factor that impaired photosynthetic performance during S. sclerotiorum infection. K2C2O4 treatment did not further decrease photosynthetic performance when D1 protein synthesis was fully inhibited, indicating that C2O42− inhibited PSII by repressing D1 protein synthesis. It was observed that K2C2O4 treatment inhibited the rate of RuBP regeneration and carboxylation efficiency. In the presence of a carbon assimilation inhibitor, K2C2O42 treatment did not further decrease photosynthetic performance, which infers that C2O42− inhibited PSII activity partly by repressing the carbon assimilation. In addition, it was showed that C2O42− treatment inhibited the PSII activity but not the PSI activity.ConclusionsThis study demonstrated that the damage to the photosynthetic apparatus induced by S. sclerotiorum is not only caused by the acidity of H2C2O4, but also by C2O42− which plays a much more important role in damaging the photosynthetic apparatus. C2O42− inhibits PSII activity, as well as the rate of RuBP regeneration and carboxylation efficiency, leading to the over production of reactive oxygen species (ROS). By inhibiting the synthesis of D1, ROS may further accelerate PSII photoinhibition.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-014-0240-4) contains supplementary material, which is available to authorized users.

Photoinhibition in a C4 plant, Zea mays L.: a minireview

Light and oxygen are both essential to life but can also cause some damage to plant photosynthesis and productivity, particularly excess light in the presence of oxygen. This phenomenon is called photoinhibition, i.e. the photooxidative damage of photosystems due to excess light associated or not with abiotic stresses. To tolerate photoinhibition, the plant needs to be able to avoid or tolerate excess light or to repair the photodamage by maintaining the de novo D1 protein synthesis that is essential for PS II complex activity. When the electron transport of the photosystems is diminished because the use of final electron acceptors, ferredoxin or NADPH2, via the Calvin cycle is reduced, electrons from water photolysis are captured by O2 forming ROS, which inhibits protein synthesis, even in C4 plants. To tolerate excess light or repair photodamage under photoinhibitory conditions, several mechanisms exist to reduce ROS and/or maintain ATP production and use for protein synthesis and growth: the scavenging systems (reducing ROS activity), such as the xanthophyll (thermal dissipation of excitation energy) and ascorbate cycle (associated with the Mehler reaction, in the water–water cycle) or the activity of antioxidants compounds, such as carotenoids; the maintenance of cyclic electron flow (only ATP production); and the photorespiration cycle (consuming reducing equivalents and ATP). Photoinhibition is a phenomenon that occurs in C3 and C4 plants, such as corn, under PPFD above 1,000 µmol m−2 s−1, and photooxidative damage seems to be more accentuated in mesophyll chloroplasts than in bundle sheath agranal chloroplasts.

Glycine betaine application in grain filling wheat plants alleviates heat and high light-induced photoinhibition by enhancing the psbA transcription and stomatal conductance

Wheat (Triticum aestivum) plants often suffer from heat stress combined with high irradiance during the grain filling stage. Glycine betaine (GB) is a compatible solute that accumulates rapidly to enhance stress tolerance in wheat under abiotic stress. In this study, the effect of foliar application of GB on photosystem II (PSII) photochemistry in wheat leaves under the heat and high light stresses were investigated. The results showed that GB-supplemented wheat plants maintained higher chlorophyll content, photochemical activity of PSII and net photosynthetic rate (P n) than stressed only plants. Moreover, GB-treated plants could retard the decrease of psbA gene transcription and accelerate the endogenous accumulation of GB in leaves. The results suggested that the increasing stress tolerance by GB accumulation was associated with an improvement in D1 protein synthesis, which accelerated the repair of PSII following stress-enhanced photoinhibition. Moreover, the accumulation of abscisic acid under the heat and high light stresses was retarded by foliar application of GB, and the decrease of stomatal conductance was inhibited as well. The results suggested that GB accumulation in vivo was involved in the regulation of stomatal conductance. Further work is required to elucidate the mechanism of GB-induced stomatal movement and PSII photoprotection.

LeCDJ1, a chloroplast DnaJ protein, facilitates heat tolerance in transgenic tomatoes

The roles of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ1) were investigated using wild-type (WT) and sense transgenic tomatoes. The LeCDJ1 expression was upregulated by 38 °C, 42 °C, 45 °C, NaCl, PEG, methyl viologen (MV) and hydrogen peroxide (H2O2), but not by 30 °C and 35 °C. Meanwhile, LeCDJ1 was involved in the response of plants to abscisic acid (ABA). Under heat stress, the sense plants showed better growth, higher chlorophyll content, lower malondialdehyde (MDA) accumulation and relative electrical conductivity (REC), and also less PSII photoinhibition than WT. Interestingly, the sense plants treated with streptomycin (SM), an inhibitor of organellar translation, still showed higher maximum photochemistry efficiency of PSII (Fv/Fm) and D1 protein levels than the SM-untreated WT, suggesting that the protective effect of LeCDJ1 on PSII was, at least partially, independent of D1 protein synthesis. Furthermore, the relatively lower superoxide radical (O2(•-)) and H2O2 levels in the sense plants were considered to be due to the higher ascorbate peroxidase (APX) and superoxide dismutase (SOD) activity, which seemed unlikely dependent on their transcription level. These results indicated that LeCDJ1 overexpression facilitated heat tolerance in transgenic tomatoes.

The chloroplast protein LTO1/AtVKOR is involved in the xanthophyll cycle and the acceleration of D1 protein degradation

The thylakoid protein LTO1/AtVKOR-DsbA is recently found to be an oxidoreductase involved in disulfide bond formation and the assembly of photosystem II (PSII) in Arabidopsis thaliana. In this study, experimental evidence showed that LTO1 deficiency caused severe photoinhibition which was related to the xanthophyll cycle and D1 protein degradation. The lto1-2 mutant was more sensitive to intense irradiance than wild type. When treated with different concentrations of dithiothreitol (DTT), an inhibitor of violaxanthin de-epoxidase (VDE) in the xanthophyll cycle, there was a larger reduction in NPQ in the wild type than in the lto1-2 mutant under high irradiance, indicating that lto1-2 had a lower sensitivity to DTT gradients than did the wild type. Zeaxanthin in the xanthophyll cycle, which participates in the thermal dissipation of excess absorbed light energy, was much less active in lto1-2 than in the wild type under intense light levels, and the de-epoxidation state of the xanthophyll cycle was consistent with the susceptibility of NPQ. Together these observations indicated that aggravated photoinhibition in lto1-2 was related to a reduction in xanthophyll cycle-associated energy dissipation. When D1 protein synthesis was suppressed by an inhibitor of chloroplast protein synthesis (streptomycin sulfate), the levels of D1 protein decreased more in the lto1-2 mutant than in the wild type when exposed to intense light levels, implying that a deficiency in LTO1 accelerated the degradation of D1 and thus affected D1 turnover. Transgenic complementation of plants with lto1-2 ultimately allowed for the recovery of the photoinhibition properties of leaves.

A chloroplast-targeted DnaJ protein contributes to maintenance of photosystem II under chilling stress

DnaJ proteins act as essential molecular chaperones in protein homeostasis and protein complex stabilization under stress conditions. The roles of a tomato (Lycopersicon esculentum) chloroplast-targeted DnaJ protein (LeCDJ1), whose expression was upregulated by treatment at 4 and 42 °C, and with high light, NaCl, polyethylene glycol, and H2O2, were investigated here using sense and antisense transgenic tomatoes. The sense plants exhibited not only higher chlorophyll content, fresh weight and net photosynthetic rate, but also lower accumulation of reactive oxygen species and membrane damage under chilling stress. Moreover, the maximal photochemistry efficiency of photosystem II (PSII) (F v/F m) and D1 protein content were higher in the sense plants and lower in the antisense plants, and the photoinhibitory quenching was lower in the sense plants and higher in the antisense plants, suggesting that the inhibition of PSII was less severe in the sense plants and more severe in the antisense plants compared with the wild type. Furthermore, the PSII protein complexes were also more stable in the sense plants. Interestingly, the sense plants treated with streptomycin (SM), an inhibitor of organellar translation, still showed higher F v/F m, D1 protein content and PSII stability than the SM-untreated antisense plants. This finding suggested that the protective effect of LeCDJ1 on PSII was, at least partially, independent of D1 protein synthesis. Furthermore, chloroplast heat-shock protein 70 was identified as the partner of LeCDJ1. These results indicate that LeCDJ1 has essential functions in maintaining PSII under chilling stress.

Effects of High Temperature Stress on PSII Function and Its Relation to D1 Protein in Chloroplast Thylakoid in Rice Flag Leaves

On the basis of the artificial controlled temperature treatments in growth chambers, influences of high temperature stress at rice filling stage on the expression and distribution of D1 protein in leaf chloroplast thylakoid, as well as their relations to the decline of leaf photosynthetic rate and the damage of PSII membrane system were investigated by detecting the temporal pattern of leaf photosynthetic rate and chlorophyll fluorescence parameters, with SDS-PAGE electrophoresis, western-blotting and immunogold labeling methods. The results showed that rice flag leaves exposed high temperature generally had lower net photosynthetic rate (Pn), PSII potential efficiency(Fv /Fo) and solar energy transmitting efficiency(Fv/Fm) compared with those under normal temperature, with more severe damage and remarkable function dropping in chloroplast thylakoid due to prolonging stress times and accelerating leaves senescence.The amount of D1 protein in rice leaves under high temperature stress decreased significantly. In the two types of D1 protein, the amount of non-phosphorylated D1 protein was more sensitive to high temperature stress than the phosphorylated D1 protein.The RNA transcription of psbA gene in chloroplast, controlling D1 protein synthesis, was down-regulated by high temperature stress, inhabiting mRNA transcription and protein translation in D1 protein synthesis, thereby causing the functional damage of PSII reaction centre in thylakoid and dropping leaf photosynthetic rate under high temperature stress. It could be suggested that the functional damage of PSII reaction centre under high temperature stress be caused by the combined action of D1 protein hosphorylation and psbA gene expression, resulting in the decrease of photosynthetic rate and high temperature tolerance in two rice genotypes.

Photosystem II Function and Dynamics in Three Widely Used Arabidopsis thaliana Accessions

Columbia-0 (Col-0), Wassilewskija-4 (Ws-4), and Landsberg erecta-0 (Ler-0) are used as background lines for many public Arabidopsis mutant collections, and for investigation in laboratory conditions of plant processes, including photosynthesis and response to high-intensity light (HL). The photosystem II (PSII) complex is sensitive to HL and requires repair to sustain its function. PSII repair is a multistep process controlled by numerous factors, including protein phosphorylation and thylakoid membrane stacking. Here we have characterized the function and dynamics of PSII complex under growth-light and HL conditions. Ws-4 displayed 30% more thylakoid lipids per chlorophyll and 40% less chlorophyll per carotenoid than Col-0 and Ler-0. There were no large differences in thylakoid stacking, photoprotection and relative levels of photosynthetic complexes among the three accessions. An increased efficiency of PSII closure was found in Ws-4 following illumination with saturation flashes or continuous light. Phosphorylation of the PSII D1/D2 proteins was reduced by 50% in Ws-4 as compared to Col-0 and Ler-0. An increase in abundance of the responsible STN8 kinase in response to HL treatment was found in all three accessions, but Ws-4 displayed 50% lower levels than Col-0 and Ler-0. Despite this, the HL treatment caused in Ws-4 the lagest extent of PSII inactivation, disassembly, D1 protein degradation, and the largest decrease in the size of stacked thylakoids. The dilution of chlorophyll-protein complexes with additional lipids and carotenoids in Ws-4 may represent a mechanism to facilitate lateral protein traffic in the membrane, thus compensating for the lack of a full complement of STN8 kinase. Nevertheless, additional PSII damage occurs in Ws-4, which exceeds the D1 protein synthesis capacity, thus leading to enhanced photoinhibition. Our findings are valuable for selection of appropriate background line for PSII characterization in Arabidopsis mutants, and also provide the first insights into natural variation of PSII protein phosphorylation.

Mitochondrial alternative oxidase pathway protects plants against photoinhibition by alleviating inhibition of the repair of photodamaged PSII through preventing formation of reactive oxygen species in Rumex K‐1 leaves

The purpose of this study was to explore how the mitochondrial AOX (alternative oxidase) pathway alleviates photoinhibition in Rumex K-1 leaves. Inhibition of the AOX pathway decreased the initial activity of NADP-malate dehydrogenase (EC 1.1.1.82, NADP-MDH) and the pool size of photosynthetic end electron acceptors, resulting in an over-reduction of the photosystem I (PSI) acceptor side. The over-reduction of the PSI acceptor side further inhibited electron transport from the photosystem II (PSII) reaction centers to the PSII acceptor side as indicated by an increase in V(J) (the relative variable fluorescence at J-step), causing an imbalance between photosynthetic light absorption and energy utilization per active reaction center (RC) under high light, which led to the over-excitation of the PSII reaction centers. The over-reduction of the PSI acceptor side and the over-excitation of the PSII reaction centers enhanced the accumulation of reactive oxygen species (ROS), which inhibited the repair of the photodamaged PSII. However, the inhibition of the AOX pathway did not change the level of photoinhibition under high light in the presence of the chloroplast D1 protein synthesis inhibitor chloramphenicol, indicating that the inhibition of the AOX pathway did not accelerate the photodamage to PSII directly. All these results suggest that the AOX pathway plays an important role in the protection of plants against photoinhibition by minimizing the inhibition of the repair of the photodamaged PSII through preventing the over-production of ROS.

D1 protein turnover is involved in protection of Photosystem II against UV-B induced damage in the cyanobacterium Arthrospira ( Spirulina) platensis

By using two strains of Arthrospira (Spirulina) platensis, an economically important filamentous cyanobacterium, we compared the impairment of PSII activity and loss of D1 protein content under UV-B radiation. Our study showed that UV-B radiation induced a gradual loss of the oxygen-evolving activity to about 56% after 180 min UV-B irradiation both in strains 439 and D-0083, which have been kept under indoor and an outdoor culturing conditions, respectively for a prolonged period of time. The loss of oxygen evolution was accelerated in both strains in the presence of lincomycin, an inhibitor of protein synthesis, and the amount of D1 protein showed a decrease comparable to that of oxygen evolution during the UV-B exposure. However, the UV-B induced loss of oxygen-evolving activity and D1 protein amount was largely prevented when A. platensis cells were exposed to UV-B irradiance supplemented with visible light. Comparison of the two strains also showed a smaller extent of D1 protein synthesis dependent PSII repair in the indoor strain. Our results show that turnover of the D1 protein is an important defense mechanism to counteract the UV-B induced damage of PSII in A. platensis, and also that visible light plays an important role in maintaining the function of PSII under simultaneous exposure to UV-B and visible light.

PSII photoinhibition and photorepair in Symbiodinium (Pyrrhophyta) differs between thermally tolerant and sensitive phylotypes

Cnidarians containing symbiotic microalgae often inhabit highly variable light environments where successful growth requires that, during transient (potentially stressful) periods of high light (HL), the microalgal cells invest energy in photoprotection to minimise photodamage, or allow for photodamage to occur and invest in photorepair; however, the relative contribution of photoprotection and photorepair remains uncharacterised. Here we determined the light dependence of Photosystem II (PSII) photoinhibition and photorepair in 2 phylotypes of Symbiodinium displaying different susceptibilities to thermal stress. Upon exposure to photon flux densities (PFDs) >500 μmol photons m-2 s-1 the thermally 'sensitive' Strain A1.1 displayed higher net photoinhibition, measured as a decrease in maximum PSII efficiency (Fv/Fm), than the thermally 'tolerant' Strain A1. In contrast, gross photoinhibition, assessed as the decline of Fv/F m in the presence of an inhibitor of D1 protein synthesis, was similar in the 2 strains. Therefore, photorepair was considered to be the key mechanism minimising net photoinhibition in Strain A1. Consistent with this conclusion, the 2 strains displayed similar capacities for other mechanisms of avoiding photodamage, specifically, photochemical (qP) and non-photochemical (NPQ) excitation energy quenching. Measurements on Strain A1 grown under 2 PFDs (100 and 650 μmol photons m-2 s-1) revealed that photoacclimation to HL involved the upregulation of qP, which minimised gross photoinhibition by maintaining PSII in a more oxidised state. We conclude that both interspecific (e.g. phylotype diversity) and intraspecific (e.g. photoacclimation state) factors affect the susceptibility of Symbiodinium to light stress. © Inter-Research 2010.

Phosphatidylglycerol depletion affects photosystem II activity in Synechococcus sp. PCC 7942 cells

The role of phosphatidylglycerol (PG) in photosynthetic membranes of cyanobacteria was analyzed in a Synechococcus sp. PCC 7942 mutant produced by inactivating its cdsA gene presumably encoding cytidine 5'-diphosphate-diacylglycerol synthase, a key enzyme in PG synthesis. In a medium supplemented with PG the Synechococcus sp. PCC 7942/DeltacdsA cells grew photoautotrophically. Depletion of PG in the medium resulted (a) in an arrest of cell growth and division, (b) in a suppression of O(2) evolving activity, and (c) in a modification of Chl fluorescence induction curves. Two-dimensional PAGE showed that in the absence of PG (a) the amount of the PSI monomers increased at the expense of the PSI trimers and (b) PSII dimers were decomposed into monomers. [(35)S]methionine labeling confirmed that PG depletion did not block the de novo synthesis of PSII proteins but slowed down the assembly of the newly synthesized D1 protein into PSII core complexes. Retailoring of PG was observed during PG depletion: the exogenously added artificial dioleoyl PG was transformed into photosynthetically more essential PG derivatives. Concomitantly with a decrease in PG content, SQDG content increased, but it could not restore photosynthetic activity.

The Psb27 Protein Facilitates Manganese Cluster Assembly in Photosystem II

Photosystem II (PSII) is a large membrane protein complex that uses light energy to convert water to molecular oxygen. This enzyme undergoes an intricate assembly process to ensure accurate and efficient positioning of its many components. It has been proposed that the Psb27 protein, a lumenal extrinsic subunit, serves as a PSII assembly factor. Using a psb27 genetic deletion strain (Deltapsb27) of the cyanobacterium Synechocystis sp. PCC 6803, we have defined the role of the Psb27 protein in PSII biogenesis. While the Psb27 protein was not essential for photosynthetic activity, various PSII assembly assays revealed that the Deltapsb27 mutant was defective in integration of the Mn(4)Ca(1)Cl(x) cluster, the catalytic core of the oxygen-evolving machinery within the PSII complex. The other lumenal extrinsic proteins (PsbO, PsbU, PsbV, and PsbQ) are key components of the fully assembled PSII complex and are important for the water oxidation reaction, but we propose that the Psb27 protein has a distinct function separate from these subunits. We show that the Psb27 protein facilitates Mn(4)Ca(1)Cl(x) cluster assembly in PSII at least in part by preventing the premature association of the other extrinsic proteins. Thus, we propose an exchange of lumenal subunits and cofactors during PSII assembly, in that the Psb27 protein is replaced by the other extrinsic proteins upon assembly of the Mn(4)Ca(1)Cl(x) cluster. Furthermore, we show that the Psb27 protein provides a selective advantage for cyanobacterial cells under conditions such as nutrient deprivation where Mn(4)Ca(1)Cl(x) cluster assembly efficiency is critical for survival.

Synthesis of the D2 protein of photosystem II in Chlamydomonas is controlled by a high molecular mass complex containing the RNA stabilization factor Nac2 and the translational activator RBP40.

Gene expression in chloroplasts is regulated mainly at the posttranscriptional level. In the green alga Chlamydomonas reinhardtii, synthesis of the D2 protein (PsbD), which is the rate-determining subunit for the assembly of photosystem II, depends on the RNA stability factor Nac2. In addition, the RNA binding protein RBP40 has been implicated in translational control via a U-rich element in the 5' untranslated region (5'UTR) of the psbD mRNA. Here, we report the identification of the RBP40 gene based on mass spectrometric analysis of its purified product. Unexpectedly, this was found to be identical to the previously described RNA binding protein RB38, which had been suggested to be involved in the regulation of D1 protein synthesis. However, we show that RBP40 binds to the psbD 5'UTR in a Nac2-dependent fashion both in vitro and in vivo. Molecular characterization of RBP40 RNA interference lines confirmed that RBP40 specifically affects the initiation of D2 synthesis. Native polyacrylamide gel electrophoresis, coimmunoprecipitation, and sedimentation analyses revealed that Nac2 and RBP40 form parts of a complex of 550 kD that is displaced from the psbD mRNA prior to polysome assembly. Together, these data indicate that the processes of 5'UTR-mediated RNA stabilization and translation initiation are tightly coupled in Chlamydomonas.

Expression of psbA Genes is Regulated at Multiple Levels in the Cyanobacterium Synechococcus sp. PCC 7942

In cyanobacterium Synechococcus sp. PCC 7942 the photosystem II reaction-center protein D1 is encoded by three psbA genes. The psbAI gene encodes D1:1 protein, the form used for acclimated growth, and psbAII and psbAIII genes encode the stress-induced form, D1:2 protein. Strong light and low temperature have been shown to induce the expression of psbAII/III genes and down-regulate the expression of psbAI gene. Recently, we reported the involvement of reduced thiols in the up-regulation of psbAII/III genes. In this study, we have analyzed the regulation of psbA gene expression in Synechococcus further, at both the transcriptional and post-transcriptional levels. We show that the inhibitors of the photosynthetic electron-transfer chain, which have different effects on the redox state of the plastoquinone (PQ) pool, have similar effect on the transcription of psbA genes. The inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea (DCMU) and 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) do not cause any changes in psbA gene expression when added under low-light conditions, but dramatically reduce the high-light induction of psbAII/III genes when added upon a high-light shift. Moreover, when the thiol reductant, dithiothreitol, is added to Synechococcus cells together with DCMU concomitant with the high-light shift, no inhibition of psbAII/III gene up-regulation takes place, indicating that the thiol redox state rather than the redox state of the PQ pool regulates psbA gene transcription. We also provide evidence for post-transcriptional regulation of psbA gene expression, particularly, inhibition of translation of psbAI transcripts at high light, and demonstrate that the D1 protein synthesis and degradation processes are coregulated in Synechococcus.

Formation of DEG5 and DEG8 Complexes and Their Involvement in the Degradation of Photodamaged Photosystem II Reaction Center D1 Protein in Arabidopsis

The widely distributed DEGP proteases play important roles in the degradation of damaged and misfolded proteins. Arabidopsis thaliana contains 16 DEGP-like proteases, four of which are located in the chloroplast. Here, we show that DEG5 and DEG8 form a hexamer in the thylakoid lumen and that recombinant DEG8 is proteolytically active toward both a model substrate (beta-casein) and photodamaged D1 protein of photosystem II (PSII), producing 16-kD N-terminal and 18-kD C-terminal fragments. Inactivation of DEG5 and DEG8 resulted in increased sensitivity to photoinhibition. Turnover of newly synthesized D1 protein in the deg5 deg8 double mutant was impaired, and the degradation of D1 in the presence of the chloroplast protein synthesis inhibitor lincomycin under high-light treatment was slowed in the mutants. Thus, DEG5 and DEG8 are important for efficient turnover of the D1 protein and for protection against photoinhibition in vivo. The deg5 deg8 double mutant showed increased photosensitivity and reduced rates of D1 degradation compared with single mutants of deg5 and deg8. A 16-kD N-terminal degradation fragment of the D1 protein was detected in wild-type plants but not in the deg5 deg8 mutant following in vivo photoinhibition. Therefore, our results suggest that DEG5 and DEG8 have a synergistic function in the primary cleavage of the CD loop of the PSII reaction center protein D1.

Ribosomal Stress Couples the Unfolded Protein Response to p53-dependent Cell Cycle Arrest

Protein misfolding in the endoplasmic reticulum (ER) triggers a signaling pathway termed the unfolded protein response path-way (UPR). UPR signaling is transduced through the transmembrane ER effectors PKR-like ER kinase (PERK), inositol requiring kinase-1 (IRE-1), and activating transcription factor 6 (ATF6). PERK activation triggers phosphorylation of eIF2alpha leading to repression of protein synthesis, thereby relieving ER protein load and directly inhibiting cyclin D1 translation thereby contributing to cell cycle arrest. However, PERK(-/-) murine embryonic fibroblasts have an attenuated G(1)/S arrest that is not attributable to cyclin D1 loss, suggesting a cyclin D1-independent mechanism. Here we show that the UPR triggers p53 accumulation and activation. UPR induction promotes enhanced interaction between the ribosome proteins (rpL5, rpL11, and rpL23) and Hdm2 in a PERK-dependent manner. Interaction with ribosomal proteins results in inhibition of Hdm2-mediated ubiquitination and degradation of p53. Our data demonstrate that ribosomal subunit:Hdm2 association couples the unfolded protein response to p53-dependent cell cycle arrest.

Cyclin D3 is down-regulated by rapamycin in HER-2-overexpressing breast cancer cells

Rapamycin and its analogues are being tested as new antitumor agents. Rapamycin binds to FKBP-12 and this complex inhibits the activity of FRAP/mammalian target of rapamycin, which leads to dephosphorylation of 4EBP1 and p70 S6 kinase, resulting in blockade of translation initiation. We have found that RAP inhibits the growth of HER-2-overexpressing breast cancer cells. The phosphorylation of mammalian target of rapamycin, p70 S6 kinase, and 4EBP1 is inhibited by rapamycin and cells are arrested in the G1 phase, as determined by growth assays, fluorescence-activated cell sorting analysis, and bromodeoxyuridine incorporation studies. Rapamycin causes down-regulation of cyclin D3 protein, retinoblastoma hypophosphorylation, loss of cyclin-dependent kinase (cdk) 4, cdk6, and cdk2 activity. The half-life of cyclin D3 protein decreases after rapamycin treatment, but not its synthesis, whereas the synthesis or half-life of cyclin D1 protein is not affected by the drug. Additionally, rapamycin caused accumulation of ubiquitinated forms of cyclin D3 protein, proteasome inhibitors blocked the effect of rapamycin on cyclin D3, and rapamycin stimulated the activity of the proteasome, showing that the effect of rapamycin on cyclin D3 is proteasome proteolysis dependent. This effect depends on the activity of HER-2 because Herceptin, a neutralizing antibody against HER-2, is able to block both the induction of proteasome activity and the cyclin D3 down-regulation due to rapamycin. Furthermore, inhibition of HER-2 gene expression by using small interfering RNA blocked the rapamycin effects on cyclin D3. These data indicate that rapamycin causes a G1 arrest in HER-2-overexpressing breast cancer cells that is associated with a differential destabilization and subsequent down-regulation of cyclin D3 protein.

Glycinebetaine Counteracts the Inhibitory Effects of Salt Stress on the Degradation and Synthesis of D1 Protein during Photoinhibition in Synechococcus sp. PCC 7942

Glycinebetaine (hereafter referred to as betaine) is a compatible solute that accumulates in certain plants and microorganisms in response to various types of stress. We demonstrated previously that when the cyanobacterium Synechococcus sp. PCC 7942 (hereafter Synechococcus) is transformed with the codA gene for choline oxidase, it can synthesize betaine from exogenously supplied choline, exhibiting enhanced tolerance to salt and cold stress. In this study, we examined the effects of salt stress and betaine synthesis on the photoinhibition of photosystem II (PSII). Salt stress due to 220 mm NaCl enhanced photoinhibition of PSII and betaine protected PSII against photoinhibition under these conditions. However, neither salt stress nor betaine synthesis affected photodamage to PSII. By contrast, salt stress inhibited repair of photodamaged PSII and betaine reversed this inhibitory effect of salt stress. Pulse-chase-labeling experiments revealed that salt stress inhibited degradation of D1 protein in photodamaged PSII and de novo synthesis of D1. By contrast, betaine protected the machinery required for degradation and synthesis of D1 under salt stress. Neither salt stress nor betaine affected levels of psbA transcripts. These observations suggest that betaine counteracts the inhibitory effects of salt stress, with resultant accelerated repair of photodamaged PSII.

Thermal inhibition and recovery of the maximum quantum yield of photosystem II and the maximum electron transport rate in zooxanthellae of a reef-building coral

To examine whether damage to zooxanthellar photosystem II (PSII) is the primary step of thermally induced coral bleaching, we first assessed the relationship between the maximum quantum yield of PSII (Fv/Fm) or active PSII centers (Fv/Fo) and maximum electron transport rate (ETRmax), which represent the PSII activity and electron flow beyond the PSII, respectively, in the symbiotic algae of the coral Pachyseris rugosa. Next, the photo-physiological responses of the symbionts to stress treatments of 1h high temperature (33.5°C) and/or high light (1030μmol quanta m-2 s-1) stress and 6h recovery were investigated. The 1h high temperature treatment in darkness produced a significant, but reversible (6h recovery) decline in ETRmax without any change in Fv/Fm. The 1h high light treatment reduced Fv/Fm, but not ETRmax. High temperature in combination with high light resulted in a more pronounced reduction in Fv/Fm, along with a significant decline in ETRmax. Neither Fv/Fm nor ETRmax recovered fully within 6h. Reversible increases in non-photochemical quenching (NPQ), energy dissipation in PSII, were also recorded. Chloramphenicol (CAP), an inhibitor of synthesis of D1 protein of PSII reaction center, revealed that a minimum of -60% of the Fv/Fm or -30% of active PSII centers (Fv/Fo) is required to maintain ETRmax in the in hospite symbionts. These results suggest that the primary step of heat-induced damage in the symbiont's photosynthetic apparatus involves a component beyond the PSII, probably at the level of the dark reaction as indicated by reduced ETRmax and the PSII damage is secondary.