Immunoelectron Microscopy for Locating Calvin Cycle Enzymes in the Thylakoids of Synechocystis 6803

Unicellular cyanobacteria Synechocystis 6803 were fixed using high-pressure freezing （HPF） and freeze substitution without any chemical cross-linkers. Immunoelectron microscopy of these cells showed that five sequential enzymes of the Calvin cycle （phosphoriboisomerase, phosphoribulokinase, ribulose-1,5-bisphosphate carboxylase/oxygenase （RuBisCO）, 3-phosphoglyceratekinase and glyceraldehyde-3-phosphate dehydrogenase） and the catalytic portion of the chloroplast H^＋-ATP synthase （CF1） are located adjacent to the thylakoid membranes. Cell-free extracts of Synechocystis were processed by ultracentrifugation to isolate thylakoid fractions sedimenting at 40 000, 90 000, and 150 000 g. Among these, the 150 000-g fraction showed the highest linked activity of the above five sequential Calvin cycle enzymes and also the highest coordinated activity of light and dark reactions as assessed by ribose-5-phosphate （R-5-P） ＋ADP dependent CO2 fixation. Immunogold labeling of this membrane fraction confirmed the presence of the above five enzymes as well as the catalytic portion of the CF1 ATP synthase. Notably, the protein A-gold labeling of the thylakoids was observed without use of chemical cross-linkers and in spite of the normal washing steps used during standard immunolabeling. The results showed that soluble Calvin cycle enzymes might be organized along the thylakoid membranes.

STUDY ON SHRINKAGE OF THYLAKOIDS RELATED TO H^+-UPTAKE

It has been proved that the light-induced apparent absorbance change of thylakoid suspension at 540 nm (ΔA540)reflects the shrinkage of thylakoids. Recently, this method has been used to detect the energized state of thylakoids in vivo under various circumstances.

Chloroplast Composition and Structural Differences in a Chlorophyll-reduced Mutant of Oilseed Rape Seedlings

对黄化突变体Cr352 9和野生型油菜 (BrassicanapusL .) 352 9叶绿体的超微结构和组成进行了比较。与野生型相比 ,突变体Cr352 9叶片具有较少的类囊体、较少的垛叠膜区和较少的叶绿素含量。突变体的Chla/Chlb比值较高 ,是野生型的 2倍。电泳结果表明 ,突变体类囊体膜中LHCⅡ和其三聚体LHCⅡ 的含量减少。SDS_PAGE分析显示 ,LHCⅡ的脱辅基蛋白在突变体类囊体膜中明显减少。免疫印迹进一步表明 ,所有LHCⅡ组分的含量仅为野生油菜的类囊体膜的 1 / 3。突变体Cr352 9的天线系统比野生型 352 9的小。

Changes in Violaxanthin Deepoxidase Activity and Unsaturation of Thylakoid Membrane Lipids in Indica and Japonica Rice Under Chilling Condition and Strong Light

To explore the differences of sensitivities to chilling and strong light in indica and japonica rice (Oryza sativa L), the changes in unsaturation of thylakoid membrane lipids and xanthophyll cycle were studied under chilling condition and strong light. The contents of unsaturated fatty acids of thylakoid membrane lipids decreased and that of the saturated ones increased with the time of chilling and strong light treatment, resulting in the reduction of the index of unsaturation of fatty acids (IUFA). The activity of violaxanthin deepoxidase (VDE), a key enzyme of xanthophyll cycle, also reduced. The content of violaxanthin (V) increased, and the contents of antheraxanthin (A) and zeaxanthin M decreased, the ratio of (A+Z)/ (A+Z+V) decreased correspondingly. Arrhenius analysis showed that VDE was sensitive to both chilling and unsaturation level of thylakoid membrane lipids. Correlation analysis showed that there was distinctly positive relationships between IUFA of thylakoid membrane lipids and the activity of VDE, Fv/Fm, and D, protein content. Lower IUFA values, less fluidity and stability of thylakoid membrane lipids, lower VDE activity and (A+Z)/(A+Z+V) ratio were found in indica rice cv. Shanyou 63 than in japonica rice cv. 9516 under chilling and strong light.

Effects of Cadmium on Growth, Photosynthetic Pigments, Photosynthetic Performance, Biochemical Parameters and Structure of Chloroplasts in the Agarophyte <i>Gracilaria domingensis</i>(Rhodophyta, Gracilariales)

This paper aimed to evaluate the effects of different concentrations of cadmium on growth rates, photosynthetic pigments, photosynthetic performance, biochemical parameters and structure of chloroplasts in G. domingensis. To accomplish this, apical segments of G. domingensis were cultivated with different concentrations of cadmium, ranging from 100 to 300 μM, over a period of 16 days, and were processed for transmission electron microscopy analysis. The plants exposed to cadmium showed chloroplast alteration, especially degeneration of thylakoids and a decrease in the concentration of photosynthetic pigments, such as chlorophyll a and phycobiliproteins. However, the volume of plastoglobuli increased. As a defense mechanism, the plants treated with cadmium showed an increase in glutathione reductase activity. These results agree with the decreased photosynthetic performance and relative electron transport rate observed after exposure of algae to cadmium. Taken together, these findings strongly indicate that cadmium negatively affects the ultrastructure and metabolism of the agarophyte G. domingensis, thus posing a threat to the economic vitality of this red macroalga.

Characterization of Energy_Transduction in Thermal Pretreated Chloroplast from Spinach

Characterization of energy-transduction on die chloroplast thylakoid membranes from spinach (Spinacia oleracca L.) after thermal pretreatment was investigated. The related reactions of energy-transduction in chloroplasts were seriously affected by thermal pretreatment. The results were obtained as following: (1) The rate of cyclic photophosphorylation declined when the pretreatment temperature increased in the range of 25 to 45 degreesC. (2) The thermal pretreatment led to a decrease of the activity of thylakoid membrane-bounded ATPase. (3) Proton uptake of chloroplasts acid the fluorescence quenching of 9-aminoacridine (9-AA) in thylakoid membrane decreased after the thermal pretreatment, but addition of dicyclohexylcarbodiimide (DCCD) could partially restore the fluorescence quenching of 9-AA. (4) Both the rates of fast phase in electrochroism absorption change at 515 nm and the millisecond delayed light emission (ms-DLE) of chloroplast showed a progressive decrease upon raising the temperature of pretreatment. (5) Immunbloting analysis showed that the thermal pretreatment caused the changes of protein content and the electrophoresis mobility of thylakoid membrane-bound ATPase and its alpha -subunit. (6) If the temperature of pretreatment were higher than 33 degreesC, oxygen uptake of PS I -mediated in the samples was rapidly inhibited, but addition of sinapine into the reaction medium could partially restore the ability of oxygen uptake in the samples. These results are briefly discussed in relation to the change of permeability of thylakoid membranes, the dissociation of coupling factor complex as well as accumulation of the radicals in the thylakoid membranes after thermal pretreatment.

Isolation of Plasma and Thylakoid Membranes from the Heterocystous Cyanobacterium Anabaena sp. PCC 7120

利用水溶性多聚体双相法分离蓝细菌Anabaenasp .PCC 712 0质膜和类囊体膜两种膜系统。吸收光谱分析表明 ,质膜相和类囊体膜相的主要色素分别为类胡萝卜素和叶绿素。SDS_凝胶电泳显示这两种膜系统蛋白组成有很大差别。这种分离方法容易操作 ,对研究蓝细菌的膜蛋白和膜脂非常有用。

On the Role of Chloroplasts in the Polymerization of Tannins in Tracheophyta: A Monograph

Proanthocyanidins are formed in the chlorophyllous organs of Tracheophyta from a redifferentiation of chloroplasts involving the thylakoidal membrane and lumen. With the purpose to help researchers of concerned disciplines to identify such chloroplasts, we described herein the morphologies of functional and redifferentiating chloroplasts in various members of Tracheophyta. The most obvious sign of redifferentiation is a tremendous swelling of the chloroplast which turns obese. De novo genesis of osmiophilic materials is also characteristic, either as single dots attached to the inner face of the swollen thylakoidal membrane which will yield the tannosomes, or as pearl necklace-shaped structures protruding into the lumen;this last formation can be viewed as a giant tannosome forming finally stromal chlorotannic accretions. Whatever their mode of formation is, tannosomes are expulsed from the chloroplast as shuttles.

Effects of Membrane Lipids on the Electron Transfer Activity of Cytochrome b_6f Complex from Spinach

A lipid_depleted cytochrome b 6f (Cyt b 6f) preparation was obtained from spinach (Spinacia oleracea L.) chloroplasts. Upon reconstitution of this preparation with the membrane lipids purified from spinach thylakoid, the effects of different membrane lipids on the electron transfer activity were studied. The results show that the electron transfer activity of Cyt b 6f is obviously stimulated to different extents, respectively, by monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), phosphatidylcholine (PC), phosphatidylglycerol (PG) and sulfoquinovosyldiacylglycerol (SQDG), and that the extents of stimulation may be closely related to the charge of the membrane lipids. The stimulation of non_charged lipids (MGDG, DGDG) and neutrally_charged lipid (PC) was high with a maximum enhancement of 89%, 75% and 77%, respectively; but the stimulation of two kinds of negatively_charged lipid (PG and SQDG) was relatively low with a maximum enhancement of 43% and 26%, respectively.

Overaccumulation of glycine betaine makes the function of the thylakoid membrane better in wheat under salt stress

Wheat(Triticum aestivum L.) lines T1, T4, and T6 were genetically modified to increase glycine betaine(GB) synthesis by introduction of the BADH(betaine aldehyde dehydrogenase, BADH)gene from mountain spinach(Atriplex hortensis L.). These transgenic lines and WT of wheat(T. aestivum L.) were used to study the effect of increased GB synthesis on wheat tolerance to salt stress. Salt stress due to 200 mmol L-1Na Cl impaired the photosynthesis of the four wheat lines, as indicated by declines in net photosynthetic rate(Pn), stomatal conductance(Gs),maximum photochemical efficiency of PSII(Fv/Fm), and actual photochemical efficiency of PSII(ФPSII) and an increase in intercellular CO2concentration(Ci). In comparison with WT, the effect of salinity on the three transgenic lines was mild. Salt stress caused disadvantageous changes in lipids and their fatty acid compositions in the thylakoid membrane of the transgenic lines and WT. Under salt stress, the three transgenic lines showed slightly higher chlorophyll and carotenoid contents and higher Hill reaction activities and Ca2+-ATPase activity than WT. All the results suggest that overaccumulation of GB resulting from introduction of the BADH gene can enhance the salt tolerance of transgenic plants, especially in the protection of the components and function of thylakoid membranes, thereby making photosynthesis better. Changes in lipids and fatty acid compositions in the thylakoid membrane may be involved in the increased salt stress tolerance of the transgenic lines.

Bicarbonate use and carbon dioxide concentrating mechanisms in photosynthetic organisms

Photosynthesis is crucial to the reduction of carbon dioxide in the atmosphere.The key enzyme of photosynthesis,Ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco),has two mutably competing substrates,CO2 and O2.It has features of carboxylase and oxygenase.Rubisco performs the function of carboxylase to reduce inorganic carbon to form organic substances,which precondition is that more carbon dioxide accumulates around it.Carbon dioxide concentrating mechanisms(CCMs)are vital to cope with the limit of carbon dioxide.Various bicarbonate use pathway has a differential contribution to inorganic carbon assimilation.Bicarbonate transport,extracellular bicarbonate dehydration,or H+-ATPase-driven bicarbonate uptake,which induced CCMs,can support a considerable share of photosynthesis in photosynthetic organisms.However,CCMs in thylakoid membranes may be the most important.The CCMs occurred in the plasma membrane were secondary,evolutionary,and inducible,while CCMs coupled with photosynthetic oxygen evolution in thylakoid membranes,were primitive,major,and indispensable.A hypothetical schematic model of CCMs occurred in the plasma membrane and thylakoid membranes being proposed.

Thylakoid Transit Peptide Is Related to the Expression and Localization of NdhB Subunits in Soybean

The chloroplast NAD(P)H dehydrogenase(NDH)complex,as one of the most important photosynthesis protein complexes in thylakoid membrane,is involved in photosystem I(PSI)cyclic electron transport(CEF).Under abiotic environmental stress,the photosynthetic apparatus is susceptible to the damage caused by the strong light illumination.However,the enhancement of NDHdependent CEF could facilitate the alleviation of the damage to the photosynthetic apparatus.The NdhB subunit encoded by chloroplast genome is one of most important subunits of NDH complex and consists of 510 amino acids.Here,according to cloning ndhB from Melrose(cultivated soybean),ACC547(wild salt-tolerant soybean),S113-6 and S111-9(hybrid descendant),based on the comparison and analysis of the sequences of NdhB subunits,we found that there is a novel thylakoid transit peptide of NdhB subunit in S111-9.In addition,crosslink immunoprecipitation,immunogold labeling and co-expression of GFP fusion protein indicated that the novel thylakoid transit peptide is favorable to the expression and localization of NdhB subunit in chloroplast.Therefore,we suggest that this novel thylakoid transit peptide plays the same role as chaperonin and contributes to facilitating the expression and localization of NdhB subunit.

Thylakoid engineered M2 macrophage for sonodynamic effect promoted cell therapy of early atherosclerosis

Atherosclerosis is the most common cause of cardiovascular diseases that contribute to the major morbidity worldwide,but still lacking of effective treatment strategy.Here,a hybrid cell is constructed for the sonodynamic effect promoted cell therapy of early atherosclerosis by fusing M2 macrophages with thylakoid(TK)membranes.After systemic administration,the obtained TK-M2 actively accumulates in the early atherosclerotic plaques,wherein M2 macrophages relieve the cholesterol accumulation and the inflammation in the foam cells.Meanwhile,the TK membranes decorated on the M2 macrophages exhibit both type I and type II sonodynamic effects under ultrasound(US)activation,inducing the direct apoptosis of foam cells.The cooperation of M2 and TK leads to significant outcome in eliminating atherosclerotic plaques without obvious side-effects,providing a new avenue for atherosclerosis treatment.

Self-Assembled Protein Hybrid Nanofibrils for Photosynthetic Hydrogen Evolution

In artificial photosynthesis systems,synthetic diiron complexes are popular[FeFe]-hydrogenase mimics,which are attractive for the fabrication of photocatalyst-protein hybrid structures to amplify hydrogen(H2)generation capability.However,constructing a highly bionic and efficient catalytic hybrid system is a major challenge.Notably,we designed an ideal hybrid nanofibrils system that incorporates the crucial components:(1)a[FeFe]-H2ase mimic,which has a three-arm architecture(named triFeFe)for more interaction sites and higher catalytic activity and(2)uniform hybrid nanofibrils as the biological environment in which cysteine-catalyst coordination and the hydrogen-bonding network play a vital role in both catalyst binding and hydrogen evolution reaction activity.The assembled hybrid nanofibrils achieve efficient H2 generation with a turnover number of 2.3×103,outperforming previously reported diiron catalyst-protein hybrid systems.Additionally,the hybrid nanofibrils work with photosynthetic thylakoids to produce H2,without extra photosensitizers or electron shuttle proteins,which advances the bioengineering of living systems for solar-driven biofuel production.

Light-Harvesting Artificial Cells for the Generation of ATP and NADPH

The bottom-up construction of self-powered artificial cells is significant to understand the energy supply and metabolism of nature cells.Here,we demonstrate an efficient manner to build thylakoid-containing artificial cells,which continuously convert light energy into chemical energy to supply adenosine 5'-triphosphate(ATP)under light illumination.The production of ATP supplies energy to promote the biological enzyme cascade reactions,where glucose is transformed into glucose 6-phosphate(G6P)under the catalysis of hexokinase(HK).G6P was further converted to gluconolactone 6-phosphate(PG)in the presence of 6-phosphate dehydrogenase(G6PDH),meanwhile NADP^(+) was converted to nicotinamide adenine dinucleotide phosphate(NADPH).The self-powered artificial cells were demonstrated to generate ATP and NADPH successively,which provided a way for building more complicated artificial cells.

Engineering Multifunctional Thylakoid as an Oxygen Self-Supplying Photosensitizer for Esophageal Squamous Cell Carcinoma-Targeted Photodynamic Therapy

Photodynamic therapy(PDT)has emerged as an alternative treatment strategy for esophageal squamous cell carcinoma(ESCC).However,the clinical therapeutic efficiency of PDT is severely limited by poorly targeted photosensitizer delivery,insufficient oxygen supply,and neutralization by excessive glutathione(GSH)in tumor tissue.Herein,an engineered multifunctional thylakoid nanostructure,TMEM@PLGA@GA(abbreviated as TEPG),composed of a thylakoid membrane(TM)and ESCC cell membrane(EM)-fused biomembrane(TM-EM)shell and gambogic acid(GA)-loaded poly(lactic-co-glycolic acid)nanocore,was designed for enhanced PDT for ESCC.When fused with EM,TM-EM exhibits a tumor targeting advantage due to the homologous affinity of tumor membrane camouflage.The catalase present on TM-EM catalytically decomposes endogenous hydrogen peroxide into oxygen to alleviate hypoxia in the tumor tissue.Moreover,when TEPG was selectively internalized by ESCC cells,GA was released to consume the excessive intracellular GSH.Under infrared irradiation,the PDT effects were enhanced by the self-oxygen supply and GSH scavenging ability provided by TEPG.An in vivo study showed that TEPG effectively induced ESCC tumor cell apoptosis and greatly inhibited the growth of ESCC tumors under infrared irradiation.This study constructed an engineered multifunctional thylakoid-based nanomedicine as an integrated solution to enhance PDT for ESCC.

FtsH4 protease controls biogenesis of the PSII complex by dual regulation of high light-inducible proteins

FtsH proteases are membrane-embedded proteolytic complexes important for protein quality control and regulation of various physiological processes in bacteria,mitochondria,and chloroplasts.Like most cyanobacteria,the model species Synechocystis sp.PCC 6803 contains four FtsH homologs,FtsH1–FtsH4.FtsH1–FtsH3 form two hetero-oligomeric complexes,FtsH1/3 and FtsH2/3,which play a pivotal role in acclimation to nutrient deficiency and photosystem Ⅱ quality control,respectively.FtsH4 differs from the other three homologs by the formation of a homo-oligomeric complex,and together with Arabidopsis thaliana AtFtsH7/9 orthologs,it has been assigned to another phylogenetic group of unknown function.Our results exclude the possibility that Synechocystis FtsH4 structurally or functionally substitutes for the missing or non-functional FtsH2 subunit in the FtsH2/3 complex.Instead,we demonstrate that FtsH4 is involved in the biogenesis of photosystem Ⅱ by dual regulation of high light-inducible proteins(Hlips).FtsH4 positively regulates expression of Hlips shortly after high light exposure but is also responsible for Hlip removal under conditions when their elevated levels are no longer needed.We provide experimental support for Hlips as proteolytic substrates of FtsH4.Fluorescent labeling of FtsH4 enabled us to assess its localization using advanced microscopic techniques.Results show that FtsH4 complexes are concentrated in well-defined membrane regions at the inner and outer periphery of the thylakoid system.Based on the identification of proteins that co-purified with the tagged FtsH4,we speculate that FtsH4 concentrates in special compartments in which the biogenesis of photosynthetic complexes takes place.

Chloroplast Proteomics and the Compartmentation of Plastidial Isoprenoid Biosynthetic Pathways

Recent advances in the proteomic field have allowed high-throughput experiments to be conducted on chloroplast samples. Many proteomic investigations have focused on either whole chloroplast or sub-plastidial fractions. To date, the Plant Protein Database （PPDB, Sun et al., 2009） presents the most exhaustive chloroplast proteome available online. However, the accurate localization of many proteins that were identified in different sub-plastidial compartments remains hypothetical. Ferro et al. （2009） went a step further into the knowledge of Arabidopsis thaliana chloroplast proteins with regards to their accurate localization within the chloroplast by using a semi-quantitative proteomic approach known as spectral counting. Their proteomic strategy was based on the accurate mass and time tags （AMT） database approach and they built up AT_CHLORO, a comprehensive chloroplast proteome database with sub-plastidial localization and curated information on envelope proteins. Comparing these two extensive databases, we focus here on about 100 enzymes involved in the synthesis of chloroplast-specific isoprenoids. Well known pathways （i.e. compartmentation of the methyl erythritol phosphate biosynthetic pathway, of tetrapyrroles and chlorophyll biosynthesis and breakdown within chloroplasts） validate the spectral counting-based strategy. The same strategy was then used to identify the precise localization of the biosynthesis of carotenoids and prenylquinones within chloroplasts （i.e. in envelope membranes, stroma, and/or thylakoids） that remains unclear until now.

Biochemical Properties and Inhibition Kinetics of Phosphatase from Wheat Thylakoid Membranes

A phosphetase that hydrolyses phosphate monoesters has been Isolated from wheat thylakold membranes. Biochemical properties and inhibition kinetics of the phosphatase were Investigated using several Ions, organlc solvents, and Inhlbltors. Wheat （Trltlcum aestivum L. cv. PH82-2-2） thylakold membrane phosphatase activity was activated by Mg^2＋, Ca^2＋, and Fe^2＋ and was inhibited by Mn^2＋ and Cu^2＋. For example, enzyme activity was acUvated 34.81% by 2 mmol/l. Mg^2＋, but was Inhibited 22.3% and 8.5% by 2 and 1 mmol/L Cu^2＋, respectively. Methanol, ethanol and glycol were all able to activate enzyme activity. Enzyme activity was activated 58.5%, 48.2%, and 8.7% by 40% ethanol, methanol and glycol, respectively. From these results, It can be seen that the degree of actlvetlon of the phosphetase was greatest for ethanol and the type of acUvatlon was uncompetltlve. Moreover, the activity of the thylakold membrane phosphetase was Inhibited by molybdate, vanadete, phosphate, and fluoride and the type of Inhibition produced by these elements was uncompetltlve, non-competitive, competltlve and mixed, respectively.

Overexpression of Sweet Pepper Glycerol-3-Phosphate Acyltransferase Gene Enhanced Thermotolerance of Photosynthetic Apparatus in Transgenic Tobacco

In order to investigate the relationship between the lipid composition in thylakoid membrane and thermostability of photosynthetic apparatus, tobacco transformed with sweet pepper sense glycerol-3-phosphate acyltransferase （GPAT） gene were used to analyze the lipid composition in thylakoid membrane, the net photosynthetic rate and chlorophyll fluorescence parameters under high temperature stress. The results showed that the saturated extent of monogalactosyldiacylglycerol （MGDG）, sulfoquinovosyldiacylglycerol, digalactosyldiacylglycerol and phosphatidylglycerol in thylakoid membrane of transgenic tobacco T1 lines increased generally. Particularly, the saturated extent in MGDG increased obviously by 16.2% and 12.6% in T1-2 and T1-1, respectively. With stress temperature elevating, the maximum efficiency of photosystem Ⅱ （PSⅡ） photochemistry （Fv/Fm）, actual photochemical efficiency of PSll in the light （ФPSⅡ） and net photosynthetic rate （Pn） of the two lines and wild type tobacco plants decreased gradually, but those parameters decreased much less in transgenic plants. Even though the recovery process appeared differently in the donor and acceptor side of PSⅡ in transgenic tobacco compared with wild-type plants, the entire capability of PSⅡ recovered faster in transgenic tobacco, which was shown in the parameters of PI, Fv/Fm and ФPSⅡ, as a result, the recovery of Pn was accelerated. Conclusively, we proposed that the increase in saturated extent of thylakoid membrane lipids in transgenic plants enhanced the stability of photosynthetic apparatus under high temperature stress.