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。

Conformational changes in photosynthetic pigment proteins on thylakoid membranes can lead to fast non-photochemical quenching in cyanobacteria

A high non-photochemical quenching(NPQ) appeared below the phase transition temperature when Microcystis aeruginosa PCC7806 cells were exposed to saturated light for a short time.This suggested that a component of NPQ,independent from state transition or photo-inhibition,had been generated in the PSII complex;this was a fast component responding to high intensity light.Glutaraldehyde(GA),commonly used to stabilize membrane protein conformations,resulted in more energy transfer to PSII reaction centers,affecting the energy absorption and dissipation process rather than the transfer process of phycobilisome(PBS).In comparison experiments with and without GA,the rapid light curves(RLCs) and fluorescence induction dynamics of the fast phase showed that excess excitation energy was dissipated by conformational change in the photosynthetic pigment proteins on the thylakoid membrane(PPPTM).Based on deconvolution of NPQ relaxation kinetics,we concluded that the fast quenching component(NPQ f) was closely related to PPPTM conformational change,as it accounted for as much as 39.42% of the total NPQ.We hypothesize therefore,that NPQ f induced by PPPTM conformation is an important adaptation mechanism for Microcystis blooms under high-intensity light during summer and autumn.

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.

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.

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.

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。

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.

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.

Changes in Unsaturated Levels of Fatty Acids in Thylakoid PSII Membrane Lipids During Chilling-induced Resistance in Rice

Temperature is one of the abiotic factors limiting growth and productivity of plants. In the present work, the effect of low non-freezing temperature, as an inducer of ＂chilling resistance＂, was studied in three cultivars of rice （Oryza sativa L.）, japonica cv. 9516 （j-9516）, the two parental lines of superhigh-yield hybrid rice between subspecies, Peiai/E32 （ji-PE）, and the traditional indica hybrid rice Shanyou 63 （i-SY63）. Leaves of chill-treated rice showed chilling-induced resistance, as an increase of their low-temperature tolerance was measured using chlorophyll fluorescence measurements, revealing a change in photosystem II （PSII） efficiency. After 5 d of exposure to 11 ~C under low light （100 pmol·m^-2·s^-1）, levels of unsaturated fatty acids in PSII thylakoid membrane lipids decreased during the initial 1-2 d, then increased slowly and reached 99.2%, 95.3% and 90.1% of the initial value （0 d） in j-9516, ji-PE and i-SY63, respectively, on the fifth day. However, under medium light （600 μmol·m^-2·s^-1）, all cultivars experienced similar substantial photoinhibition, which approached steady state levels after a decline in levels of unsaturated fatty acids in PSII thylakoid membrane lipids to about 57.1%, 53.8% and 44.5% of the initial values （0 d） in j-9516, ji-PE and i-SY63 on the fifth day. Under either chilling-induced resistance （the former） or low temperature photoinhibition （the latter） conditions, the changes of other physiological parameters such as D1 protein contents, electron transport activities of PSII （ETA）, Fv/Fm, xanthophyl cycle activities expressed by DES （deepoxide state） were consistent with that of levels of unsaturated fatty acids in PSII thylakoid membrane lipids. So there were negative correlations between saturated levels of fatty acids （16：1（3t）, 16：0, 18：0）, especially the 16：1（3t） fatty acid on thylakoid membrane and other physiological parameters, such as D1 protein contents, ETA and （A＋Z）/（A＋V＋Z）. A specific role of desaturation of fatty acids and the photoprotective pigments of the xanthophyl cycle, leading to an acclimation response in thylakoid membrane lipids may be involved. We conclude that chilling-induced resistance is accelerated by the unsaturation of thylakoid membranes, and the ability of rice plants to cold-harden can be enhanced by genetic engineering.

Pitt, a Novel Tetratricopeptide Repeat Protein Involved in Light-Dependent Chlorophyll Biosynthesis and Thylakoid Membrane Biogenesis in Synechocystis sp. PCC 6803

Biogenesis of photosynthetic pigment/protein complexes is a highly regulated process that requires various assisting factors. Here, we report on the molecular analysis of the Pitt gene （sir1644） from the cyanobacterium Synechocystis sp. PCC 6803 （Synechocystis 6803） that encodes a membrane-bound tetratricopeptide repeat （TPR） protein of formerly unknown function. Targeted inactivation of Pitt affected photosynthetic performance and light-dependent chlorophyll synthesis. Yeast two-hybrid analyses and native PAGE strongly suggest a complex formation between Pitt and the light-dependent protochlorophyllide oxidoreductase （POR）. Consistently, POR levels are approximately threefold reduced in the pitt insertion mutant. The membrane sublocalization of Pitt was found to be dependent on the presence of the periplasmic photosystem Ⅱ （PSⅡ） biogenesis factor PratA, supporting the idea that Pitt is involved in the early steps of photosynthetic pigment/protein complex formation.

A Thylakoid Membrane Protein Functions Synergistically with GUN5 in Chlorophyll Biosynthesis

Chlorophyll(Chl)is essential for photosynthetic reactions and chloroplast development.While the enzymatic pathway for Chl biosynthesis is well established,the regulatory mechanism underlying the homeostasis of Chl levels remains largely unknown.In this study,we identified CBD1(Chlorophyll Biosynthetic Defect1),which functions in the regulation of chlorophyll biosynthesis.The CBD1 gene was expressed specifically in green tissues and its protein product was embedded in the thylakoid membrane.Furthermore,CBD1 was precisely co-expressed and functionally correlated with GUN5(Genome Uncoupled 5).Analysis of chlorophyll metabolic intermediates indicated that cbd1 and cbd1gun5 mutants over-accumulatedmagnesium protoporphyrin Ⅸ(Mg-Proto Ⅸ).In addition,the cbd1 mutant thylakoid contained less Mg than the wild type not only as a result of lower Chl content,but also implicating CBD1 in Mg transport.This was supported by the finding that CBD1 complemented a Mg^(2+)uptake-deficient Salmonella strain under low Mg conditions.Taken together,these results indicate that CBD1 functions synergistically with CHLH/GUN5 in Mg-Proto IX processing,and may serve as a Mg-transport protein to maintain Mg homeostasis in the chloroplast.

Nucleus-Encoded Light-Harvesting Chlorophyll a/b Proteins are Imported Normally into Chlorophyll b-Free Chloroplasts of Arabidopsis

Chloroplast-located proteins which are encoded by the nuclear genome have to be imported from the cytosol into the organelle in a posttranslational manner. Among these nuclear-encoded chloroplast proteins are the light- harvesting chlorophyll a/b-binding proteins （LHCPs）. After translation in the cytosol, precursor proteins of LHCPs are imported via the TOC/TIC translocase, processed to their mature size to insert into thylakoid membranes where they recruit chlorophylls a and b to form pigment-protein complexes. The translocation of proteins is a highly regulated process which employs several regulators. To analyze whether CAO （chlorophyll a oxigenase） which converts chlorophyll a to chlorophyll b at the inner chloroplast membrane, is one of these regulators, we performed import reactions utilizing a homozygous loss-of-function mutant （cao-1）. We imported in vitro translated and 35S-labeled precursor proteins of light- harvesting proteins of photosystem II LHCB1, LHCB4, and LHCB5 into chloroplasts isolated from cao-1 and show that import of precursor proteins and their processing to mature forms are not impaired in the mutant. Therefore, regulation of the import machinery cannot be responsible for the decreased steady-state levels of light-harvesting complex （LHC） proteins. Regulation does not take place at the transcriptional level either, because Lhcb mRNAs are not down-regulated. Additionally, reduced steady-state levels of LHCPs also do not occur due to posttranslational turnover of non-functional LHCPs in chloroplasts. Taken together, our data show that plants in the absence of CAO and therefore devoid of chlorophyll b are not influenced in their import behavior of LHC proteins.

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.

Towards Characterization of the Chloroplast NAD（P）H Dehydrogenase Complex

The NAD（P）H dehydrogenase （NDH） complex in chloroplast thylakoid membranes functions in cyclic electron transfer, and in chlororespiration. NDH is composed of at least 15 subunits, including both chloroplast- and nuclear-encoded proteins. During the past few years, extensive proteomic and genetic research on the higher plant NDH complex has been carried out, resulting in identification of several novel nuclear-encoded subunits. In addition, a number of auxiliary proteins, which mainly regulate the expression of chloroplast-encoded ndh genes as well as the assembly and stabilization of the NDH complex, have been discovered and characterized. In the absence of detailed crystallographic data, the structure of the NDH complex has remained obscure, and therefore the role of several NDH-associated nuclear-encoded proteins either as auxiliary proteins or structural subunits remains uncertain. In this review, we summarize the current knowledge on the subunit composition and assembly process of the chloroplast NDH complex. In addition, a novel oligomeric structure of NDH, the PSI/NDH supercomplex, is discussed.

Alb4 of Arabidopsis Promotes Assembly and Stabilization of a Non Chlorophyll-Binding Photosynthetic Complex, the CF1CF0-ATP Synthase

All members of the YidC/Oxal/Alb3 protein family are evolutionarily conserved and appear to function in membrane protein integration and protein complex stabilization. Here, we report on a second thylakoidal isoform of Alb3, named Alb4. Analysis of Arabidopsis knockout mutant lines shows that AIb4 is required in assembly and/or stability of the CF1CF0-ATP synthase （ATPase）. alb4 mutant lines not only have reduced steady-state levels of ATPase subunits, but also their assembly into high-molecular-mass complexes is altered, leading to a reduction of ATP synthesis in the mutants. Moreover, we show that Alb4 but not AIb3 physically interacts with the subunits CF1β and CF0ll. Summarizing, the data indicate that AIb4 functions to stabilize or promote assembly of CF1 during its attachment to the membrane-embedded CF0 part.

Response of linear and cyclic electron flux to moderate high temperature and high light stress in tomato

Objective： To evaluate the possible photoprotection mechanisms of cyclic and linear electron flux （CEF and LEF） under specific high temperature and high light （HH） stress. Methods： Six-leaf-stage tomato seedlings （＂Liaoyuanduoli＂, n=160） were divided into four parts： Part 1, served as control under 25 ℃, 500 pmol/（m2-s）; Part 2, spayed with distilled water （H20） under 35 ℃, 1000 pmol/（m2.s） （HH）; Part 3, spayed with 100 μmol/L diuron （DCMU, CEF inhibitor） under HH; Part 4, spayed with 60 pmol/L methyl viologen （MV, LEF inhibitor） under HH. Energy conversion, photosystem I （PSI）, and PSII activity, and trans-thylakoid membrane proton motive force were monitored during the treatment of 5 d and of the recovering 10 d. Results： HH decreased photochemical reaction dissipation （P） and the maximal photochemical efficiency of PSII （Fv/Fm）, and increased the excitation energy distribution coefficient of PSII （,8）; DCMU and MV aggravated the partition imbalance of the excitation energy （y） and the photoinhibition degree. With prolonged DCMU treatment time, electron transport rate and quantum efficiency of PSI （ETR= and Y1） significantly decreased whereas acceptor and donor side limitation of PSI （YNA and YND） increased. MV led to a significant decline and accession of yield of regulated and non-regulated energy Y NPQ and YNO, respectively. Membrane integrity and ATPase activity were reduced by HH stress, and DCMU and MV enhanced inhibitory actions. Conclusions： The protective effects of CEF and LEF were mediated to a certain degree by meliorations in energy absorption and distribution as well as by maintenance of thylakoid membrane integrity and ATPase activity.

Dissecting the Native Architecture and Dynamics of Cyanobacterial Photosynthetic Machinery

The structural dynamics and flexibility of cell membranes play fundamental roles in the functions of the cells, i.e., signaling, energy transduction, and physiological adaptation. The cyanobacterial thylakoid membrane represents a model membrane that can conduct both oxygenic photosynthesis and respiration simultaneously. In this study, we conducted direct visualization of the global organization and mobility of photosynthetic complexes in thylakoid membranes from a model cyanobacterium, Synechococcus elongatus PCC 7942, using high-resolution atomic force, confocal, and total internal reflection fluorescence microscopy. We visualized the native arrangement and dense packing of photosystem I （PSI）, photosystem II （PSlI）, and cytochrome （Cyt） befwithin thylakoid membranes at the molecular level. Furthermore, we func- tionally tagged PSI, PSlI, Cyt bef, and ATP synthase individually with fluorescent proteins, and revealed the heterogeneous distribution of these four photosynthetic complexes and determined their dynamic features within the crowding membrane environment using live-cell fluorescence imaging. We characterized red light-induced clustering localization and adjustable diffusion of photosynthetic complexes in thylakoid membranes, representative of the reorganization of photosynthetic apparatus in response to environmental changes. Understanding the organization and dynamics of photosynthetic membranes is essential for rational design and construction of artificial photosynthetic systems to undarpin bioenergy development. Knowledge of cyanobacterial thylakoid membranes could also be extended to other cell membranes, such as chloroplast and mitochondrial membranes.

Induction of Apoptosis in Purified Nuclei from Tobacco-Suspension Cells by Cytochrome b_6/f Complex

An apoptotic cell-free system containing cytosol and nuclei from normally cultured tobacco sus-pension cells was used to show that a spinach chloroplast preparation can induce apoptosis in nuclei, evi-denced by DNA electrophoresis and fluorescence microscopy observations. Further study showed that the chloroplast preparation or its pellet (thylakoid membrane) after hypoosmotic or supersonic treatment still ex-hibited the apoptosis-inducing activity, but the supernatant had no effect, which indicates that the apoptosis-inducing effector in the chloroplast preparation is water-insoluble. The induction of apoptosis by chloroplast preparation could be attenuated by Ac-DEVD-CHO, the specific inhibitor of Caspase-3, implying involve-ment of a Caspase-3-like protease during the process. Furthermore, extensive apoptosis in nuclei was in-duced by cytochrome b6/f on the thylakoid membrane, indicating that this important cytochrome complex may have an important role in the chloroplast-related apoptotic pathway.