融合膜中PSⅡ激发的电子推动嵴膜的电子传递和磷酸化

利用毛地黄苷从菠菜叶绿体类囊体膜制备了PSⅡ颗粒,氧化还原差示光谱及SDS-聚丙烯酰胺凝胶电泳结果表明其具备PSⅡ的典型特征,它具有从水氧化到质醌还原的酶活性。从大豆磷脂用超声法制备了脂质体。从鼠肝线粒体分离了嵴膜。将制备的PSⅡ颗粒预组装于脂质体,然后将此预组装物(PSⅡ—PL)再与嵴膜组合,此膜系于光下获得了相当量的ATP合成,证明了融合膜中PSⅡ电子传递可推动嵴膜的电子传递和磷酸化机构合成ATP。     

铜离子在光系统Ⅱ电子传递中的作用部位和方式

铜离子对PS Ⅱ电子传递有明显的抑制作用,并且不能被加入人工电子供体DPC而恢复电子传递。铜离子表现出对胰蛋白酶消化叶绿体膜后使PS Ⅱ电子传递所受抑制有加成作用,并且铜离子又可拮抗胰蛋白酶对被DCMU阻止的PS Ⅱ电子传递的部分恢复作用。因而推测铜离子在PS Ⅱ的作用部位是在DPC供电子处至PS Ⅱ作用中心之间,其作用方式可能在于钝化了参与PS Ⅱ电子传递的膜蛋白。用SDS-PAGE对叶绿体膜蛋白的分离结果,也符合于这一假设。     

毕氏海蓬子类囊体膜与卵磷脂重组脂质体的耐热性研究

采用卵磷脂(PC)构建脂质体,然后将毕氏海蓬子类囊体膜蛋白复合物重组到脂质体中.分析不同温度(25℃、35℃、45℃和55℃)处理后蛋白脂质体的电子传递活性、吸收光谱和荧光光谱的变化,以探讨膜脂与膜蛋白在高温胁迫下的交互作用.结果显示:蛋白脂质体光系统Ⅱ(PSⅡ)的放氧活性和光系统Ⅰ(PSⅠ)的耗氧活性随着PC比例的提高而增加,在PC与类囊体膜比例为4∶1(Lipid∶Chl,w/w)时达到最高,同时蛋白脂质体的吸收光谱和荧光光谱也呈上升趋势;在PC与类囊体膜重组比例为4∶1条件下,高温处理后的蛋白脂质体的PSⅡ放氧活性和PSⅠ耗氧活性显著大于未经重组的,其吸收光谱和荧光光谱峰值下降幅度低于未经重组的,且峰位基本没有变化.研究表明,PC可能通过增加结合天线的大小来促进蛋白脂质体对光能的吸收和能量从外周天线到PSⅡ和PSⅠ核心复合物的传递;在脂质体中,PC与类囊体膜的交互作用提高了PSⅡ和PSⅠ在高温胁迫下的光化学效率,增强了PSⅡ和PSⅠ的耐热性.     

水分胁迫对玉米类囊体膜组分的影响

研究了水分胁迫对玉米（ZeamaysL,新玉4号）叶片类囊体膜的蛋白、色素组分,以及PSⅠ、PSⅡ和Cytb／f复合物的影响。类囊体膜蛋白电泳显示水分胁迫使总膜蛋白含量下降。高效液相色谱分析表明水分胁迫也引起类囊体膜色素成分的改变。通过测定P700及各种细胞色素的含量计算所得的PSⅠ、PSⅡ和Cytb／f复合物成分表明PSⅠ对水分胁迫不敏感,Cytb／f复合物在严重水分胁迫时才受到影响,而PSⅡ在中度和严重水分胁迫时均受到较明显的影响。     

黄瓜叶片光合电子传递对水分胁迫的响应

黄瓜叶片在水分胁迫下叶片相对含水量减少,类囊体室温吸收光谱的吸收峰降低,同时其NADP光还原活性、Ca^2 -ATPase活性也相应降低,全链电子传递明显受阻。类囊体膜蛋白电泳分析结果显示：类囊体膜色素蛋白复合体含量有不同程度的降低,其中PSⅡ色素蛋白复合体含量下降较多,试验结果表明水分胁迫通过限制光能的吸收,传递双及转换效率,抑制了光合电子传递过程。     

扬麦5号旗叶光合功能衰退进程中光合膜特性的变化

旗叶自然衰退过程中光合膜特性变化的结果表明,光合功能高值持续期类囊体膜电子传递活性均维持较高水平,多肽组分也维持相对稳定;进入光合功能的速降期后,活性呈快速下降趋势,类囊体膜小分子多肽等组分均出现不同降解。旗叶全展后叶绿体ＡＴＰ含量在高值持续期维持一定水平;进入速降期后,对应于光合膜电子传递活性及Ｐ／Ｏ值,叶绿体ＡＴＰ含量变化存在“滞后”的现象;强光逆境下,速降期类囊体电子传递活性受抑制程度比高值     

解联剂促进BBY膜颗粒电子传递机理的研究

透射电镜结果表明,具有ＰＳⅡ理化特征的ＢＢＹ膜颗粒在结构上不具备完整的类囊体膜特征．９－ＡＡ荧光猝灭与毫秒级Ｃｈｌα延迟发光的测定表明,ＢＢＹ膜颗粒在功能上难以形成．光致跨膜质子浓度差．在ＢＢＹ膜颗粒中,解联剂ｇｒａｍｉｃｉｄｉｎＤ（短杆菌肽〕和ＮＨ４Ｃｌ仅在低ｐＨ值时对ＰＳⅡ电子传递有所促进,ｐＨ６．０时促进尤为显著。两种解联剂促进的数值和对ｐＨ值的依赖特征基本一致,表明两者促进机制相同．综上所述,我们推测,解联剂在ＢＢＹ膜颗粒中并不促进跨越类囊体模的质子运转,而只是加速膜上微区内的质子转移,从而促进相关的电子传递。     

毕氏海蓬子光系统Ⅱ颗粒的分离与鉴定

采用去污剂TritonX-100增溶类囊体膜和高速离心的方法,首次分离和纯化了毕氏海蓬子的光系统Ⅱ（photosystemⅡ,PSⅡ）颗粒,通过光谱学和SDS-PAGE对其进行鉴定并与类囊体膜进行比较。室温吸收光谱结果表明,PSⅡ颗粒在蓝区的叶绿素（chlorophyll,ChOb和胡萝卜素类吸收峰为485nm,在红区的Ch1b吸收峰为655nm,这两个峰值均低于类囊体膜中的。77K荧光发射光谱结果表明,提取的PSⅡ颗粒基本不含光系统Ⅰ（photosystemⅠ,PSI）的低温荧光反射峰737nm。77K荧光激发光谱结果显示,海蓬子PSⅡ颗粒在470-485am之间的Ch1b 和胡萝卜素类的荧光发射峰明显低于类囊体膜的。这说明在PSⅡ中大部分的PSI已被除去。电泳结果显示,海蓬子PSⅡ颗粒缺少PSI反应中心蛋白质亚基PsaA和PsaB,这说明提取到的PSⅡ纯度较高,这为进一步研究毕氏海蓬子PSⅡ的结构与功能奠定基础。     

CaCl_2对UV-B辐射下菠菜叶片光合特性的影响

以"丹麦旺盛菠菜"为材料,通过UV-B和CaCl2复合处理,测定光合色素含量、Hill反应活力、叶绿素荧光、MDA含量和抗氧化酶活性等参数,探讨了CaCl2对UV-B辐射下菠菜叶片电子传递链和光合膜酶保护系统的影响。结果表明,UV-B处理下,光合色素含量、chl/car、类囊体膜上PSII潜在活性(Fv/Fo)、光化学淬灭系数(qP)、非光化学淬灭系数(qN)、PSII光量子产量(ΦPSⅡ)、原初光能转化效率(Fv/Fm),以及Hill反应活力等降低,chla/chlb和MDA含量升高;喷洒CaCl2可不同程度缓解UV-B的伤害。不同处理下,POD、SOD和CAT活性的变化呈现补偿效应。UV-B强度与菠菜叶片PSII功能受损程度呈正相关,CaCl2则主要通过提高chlb含量、类囊体膜上的光量子产量和POD活性,以缓解伤害。重度UV-B辐射下,CaCl2使chlb含量显著提高可能是导致PSII捕光效率提高的重要因素。     

睡莲和菠菜光合膜光化学活性及多肽组分的比较

比较分析了水生植物睡莲及陆生植物波菜类囊体膜ＰＳＩ,ＰＳⅡ电子传递活性,吸收光谱,室温荧光发射光谱等光化学特性及类囊体膜的多肽组分。结果显示：睡莲类囊体膜ＰＳＩ,ＰＳⅡ电子传递活性相对较弱,分别对菠菜的６０．２１％和７０．８２％,其室温吸收光谱蓝紫光区域吸收较弱,没有明显的吸收峰,红光区域的吸收光谱和菠菜相似;     

Photosynthetic Electron Transport During Senescence of the Primary Leaves of Phaseolus vulgaris L.: II. THE ACTIVITY OF PHOTOSYSTEMS ONE AND TWO, AND A NOTE ON THE SITE OF REDUCTION OF FERRICYANIDE

The activity of photosystems one and two (PS I and PS II) wasmeasured in chloroplasts isolated from the primary leaves ofPhaseolus vulgaris. During foliar senescence, the rates of electrontransport through PS I and PS II declined by approximately 25%and 33% respectively. These losses of activity could not accountfor the decrease of 80% in the rate of coupled, non-cyclic electrontransport during senescence. It is therefore suggested thatan impairment of electron flow between the photosystems limitednon-cyclic electron transport in chloroplasts from older leaves.In this study the activity of PS II was measured using oxidizedp-phenylenediamine as the electron acceptor, and trifluralinas an inhibitor of electron transport between PS II and PS I.In chloroplasts from young leaves the reduction of ferricyanidewas a measure of non-cyclic electron transport, but in preparationsfrom older leaves ferricyanide received a large proportion ofelectrons from PS II.     

Regulation of Cyclic Photophosphorylation during Ferredoxin-Mediated Electron Transport : Effect of DCMU and the NADPH/NADP Ratio

Addition of ferredoxin to isolated thylakoid membranes reconstitutes electron transport from water to NADP and to O₂ (the Mehler reaction). This electron flow is coupled to ATP synthesis, and both cyclic and noncyclic electron transport drive photophosphorylation. Under conditions where the NADPH/NADP⁺ ratio is varied, the amount of ATP synthesis due to cyclic activity is also varied, as is the amount of cyclic activity which is sensitive to antimycin A. Partial inhibition of photosystem II activity with DCMU (which affects reduction of electron carriers of the interphotosystem chain) also affects the level of cyclic activity. The results of these experiments indicate that two modes of cyclic electron transfer activity, which differ in their antimycin A sensitivity, can operate in the thylakoid membrane. Regulation of these activities can occur at the level of ferredoxin and is governed by the NADPH/NADP ratio.     

Growth and development of winter rye at cold-hardening temperatures results in thylakoid membranes with increased sensitivity to low concentrations of osmoticum

Chloroplasts developed at cold-hardening (5°C) and non-hardening temperatures (20°C) were compared with respect to the stability of photosynthetic electron transport activities, the capacity to produce and maintain a H⁺ gradient and the capacity fat photophosphorylation as a function of resuspension in the presence or absence of osmoticum. The results for electron transport indicate that whole chain, photosystem I and pfaotosystem II activities in non-hardened chloroplast thyalkoids were unaffected by resuspension in the presence of high or low osmoticum. In contrast, the same electron transport activities in cold-hardened chloroplast thylakoids exhibited a 3- to 4-fold decrease in activity when resuspended in the presence of low osmoticum. Impairment of electron transport through photosystem II of cold-hardened thylakoids resuspended in the presence of low osmoticum was supported by room temperature fluorescence induction kinetics. Since the presence of Mn²⁺ partially overcame this inhibition, it is concluded that this osmotically-induced inhibition of PSII activity in cold-hardened chloroplast thylakoids may, in part, be due to damage to the H₂O-splitting side of photosystem II. Both the initial rate and the maximum capacity for cyclic photophosphorylation were significantly inhibited in cold-hardened as compared to non-hardened thylakoids upon resuspension in the presence of low concentrations of osmoticum. This was correlated with an inability of the cold-hardened chloroplast thylakoids to maintain a significant transrnembrane H⁺ gradient. The results indicate that cold-hardened thylakoid membranes required an osmotic concentration (0.8 M) twice as high as non-hardened thylakoids (0.4 M) to produce the same initial rate of H⁺ uptake. In addition, the capacity to produce a proton gradient in cold-hardened thylakoids was less stable than that in non-hardened thylakoids regardless of the osmotic concentration tested. It is concluded that development of rye thylakoid membranes at low temperature results in a differential sensitivity to low osmoticum and thus extreme caution should be exercised when comparing the structure and function of isolated thylakoids developed under contrasting thermal regimes.     

Enhanced thermal tolerance of photosynthesis and altered chloroplast ultrastructure in a mutant of Arabidopsis deficient in lipid desaturation

A mutant of Arabidopsis thaliana, deficient in activity of the chloroplast n-6 desaturase, accumulated high levels of C_16:1 and C_18:1 lipids and had correspondingly reduced levels of polyunsaturated lipids. The altered lipid composition of the mutant had pronounced effects on chloroplast ultrastructure, thylakoid membrane protein and chlorophyll content, electron transport rates, and the thermal stability of the photosynthetic membranes. The change in chloroplast ultrastructure was due to a 48% decrease in the amount of appressed membranes that was not compensated for by an increased amount of nonappressed membrane. This resulted in a net loss of 36% of the thylakoid membrane per chloroplast and a corresponding reduction in chlorophyll and protein content. Electrophoretic analysis of the chlorophyll-protein complexes further revealed a small decrease in the amount of light-harvesting complex. Relative levels of whole chain and protosystem II electron transport rates were also reduced in the mutant. In addition, the mutation resulted in enhanced thermal stability of photosynthetic electron transport. These observations suggest a central role of polyunsaturated lipids in determining chloroplast structure and maintaining normal photosynthetic function and demonstrate that lipid unsaturation directly affects the thermal stability of photosynthetic membranes.     

Kinetics of electron transport, proton transfer and photophosphorylation in chloroplasts and their relation to temperature-induced structural changes in the thylakoid membrane

Effects of various temperatures on the rates of electron transport between two photosystems, the light-induced uptake of protons, kinetics of proton efflux from the chloroplasts in the dark and photophosphorylation were studied in isolated chloroplasts. There are correlations between the physical state of thylakoid membrane and the rates of electron- and proton transport processes. The temperature dependence of "structural" parameter (fluidity of lipids in membrane) as well as the rates of electron- and proton transport processes reveal the breaks under the same temperatures. Stimulation of photophosphorylation by temperature increasing correlates with the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the heat activation of chloroplasts latent ATPase due to thermoinduced structural changes in the protein part of CF0-CF1 complex. The rate of photophosphorylation also correlates with the physical state of membrane lipids. Thermoinduced "melting" of the thylakoid membrane inhibits the ATP formation because of a decrease in photosystem 2 photochemical activity and stimulation of membrane conductivity for protons.     

Photosynthetic Electron Transport During Senescence of the Primary Leaves of Phaseolus vulgaris L.: I. NON-CYCLIC ELECTRON TRANSPORT

Coupled, non-cyclic electron transport was measured for chloroplastsisolated from the primary leaves of Phaseolus vulgaris. Preparationsfrom young, fully expanded leaves gave good rates of electrontransport, but the rates obtained decreased by approximately80% during leaf senescence. Higher rates of electron transportwere recorded for chloroplasts isolated from primary leaveswhich had regreened following removal of the remainder of theshoot. With preparations from leaves of all ages, photophosphorylationwas coupled to electron transport with a mean P/2e ratio ofapproximately 1.3. No evidence was obtained for inactivationof chloroplasts from older leaves during isolation or assay,and it is suggested that the decrease in rate of electron transportover the period of senescence, and its increase during regreening,were consequences of changes in the composition and physicalproperties of the thylakoid membrane which occur in vivo. Thedecrease in rate of non-cyclic electron transport may be importantin limiting the rate of photosynthesis in the senescing leaves.     

Influence of photoinhibition on electron transport and photophosphorylation of isolated chloroplasts

The effects of a photoinhibition treatment (PIT) on electron transport and photophosphorylation reactions were measured in chloroplasts isolated from triazine-resistant and susceptible Chenopodium album plants grown under high and low irradiance. Electron transport dependent on photosystem I (PSI) alone was much less affected by PIT than that dependent on both photosystem II (PSII) and PSI. There was a smaller difference in susceptibility to PIT between the photophosphorylation activitity dependent on PSI alone and that dependent on both PSII and PSI. Because in all cases photophosphorylation activity decreased faster upon PIT than the rate of electron transport, we conclude that photoinhibition causes a gradual uncoupling of electron transport with phosphorylation. Since the extent of the light-induced proton gradient across the thylakoid membrane decreased upon PIT, it is suggested that photoinhibiton causes a proton leakiness of the membrane. We have found no significant differences to PIT of the various reactions measured in chloroplasts isolated from triazine-resistant and susceptible plants. We have also not observed any significant differences to PIT of the photophosphorylation reactions in chloroplasts of plants grown under low irradiance, compared with those grown under high irradiance. However, the electron transport reactions in chloroplasts from plants grown under low irradiance appeared to be somewhat less sensitive to PIT than those grown under high irradiance.     

Damage to the chloroplast H+-ATPase complex by low concentrations of glutaraldehyde

The energy-dependent processes coupled to electron transport were studied in isolated pea chloroplasts treated with low concentrations (1-5 mM) of glutaraldehyde (GA) in the dark and in the light sufficient to cause energization of the membrane. After GA treatment the chloroplasts exhibited a strong suppression of cyclic and non-cyclic phosphorylation, coupled (+ADP+Pi) electron transport and diminution of the light-activated Mg2+-ATPase activity. The rate of basal electron transport was unaffected. The GA-treated chloroplasts were found to retain the capacity to form the osmotic component of the transmembrane potential. These data and the results of the effect of florizine and ATP on electron transport suggest that the effect of GA on energy transduction processes associated with photophosphorylation may consist in its action on reversible H+-ATPase. In light-adapted samples treated with GA the data characterizing the formation of a high energy state (rate of photophosphorylation, steady-state level of photo-induced quenching of atebrin fluorescence and its dark recovery; photo-induced absorbance changes at 520 nm; rate of the slow phase of delayed fluorescence increment) appear to be changed to a greater extent as compared to the dark-adapted samples. The observed changes may arise from a greater conductivity of thylakoid membranes due to fixation of the H+-ATPase proton channel in the "open" state.     

Demonstration of Two Sites of Inhibition of Electron Transport by Protein Phosphorylation in Wheat Thylakoids

The effect of protein phosphorylation on electron transportactivities of thylakoids isolated from wheat leaves was investigated.Protein phosphorylation resulted in a reduction in the apparentquantum yield of whole chain and photosystem II (PSII) electrontransport but had no effect on photosystem I (PSI) activity.The affinity of the D1 reaction centre polypeptide of PSII tobind atrazine was diminished upon phosphorylation, however,this did not reduce the light-saturated rate of PSII electrontransport. Phosphorylation also produced an inhibition of thelight-saturated rate of electron transport from water or durohydroquinoneto methyl viologen with no similar effect being observed onthe light-saturated rate of either PSII or PSI alone. This suggeststhat phosphorylation produces an inhibition of electron transportat a site, possibly the cytochrome b₆/f complex, between PSIIand PSI. This inhibition of whole-chain electron transport wasalso observed for thylakoids isolated from leaves grown underintermittent light which were deficient in polypeptides belongingto the light-harvesting chlorophyll-protein complex associatedwith photosystem II (LHCII). Consequently, this phenomenon isnot associated with phosphorylation of LCHII polypeptides. Apossible role for cytochrome b₆/f complexes in the phosphorylation-inducedinhibition of whole chain electron transport is discussed. Key words: Electron transport, light harvesting, photosystem 2, protein phosphorylation, thylakoid membranes, wheat (Triticum aestivum)     

Freezing damage and frost tolerance of the photosynthetic apparatus studied with isolated mesophyll protoplasts of Valerianella locusta L.

Mesophyll protoplasts were isolated from unhardened and cold-acclimated leaves of Valerianella locusta L. and subjected to freeze-thaw treatment. To evaluate the extent and course of freezing injury, photosynthetic reactions of whole protoplasts and of free thylakoid membranes, liberated from protoplasts by osmotic lysis, were measured. In addition, the integrity of the protoplasts was determined by microscopy. The results reveal an increased frost tolerance of protoplasts isolated from acclimated leaves with respect to all parameters measured. CO₂-dependent O₂ evolution (representing net photosynthetic CO₂ fixation of protoplasts) was the most freezing-sensitive reaction; its inhibition due to freeze-thaw treatment of protoplasts was neither correlated with disintegration of the plasma membrane, nor was it initiated by inactivation of the thylakoid membranes. The frost-induced decline of protoplast integrity was not closely correlated to thylakoid damage either. Freezing injury of the thylakoid membranes was manifested by inhibition of photosynthetic electron transport and photophosphorylation. Both photosystems were affected by freezing and thawing with strongest inhibition occurring in the water-oxidation system or at the oxidizing site of photosystem II. Photophosphorylation responded more sensitively to freezing stress than electron transport, although uncoupling (increased permeability of the thylakoid membranes to protons) was not a conspicuous effect. The data are discussed in relation to freezing injury in leaves and seem to indicate that frost damage in vivo is initiated at multiple sites.Abbreviations Chl chlorphyll - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DCIP 2,6-dichlorophenolindophenol - DPC 1,5-diphenylcarbazide - Hepes 2-[4-(2-hydroxyethyl)-1-piperazinyl]-ethanesulfonic acid - MES 2-(N-morpholino)-ethanesulfonic acid - PS I photosystem I - PS II photosystem II