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.     

杂色藻类硅藻和金藻的77K荧光特性

实验测定了7种杂色藻包括:4种硅藻三角褐指藻(Phaeodactylum tricornutum)、牟氏角毛藻(Cheatoceros mulleri)、中肋骨条藻(Skeletonema costatum)、尖刺拟菱形藻(Pseudo-nitzschia pungens)和3种金藻湛江等鞭金藻(Isochrysis zhanjiangensis)、绿色巴夫藻(Pavlova viridis)、球形棕囊藻(Phaeocystisglobosa)的77 K荧光光谱,并与其它杂色藻以及高等植物菠菜进行了比较。结果表明,4种硅藻和3种金藻的77 K低温荧光发射光谱相似,只有一个明显的荧光发射主峰位于684~686 nm,类囊体膜的荧光光谱中在700 nm附近有个不明显的肩峰,它们均没有高等植物作为PSⅠ特征的730 nm长波荧光发射峰,这个结果与杂色藻类褐藻的荧光特性一致。说明,杂色藻类PSⅠ缺少730 nm长波荧光峰可能具有其普遍性,预示杂色藻类在PSⅠ结构以及能量传递等方面与高等植物具有很大的差异。     

单半乳糖甘油二脂缺失对烟草光合特性的影响

以单半乳糖甘油二脂(MGDG)相对含量比野生烟草显著降低的突变体(M18)及野生型烟草为材料,通过对转基因烟草叶绿体类囊体膜的低温荧光、放氧活性以及叶片的叶绿素荧光分析,研究MGDG部分缺失对烟草叶片光合特性的影响。结果表明,在低温下(77K)MGDG部分缺失并不影响烟草叶绿素荧光发射峰(F683和F730)的位置,但使光系统Ⅱ(PSⅡ)及光系统Ⅰ(PSⅠ)的荧光发射峰的强度减弱,F683/F730比值降低,直接影响激发能在PSⅡ和PSⅠ之间的均衡分配,使叶绿素a和叶绿素b之间的能量传递受阻,降低光能转化效率;MGDG部分缺失使PSⅡ放氧活性下降了72.9%;转基因烟草叶绿素荧光参数中最大光化学效率(Fv/Fm)、暗适应最大荧光(Fm)、实际光化学效率(Yield)、光化学猝灭系数(qP)比野生型烟草分别降低了7%、49%、32%和18%,并以Fm降幅最大。研究证明,MGDG在维持植物叶绿体类囊体膜的功能,特别是PSⅡ的功能方面起着重要的作用。     

叶绿体膜的结构与功能——ⅩⅦ.LHCP的蛋白磷酸化及其在膜上的横向移动

捕光叶绿素a/b蛋白复合体的蛋白磷酸化使叶绿体低温荧光(77K)在735 nm处的发射增强,间质片层膜的叶绿素a/b比值降低。聚丙烯酰胺凝胶电泳分析表明:LHCP蛋白磷酸化引起部分LHCP在膜上不仅以单体,而且以二聚体、三聚体的形式从富含PSⅡ的基粒膜横向移动到富含PS Ⅰ的间质膜,并与PS Ⅰ相结合,作为它的外周天线,扩大了PS Ⅰ的捕光面积,从而使激发能分配有利于PS Ⅰ。     

褐藻裙带菜光系统Ⅰ的能量传递特性

高等植物的PSⅠ复合物在 77K条件下有 730nm长波发射峰,这个荧光峰是PSⅠ的重要表征。       

低温对水稻类囊体膜蛋白磷酸化及光合机构光能分配的影响

研究了低温胁迫对水稻类囊体膜蛋白磷酸化和光能分配的影响。类囊体膜蛋白组分的SDS-PAGE和免疫印迹分析结果显示,低温弱光条件下光系统Ⅱ(PSⅡ)功能蛋白的稳态水平均有所降低。低温(77K)荧光分析表明,低温处理后类囊体膜光能吸收明显下降,而且FPSⅡ/FPSⅠ的比值均较对照组下降,表明低温弱光条件下有更多的激发能被分配到PSⅠ。低温处理同时还改变了类囊体膜蛋白磷酸化水平,捕光天线LHCⅡ蛋白中lhcb1的磷酸化水平明显降低,lhcb2的磷酸化水平增加,进一步证实lhcb2向PSⅠ移动,改变光能分配。PSⅡ反应中心D1、D2蛋白和核心天线CP43的磷酸化水平增高,有利于PSⅡ二聚体的稳定。     

褐藻77 K荧光特异性研究

测定了裙带菜、叉开网地藻、海带、囊藻、海蒿子、鼠尾藻、萱藻和水云等8种褐藻的77K荧光光谱并同菠菜和红藻条斑紫菜作了比较。结果表明与红藻和高等植物明显不同,褐藻没有作为PSⅠ特征的730 nm荧光峰。按荧光主峰的波长,可以分为二种类型：裙带菜、叉开网地藻、海带和囊藻的荧光主峰位于690 nm,海蒿子、萱藻、水云和鼠尾藻的荧光主峰在705-720 nm。这种77K荧光特异性预示褐藻同高等植物之间在PSⅠ结构上的差异。     

光系统Ⅱ超分子复合物的放氧活性分析

以高等植物大量捕光色素蛋白复合物(major light harvesting complex Ⅱ,LHCⅡb)对光系统Ⅱ(photosystem Ⅱ,PSⅡ)放氧活性的影响为研究对象,从豌豆类囊体膜中分步提取出三种PSⅡ蛋白复合物,结合电泳和光谱分析,鉴定出三种复合物的主要区别是LHCⅡb含量不同.对于三种PSⅡ色素...     

尖叶拟船叶藓的77K荧光光谱及对强光照的短期适应

报道了东亚特有濒危植物尖叶拟船叶藓(Dolichomitriopsis diversiformis)在不同光质的光照诱导下的低温77K荧光光谱及状态转移的初步研究结果,实验中,尖叶拟船叶藓在77K下出现了3条发射带,分别是F680、F685、F720nm,并没有出现存在于大部分高等植物中的F695nm和F740nm两个峰．经过PSⅡ光诱导后、在77K下出现了F680nm,这个峰在77K下出现是首次报道,而以前的研究认为只在4K下才出现这一条光谱带,这一结果表明尖叶拟船叶藓叶绿体的两个光系统结构与其他高等植物存在着差异。在自然光下,PSⅡ与PSⅠ的总能量比是2.04,经过15min的PSⅡ光(670nm)诱导后,PSⅡ与PSⅠ的总能量比变成了1．28(状态2),当用15min的PSⅠ光(716nm)照射后,PSⅡ与PSⅠ的总能量比从2．04变成了3．4l(状态1)。在自然光下,由尖叶拟船叶藓的光系统的外部LHCⅡ所吸收的激发能是整个光系统激发能的21．19％．这说明尖叶拟船叶藓对光的短期调节能力是21．19％．尖叶拟船叶藓的光系统的外部LHCⅡ有51．7％位于PSⅡ中,48．3％在PSⅠ中.     

瞬时与延迟叶绿素荧光及820nm光反射动力学同步测量技术在光合作用研究中的应用

在光合作用研究中,通过分析瞬时叶绿素荧光诱导动力学曲线,可以获得围绕光系统Ⅱ(photosystemⅡ,PSⅡ)发生的原初光化学反应的信息。通过分析延迟叶绿素荧光诱导动力学和衰减动力学曲线,可以探寻发生电荷重组而产生延迟荧光的不同基团,从而更加直接地了解PSⅡ的状态。而820 nm光反射可以用来检测发生在光系统Ⅰ(photosystemⅠ,PSⅠ)的原初光化学反应。文章简要介绍了这三种动力学的发生原理及其在光合作用研究中的优缺点,并举例说明了瞬时荧光、延迟荧光及820 nm光反射动力学同步测量技术在光合作用研究中的应用,以及三者之间的互补与印证作用。     

The Chlorophyll-Protein Complexes Resolved from Ultrasonically Broken Thylakoid Memb-ranes of Blue-Green Algae

When the thylakoid membranes of blue-green algae were broken by ultrasonic vibrations and subjected to polyacrylamide gel electrophoresis at 4℃, six green zones were resolved. They were designated as CPIa, CPlb, CPI; CPal, CPa2, and FC. The absorption spectrum of CPI had a red maximum at 674 nm and a peak in the blue at 435 nm. It was identified as PS chlorophyll a-protein Complex, but was contaminated with minor PSⅡ which was implied by the appearance of fluorescence emission peak at 680 nm besides the main one at 725 nm at 77 K. The spectral properties of CPIa and CPlb were similar to that of CPl. The absorption spectra of CPa1 and CPa2 were similar, both having red maxima at 667 nm and peaks in the blue at 431.5 nm. Their fluorescence emission had the same peaks at 684 nm at 77 K indicating that they belonged to PSⅡ. It was recognized that CPal of 47 kD is the reaction center complex of photosystem Ⅱ and CPa2 of 40 kD is the internal antenna complex of photosystem Ⅱ. The spectral characteristics of the chlorophyll-protein complexes resolved by ultrasonic method were similar to those of the same complexes resolved by SDS solubilization, except the absorbance positions of CPa1 and CPa2 in the blue peak and the red one which shifted to blue about 3–5 nm. It was calculated that in thylakoid membranes of blue-green algae 40.93% chlorophyll was in PSⅠ, while 38.78% of chlorophyll in PSⅡ. The difference of chlorophyll contents between PSⅠ and PSⅡ was only 2.15%. Concerning the fact that minor PSⅡ compound remained in the part of PSⅠ zones, it might be concluded that the distribution of chlorophyll between PSⅠ and PSⅡ in blue-green algae was equal. This result was in agreement with the hypothesis that PSⅠ and PSⅡ operates in series in photosynthetic electron transport.     

Hg2+对菠菜离体类囊体膜光化学活性和多肽组分的影响

重金属Ｈｇ＾２＋对菠菜（Ｓｐｉｎａｃｉａ ｏｌｅｒａｃｅａ Ｌ．）离体类囊体膜的光合电子传递活性、室温吸收光谱、室温荧光发射光谱以及多肽组分影响的研究结果表明：Ｈｇ＾２＋对两个光系统的电子传递活性都有抑制作用,且Ｈｇ＾２＋对ＰＳＩ的抑制作用较ＰＳⅡ大;Ｈｇ＾２＋处理使类囊体膜的室温吸收光谱峰及室温荧光发射峰降低,但未使类囊体膜的多肽组分发生改变。     

Interaction of phycobilisomes with photosystem II dimers and photosystem I monomers and trimers in the cyanobacterium Spirulina platensis.

Distribution of phycobilisomes between photosystem I (PSI) and photosystem II (PSII) complexes in the cyanobacterium Spirulina platensis has been studied by analysis of the action spectra of H2 and O2 photoevolution and by analysis of the 77 K fluorescence excitation and emission spectra of the photosystems. PSI monomers and trimers were spectrally discriminated in the cell by the unique 760 nm low-temperature fluorescence, emitted by the trimers under reductive conditions. The phycobilisome-specific 625 nm peak was observed in the action spectra of both PSI and PSII, as well as in the 77 K fluorescence excitation spectra for chlorophyll emission at 695 nm (PSII), 730 nm (PSI monomers), and 760 nm (PSI trimers). The contributions of phycobilisomes to the absorption, action, and excitation spectra were derived from the in vivo absorption coefficients of phycobiliproteins and of chlorophyll. Analyzing the sum of PSI and PSII action spectra against the absorption spectrum and estimating the P700:P680 reaction center ratio of 5.7 in Spirulina, we calculated that PSII contained only 5% of the total chlorophyll, while PSI carried the greatest part, about 95%. Quantitative analysis of the obtained data showed that about 20% of phycobilisomes in Spirulina cells are bound to PSII, while 60% of phycobilisomes transfer the energy to PSI trimers, and the remaining 20% are associated with PSI monomers. A relevant model of organization of phycobilisomes and chlorophyll pigment-protein complexes in Spirulina is proposed. It is suggested that phycobilisomes are connected with PSII dimers, PSI trimers, and coupled PSI monomers.     

Chlorophyll-protein composition of the thylakoid membrane from Prochlorothrix hollandica, a prokaryote containing chlorophyll b

The chlorophyll-protein complexes of the thylakoid membrane from Prochlorothrix hollandica were identified following electrophoresis under nondenaturing conditions. Five complexes, CP1-CP5, were resolved and these green bands were analyzed by spectroscopic and immunological methods. CP1 contains the photosystem I (PSI) reaction center, as this complex quenched fluorescence at room temperature, and had a 77 K fluorescence emission peak at 717 nm. CP4 contains the major chlorophyll-a-binding proteins of the photosystem II (PSII) core, because this complex contained polypeptides which cross-reacted to antibodies raised against Chlamydomonas PSII proteins 5 and 6. Furthermore, fluorescence excitation studies at 77 K indicated that only a Chl a is bound to CP4. Complexes CP2, CP3 and CP5 contained functionally bound Chl a and b as judged by absorption spectroscopy at 20 degrees C and fluorescence excitation spectra at 77 K. CP2, CP3 and CP5 all contain polypeptides of 30-33 kDa which are immunologically distinct from the LHC-II complex of higher plant thylakoids.     

Quantitative analysis of 77K fluorescence emission spectra in Synechocystis sp. PCC 6714 and Chlamydomonas reinhardtii with variable PS I/PS II stoichiometries

Low-temperature (77 K) fluorescence emission spectra of intact cells of a cyanobacterium, Synechocystis sp. PCC 6714, and a green alga, Chlamydomonas reinhardtii, were quantitatively analyzed to examine differences in PS I/PS II stoichiometries. Cells cultured under different spectral conditions had various PS I/PS II molar ratios when estimated by oxidation-reduction difference absorption spectra of P700 (for PS I) and Cyt b-559 (for PS II) with thylakoid membranes. The fluorescence emission spectra under the Chl a excitation at 435 nm were resolved into several component bands using curve-fitting methods and the relative band area between PS II (F685 and F695) and PS I (F710 or F720) emissions was compared with the PS I/PS II stoichiometries of the various cell types. The results indicated that the PS I/PS II fluorescence ratios correlated closely with photosystem stoichiometries both in Synechocystis sp. PCC 6714 and in C. reinhardtii grown under different light regimes. Furthermore, the correlation between the PS I/PS II fluorescence ratios and the photosystem stoichiometries is also applicable to vascular plants.     

Effect of phosphatidylglycerol on molecular organization of photosystem I

Phosphatidylglycerol (PG) is the only anionic phospholipid in photosynthetic membrane. In this study, photosystem I (PSI) particles obtained from plant spinach were reconstituted into PG liposomes at a relatively high concentration. The results from visible absorption, fluorescence emission, and circular dichroism (CD) spectra reveal an existence of the interactions of PSI with PG. PG effect causes blue-shift and intensity decrease of Chl a peak bands in the absorption and 77 K fluorescence emission. The visible CD spectra indicate that the excitonic interactions for Chl a and Chl b molecules were enhanced upon reconstitution. Furthermore, more or less blue- or red-shift of the peaks characterized by Chl a, Chl b, and carotenoid molecules are also occurred. Simultaneously, an increase in alpha-helix and a decrease particularly in the disordered conformations of protein secondary structures are observed. In addition, the same effect also leads to somewhat more tryptophan (Trp) residues exposed to the polar environment. These results demonstrate that some alteration of molecular organization occurs within both the external antenna LHCI and PSI core complex after PSI reconstitution.     

盐酸胍对毕氏海蓬子类囊体膜蛋白光谱特征的影响

研究了盐酸胍（GuHCl）处理对毕氏海蓬子类囊体膜蛋白亚基和光谱特征的影响。结果显示,随着GuHCl处理浓度的增高,类囊体膜蛋白的吸收光谱和荧光发射光谱明显下降,峰位发生蓝移。这表明GuHCl处理下,类囊体膜蛋白色素微环境发生明显变化,色素蛋白结构遭到破坏;较低浓度2mmol.L-1GuHCl处理下,随着GuHCl处理时间的延长,类囊体膜蛋白的吸收光谱和荧光发射光谱也呈下降趋势,但与GuHCl浓度梯度处理比,下降程度略缓,峰位也基本没有变化。这说明类囊体膜在2mmol.L-1GuHCl不同处理时间下表现出的耐受性比在GuHCl浓度梯度处理条件强。     

Heat-induced changes in photosystem I activity as measured with different electron donors in isolated spinach thylakoid membranes.

Heat-induced changes in photosystem I (PSI) have been studied in terms of rates of oxygen consumption using various donors (DCPIPH2, TMPDred and DADred), formation of photo-oxidized P700 and changes in Chl a fluorescence emission at 77 K. Linear heating of thylakoid membranes from 35 degrees C to 70 degrees C caused an enhancement in PSI-mediated electron transfer rates (DCPIPH2-->MV) up to 55 degrees C. However, no change was observed in PSI rates when other electron donors were used (TMPDred and DADred). Similarly, Chl a fluorescence emission spectra at 77 K of heat-treated thylakoid membranes did not show any increase in peak at 735 nm, however, a significant decrease was observed as a function of temperature in the peaks at 685 and 694 nm. In DCMU-treated control thylakoid membranes maximum photo-oxidized P700 was generated at g = 2.0025. In heat-treated thylakoid membranes maximum intensity of photo-oxidized P700 signal was observed at approximately 50-55 degrees C without DCMU treatment. The steady-state signal of the photo-oxidized P700 was studied in the presence of DCPIPH2 and TMPDred as electron donors in DCMU-treated control and in 50 degrees C treated thylakoid membranes. We present here the first of such comparative study of PSI activity in terms of the rates of oxygen consumption and re-reduction kinetics of photo-oxidized P700 in the presence of different electron donors. It appears that the formation of the P700+ signal in heat-treated thylakoid membranes is due to an inhibited electron supply from PSII and not due to spillover or antenna migration.     

Differential changes in degradation of chlorophyll-protein complexes of photosystem I and photosystem II during flag leaf senescence of rice.

The stability of chlorophyll-protein complexes of photosystem I (PSI) and photosystem II (PSII) was investigated by chlorophyll (Chl) fluorescence spectroscopy, absorption spectra and native green gel separation system during flag leaf senescence of two rice varieties (IIyou 129 and Shanyou 63) grown under outdoor conditions. During leaf senescence, photosynthetic CO(2) assimilation rate, carboxylase activity of Rubisco, chlorophyll and carotenoids contents, and the chlorophyll a/b ratio decreased significantly. The 77 K Chl fluorescence emission spectra of thylakoid membranes from mature leaves had two peaks at around 685 and 735 nm emitting mainly from PSII and PSI, respectively. The total Chl fluorescence yields of PSI and PSII decreased significantly with senescence progressing. However, the decrease in the Chl fluorescence yield of PSI was greater than in the yield of PSII, suggesting that the rate of degradation in chlorophyll-protein complexes of PSI was greater than in chlorophyll-protein complexes of PSII. The fluorescence yields for all chlorophyll-protein complexes decreased significantly with leaf senescence in two rice varieties but the extents of their decrease were significantly different. The greatest decrease in the Chl fluorescence yield was in PSI core, followed by LHCI, CP47, CP43, and LHCII. These results indicate that the rate of degradation for each chlorophyll-protein complex was different and the order for the stability of chlorophyll-protein complexes during leaf senescence was: LHCII>CP43>CP47>LHCI>PSI core, which was partly supported by the green gel electrophoresis of the chlorophyll-protein complexes.