高温胁迫对螺旋藻光系统II影响的研究

高温胁迫是限制高等植物和藻类生长和产量的主要环境因子之一。光系统II（PSII）对环境胁迫的响应被认为是光合作用适应逆境过程中最重要的一个环节。高温胁迫对螺旋藻PSII的研究相对较少，对PSII受体侧的研究更加少了。我们借助热致发光及QA-再氧化动力学，这两种检测完整光合生物PSII供体侧和受体侧电子传递的有效、简单、无损伤的方法，为高温胁迫如何影响供体侧和受体侧的电子传递提供更直接的依据，获得高温胁迫对PSII功能影响的更精确的消息。另外，有关螺旋藻在高温胁迫下的能量传递过程研究较少，希望在荧光光谱研究的结果上探求其对高温胁迫的适应机理。主要研究结果如下： 1.高温胁迫抑制螺旋藻PS II的活性， PSII原初光能转化效率Fv/Fm随处理温度的提高而下降。高温去除后Fv/Fm可以得到部分恢复（5-15%）。 2.高温胁迫对闪光诱导的可变荧光衰减动力学有显著影响，分别代表QA-到QB 的直接电子传递和PQ分子扩散到空的QB位点后QA-到QB电子传递的快相(半衰期160 ms)和中相(半衰期2 ms)占整个可变荧光的比例，随处理温度的升高显著降低，而代表S2QA- 电荷重组的慢相（半衰期约4s）显著增加。显示高温导致QA到QB的电子传递以及PQ与QB位点的结合受阻，从而促进了QA-与放氧复合体S2态的重组过程。同时我们发现，经过5分钟的恢复，这些光系统II还原侧电子传递的功能抑制能够大部分得到恢复，显示高温胁迫对受体侧电子传递的影响具有可逆性。 3. 通过采用77K低温荧光光谱等手段，我们研究了高温胁迫对螺旋藻细胞光合能量传递的影响。研究显示，高温胁迫对580nm和436nm激发的低温荧光光谱都有显著影响。高温胁迫对PS I的发射峰F725和F751没有显著影响，显示高温没有影响藻胆体到光系统I的激发能传递。而高温胁迫引起了PBS对PS II荧光发射比值的上升，说明高温抑制了藻胆体到光系统II之间的激发能传递。但藻蓝蛋白的发射峰643nm在高温处理后基本没有变化，显示高温抑制PBS到PS II的能量传递不是由于藻蓝蛋白到别藻蓝蛋白之间的能量传递受阻造成的。结果还显示，高温胁迫对藻胆体到光系统II能量传递的抑制也不是由于藻胆体与光系统II发生分离，而是抑制了别藻蓝蛋白到CP43和CP47的能量传递，原因可能是由于藻胆体内部结构的改变引起的。 ４.热致发光（TL）和荧光衰减动力学的测定和分析结果显示，高温胁迫改变了S2QA-和S2QB-重组体的稳定性，其中S2QA-的稳定性降低，S2QB-的稳定性升高。根据具有异质性的TL信号，我们推测有活性PSII中有可能存在对高温胁迫敏感度不同的两种亚基，它们具有不同的QB/QB-氧化还原势能。当高温胁迫造成相当数量的反应中心失活，QA到QB正常的电子传递受阻时，光系统有可能通过保存更多的具有较高能量的反应中心亚基，达到促进QA到QB的电子传递的目的。 5. OJIP荧光瞬态上升曲线在高温胁迫后出现标志性K峰，说明螺旋藻的放氧复合物受到伤害，放氧S态引起的多次闪光下的TL振荡，显示这个高温处理对S1→S2的转变没有影响，却抑制了S2→S3的转变，这同OJIP荧光瞬态上升曲线的结果相一致，说明高温对螺旋藻放氧复合体造成了伤害。

LPA2蛋白调控PSII组装的分子机理研究

光系统II（PSII）是叶绿体类囊体膜上电子传递链中第一个色素蛋白复合体，由20多个蛋白亚基组成。它催化光驱动的水的裂解和醌的氧化。由于其结构的复杂性，PSII的生物发生和组装是核基因与叶绿体基因编码的蛋白以一定次序多步骤合成、组装的复杂过程，并需要大量的核基因编码的调节组装因子的参与。分离、鉴定拟南芥中这些核基因编码的叶绿体蛋白并研究它们的作用机制有助于我们认识高等植物PSII复合物组装和功能调控的分子机理。因此，我们从T-DNA插入的拟南芥突变体库中筛选到PSII突变体lpa2（low photosystemII accumulation），对LPA2蛋白调控光系统II复合物组装的功能进行了研究，并进一步探讨了LPA2和其他调节因子协同作用参与PSII组装的模式。 突变体lpa2具有高叶绿素荧光表型，与野生型相比生长量、色素含量均显著降低。蛋白免疫印记发现在lpa2突变体中光系统II复合物的累积量明显降低，仅有野生型的30%左右，而其他复合物的含量变化不大。核酸杂交和与多聚核糖体结合的检测表明光系统II亚基在转录及翻译启始水平没有受到影响。拟南芥叶片蛋白标记实验证明在突变体中CP43的合成量明显降低而其他光系统II主要蛋白CP47, D1 和 D2的合成正常，但相对于野生型这些蛋白的周转速率加快。在突变体中，新合成的蛋白亚基可以组装进入光系统II复合物，但新合成的CP43蛋白组装效率降低。以上的结果表明LPA2对光系统II的正常组装起着重要的作用，LPA2的缺失导致CP43不能有效组装进入光系统II，从而引起其他核心蛋白周转加快，光系统 II复合物累积量降低，最终植株光合效率降低。 基因克隆和蛋白定位分析表明LPA2基因编码一个内在的类囊体膜蛋白，但并不是光系统II的亚基组分。进一步采用酵母双杂分析证实了LPA2蛋白与光系统II核心蛋白CP43有相互作用，而与中心蛋白D1和D2没有相互作用。此外实验还表明LPA2蛋白与参与类囊体膜生物发生有关的Alb3蛋白有相互作用。因此LPA2可能是与Alb3形成复合物来协助CP43有效的整合进入光系统II。 另外，我们实验室已鉴定，LPA3，LPA4也是分别特异地参与CP43和D1组装的光系统II分子伴侣。LPA2，LPA3基因共同缺失会使幼苗不能光合自养而致死，因而LPA2和LPA3共同相互作用促进CP43的组装。体内和体外实验证明LPA2，LPA3和LPA4都和Alb3相互作用，而参与D1组装的分子伴侣LPA1不和Alb3以及上述这些伴侣因子作用。因此，Alb3 很有可能与LPA2、LPA3和LPA4形成多蛋白复合物在D1蛋白合成之后的组装过程中起作用。这些结果表明光系统II多亚基复合物组装是多步骤的，并通过一个精确复杂的调控网络确保复合物的有效组装以及功能行使。

Isolation and characterization of photosystem II of Porphyra yezoensis ueda

The thylakoid membranes were isolated and purified from gametophyte of Porphyrayezoensis Ueda (P yezoensis) by sucrose density gradient ultracentrifugation. After R yezoensis gametophyte thylakoid membranes were solubilized with SDS, the photosystem 11 (PSII) particles were isolated and purified. The activity of PSII particles was determined with DCIP (2,6-dichloroindophenol) photoreduction reaction. The composition of purified PSII particles was detected by SDS-PAGE. As a result, seven proteins including 55 kD protein, 47 kD protein, 43 kD protein, 33 kD protein, 31 kD protein, 29 kD protein, and 18 kD protein were found. Compared with PSII particles of higher plants and other algae, they were identified as D1/D2 complex, CP47, CP43, 33 kD protein, D1, D2 and cyt c-550 respectively. Besides, other three new proteins of 20 kD, 16 kD and 14 kD respectively were found. Among these extrinsic proteins, the 16 kD and 14 kD proteins had not been reported previously, and the 20 kD protein was found for the first time in multicellular red algae.

Isolation and characterization of photosystem II from the filamentous sporophyte of Porphyra yezoensis

Thylakoid membranes were isolated and purified from diploid filamentous sporophytes of Porphyra yezoensis Ueda using sucrose density gradient ultracentrifugation (SDGUC). After thylakoid membranes were solubilized with SDS, the photosystem II (PSII) particles with high 2, 6-dichloroindophenol (DCIP) photoreduction activity were isolated by SDGUC. The absorption and fluorescence spectra, DCIP photoreduction activity and oxygen evolution activity of the thylakoid membranes and PSII particles were determined. The polypeptide composition of purified PSII particles was distinguished by SDS-PAGE. Results showed that PSII particles of sporophytes differed from the gametophytes in spectral properties and polypeptide composition. Apart from 55 kDa D1-D2 heterodimer, CP47, CP43, 33 kDa protein was also detected. However, cyt c-550, 20 kDa, 14 kDa and 16 kDa proteins found in PSII particles from gametophytes were not detected in the sporophytes.

Isolation and characterization of the oxygen-evolving photosystem II complex from the economical red alga Bangia fusco-purpurea

The highly pure and active photosystem II (PSII) complex was isolated from Bangia fusco-purpurea (Dillw) Lyngb., an important economic red alga in China, through two steps of sucrose density gradient ultracentrifugation and characterized by the room absorption and fluorescence emission spectra, DCIP (2,6-dichloroindophenol) reduction, and oxygen evolution rates. The PSII complex from B. fusco-purpurea had the characteristic absorption peaks of chlorophyll (Chl) a (436 and 676 nm) and typical fluorescence emission peak at 685 nm (Ex = 436 nm). Moreover, the acquired PSII complex displayed high oxygen evolution (139 mu mol O-2/(mg Chl h) in the presence of 2.5 mM 2,6-dimethybenzoqinone as an artificial acceptor and was active in photoreduction of DCIP (2,6-dichloroindophenol) by DPC (1,5-diphenylcarbazide) at 163 U/(mg Chl a h). SDS-PAGE also suggested that the purified PSII complex contained four intrinsic proteins (D1, D2, CP43, and CP47) and four extrinsic proteins (33-kD protein, 20-kD protein, cyt c-550, and 14-kD protein).

Competing demands for a complex system : photosystem II repair, photoprotection and quantum yield

Photosynthesis in general is a key biological process on Earth and Photo system II (PSII) is an important component of this process. PSII is the only enzyme capable of oxidizing water and is largely responsible for the primordial build-up and present maintenance of the oxygen in the atmosphere. This thesis endeavoured to understand the link between structure and function in PSII with special focus on primary photochemistry, repair/photodamage and spectral characteristics. The deletion of the PsbU subunit ofPSII in cyanobacteria caused a decoupling of the Phycobilisomes (PBS) from PSII, likely as a result of increased rates of PSII photodamage with the PBS decoupling acting as a measure to protect PSII from further damage. Isolated fractions of spinach thylakoid membranes were utilized to characterize the heterogeneity present in the various compartments of the thylakoid membrane. It was found that the pooled PSIILHCII pigment populations were connected in the grana stack and there was also a progressive decrease in the reaction rates of primary photochemistry and antennae size of PSII as the sample origin moved from grana to stroma. The results were consistent with PSII complexes becoming damaged in the grana and being sent to the stroma for repair. The dramatic quenching of variable fluorescence and overall fluorescent yield of PSII in desiccated lichens was also studied in order to investigate the mechanism by which the quenching operated. It was determined that the source of the quenching was a novel long wavelength emitting external quencher. Point mutations to amino acids acting as ligands to chromophores of interest in PSII were utilized in cyanobacteria to determine the role of specific chromophores in energy transfer and primary photochemistry. These results indicated that the Hl14 ligated chlorophyll acts as the 'trap' chlorophyll in CP47 at low temperature and that the Q130E mutation imparts considerable changes to PSII electron transfer kinetics, essentially protecting the complex via increased non-radiative charge Photosynthesis in general is a key biological process on Earth and Photo system II (PSII) is an important component of this process. PSII is the only enzyme capable of oxidizing water and is largely responsible for the primordial build-up and present maintenance of the oxygen in the atmosphere. This thesis endeavoured to understand the link between structure and function in PSII with special focus on primary photochemistry, repair/photodamage and spectral characteristics. The deletion of the PsbU subunit ofPSII in cyanobacteria caused a decoupling of the Phycobilisomes (PBS) from PSII, likely as a result of increased rates of PSII photodamage with the PBS decoupling acting as a measure to protect PSII from further damage. Isolated fractions of spinach thylakoid membranes were utilized to characterize the heterogeneity present in the various compartments of the thylakoid membrane. It was found that the pooled PSIILHCII pigment populations were connected in the grana stack and there was also a progressive decrease in the reaction rates of primary photochemistry and antennae size of PSII as the sample origin moved from grana to stroma. The results were consistent with PSII complexes becoming damaged in the grana and being sent to the stroma for repair. The dramatic quenching of variable fluorescence and overall fluorescent yield of PSII in desiccated lichens was also studied in order to investigate the mechanism by which the quenching operated. It was determined that the source of the quenching was a novel long wavelength emitting external quencher. Point mutations to amino acids acting as ligands to chromophores of interest in PSII were utilized in cyanobacteria to determine the role of specific chromophores in energy transfer and primary photochemistry. These results indicated that the Hl14 ligated chlorophyll acts as the 'trap' chlorophyll in CP47 at low temperature and that the Q130E mutation imparts considerable changes to PSII electron transfer kinetics, essentially protecting the complex via increased non-radiative charge.

Molecular characterization of PsbW, a nuclear-encoded component of the photosystem II reaction center complex in spinach.

We describe the isolation and characterization of cDNAs encoding the precursor polypeptide of the 6.1-kDa polypeptide associated with the reaction center core of the photosystem II complex from spinach. PsbW, the gene encoding this polypeptide, is present in a single copy per haploid genome. The mature polypeptide with 54 amino acid residues is characterized by a hydrophobic transmembrane segment, and, although an intrinsic membrane protein, it carries a bipartite transit peptide of 83 amino acid residues which directs the N terminus of the mature protein into the chloroplast lumen. Thylakoid integration of this polypeptide does not require a delta pH across the membrane, nor is it azide-sensitive, suggesting that the polypeptide chain inserts spontaneously in an as yet unknown way. The PsbW mRNA levels are light regulated. Similar to cytochrome b559 and PsbS, but different from the chlorophyll-complexing polypeptides D1, D2, CP43, and CP47 of photosystem II, PsbW is present in etiolated spinach seedlings.