不同膜脂环境中PSII核心复合物和外周天线之间的功能关系

光合作用过程中光能的吸收、传递和转化都是在类囊体膜中进行的，它是由脂质双层膜和色素蛋白复合物构成的。光系统II（PSII）是存在于类囊体膜中的多亚基色素蛋白复合物，主要功能是吸收光能，进行光诱导的电荷分离，产生电子传递并催化水的光解。光系统II捕光天线复合物（LHCII）与PSII核心复合物结合形成的PSII-LHCII超分子复合物，是PSII在体内的基本结构和功能单元，这一结构保证了LHCII吸收的能量快速有效的传递到PSII反应中心，进行原初光化学反应。膜脂与膜蛋白的相互作用在调节PSII-LHCII超分子复合物各亚基之间的结构和功能方面起着重要作用，而在类囊体膜脂中，非双层脂单半乳糖甘油二脂（MGDG）含量最多，约占50%，在光合膜蛋白的结构和功能中具有重要作用。 本论文利用脂质体重组等技术研究了LHCII和放氧核心超分子复合物（OECC）之间的功能关系，MGDG的作用以及微量天线的功能。主要结果如下： 1. MGDG能和Chl a、PC或其它类囊体膜脂一起与PSII蛋白构建蛋白脂质体，脂质体形状较规则统一，基本呈圆球状，阻止了MGDG反六角相结构的形成。脂质体的直径大小在100-500 nm之间，属于小单层脂质体。PSII膜蛋白LHCII和OECC能在MGDG脂质体中实现重组，形成LHCII-OECC超分子复合物，在结构上相互偶联，LHCII-OECC蛋白颗粒直径在15-25 nm之间。LHCII吸收的能量能够传递到核心复合物OECC中，形成功能上的偶联，而且LHCII的结合增加了功能天线的大小和捕光截面积，从而提高了PSII的光化学活性。 2. MGDG对蛋白脂质体的结构和功能有影响。低温荧光发射光谱和PSII光化学活性的结果显示，MGDG影响了PSII复合物色素和蛋白的存在状态；MGDG能增强LHCII和OECC之间的相互作用，促进能量从LHCII到核心复合物的传递，提高PSII的光化学活性。 3. MGDG促进类囊体膜脂和PC-MGDG蛋白脂质体的放氧活性的原因不同。在类囊体膜脂脂质体中，MGDG主要通过膜蛋白疏水部分的横向压力增加PSII偶合的天线量，提高PSII的光化学活性；而在PC-MGDG蛋白脂质体中，MGDG不能加强PSII与天线的偶合，可能是通过MGDG与LHCII的相互作用，增加PSII的光化学活性。 4. 微量天线不是大量天线和核心复合物重组和相互作用所必需的，但微量天线的存在，能促进大量天线与PSII核心复合物之间的能量传递和放氧活性，大量天线与PSII核心复合物之间的偶联作用得到增强。而且蛋白脂质体放氧活性的证据表明，MGDG能促进微量天线的这种作用。

拟南芥光系统复合物组装调控因子的功能研究

光系统I与光系统II ( PSI和PSII ) 是由核基因与叶绿体基因共同编码的蛋白组成的多亚基色素蛋白复合体，其复合物组装过程中蛋白以一定地次序合成并组装。现有研究表明光合膜多亚基复合物形成的每一个过程都需要一个或多个调节因子的参与。发现这些调节因子，并研究它们的作用机制将有助于我们认识高等植物两个光系统复合物组装和功能调控的分子机理。因此，我们采用正向遗传学和反向遗传学方法去寻找这些调控因子。我们一方面应用Gateway技术构建拟南芥cDNA表达文库，采用酵母双杂交技术从中筛选与Alb3互作的蛋白，称为ALIP ( Albino3 Interacting Protein )；从ABRC订购编码这些互作蛋白的基因的T-DNA插入突变株系，其中发现了一个影响PSI功能的突变体alip1；另一方面，通过对拟南芥T-DNA插入突变体库进行筛选，发现了一批影响PSII功能的突变体 ( low photosystem II accumulation )，其中包括lpa1、lpa2和lpa66-1。本实验对alip1和lpa66-1突变体进行了深入研究，初步探讨了这两个基因编码的蛋白参与调控PSI以及PSII的组装机理。 突变体lpa66-1是一个高叶绿素荧光突变体，与野生型比较生长缓慢，叶色黄，叶绿素含量低。叶绿素荧光慢诱导曲线显示它是一个影响PSII功能的突变体。类囊体膜蛋白的免疫印迹发现lpa66-1突变体中PSII复合物的累积量降低到野生型的30%左右，其他复合物的含量变化不大。体内蛋白标记实验显示，PSII反应中心蛋白D1，D2的合成速率下降，PSII核心蛋白的周转加快。新合成的蛋白组装进PSII的效率比野生型显著降低。LPA66是一个定位于叶绿体的PPR蛋白。因为野生型拟南芥LPA66蛋白能够特异性的编辑psbF转录本，故野生型psbF转录本中第77C被编辑为77U，从而使相应的氨基酸序列中第26个氨基酸丝氨酸被编辑为苯丙氨酸，而lpa66-1突变体中，LPA66蛋白的缺失导致该位点不能被编辑，PSII复合体也不能有效组装。 Alb3/Oxa1p/YidC蛋白家族广泛的参与蛋白质转运和多亚基复合物组装，采用分裂泛素化酵母双杂交发现与Alb3相互作用蛋白ALIP1。突变体alip1也是一个高叶绿素荧光突变体，叶色黄，在土里生长极为缓慢，且不能开花，不育。叶绿素荧光慢诱导曲线显示，突变体中PSII功能基本没有受影响；而P700显示alip1是一个影响PSI功能的突变体。类囊体膜蛋白的免疫印迹发现突变体中PSI核心蛋白PsaA/B的累积量为野生型的40%左右，而PSII及其他复合物的含量无明显变化。Northern印迹结果显示PsaA/B在转录水平不受影响，而体内蛋白标记实验显示，PSI反应中心蛋白PsaA/B的合成速度下降。蔗糖密度梯度离心分析类囊体膜蛋白的组分显示ALIP1能够与Alb3共迁移。而Alb3对于类囊体膜上大分子复合体的组装有重要作用，我们推测，ALIP1可能与Alb3形成一个复合物，或者作为一个中间体介导Alb3参与PSI的组装。

Metal Retention Experiments for the Design of Soil-Mix Technology Permeable Reactive Barriers

Soil-mix technology is effective for the construction of permeable reactive barriers (PRBs) for in situ groundwater treatment. The objective of this study was to perform initial experiments for the design of soil-mix technology PRBs according to (i) sorption isotherm, (ii) reaction kinetics and (iii) mass balance of the contaminants. The four tested reactive systems were: (i) a granular zeolite (clinoptilolite-GZ), (ii) a granular organoclay (GO), (iii) a 1:1-mixture GZ and model sandy clayey soil and (iv) a 1:1:1-mixture of GZ, GO and model soil. The laboratory experiments consisted of batch tests (volume 900mL and sorbent mass 18g) with a multimetal solution of Pb, Cu, Zn, Cd and Ni. For the adsorption experiment, the initial concentrations ranged from 0.01 to 0.5mM (2.5 to 30mg/L). The maximum metal retention was measured in a batch test (300mg/L for each metal, volume 900mL, sorbent mass 90-4.5g). The reactive material efficiency order was found to be GZ>GZ-soil mix>GZ-soil-GO mix>GO. Langmuir isotherms modelled the adsorption, even in presence of a mixed cations solution. Adsorption was energetically favourable and spontaneous in all cases. Metals were removed according to the second order reaction kinetics; GZ and the 1:1-mix were very similar. The maximum retention capacity was 0.1-0.2mmol/g for Pb in the presence of clinoptilolite; for Cu, Zn, Cd and Ni, it was below 0.05mmol/g for the four reactive systems. Mixing granular zeolite, organoclay and model soil increased the chemisorption. Providing that GZ is reactive enough for the specific conditions, GZ can be mixed to obtain the required sorption. Granular clinoptilolite addition to soil is recommended for PRBs for metal contaminated groundwater. The laboratory experiments consisted of batch tests with a multimetal solution of Pb, Cu, Zn, Cd and Ni. The four reactive materials chosen were granular zeolite, clinoptilolite and model sandy clayey soil, granular organoclay and a mix of clinoptilolite, model soil and organoclay. The reactive material efficiency order was found to be granular clinoptilolite>clinoptilolite-soil mix>clinoptilolite-soil-organoclay mix>granular organoclay. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Increasing the open-circuit voltage of photoprotein-based photoelectrochemical cells by manipulation of the vacuum potential of the electrolytes.

The innately highly efficient light-powered separation of charge that underpins natural photosynthesis can be exploited for applications in photoelectrochemistry by coupling nanoscale protein photoreaction centers to man-made electrodes. Planar photoelectrochemical cells employing purple bacterial reaction centers have been constructed that produce a direct current under continuous illumination and an alternating current in response to discontinuous illumination. The present work explored the basis of the open-circuit voltage (V(OC)) produced by such cells with reaction center/antenna (RC-LH1) proteins as the photovoltaic component. It was established that an up to ~30-fold increase in V(OC) could be achieved by simple manipulation of the electrolyte connecting the protein to the counter electrode, with an approximately linear relationship being observed between the vacuum potential of the electrolyte and the resulting V(OC). We conclude that the V(OC) of such a cell is dependent on the potential difference between the electrolyte and the photo-oxidized bacteriochlorophylls in the reaction center. The steady-state short-circuit current (J(SC)) obtained under continuous illumination also varied with different electrolytes by a factor of ~6-fold. The findings demonstrate a simple way to boost the voltage output of such protein-based cells into the hundreds of millivolts range typical of dye-sensitized and polymer-blend solar cells, while maintaining or improving the J(SC). Possible strategies for further increasing the V(OC) of such protein-based photoelectrochemical cells through protein engineering are discussed.

Depletion of Vipp1 in Synechocystis sp PCC 6803 affects photosynthetic activity before the loss of thylakoid membranes

A vipp1 mutant of Synechocystis sp. PCC 6803 could not be completely segregated under either mixotrophic or heterotrophic conditions. A vipp1 gene with a copper-regulated promoter (P-petE-vipp1) was integrated into a neutral platform in the genome of the merodiploid mutant. The copper-induced expression of P-petE-vipp1 allowed a complete segregation of the vipp1 mutant and observation of the phenotype of Synechocystis 6803 with different levels of vesicle-inducing protein in plastids 1 (Vipp1). When P-petE-vipp1 was turned off by copper deprivation, Synechocystis lost Vipp1 and photosynthetic activity almost simultaneously, and at a later stage, thylakoid membranes and cell viability. The photosystem II (PSII)-mediated electron transfer was much more rapidly reduced than the PSI-mediated electron transfer. By testing a series of concentrations, we found that P-petE-vipp1 cells grown in medium with 0.025 mu M Cu2+ showed no reduction of thylakoid membranes, but greatly reduced photosynthetic activity and viability. These results suggested that in contrast to a previous report, the loss of photosynthetic activity may not have been due to the loss of thylakoid membranes, but may have been caused more directly by the loss of Vipp1 in Synechocystis 6803.

Bifunctional enzyme FBPase/SBPase is essential for photoautotrophic growth in cyanobacterium Synechocystis sp PCC 6803

From a random insertion mutant library of Synechocystis sp. PCC 6803, a mutant defective in photoautotrophic growth was obtained. The interrupted gene was identified to be slr2094 (rbpl), which encodes the fructose-1,6-biphosphatase (FBPase)/sedoheptulose-1,7-biphosphatase (SBPase) bifunctional enzyme (F-I). Two other independently constructed slr2094 mutants showed an identical phenotype. The FBPase activity was found to be virtually lacking in an slr2094 mutant, which was sensitive to light under mixotrophic growth conditions. These results indicate that slr2094 is the only active FBPase-encoding gene in this cyanobacterium. Inactivation of photosystem II by interrupting psbB in slr2094 mutant alleviated the sensitiveness to light. This report provides the direct genetic evidence for the essential role of F-I in the photosynthesis of Synechocystis sp. PCC 6803. (c) 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

Effects of sand burial on biomass, chlorophyll fluorescence and extracellular polysaccharides of man-made cyanobacterial crusts under experimental conditions

Soil cyanobacterial crusts occur throughout the world, especially in the semiarid and arid regions. It always encounters sand burial, which is an important feature of mobile sand dunes. A greenhouse 41 study was conducted to determine the effects of sand burial on biomass, chlorophyll fluorescence and extracellular polysaccharides of man-made cyanobacterial crusts in six periods of time (0, 5, 10, 15, 20 and 30 d after burying) and at five depths (0, 0.2, 0.5, 1 and 2cm). The results indicated that with the increase of the burial time and burial depth extracellular polysaccharides content and Fv/Fm decreased correspondingly and there were no significant differences between 20 and 30 burial days under different burial depths. The degradation of chlorophyll a content appeared only at 20 and 30 burial days and there was also no significant difference between them under different burial depths. It was also observed a simultaneous decrease of the values of the Fv/Fm and the content of extracellular polysaccharides happened in the crusted cyanobacterium Microcoleus vaginatus Gom. It may suggest that there exists a relationship between extracellular polysaccharides and recovery of the activity of photosystem II (PS II) after rehydration.

Solar UV radiation drives CO2 fixation in marine phytoplankton: A double-edged sword

Photosynthesis by phytoplankton cells in aquatic environments contributes to more than 40% of the global primary production (Behrenfeld et al., 2006). Within the euphotic zone (down to 1% of surface photosynthetically active radiation [PAR]), cells are exposed not only to PAR (400-700 nm) but also to UV radiation (UVR; 280-400 nm) that can penetrate to considerable depths (Hargreaves, 2003). In contrast to PAR, which is energizing to photosynthesis, UVR is usually regarded as a stressor (Hader, 2003) and suggested to affect CO2-concentrating mechanisms in phytoplankton (Beardall et al., 2002). Solar UVR is known to reduce photosynthetic rates (Steemann Nielsen, 1964; Helbling et al., 2003), and damage cellular components such as D1 proteins (Sass et al., 1997) and DNA molecules (Buma et al., 2003). It can also decrease the growth (Villafane et al., 2003) and alter the rate of nutrient uptake (Fauchot et al., 2000) and the fatty acid composition (Goes et al., 1994) of phytoplankton. Recently, it has been found that natural levels of UVR can alter the morphology of the cyanobacterium Arthrospira (Spirulina) platensis (Wu et al., 2005b). On the other hand, positive effects of UVR, especially of UV- A (315-400 nm), have also been reported. UV- A enhances carbon fixation of phytoplankton under reduced (Nilawati et al., 1997; Barbieri et al., 2002) or fast-fluctuating (Helbling et al., 2003) solar irradiance and allows photorepair of UV- B-induced DNA damage (Buma et al., 2003). Furthermore, the presence of UV-A resulted in higher biomass production of A. platensis as compared to that under PAR alone (Wu et al., 2005a). Energy of UVR absorbed by the diatom Pseudo-nitzschia multiseries was found to cause fluorescence (Orellana et al., 2004). In addition, fluorescent pigments in corals and their algal symbiont are known to absorb UVR and play positive roles for the symbiotic photosynthesis and photoprotection (Schlichter et al., 1986; Salih et al., 2000). However, despite the positive effects that solar UVR may have on aquatic photosynthetic organisms, there is no direct evidence to what extent and howUVR per se is utilized by phytoplankton. In addition, estimations of aquatic biological production have been carried out in incubations considering only PAR (i. e. using UV-opaque vials made of glass or polycarbonate; Donk et al., 2001) without UVR being considered (Hein and Sand-Jensen, 1997; Schippers and Lurling, 2004). Here, we have found that UVR can act as an additional source of energy for photosynthesis in tropical marine phytoplankton, though it occasionally causes photoinhibition at high PAR levels. While UVR is usually thought of as damaging, our results indicate that UVR can enhance primary production of phytoplankton. Therefore, oceanic carbon fixation estimates may be underestimated by a large percentage if UVR is not taken into account.

硫手性有机小分子催化剂的设计、合成及其在不对称亚胺氢转移还原中的应用

过去十多年，世界手性药物市场需求迅速增长，手性制药工业的发展壮大，已经引起了各国政府、学术界，特别是企业界的高度重视。手性药物中含有大量的手性胺单元，因此研究高效构建手性胺结构单元的方法具有重要的意义和实用价值，而亚胺的不对称还原是合成手性胺最便捷的方法。 手性有机小分子路易斯碱催化三氯氢硅不对称还原亚胺是最近几年才发展起来的一类新的亚胺不对称还原方法。尽管在对映选择性和底物适用范围等方面已经获得了突破性的进展，但是，高性能的路易斯碱催化剂仅局限于N-甲酰氨基酸酰胺一种类型，而且其底物适用范围和催化活性仍不够理想。因此，发展新型催化剂很有必要。 手性硫氧化物作为手性诱导剂的应用已经有数十年的时间，广泛应用在不对称合成及天然产物的全合成中。理论上，硫氧结构单元也可以作为路易斯碱，对硅烷类试剂进行活化，而且硫氧键还有碳氧键难以比拟的先天优势，硫原子自带手性特征，在反应过程中，手性中心离反应位点更近，因此，从手性硫氧化合物出发，极有可能开发出新的高效手性路易斯碱催化剂。最近，Kobayashi和Khiar在亚胺的不对称烯丙基化反应中用手性亚砜活化烯丙基三氯硅烷，获得了较好的ee值，但反应中手性亚砜的用量都需要化学计量以上，因此还不能算做真正意义上的催化剂，进一步的文献调研也未见真正意义上的硫手性有机小分子催化剂。 本文首次成功将硫手性亚磺酰胺衍生物应用于催化三氯氢硅对亚胺的不对称还原，在经过对亚磺酰胺衍生物的多次结构优化，开发出了合成容易，催化活性和立体选择性都很优良，并且有着前所未有的底物普适性的新型手性路易斯碱催化剂。 我们首先尝试将商品化的20mol%叔丁基亚磺酰胺和对甲基亚磺酰胺直接用作催化剂催化三氯氢硅对亚胺的不对称还原，尽管仅获得中等的收率和很低的对映选择性，但证明我们的设计思路是可行的。在此基础上，我们以叔丁基亚磺酰胺为原料和基本骨架，设计合成了一系列的亚磺酰胺类催化剂，通过对催化剂的结构改造，发现当催化剂中存在较强酸性的酚羟基时，催化效果得到大幅提高。随着对催化剂的进一步结构优化，我们找到了一个结构简单，催化效果还不错的催化剂，经过反应条件优化以后，催化反应的收率最高能达到98%，对映选择性最高达93%，并且这个催化剂的底物适应范围比之前报道的催化剂都要广泛。针对酚羟基在催化剂中的重要作用，我们进行了仔细的机理研究后发现，在催化反应中，催化剂极有可能是通过双分子机理去活化三氯氢硅从而实现不对称催化的，而酚羟基的作用就是通过分子间氢键促进双分子催化剂与三氯氢硅的络合。受此启发，我们设计了一系列具有双齿结构的催化剂，通过对双齿催化剂的结构优化，最终筛选出了一个结构更加简单，但催化效果更好的双齿催化剂。10mol%该催化剂催化亚胺还原最高获得95%的收率和96%的ee值。这一结果也进一步验证了我们先前对催化剂机理的推测。 随后，我们还尝试将这些催化剂用于二级胺和芳香酮的直接还原胺化反应中，虽然能获得不错的收率，但对映选择性却很差，我们对反应条件进行了仔细的摸索，仍然没有获得突破。但这些实验为进一步研究二级胺和酮的不对称直接还原胺化反应奠定了良好的基础。 In the past decade, the rapid growth of the global chiral drug market and the significant development of the chiral pharmaceutical industry have attracted a great deal of attention from government, academia and enterprises. Chiral amine is an important structural motif of chiral drugs. Therefore, development of methods for the construction of this motif is of great importance. Catalytic enantioselective reduction of imines represents one of the most straightforward and efficient methods for the preparation of chiral amines. The chiral Lewis base organocatalysts promoted asymmetric reduction of imines by HSiCl3 has recently achieved significant advancements. Although big breakthroughs have been made in terms of substrate generality and enantioselectivity, the highly effective catalysts are limited to N-formyl amino acid amides, of which the efficiency and substrate scope remain unsatisfactory. Therefore, development of novel organocatalysts for this transformation is in great demand. Chiral sulfoxides have been well established as efficient and versatile stereocontrollers and have been extensively used in asymmetric synthesis and total synthesis of natural products. The S=O structural motif of sulfoxide could also behave as Lewis base activator for cholorsilane reagents, which, moreover, could be even better than caboxamide considering that the sulfur atom is chiral and thus the chirality center is closer to the reaction center. There exist great potentials that highly effective novel Lewis base organocatalysts could be developed starting from S-chiral sulfoxides. Recently, several S-chiral sulfoxides were reported by Kobayashi and Khiar to be used as Lewis base catalyst to activate allyltrichlorosilanes in asymmetric allylations and good enantioselectivities were obtained. However, these S-chiral sulfoxides were all used at a more than stoichiometric amount and were thus not authentically catalytic. A careful literature survey further revealed that there has been so far no S-chiral organocatalyst available. In this study, we, for the first time, successfully used S-chiral sulfinamides as Lewis base organocatalysts for the asymmetric reduction of ketimines by HSiCl3. After several rounds of structural optimization, we developed the first example of highly effective S-chiral organocatalysts, which promoted the asymmetric reduction of ketimines with trichlorosilane in high yield and excellent enantioselectivity with unprecedented substrate spectrum. In our initial practice, we examined 20mol% of the commercially available (R)-tert-butanesulfinamide and (S)-toluenesulfinamide as the catalyst in the hydrosilylation of ketimine. Although the product was only furnished in moderate yield and low ee, these results demonstrated that our strategy of catalyst design is on the right way. Next, starting from chiral tert-butanesulfinamide, we prepared a series of tert-butanesulfinamide derivatives via simple reductive amination and examined their catalytic efficiencies in the reduction of ketimine. We found that the catalyst bearing a phenolic hydroxyl group exhibited good reactivity and enantioselectivity. On the basis of which, we obtained a structurally simple and highly effective novel organocatalyst, affording the product in 98% yield and 93% ee under optimal reaction conditions. After careful exploration on the role of phenolic hydroxyl group in the catalyst, we speculated that two molecules of the catalyst be involved in the course of reaction, of which the assembly around the silicon center is facilitated by the intermolecular hydrogen bonding through the phenolic hydroxyl groups. Thus, we incorporated two units of sulfonamide into one molecular and prepared a new type of bissulfinamides organocatalysts and examined their catalytic efficiencies in the reduction of ketimine. After optimizing the structure of these catalysts, we finally obtained a novel organocatalyst which has even simpler molecular structure but showed better efficacies, 10mol% of which afforded up to 97% yield and 96% ee under optimal reaction conditions. These results further proved our speculation about the catalytic mechanism. We also examined the newly developed S-chiral organocatalysts in direct asymmetric reductive amination of secondary amines with aromatic ketone. The product was furnished in good yield but in low ee. No better results could be obtained despite our intense opimization efforts. Nevertheless, these experiments laid excellent foundations for eventual success.

沙棘(Hippophae rhamnoides L.)在低温和增强UV-B胁迫下的生态生理反应

植物生长和生产力受到自然界各种形式的生物和非生物胁迫因子的影响。这些胁迫包括低温、高温、盐碱、干旱、洪水、重金属、虫害、病害和紫外线辐射等等。而人类活动大大加剧了这些胁迫所带来的影响。由于人类污染而导致臭氧层衰减以及由此产生的地球表面紫外辐射增强已经成为全球气候变化的一个主要方面。UV-B胁迫，甚至当前的辐射水平，所带来的影响已经引起科学工作者的广泛关注。 为了生存和繁殖，植物不得不面临环境中各种潜在胁迫所带来的负面影响。然而，植物生活型的不可移动性决定了其逃避胁迫的局限性。因此，绝大多数植物都是通过对胁迫作出反应，通过修复或者更新组织来降低伤害。而植物应对环境变化的能力则是由其生长模式的种属特异性和本身的遗传组成所决定。在自然界，植物常常同时面临多种胁迫，这些胁迫所引发的植物反应可能具有叠加、协同或者拮抗作用。沙棘是一种具刺、具有固氮功能的多年生雌雄异株灌木，广泛分布于亚欧大陆的温带地区和亚洲亚热带的高海拔地区。在中国，沙棘常常被用作植被恢复中的先锋树种而大量栽培。本文采用沙棘作为模式植物，试图探索木本植物对低温，UV-B辐射增强以及其与干旱的复合胁迫的响应以及沙棘对这些胁迫响应是否具有种群差异性。 对来自南北两个种群的沙棘进行短日照和低温处理，检测了其在抗寒锻炼和抗寒性发育过程中存在的性别差异。结果表明，短日照和低温都分别能够诱导抗寒锻炼的发生，而两者同时存在对所有实验植株抗寒性的大小具有叠加效应。然而，短日照和低温所诱导的抗寒性在两个种群中都具有性别差异性，雄性植株比雌雄植株对短日照和低温更为敏感。同时，南北种群间也存在差异性，北方种群的植物比南方种群的植物对短日照和低温敏感，从而在短日照下抗寒锻炼的发生时间更早，低温诱导的抗寒性更大。短日照和低温诱导植物增加抗寒性的同时伴随着脱落酸的变化。脱落酸的变化因处理，种群和性别的不同而不同。这些生理反应表明不同的沙棘种群，不同的植株性别对同一环境胁迫可能存在不同的生存策略。 比较了来自高低两个海拔的沙棘种群对于干旱和UV-B辐射增强以及两者复合胁迫条件下的生理生态反应。干旱使两个种群中植株总的生物量，总叶面积，比叶面积，叶片含碳量，含磷量，木质素含量和碳氮比显著降低，使根冠比，粗根细根比和叶片脱落酸含量显著增加。干旱而非UV-B使得δ13C 值显著增加。但是，比较而言，来自高海拔的种群对干旱反应更为强烈，而来自低海拔的种群对UV-B更敏感。在UV-B辐射增强的处理下，干旱所诱导的脱落酸的积累被显著抑制。而且我们检测到在一些指标上存在显著的干旱×UV-B交互作用，如两个种群中在总生物量上，低海拔种群中在总叶面积，粗根细根比上，高海拔种群中在比叶面积，δ13C值，木质素含量上都存在明显的交互作用。这些结果表明这两个种群对胁迫具有不同的适应性反应，来自高海拔的种群比来自低海拔的种群更能够抵御干旱和UV-B胁迫。 室外实验表明，UV-B 去除/增补对沙棘高低两个海拔种群的影响都不大。对生物量的积累，植株高度以及一些常见的胁迫反应生理指标比如丙二醛、ABA 和游离脯氨酸都没有显著影响。UV-B 的效应比UV-A 大，植物反应在无UV 和仅有UV-A 的处理间没有什么区别。然而，UV-B 去除的两个处理和UV-B 存在的两个处理间存在显著区别。UV-B 使得两个种群都显著降低了比叶面积(SLA)，但却使长期用水效率增加。但UV-B对光合色素和光合系统II 的影响不大。总体看来，来自低海拔的种群对UV-B 更为敏感。 Plant is adversely affected by various abiotic and biotic stress factors. These stressors includelow temperature, heat, salt, drought, flooding, heavy metal toxicity, wounding by herbivores,infecting by pathogenic microorganisms, ultraviolet (UV) radiation and so on. Variousanthropogenic activities have accentuated the existing stress factors. One of the mostimportant aspects of global change is that of stratospheric ozone depletion caused by seriousanthropogenic pollution and the resulting increase in UV radiation reaching the surface of theEarth. Scientists have become concerned about the effects that considerable UV-B stress, evenat current levels. In order to survive and reproduce, plants have to be able to cope with lots of potentiallyharmful stress factors that are almost constantly present in their environment. Most plants’responses under stress are to neutralize the stress, repairing the damage or regrowing newtissue rather than to avoid it due to their sessile life style. The plant defense capacity dependson plant-specific modular growth patterns and genetic make-up that allows for flexibleresponses to changing environments. Plants usually encounter several stresses simultaneouslyunder field conditions, and the stresses may cause a variety of plant responses, which can beadditive, synergistic or antagonistic. Sea buckthorn (Hippophae rhamnoides L.), a thorny nitrogen fixing deciduously perennialshrub, which is widely distributed throughout the temperate zones of Asia and Europe and thesubtropical zones of Asia at high altitudes. It has been widely used in forest restoration as thepioneer species in China. In this paper, we used sea buckthorn as a model, tried to get some understand of how plants fight low temperature, enhanced UV-B radiation level and thatcombination of drought. And also, want to know whether does there exist some populationspecific responses to such stressors. Sexual differences in cold acclimation and freezing tolerance development of two contrastingsea buckthorn (Hippophae rhamnoides L.) ecotypes from northern and southern regions inChina were recorded after exposure to short day photoperiod (SD) and low temperature (LT).The results demonstrated that cold acclimation could be triggered by exposing the plants toSD or LT alone, and that a combination of both treatments had an additive effect on freezingtolerance in all plants tested. However, development of freezing tolerance was dependent onthe sex of plants under SD and LT, the males were clearly more responsive to SD and LT thanthe females in both ecotypes studied. On the other hand, development of freezing tolerancewas also ecotype-dependent, the northern ecotype was more responsive to SD and LT than thesouthern ecotype, resulting in earlier cold acclimation under SD and higher freezing toleranceunder LT. Moreover, development of freezing tolerance induced by SD and LT wasaccompanied by changes in ABA levels. These alterations in ABA levels were different indifferent treatments, ecotypes and sexes. Therefore, the differences in SD and LT-inducedphysiological responses showed that the different ecotypes and the different sexes mightemploy different survival strategies under environmental stress. Two contrasting populations from the low and high altitudinal regions were employed toinvestigate the effects of drought, UV-B and their combination on sea buckthorn. Droughtsignificantly decreased total biomass, total leaf area, specific leaf area，leaf carbon (C),phophous (P), lignin content and the ratio of C: N in both populations, and increasedroot/shoot ratio, fine root/coarse root ratio and abscisic acid content (ABA), in bothpopulations. Drought but not UV-B resulted in significantly greater carbon isotopecomposition (δ13C) values in both populations. However, the high altitudinal population wasmore responsive to drought than the low altitudinal population. The drought-inducedenhancement of ABA in the high altitudinal population was significantly suppressed in thecombination of drought and elevated UV-B. Moreover, significant drought × UV-B interactionwas detected on total biomass in both populations, total leaf area and fine root/coarse root inthe low altitudinal population, specific leaf area, δ13C value and leaf lignin content in the high altitudinal population. These results demonstrated that there were different adaptive responsesbetween two contrasting populations, the high altitudinal population exhibited highertolerance to drought and UV-B than the low altitudinal population. A field experiment was conducted to investigate effects of UV-B exclusion/supplementationon two altitudinal populations of sea buckthorn. UV-B exclusion or supplementation had littleeffects on both populations investigated. For instance, the total biomass, plant height andsome physiological index such as Malondialdehyde (MDA), ABA and free proline were notchanged significantly. The UV-B effects are more significant than that of UV-A, nodifferences were found between treatments of excluded UV and excluded UV-B. However,compared with treatments of UV-B exclusion (including absent of UV-B and all UV band),the present of UV-B (including near ambient environment and enhanced UV-B) significantdecreased specific leaf area, and increased long time water use efficiency as evaluated by δ13Cvalue. UV-B had little effects on photosynthetic pigments and Photosystem II (PSII). The lowaltitude population is more sensitive to UV-B than that of the high altitude population.

川西亚高山两种针叶树幼苗对增温和施氮的响应

人类活动引起全球大气中温室气体（CO2、CH4、NOx）浓度不断增加，致使地球表面温度在过去的100 年中已经增长了0.74 ± 0.18℃，预计到本世纪末将会增加1.1-6.4℃。此外，氮沉降也是当今社会的重要环境问题，随着经济发展的全球化, 高氮沉降也呈现出全球化趋势。全球气候变暖和氮沉降给陆地生态系统的地上、地下生物学和生物地球化学过程所带来巨大影响越来越引起人们的关注。 本文以川西亚高山针叶林的两个重要树种云杉和油松幼苗为研究对象，采用红外辐射增温（空气增温2.1℃，土壤增温2.6℃）和根部施氮（施氮量25 g N m-2yr-1）的方法，从生长形态、光合作用、抗氧化能力和矿质营养等方面研究这两种幼苗对气候变暖和氮沉降的响应。该实验为室外控制实验，包括四个处理：(1)不增温+不施氮（UU）；(2) 不增温+施氮（UF）；(3) 增温+不施氮（WU）；(4) 增温+施氮（WF）。本研究旨在从生理生化、物质代谢 、生长及形态等不同水平上研究模拟增温和施氮对两种树苗的联合效应，提高我们对全球变化下亚高山针叶林早期更新过程的理解，同时也为森林管理提供科学依据。具体研究结果如下： 单独增温处理显著提高了云杉和油松幼苗的地茎、叶重、茎重、根重以及总生物量；单独施氮处理也增加了两种幼苗的株高和总生物量。而增温和施氮联合作用对两种幼苗生长的影响并不相同，联合作用对云杉幼苗生长指标的正效应显著低于单独施氮处理，但是联合作用比单独增温或施氮更大程度的促进了油松幼苗生物量的积累。 单独增温和施氮都有利于提高云杉和油松叶片中叶绿素含量、净光合速率(A)、最大净光合速率(Amax)、表观量子效率（Φ）、最大光能转化效率（Fv/Fm）和量子产量（Y）。与对两种幼苗生长指标的影响相似，加氮和增温共同作用下油松幼苗的以上光合指标比在单独增温或施氮处理下有更大程度的提高；而联合作用下云杉幼苗叶绿素含量、净光合速率、最大净光合速率、表观量子效率、最大光能转化效率以及量子产量比单独施氮处理明显地降低。 增温和施氮都显著地降低了云杉和油松幼苗针叶组织中活性氧和丙二醛的积累。交互作用降低了云杉幼苗叶片的抗氧化酶活性、脯氨酸和ASA 的含量，却显著提高了油松幼苗SOD、POD、APX 等抗氧化酶的活性，并且对油松幼苗脯氨酸和ASA 积累的促进作用比单一因子更加明显。因此，增温和施氮共同作用下油松幼苗叶片中O2-产生速率、H2O2 及MDA 含量明显降低，而云杉叶片中只有O2-产生速率出现降低趋势。 增温和施氮都降低了云杉体内的P、Ca、Mg 元素的含量，增加了Cu、Zn、Mn 在各器官内的积累。对油松幼苗而言，增温和加氮单独作用也显著降低了Ca 含量增加了Cu、Zn、Mn 的积累，但是不同于云杉幼苗的是P、Mg 也显著增加。增温和施氮联合作用对云杉幼苗体内元素的影响与单一施氮处理或增温处理相似，不同的是比单一因子作用更为明显降低了P、Ca、Mg 含量，增加了植株中N、Cu、Zn、Mn 的含量，但是油松矿质元素含量在联合作用下并没有产生类似于云杉幼苗的双因子叠加效应。 总之，尽管单独增温或者施氮都有利于云杉和油松幼苗生长指标、光合能力以及抗氧化能力的提高。但是，增温和施氮对云杉幼苗生长生理的促进效应非但没有在交互作用下有更大的提高，反而低于单独氮处理。与此不同的是，增温和施氮联合作用比单因子作用更有利于油松幼苗生长及生理指标的提高。 With the continued increase in atmospheric concentrations of greenhouse gases (CO2、CH4、NOx), the mean global surface temperature has increased by about 0.74 ± 0.18℃ over the past century and is predicted to rise by as much as 6.4℃ during this century. Besides global warming, nitrogen deposition is another serious environmental problem caused by human activities, and high nitrogen load has become globalization as a result of global economy development. Global climate warming and nitrogen deposition have induced dramatic alternations in above - and below- ground biology and biogeochemistry process in terrestrial ecosystems, and more and more attention has been invited to those problems. This experiment mainly studies two important species Picea asperata and Pinus tabulaeformis in subalpine coniferous forest of western Sichuan, China. Infared heaters are induced to increase both air and soil temperature by 2.1℃ and 2.6 ℃, respectively. Ammonium nitrate solution (for a total equivalent to 25 g N m-2 year-1) is added to soil surface. There are four treatments in this study: (1) unwarmed unfertilized (UU); (2) unwarmed fertilized (UF); (3) warmed unfertilized (WU); (4) warmed fertilized (WF). This study is conducted to determine the influences of experimental warming and nitrogen fertilization on physiolchemistry, nutrition metabolism, growth and morphology in the two coniferous species seedlings. The current study is favorable for increasing our understanding on the early phase of regeneration behavior in subalpine coniferous forest, and it also provide scientific direction for forest management under future global changes. The results are as follows: Artificial warming alone significantly increased basal diameter, leaf mass, stem mass, root mass and total biomass for Picea asperata and Pinus tabulaeformis seedlings, and single nitrogen fertilization are also favorable for growth of the two species and stimulate plant hight and total biomass. The two species seedlings respond differently to the combination of elevated temperature and nitrogen addition. Warming combined with nitrogen fertilization weakens the positive effects of nitrogen addition for growth of Picea asperata seedlings. However, the combination of elevated temperature and nitrogen fertilization further increase biomass accumulation of Pinus tabulaeformis seedlings. Both elevated temperature alone and nitrogen fertilization alone can increase photosynthetic pigments contents, net photosynthetic rate (A), maximum net photosynthetic rate (Amax), apparent quantity yield (Φ), maximum photochemical efficiency of photosystem II (Fv/Fm) and effective quantum yield (Y). Similarly with growth parameters, the combination of warming and nitrogen addition induced more increment of these above photosynthetic parameters for Pinus tabulaeformis seedlings. However, these photosynthetic parameters of Picea asperata seedlings under the combination of warming and nitrogen addition are lower than those under nitrogen fertilization alone. The levels of active oxygen species (AOS) and malodiadehyde (MDA) in needles of the two coniferous species seedling are obviously decreased by experimental warming or additional nitrogen. Warming combined with nitrogen fertilizer reduces the activities of SOD, CAT and APX, and the contents of proline and ASA of Picea asperata seedlings, but the combination significantly increases activities of these antioxidant enzymes in needlels of Pinus tabulaeformis seedlings and further improves the accumulation of proline and ASA compared to either artificial warming or nitrogen addition. Therefore, the rate of O2 - production, the contents of H2O2 and MDA in needles of Pinus tabulaeformis seedlings are remarkably reduced by the combination of warming and nitrogen addition, but the combination only significantly decreased the rate of O2 - production of Picea asperata seedlings. Elevated temperature or nitrogen fertilization decrease the contents of P, Ca, Mg but increase Cu, Zn, Mn contents for Picea asperata seedlings. For Pinus tabulaeformis seedlings, elevated temperature alone and nitrogen fertilization alone decreased Ca, but increased P, Mg, Cu, Zn, Mn contents. The effects of the combination of warming and nitrogen addition on these element contents in needles of Picea asperata seedlings are added or multiplied the effects of warming and nitrogen addition alone, resulting in less contens of P, Ca, Mg and more contents of Cu, Zn, Mn than either elevated temperature or nitrogen fertilization. Howere, these adding or multipluing single-factor effects on contents of these elements are not observed in the case of Pinus tabulaeformis seedlings. In conclusion, growth parameters, photosynthetic capacities and antioxidant abilities of Picea tasperata and Pinus abulaeformis seedlings are improved by experimental warming or nitrogen fertilization. Interestingly, the positive effects of warming and nitrogen addition on growth and physiological performances are not multiplied by the combination of elevated temperature and nitrogen fertilization, even dempened for Picea asperata seedlings. However, for Pinus tabulaeformis seedlings, growth and physiological performances are further improved by the combination.

REDOX THERMODYNAMICS OF CYTOCHROME-C AT THE BARE GLASSY-CARBON ELECTRODE

Investigation of the redox thermodynamics of horse heart cytochrome c at bare glassy carbon electrodes has been performed using cyclic voltammetry with a nonisothermal electrochemical cell. The thermodynamic parameters of the electron-transfer reaction of cytochrome c have been estimated in different component buffer solutions. The change DELTAS(re)-degrees in reaction center entropy and the formal potential E-degrees' (at 25-degrees-C, vs. standard hydrogen electrode (SHE)) for cytochrome c are found to be -64.1 J K-1 mol-1 and 0.251 V in phosphate buffer, -64.8 J K-1 mol-1 and 0.257 V in Tris + HCl buffer, -65.6 J K-1 mol-1 and 0.261 V in Tris+CH3COOH buffer (pH 7.0, ionic strength 100 mM). The temperature dependence of the formal potential obtained in phosphate buffer with or without NaCl in the range 5-55-degrees-C shows biphase characteristics in an alkaline solution with an intersection point at ca. 44-degrees-C or 42-degrees-C, which should be due to a structural change in the protein moiety of cytochrome c. However, in acidic and neutral solutions only a monotonic relationship between E-degrees' and temperature is observed. The effect of the buffer component on E-degrees' for cytochrome c is also discussed.

TIDAL DEPENDENCE OF PHOTOINHIBITION OF PHOTOSYNTHESIS IN MARINE MACROPHYTES OF THE SOUTH CHINA SEA

PHOTOSYSTEM-II; CHLOROPHYLL FLUORESCENCE; ULVA-ROTUNDATA; ELECTRON-TRANSPORT; FIELD EXPERIMENTS; O-2 EVOLUTION; QUANTUM YIELD; TEMPERATURE; MACROALGAE; RESPONSES

The immune responses in Chinese mitten crab Eriocheir sinensis challenged with double-stranded RNA

Double-stranded RNA (dsRNA) is a virus-associated molecular pattern which induces antiviral innate immune responses and RNA interference (RNAi) in mammals. In invertebrates, RNAi phenomenon has been widely studied, but dsRNA-induced innate immune response is seldom reported. In the present study, two different dsRNAs specific for green fluorescent protein (GFP) and the putative D1 protein of photosystem II (NoPSD) from Nannochloropsis oculata, were employed to challenge Chinese mitten crab Eriocheir sinensis. The temporal changes of phenoloxidase (PO), acid phosphatase (ACP), superoxide dismutase (SOD) and malondialdehyde (MDA) content, as well as the mRNA expression of some immune-related genes were examined in order to estimate the effect of dsRNAs on the innate immunity of E. sinensis. The activities of PO, ACP and SOD significantly increased after dsRNA treatment, whereas malondialdehyde (MDA) content did not change significantly. Among the examined genes, only the mRNA expression of EsALF, an antibacterial peptide in E. sinensis, was significantly up-regulated (about 5 fold, P < 0.05) at 12 h after dsRNA treatment, while no significant expression changes were observed among the other immune genes. The increase of PO, ACP and SOD activities, and mRNA expression level of EsALF after dsRNA stimulation indicate that phenoloxidase, hydrolytic enzyme, antioxidation and EsALF were involved in dsRNA-induced innate immunity, suggesting that broad-spectrum immune responses could be induced by dsRNA in E. sinensis. (C) 2009 Elsevier Ltd. All rights reserved.

Isolation of the main light-harvesting chlorophyll a/b-protein complex from thylakoid membranes of marine alga, Bryopsis corticulans by a direct method

The main chlorophyll a/b light-harvesting complex (LHC 11) has been isolated directly from thylakoid membranes of marine green alga (Bryopsis corticulans Setch.) by two consecutive runs of anion exchange and gel-filtration chromatography. LHC 11 proteins in the membrane extracts treated with 3% n-Octyl-b-D-glucopyranoside (OG) obtained specific binding ability on Q Sepharose column, and thus were isolated from the thylakoid membranes in a highly selective fraction. The monomeric, trimeric and oligomeric subcomplexes of LHC 11 have been obtained by fractionation of the LHC 11 mixes with sucrose density gradient ultracentrifugation. The SDS-PAGE analysis of peptide composition and absorption spectrum showed that LHC 11 monomers, trimers and oligomers prepared through this work were intact and in high purity. Our report is the first to show that it is possible to purify LHC If directly from thylakoid membranes without extensively biochemical purification.