发菜类囊体膜的组成及其光合特性的研究

发菜（Nostoc flagelliforme Born. et Flah.）的细胞壁由纤维素、半纤维素、糖脂和蛋白质组成。未经破碎的细胞难以进行各种光合特性的研究。由于纯度较高的发菜类囊体膜制备比较困难，对它的光合机理的研究一直是停留在整体水平上进行。我们采用French Press低温下高压破碎细胞，建立了一种快速简便的制备方法。在提取液中加入一定浓度的Ca2+ （Ca2+既有助于维持类囊体膜的放氧活性又可以使类囊体膜在较低的离心速度下使类囊体膜得到凝集沉淀），从而在较短时间内、在高速离心的情况下得到了纯度较高并具有较高放氧活性的发菜类囊体膜。在此基础上，我们采用改进的Allen（1991）的温和绿胶系统，首次对陆生蓝藻发菜类囊体膜色素蛋白复合体进行了分离，共分离出了11条绿色的色素蛋白复合物条带和两条浅黄色的条带。7条绿色的色素蛋白复合物条带属于PSI组分，4条绿色的蛋白复合物条带属于PSII组分，其中一条浅黄色条带系未被报道过的新的色素蛋白复合物条带，经其光谱性质的分析初步鉴定为类胡萝卜素蛋白复合物，此复合物的分离有助于解释发菜独特的适应荒漠、半荒漠地带高光辐射的特性。 本文还对干燥状态、复水30分钟后和复水生长24小时后的野生发菜及人工培养的发菜藻丝体膜脂及其脂肪酸组成进行了分析。发菜的膜脂由MGDG、MGDG、SQDG和PG组成，其酯酰基部位连接有16:0、16:1、18:0、18:1、18:2和18:3六种脂肪酸。野生发菜中具有高含量的不饱和脂肪酸，其含量可达总脂的73%，其中16:1和18:3分别达到28.9mol%和34.3mol%，远远高于已报道的其它蓝藻，所以我们推测发菜具有极强的抗逆性和其膜脂不饱和程度密切相关。分析不同处理的发菜的膜脂和脂肪酸组成表明，复水对野生发菜的膜脂及其脂肪酸组成没有显著影响，说明发菜的膜脂和脂肪酸组成在干燥状态下能保持很高的稳定性。从野生发菜分离出的藻丝体在25 ℃条件下培养，其膜脂脂肪酸组成发生了显著变化，主要表现为脂肪酸的不饱和程度的大幅度降低，18:3从34.9mol%降低到8.6mol%，16:1从28.9mol%降低到13.9mol%。上述结果表明了发菜具有极强的通过改变其膜脂的脂肪酸组成而适应生存环境的能力。

发状念珠藻光合特征及其在复水－失水过程中能量传递变化的研究

摘要 "发状念珠藻（Nostoc flagelliforme Born. Et Flah.），俗名发菜，是生长于干旱、半干旱土壤表面的陆生蓝藻，具有极强的抗旱能力。发菜光合作用方面的研究大多处于整体细胞水平，且研究手段非常有限。本实验对发菜光合特征进行深入研究，并探讨了发菜在干湿交替过程中能量传递的变化情况。 叶绿素和藻胆素是发菜细胞中两种主要的光合色素。发菜复水后光合活性完全恢复时，在室温（20℃）或低温（77K）下，其绝大部分的荧光是由于藻胆素被激发而产生。在室温下，大部分荧光来自藻胆体;当叶绿素被激发后，产生的荧光非常微弱。在低温下，藻胆素被激发后，荧光发射光谱中可分辨出藻胆蛋白、光系统Ⅰ和光系统Ⅱ的发射峰;叶绿素被激发后，荧光发射光谱包括光系统Ⅰ和光系统Ⅱ的荧光。相比之下，室温荧光发射光谱不适于用做发菜细胞光合作用方面的研究。 我们设计了一种新方法，从野生发菜细胞中分离得到类囊体膜及细胞质膜，并对其性质进行分析。发菜细胞外复杂的胶质结构使得现有破碎其它蓝藻细胞的方法无法破碎发菜细胞。通过实验发现，联合使用细胞破碎仪和毛地黄皂甙（0.3%）可有效破碎发菜细胞;并且毛地黄皂甙在低浓度下（≦0.5%），对色素与蛋白的结合不会造成破坏作用。随后，通过蔗糖密度梯度离心可将细胞质膜与类囊体膜分离。发菜类囊体膜的光谱性质与其它蓝藻相似。细胞质膜除结合有类胡萝卜素外，还结合有少量叶绿素前体。类囊体膜和细胞质膜膜脂及脂肪酸组成相似。其中，十六碳烯酸[16：1(9)]和亚麻酸[18：3(9,12,15)]是含量最高的两种脂肪酸，分别占总脂肪酸含量的三分之一左右。高含量的多不饱和脂肪酸可能和发菜极强的抗旱能力有关。 我们首次对发菜捕光色素蛋白复合物－藻胆体的组成和结构进行分析。发菜藻胆体为“3核＋6杆”的半圆盘结构。组成藻胆体的藻胆蛋白包括藻蓝蛋白和别藻蓝蛋白。两个藻蓝蛋白六聚体通过连接肽组成藻胆体的“杆”结构。在“杆”结构中等量分布着两条连接肽（分子量分别为29kDa和34kDa）为杆连接肽和核杆连接肽。而“核”结构中核膜连接肽的分子量为103kDa。 发菜在无霜期，几乎每天经历一次复水－干燥过程：夜间的结露使发菜在黑暗中复水，而清晨太阳升起后，在光照下迅速失水进入休眠状态。我们研究了发菜在黑暗中的复水过程及在光照下失水过程中藻胆体与光系统能量传递的变化情况。发菜在黑暗中复水后，光系统Ⅱ活性无法恢复。藻胆体内及藻胆体与光系统Ⅰ的能量传递在5分钟内恢复;而藻胆体与光系统Ⅱ的能量传递只能部分恢复。我们设想，发菜在复水过程中通过双扳机－水和光－控制光合活性的恢复，以及在黑暗中部分恢复藻胆体与光系统Ⅱ的能量传递，将减少不必要的能量消耗与通过光合作用储备尽可能多的化学能－这两个生存策略有机的结合起来。发菜在光照下的失水过程中，光合活性在含水量降至90％前基本保持稳定，随后迅速下降。而在含水量达到150％后，藻胆体内的能量传递便开始受到抑制，并且随着含水量的降低，该抑制现象逐步加剧。这样，发菜在干燥过程中，通过抑制藻胆体内的能量传递，减少了传递到光系统Ⅱ反应中心的能量，从而避免了在光合活性下降过程中过剩光能对光系统Ⅱ产生的破坏作用。"

Characteristics and comparative study of chlorophyll-protein complexes from siphonous green algae

By mild PAGE method, 11, 11, 7 and 9 chlorophyll-protein complexes were isolated from two species of siphonous green algae ( Codium fragile (Sur.) Harlot and Bryopsis corticulans Setch.), green alga (Ulothrix flacca (Dillw.) Thur.), and spinach (Spinacia oleracea Mill.), respectively. Apparent molecular weights, Chi a/b ratios, distribution of chlorophyll, absorption spectra, low temperature fluorescence spectra of these complexes were determined, and compared with one another. PS I complexes of two siphonous green algae are larger in apparent molecular weight because of the attachment of relative highly aggregated LHC I. Four isolated light-harvesting complexes of PSII are all siphonaxanthin-Chl a/b-protein complexes, and they are not monomers and oligomers like those in higher plants. Especially, the absence of 730 nn fluorescence in PS I complexes indicates a distinct structure and energy transfer pattern.

Responses of photosynthetic activity in the drought-tolerant cyanobacterium, Nostoc flagelliforme to rehydration at different temperature

Photosynthetic activity during rehydration at four temperatures (5, 15, 25, 35 degrees C) was studied in a terrestrial, highly drought-tolerant cyanobacterium, Nostoc flagelliforme. At all the temperatures, the optimum quantum yield F-v/F-m increased rapidly within I It and then increased slowly during the process of rehydration. The increase in F-v/F-m at 25 and 35 degrees C was larger than that at 5 and 15 degrees C. In addition, the changes of initial intensity of fluorescence (F-0) and variable fluorescence (F-v) were more significant at 25 and 35 degrees C than those at 5 and 15 degrees C. Chlorophyll a content increased with the increase of temperature during the course of rehydration, with this being more pronounced at 25 and 35 degrees C. The photosynthetic rates at 25 and 35 degrees C were higher than those at 5 and 15 degrees C. Induction of chlorophyll fluorescence with sustained rewetting at 5 and 15 degrees C had two phases of transformation, whereas at 25 and 35 degrees C it had a third peak kinetic phase and showed typical chlorophyll fluorescence steps on rewetting for 24 h, representing a normal physiological state. A comparison of the chlorophyll fluorescence parameters, chlorophyll a content, and the chlorophyll fluorescence induction led to the conclusion that N. flagelliforme had a more rapid and complete recovery at 25 and 35 degrees C than that at 5 and 15 degrees C, although it could recover its photosynthetic activity at any of the four temperatures. (c) 2007 Published by Elsevier Ltd.

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.

Heterogeneity of photosystem II as it occurs in domain specific regions of the thylakoid membrane of spinach (spinacia oleracia L.)

Thylakoid membrane fractions were prepared from specific regions of thylakoid membranes of spinach (Spinacia oleracea). These fractions, which include grana (83), stroma (T3), grana core (8S), margins (Ma) and purified stroma (Y100) were prepared using a non-detergent method including a mild sonication and aqueous two-phase partitioning. The significance of PSlla and PSII~ centres have been described extensively in the literature. Previous work has characterized two types of PSII centres which are proposed to exist in different regions of the thylakoid membrane. a-centres are suggested to aggregate in stacked regions of grana whereas ~-centres are located in unstacked regions of stroma lamellae. The goal of this study is to characterize photosystem II from the isolated membrane vesicles representing different regions of the higher plant thylakoid membrane. The low temperature absorption spectra have been deconvoluted via Gaussian decomposition to estimate the relative sub-components that contribute to each fractions signature absorption spectrum. The relative sizes of the functional PSII antenna and the fluorescence induction kinetics were measured and used to determine the relative contributions of PSlla and PSII~ to each fraction. Picosecond chlorophyll fluorescence decay kinetics were collected for each fraction to characterize and gain insight into excitation energy transfer and primary electron transport in PSlla and PSII~ centres. The results presented here clearly illustrate the widely held notions of PSII/PS·I and PSlIa/PSII~ spatial separation. This study suggests that chlorophyll fluorescence decay lifetimes of PSII~ centres are shorter than those of PSlIa centres and, at FM, the longer lived of the two PSII components renders a larger yield in PSlIa-rich fractions, but smaller in PSIlr3-rich fractions.

Relationship between photosynthetic electron transport and pH gradient across the thylakoid membrane in intact leaves.

Under conditions (0.2% CO2; 1% O2) that allow high rates of photosynthesis, chlorophyll fluorescence was measured simultaneously with carbon assimilation at various light intensities in spinach (Spinacia oleracea) leaves. Using a stoichiometry of 3 ATP/CO2 and the known relationship between ATP synthesis rate and driving force (Delta pH), we calculated the light-dependent pH gradient (Delta pH) across the thylakoid membrane in intact leaves. These Delta pH values were correlated with the photochemical (qP) and nonphotochemical (qN) quenching of chlorophyll fluorescence and with the quantum yield of photosystem II (phiPSII). At Delta pH > 2.1 all three parameters (qP, qN, and phiPSII) changed very steeply with increasing DeltapH (decreasing pH in the thylakoid). The observed pH dependences followed hexacooperative titration curves with slightly different pKa values. The significance of the steep pH dependences with slightly different pKa values is discussed in relation to the regulation of photosynthetic electron transport in intact leaves.

发菜(Nostoc flagelliforme)培养条件的研究

研究了光照强度、日供水次数、CO2浓度和培养基成分对发菜生长的影响。结果显示,中度光强(114μmol.m-2.s-1)下发菜生长最快;发菜的生长基本同供水次数成正相关系;CO2浓度的升高并没有显著促进发菜生长,低光条件下(57μmol.m-2.s-1),高浓度的CO2(2800μL/L)抑制了发菜的生长;用BG11培养的发菜生物量的增长显著高于用BG110培养的;BG11培养基中K+和CO32-的缺失并没有显著影响发菜的生长。

Influence of dew on biomass and photosystem II activity of cyanobacterial crusts in the Hopq Desert, northwest China

Dew is an important water source for desert organisms in semiarid and arid regions. Both field and laboratory experiments were conducted to investigate the possible roles of dew in growth of biomass and photosynthetic activity within cyanobacterial crust. The cyanobacteria, Microcoleus vaginatus Gom. and Scytonema javanicum (Kutz.) Born et Flah., were begun with stock cultures and sequential mass cultivations, and then the field experiment was performed by inoculating the inocula onto shifting sand for forming cyanobacterial crust during late summer and autumn of 2007 in Hopq Desert, northwest China. Measurements of dew amount and Chlorophyll a content were carried out in order to evaluate the changes in crust biomass following dew. Also, we determined the activity of photosystem II(PSII) within the crust in the laboratory by simulating the desiccation/rehydration process due to dew. Results showed that the average daily dew amount as measured by the cloth-plate method (CPM) was 0.154 mm during fifty-three days and that the crust biomass fluctuated from initial inoculation of 4.3 mu g Chlorophyll a cm(-2) sand to 5.8-7.3 mu g Chlorophyll a cm(-2) crust when dew acted as the sole water source, and reached a peak value of approximately 8.2 mu g Chlorophyll a cm(-2) crust owing to rainfalls. It indicated that there was a highly significant correlation between dew amounts and crust moistures (r = 0.897 or r = 0.882, all P < 0.0001), but not a significant correlation between dew and the biomass (r = 0.246 or r = 0.257, all P > 0.05), and thus concluded that dew might only play a relatively limited role in regulating the crust biomass. Correspondingly, we found that rains significantly facilitated biomass increase of the cyanobacterial crust. Results from the simulative experiment upon rehydration showed that approximately 80% of PSII activity could be achieved within about 50 min after rehydration in the dark and at 5 degrees C, and only about 20% of the activity was light-temperature dependent. This might mean that dew was crucial for cyanobacterial crust to rapidly activate photosynthetic activity during desiccation and rehydration despite low temperatures and weak light before dawn. It also showed in this study that the cyanobacterial crusts could receive and retain more dew than sand, which depended on microclimatic characteristics and soil properties of the crusts. It may be necessary for us to fully understanding the influence of dew on regulating the growth and activity of cyanobacterial crust, and to soundly evaluate the crust's potential application in fighting desertification because of the available water due to dew. (C) 2009 Published by Elsevier Ltd.

The vertical microdistribution of cyanobacteria and green algae within desert crusts and the development of the algal crusts

Substantial amounts of algal crusts were collected from five different desert experimental sites aged 42, 34, 17, 8 and 4 years, respectively, at Shapotou ( China) and analyzed at a 0.1 mm microscale of depth. It was found that the vertical distribution of cyanobacteria and microalgae in the crusts was distinctly laminated into an inorganic-layer (ca. 0.00 - 0.02 mm, with few algae), an algae-dense-layer ( ca. 0.02 - 1.0 mm) and an algae-sparse-layer ( ca. 1.0 - 5.0 mm). It was interesting to note that in all crusts Scytonema javanicum Born et Flah ( or Nostoc sp., cyanobacterium), Desmococcus olivaceus (Pers ex Ach., green alga) Laundon and Microcoleus vaginatus Gom. ( cyanobacterium) dominated at the depth of 0.02 - 0.05, 0.05 - 0.1 and 0.1 - 1.0 mm, respectively, from the surface. Phormidium tenue Gom. ( or Lyngbya cryptovaginatus Schk., cyanobacterium) and Navicula cryptocephala Kutz.( or Hantzschia amphioxys (Ehr.) Grun. and N. cryptocephala together, diatom) dominated at the depth of 1.0 - 3.0 and 3.5 - 4.0 mm, respectively, of the crusts from the 42 and 34 year old sites. It was apparent that in more developed crusts there were more green algae and the niches of Nostoc sp., Chlorella vulgaris Beij., M. vaginatus, N. cryptocephala and fungi were nearer to the surface. If lichens and mosses accounted for less than 41.5% of the crust surface, algal biovolume was bigger when the crust was older, but the opposite was true when the cryptogams other than algae covered more than 70%. In addition to detailed species composition and biovolume, analyses of soil physicochemical properties, micromorphologies and mineral components were also performed. It was found that the concentration of organic matter and nutrients, electric conductivity, silt, clay, secondary minerals were higher and there were more micro-beddings in the older crusts than the less developed ones. Possible mechanisms for the algal vertical microdistribtion at different stages and the impact of soil topography on crust development are discussed. It is concluded that biomethods ( such as fine species distribution and biovolume) were more precise than mineralogical approaches in judging algal crust development and thus could be a better means to measure the potentiality of algal crusts in desert amelioration.

Culture of the terrestrial cyanobacterium, Nostoc flagelliforme (Cyanophyceae), under aquatic conditions

Both colonies and free-living cells of the terrestrial cyanobacterium, Nostoc flagelliforme (Berk. & Curtis) Bornet & Flahault, were cultured under aquatic conditions to develop the techniques for the cultivation and restoration of this endangered resource. The colonial filaments disintegrated with their sheaths ruptured in about 2 days without any desiccating treatments. Periodic desiccation played an important role in preventing the alga from decomposing, with greater delays to sheath rupture with a higher frequency of exposure to air. The bacterial numbers in the culture treated with seven periods of desiccation per day were about 50% less compared with the cultures without the desiccation treatment. When bacteria in the culture were controlled, the colonial filaments did not disintegrate and maintained the integrity of their sheath for about 20 days even without the desiccation treatments, indicating the importance of desiccation for N. flagelliforme to prevent them from being disintegrated by bacteria. On the other hand, when free-living cells obtained from crushed colonial filaments were cultured in liquid medium, they developed into single filaments with sheaths, within which multiple filaments were formed later on as a colony. Such colonial filaments were developed at 15, 25, and 30degreesC at either 20 or 60 mumol photons.m(-2).s(-1); colonies did not develop at 180 mumol photons.m(-2).s(-1), though this light level resulted in the most rapid growth of the cells. Conditions of 60 mumol photons.m(-2).s(-1) and 25degrees C appeared to result in the best colonial development and faster growth of the sheath-held colonies of N. flagelliforme when cultured indoor under aquatic conditions.

Daily production and photosynthetic characteristics of Nostoc flagelliforme grown under ambient and elevated CO2 conditions

Diurnal photosynthesis of Nostoc flagelliforme was investigated at varied levels of CO2 concentrations and desiccation in order to estimate the effects of enriched CO2 and watering on its daily production. Photosynthetic activity was closely correlated with the desiccated status of the algal mats, increased immediately after watering, reached a maximum at moderate water loss, and then declined with further desiccation. Increased CO2 concentration enhanced the diurnal photosynthesis and raised the daily production. Watering twice per day enhanced the daily production due to prolonged period of active photosynthesis. The values of daily net production were 1321280 mumol CO2 g (d. wt)(-1) d(-1), corresponding to about 0.6-6.1% daily increase in dry weight. High-CO2-grown mats required higher levels of photon flux density to saturate the alga's photosynthesis in air. Air-grown mats showed higher photosynthetic affinity for CO2 and higher levels of dark respiration compared with high-CO2-grown samples.

Photosynthetic bicarbonate utilization by a terrestrial cyanobacterium, Nostoc flagelliforme (Cyanophyceae)

The photosynthetic characteristics of the terrestrial cyanobacterium, Nostoc flagelliforme, after complete recovery by rewetting, was investigated to see whether it could use bicarbonate as the external inorganic carbon source when submerged. The photosynthesis-pH relationship and high pH compensation point suggested that the terrestrial alga could use bicarbonate to photosynthesize when submerged. The photosynthetic oxygen evolution rates were significantly inhibited in Na+-free and Na+ + Li+ media but were not affected by the absence of Cl-, implying that the bicarbonate uptake was associated with Na+/HCO3- symport rather than Cl-/HCO3- exchange system.