【作者】 戴国政；

【导师】 邱保胜； Karl Forchhammer；

【作者基本信息】 华中师范大学 ， 植物学， 2012， 博士

【摘要】 随着人类活动的加剧,人为氮输出量不断增加,由此对生态环境造成的负面效应也越来越明显,如水体富营养化、蓝藻水华的暴发、生物多样性的减少等。在大量人为输出的总氮中,铵占有相当大的比重。铵通常作为一种主要的氮营养被植物优先利用,但同时过高的铵也具有毒害作用。以往,铵毒害研究主要集中于高等植物,蓝藻的铵毒害机制研究还很欠缺,同时铵毒害对蓝藻的生态效应也没有引起人们的足够关注。基于以上认识,本论文对一种食用野生蓝藻资源-葛仙米(念珠藻属)减产的原因以及其铵毒害机制进行了探讨,分析了铵在蓝藻水华的暴发以及藻类的演替中所起到的作用,研究了铵毒害对模式蓝藻-集胞藻PCC6803光系统Ⅱ的作用机制,并从分子水平上探讨了D1蛋白的一个编码基因psbAl在抵御铵毒害中所起的作用,具体研究内容和结果如下：1.研究了两种化肥(氯化铵和氯化钾)对葛仙米以及其它四种常见蓝藻生长的影响。结果表明：五种藻在10mmol L-1氯化铵作用下,生长速率至少下降了65%,而相同浓度的氯化钾对所有藻生长没有抑制作用。同时,五种藻对铵毒害敏感性的顺序依次为：葛仙米>固氮鱼腥藻FACHB118>铜绿微囊藻FACHB905>铜绿微囊藻FACHB315>聚球藻FACHB805。葛仙米96小时铵处理的生长半抑制效应浓度为1.105mmol L-1,这个浓度远低于农业土壤中的铵浓度(2-20mmol L-1),说明铵肥的大量使用是葛仙米野生资源减产的一个重要原因。1mmol L-1氯化铵处理葛仙米96小时后,其光合放氧速率,PSII最大光化学效率(Fv/Fm),光合饱和光强以及PSII活性都显著降低,叶绿素合成较藻胆素合成更易受到铵抑制。捕光截面和电子受体QA后的电子传递速率在铵处理后增加,暗呼吸在5和10mmol L-1氯化铵处理下分别较对照组增加了246%和384%,快速荧光诱导动力学分析铵对葛仙米毒害作用的位点可能在PSⅡ的放氧复合体上。2.比较了葛仙米在两种限制性光强(40和100otons m-2s-1)下对铵毒害的敏感性。在2mmol L-1氯化铵处理96小时后,低光和高光下葛仙米的生长速率分别下降到对照组的44.3%和-16.0%。快速荧光诱导动力学分析发现5mmol L-1氯化铵在高光下更易对放氧复合体造成破坏,铵在高光下对葛仙米生长抑制的半效应浓度在比低光下低,说明葛仙米在高光下对铵毒害更为敏感。5。nmol L-1氯化铵处理一个小时内,高光处理较低光处理下的反应中心连接系数,Fv/Fm以及PQ后的电子传递速率下降更快,而捕光截面增加更明显。因此,高光下进行铵处理后,电子供体侧的快速损伤可能引起P680的过氧化,氧化β-胡萝卜素、破坏反应中心,而引起非光化学淬灭的降低,加重了反应中心的损伤。林肯霉素处理实验表明铵毒害对葛仙米PSⅡ的伤害主要作用在损伤过程,对PSⅡ的修复影响相对较小3.随着人类农业、工业活动的加剧,大量的还原性氮进入生物圈,引起全球水体富营养化和蓝藻水华。然而,至今还没有直接表明铵毒害影响藻类演替的相关报道。本研究探讨了19种不同营养类型水体中常见藻的铵毒害敏感性,证实铵可能是影响水体中蓝藻水华暴发以及常见藻类分布的因素之一。水华蓝藻-铜绿微囊藻PCC7806在高pH、高光条件下容易遭受铵毒害,pH9.0±0.2,1000μmol photons m-2s-1下处理1小时,0.06mmol L-1NH4Cl即能抑制PSⅡ的实际光化学效率至对照组的一半,而此浓度在自然水体中完全可以达到。此外,不同营养等级的水体中生长的常见藻种对铵毒害的耐受性顺序表现为超富营养化藻>富营养化藻>中度营养化藻>贫营养化藻。这种对铵毒害敏感性的变化正好解释了不同营养等级水体中常见藻的分布规律。同时,蓝藻(如微囊藻)水华经常出现在铵含量较低的夏季,并且随着铵浓度的降低而迅速消失。这可能是与春季的铵浓度较高带来的毒害效应有关(国内主要33个湖泊的平均和最大铵浓度分别为0.08mmol L-1和0.72mmol L-1),而夏秋季的铵浓度较低,没有毒害作用,仅作为氮营养被利用。因此,水体中的铵含量可能是决定蓝藻水华暴发和常见藻种类分布的一个重要限制因子。4.研究了铵对蓝藻Synechocystis sp. PCC6803光系统Ⅱ的损伤和修复的作用。结果表明铵对PSⅡ的伤害表现在加速PSⅡ的损伤,而对其修复没有抑制作用。铵对离体类囊体膜的处理实验直接证实放氧复合体是铵毒害的初级作用位点。D1蛋白能在光损伤下进行快速合成,早期结果表明Synechocystis sp. PCC6803的D1蛋白的一个编码基因psbA1在绝大多数条件下是一个沉默基因,仅微氧条件能诱导其表达。本研究显示,高光能诱导其表达,且敲除psbA1基因的突变株较野生型表现出更强的高光伤害和铵毒害敏感性,这说明psbA1基因在Synechocystis sp. PCC6803中并不是一个冗余基因,它在抗高光伤害和抗铵毒害上起到一定的保护作用。更多还原

【Abstract】 With the human intensification of agricultural and industrial activities, the amount of nitrogen input by human has been increasing, which consequently results in some significant negative ecological effects, such as eutrophication, cyanobacterial blooms, decline of biodiversity and so on. Ammonium accounts for a large proportion to the total nitrogen created by human. It is one of the major and preferred nutrients for plants, however, it is known to be toxic to many organisms at high concentration. Prevoius studies of ammonium toxicity were mainly focused on higher plants, relatively less attention has been paid in cyanobacteria, and the cyanobacterial ecological effects caused by ammonium toxicity had not been attracted human’s special concern. Based on above background, present study explored the cause of reduction of Nostoc sp.(Ge-Xian-Mi) resource—one kind of edible cyanbacteria and its ammonium toxicity mechanism, analyzed the role of ammonium in the succession of cyanobacterial blooms and the distribution of common algal species in shallow freshwater lakes, investigated the mechanism of ammonium toxicity on PSII of Synechocystis sp. PCC6803, and the function of psbAl gene on the resisting to ammonium toxicity damage, the detailed content and results of this study is as follows,1. Effects of two fertilizers, NH4Cl and KCl, on the growth of the edible cyanobacterium Ge-Xian-Mi (Nostoc) and other four cyanobacterial strains were compared. Their growth was decreased by at least65%at10mmol·L-1NH4Cl, but no inhibitory effect was observed at the same level of KCl. Meanwhile, they exhibited a great variation of sensitivity to NH4+toxicity in the order: Ge-Xian-Mi> Anabaena azotica FACHB118> Microcystis aeruginosa FACHB905> Microcystis aeruginosa FACHB315> Synechococcus FACHB805. The relative growth rate96-h EC50value of NH4+for Ge-Xian-Mi was1.105mmol L-1, which was much less than NH4+concentration in many agricultural soils (2to20mmol L’1). These indicated that the use of ammonium as nitrogen fertilizer was responsible for the reduced resource of Ge-Xian-Mi in the paddy field. After96h exposure to1mmol L-1NH4Cl, the photosynthetic rate, Fv/Fm value, saturating irradiance for photosynthesis and PSII activity of Ge-Xian-Mi colonies were remarkably decreased. The chlorophyll synthesis of Ge-Xian-Mi was more sensitive to NH4+toxicity than phycobiliproteins. Thus, its functional absorption cross section of PSII was increased markedly at NH4Cl levels≥1mmol·L-1and the electron transport on the acceptor side of PSII was significantly accelerated by NH4Cl addition≥3mmol·L-1. Dark respiration of Ge-Xian-Mi was significantly increased by246%and384%at5and 10mmol L-1NH4Cl respectively. The rapid fluorescence rise kinetic suggested that the oxygen-evolving complex of PSII was the inhibitory site of NH4-2. The sensitivity of Ge-Xian-Mi (Nostoc) to ammonium toxicity under two limiting light intensities (40and100μmol photons m-2s-) were compared. After96h exposure to2mmol L-1NH4Cl, their growth were decreased to44.3%and-16.0%of the control under low light and high one respectively. The rapid fluorescence rise kinetic showed that the oxygen-evolving complex of PSII was much greatly damaged upon5mmol L-1NH4+addition under high light. The relative growth rate96-h EC50value showed that Ge-Xian-Mi was more sensitive to NH4+toxicity under high light compared to low one during the limiting light climate. With the treatment of5mmol L-1NH4+during60min, the connectivity factor, Fv/Fm value and electron transport rate between PSII and PSI decreased much faster, and the functional absorption cross section of PSII increased much faster, under high light than that of low light. Therefore, much more degree of damage in donor side with the treatment of ammonium upon high light may cause the generation of highly reaction P680, which will oxidate the β-carotene, damage the reaction centre, reduce the nonphotochemical fluorescence quenching, and aggravate the damage of reaction centre. Meanwhile, the lincomycin addition experiment indicated NH4+triggered photodamage response for accelerating the damage and less inhibiton of repair the of PSII of Nostoc sp.(Ge-Xian-Mi).3. With the human intensification of agricultural and industrial activities, large amount of reduced nitrogen enter into the biosphere, which consequently results in the development of global eutrophication and cyanobacterial blooms. However, no research had reported the direct effect of ammonia toxicity on the algal succession in freshwater lakes. In this study, we investigated the ammonia toxicity to nineteen algal species or strains to test the hypothesis that ammonia may regulate the succession of cyanobacterial blooms and the distribution of common algal species in freshwater lakes. The bloom-forming cyanobacterium Microcystis aeruginosa PCC7806suffered from ammonia toxicity at high pH value and light intensity conditions. Low NH4CI concentration (0.06mmol L-1) resulted in the decrease of operational PSII quantum yield by50%compared to the control exposed to1000μmol photons m-2s-1for one hour at pH9.0±0.2, which can be reached in freshwater lakes. Furthermore, the tolerant abilities to NH3toxicity of eighteen freshwater algal species or strains were as follows:hypertrophication species> eutrophication species> mesotrophication species> oligotrophication species. The different sensitivities of NH3toxicity in the present study could well explain the distributing rule of common algal species in the freshwater lakes of different trophic states. Meanwhile, the cyanobacterial bloom (e.g. M. aeruginosa) always happened at the low concentration of ammonia in summer, and disappeared with the decrease of ammonia. This may be attributed to the toxic effect of ammonia to M. aeruginosa in spring (the average and maximum ammonia concentration were0.08mmol L-1, and0.72mmol L-1in33Chinese lakes), and the low level of NH3-N in summer and fall in the lakes might be used as preferred nitrogen nutrition by M. aeruginosa, rather than with toxicity. Therefore, ammonia could be a key factor to determine the distribution of common algal species and cyanobacterial bloom in the freshwater systems.4. The effects of ammonia on the photodamage of PSII and repair of damaged PSII in cyanobacterium Synechocystis sp. strain PCC6803were investigated in this study. Ammonia was shown to accelerate the decrease of PSII activity through a mechanism that directly caused photodamage of PSII, rather than inhibition of the repair of photodamaged PSII. Isolated thylakoid membranes lost PSII activity upon ammonia treatment, with the oxygen-evolving complex being the primary target for ammonia toxicity. The D1protein of photosynthetic organisms is known to be rapidly turned over in the light. Previous studies indicated that the psbAl gene encoded one form of Dl protein that was not expressed in wild-type Synechocystis6803. The present investigation shows that its expression is transiently induced by high light. Synechocystis wild-type cells are more resistant to high light and ammonia than a psbAl-deficient mutant, indicating that the psbAl gene is not redundant, but could play a role in protecting PSII against high light and ammonia toxicity.更多还原