【作者】 刘树文；

【导师】 邱保胜；

【作者基本信息】 华中师范大学 ， 植物学， 2012， 博士

【摘要】 铁供应不足限制了海洋初级生产力,特别是在约占全球海洋面积1/3的高营养盐低叶绿素(high-nutrient low-chlorophyll, HNLC)区域。自1988年以来,在HNLC海区进行了多次培养瓶内以及大规模的现场铁添加实验,研究结果表明铁在调节海洋初级生产力、生物地球化学循环以及全球气候方面起重要作用。在海洋生态系统中,聚球藻(Synechococcus)数量大、分布广,是全球海洋初级生产力的主要贡献者。聚球藻在海洋微食物网中周转迅速,每天被鞭毛虫和纤毛虫牧食的聚球藻占其现存量的35-100%。在HNLC海区的铁添加实验中,只有硅藻没有受到浮游动物捕食的影响,其生物量大量增加。长期以来,人们认为超微型浮游藻类聚球藻在HNLC海区并未受到铁限制。但是,也有一些证据表明,Synechococcus在海洋中同样受到铁限制。以往,有关铁限制对浮游藻类的生理生化影响研究主要集中在硅藻等真核藻类,而有关原核藻类聚球藻的研究较少。铁元素是许多酶和蛋白的辅因子,在光合和呼吸电子传递,以及硝态氮的吸收利用等许多代谢过程中起重要作用。但是,藻细胞内绝大多数的铁存在于光合机构中。近岸水体中的铁浓度比大洋水体中的铁浓度高,铁限制的选择压力导致真核硅藻的海岸株比大洋株对铁限制更敏感。根据理论推测,浮游藻类细胞以硝酸盐为氮源时比以铵盐为氮源时有更高的铁需求。人们不清楚海洋聚球藻的大洋株是否也比海岸株更能耐受铁限制,并且对于聚球藻以铵盐为氮源时是否有更低的铁需求也还存有争议。海洋中的铁浓度随着深度的增加而升高,但海洋中的光强随着深度的增加而逐渐减弱。结合理论预测与真核硅藻的研究表明,藻细胞在低光条件下生长时对铁的需求更高,但铁限制的藻细胞在高光条件下PSII更容易受到光抑制。因此,在聚球藻海岸株的绿色株系和红色株系中铁元素和光的互作关系值得我们深入研究。目前,聚球藻PCC7002已被实验证实能产生铁载体,以适应铁限制环境的海洋蓝藻。当它与那些不能合成铁载体的藻株共培养时,它是否具有竞争优势也值得我们探讨。本文采用专为研究微量金属元素对海洋藻类生理影响而设计的Aquil培养基,全程采用痕量金属洁净技术控制微量元素污染,采用Water-PAM荧光仪测定F0的方法监测藻细胞的生长,运用Water-PAM和FIRe荧光技术以及流式细胞技术研究铁元素对不同藻株的生理影响,探究铁限制生理反应的种间差异以及铁元素对氮素和光能利用的影响。研究结果将有助于我们正确评估铁限制对超微型浮游藻类初级生产的影响,揭示铁元素对海洋生态系统种群结构的影响,为进一步研究不同藻株适应低铁的生化和分子机制提供依据。主要结果如下：1.最小荧光产额是测定超微型浮游藻类生物量的可靠便捷方法。在铁浓度为4-1000nM的Aquil培养基中,培养两株海岸聚球藻(PCC7002和CC9311)和一株大洋聚球藻(WH8102),采用WATER-PAM荧光仪测定藻液的最小荧光F0,同时利用流式细胞仪进行细胞计数。结果表明,通过这两种方法得出的比生长速率没有显著差异。与铁充足条件(1000nM Fe)相比,WH8102和CC9311在15nMFe培养基中的比生长速率分别降低了59%和37%,PCC7002在4nM Fe条件下的比生长速率降低了57%。这表明,PCC7002最能耐受铁限制,而WH8102对铁限制最敏感。最小荧光F0值与细胞浓度之间存在线性关系,铁充足条件下不含藻红蛋白的PCC7002的每细胞最小荧光是富含藻红蛋白藻株(WH8102和CC9311)的100倍。在铁限制条件下,WH8102和CC9311每细胞的最小荧光分别增加128%和7%,但是PCC7002却降低了30%,这主要与各藻株的藻胆素组成差异以及铁限制对不同藻株光合色素的影响差异有关。在光暗周期中,PCC7002与CC9311的每细胞最小荧光变化较小,而WH8102的每细胞最小荧光在光照条件下更高。总之,通过测定稳态条件下藻细胞的F0值是一种可靠的比生长速率测定方法。2.聚球藻海岸株和大洋株的光合作用和流式信号对铁限制和氮源的不同反应。本研究比较了四株海洋聚球藻在不同铁浓度和氮源条件下的光合作用和流式信号的差异,结果表明大洋株比海岸株对铁限制更敏感,铁限制对两个大洋株的生长、光系统Ⅱ最大光化学效率、最大电子传递速率以及光化学淬灭的抑制程度大于两个海岸株。在铁限制条件下,两个海岸株不同光合单位之间的连接系数增加,而两个大洋株的连接系数降低,同时铁限制加快了两个大洋株QA的氧化速率以及海岸株PQ库的氧化速率。在铁限制条件下,两个大洋株的细胞大小与细胞内色素含量降低,而侧向散射光与前向散射光的比值(SS/FS)增加。与铁限制不同,氮源对四个藻株的光合作用影响相对较小。在铁充足条件下,以氨为氮源时促进了两个海岸株的生长。以氨为氮源时,在铁充足和铁限制条件下两个大洋株的细胞体积增加,SS/FS与细胞内色素含量降低。以往的研究表明,真核硅藻的海岸种较大洋种对铁限制更敏感,而本研究以海洋聚球藻得出的结论与此相反。至于聚球藻大洋株比海岸株对铁限制更敏感的生化机制,还有待进一步探讨。3.铁和光对聚球藻绿色和红色海岸株生长、光合作用及流式色素荧光的影响。在高铁和低铁以及高光和低光条件下培养绿色海岸株(PCC7002)与红色海岸株(CC9311),结果表明PCC7002在低光条件下更容易受到铁限制,而CC9311在铁限制条件下更容易受到高光抑制,另外在高光和低光条件下培养的CC9311都比PCC7002更容易受到铁限制。通过流式细胞仪分析发现,CC9311和PCC7002的叶绿素、藻蓝蛋白和藻红蛋白荧光在铁限制和高光条件下降低。PCC7002的捕光截面(OPSⅡ)在铁光共限制条件下降低,CC9311的σPSⅡ在铁限制和高光条件下降低,这可能是由于铁限制和高光导致与光系统Ⅱ反应中心结合的色素分子数量降低。在铁限制和光限制条件下,PCC7002的线性电子传递速率降低,而PQ的氧化速率(1/τPQ)加快,这可能是由于围绕PSI的循环电子传递加快。在铁限制和高光条件下,CC9311的线性电子传递降低,而QA氧化速率(1/τQa)和PQ的氧化速率(1/τPQ)都加快,这可能是由于围绕PSⅠ和PSⅡ的循环电子传递加快。不同铁浓度和光照条件下培养的PCC7002和CC9311,经过30min高光(300μmol photons·m-2·s-1)处理,然后进行低光恢复。通过比较高光处理后以及低光恢复后的Fv/Fm值,结果表明CC9311比PCC7002对高光更敏感且恢复程度较低。另外,在低光和低铁条件下培养的藻细胞更容易受到光抑制且恢复程度较低,这可能与低光低铁培养条件下的CC9311和PCC7002的非光化学淬灭(NPQ)较低有关。铁元素和光照的相互作用可能是红色藻株和绿色藻株垂直分布的决定因素之一。4.聚球藻PCC7002与WH8102和CC9311对铁限制的生理反应以及铁元素对它们之间竞争的影响。在铁限制条件下,聚球藻PCC7002能合成铁载体参与铁的吸收,因而通常认为具有竞争优势。在单培养条件下,铁限制对WH8102和CC9311的细胞数以及比生长速率的抑制程度较PCC7002大。铁限制对PCC7002的细胞内ROS相对含量以及死亡细胞比例的影响也较小,因此PCC7002比WH8102和CC9311更耐受铁限制。在铁限制条件下,PCC7002的前向散射光(与细胞大小相关)降低,CC9311和WH8102的前向散射光显著增大。铁限制影响三个藻株的DNA含量和细胞周期,WH8102和CC9311的细胞周期属于快速生长模式,PCC7002的细胞周期属于慢速生长模式。在铁限制条件下,WH8102和CC9311的G1期细胞比例增大。PCC7002的DNA含量分布图在铁充足条件下为较宽的单峰,而在铁限制条件下为较窄的单峰。与单培养时相比,在铁限制条件下分别与WH8102和CC9311混合培养的PCC7002,其比生长速率、细胞产量、叶绿素荧光、前向散射光和DNA含量降低,ROS含量升高,死亡细胞的比例增大。然而,在铁充足条件下混合培养的PCC7002与单培养时没有显著差异。无论是在铁充足还是铁限制条件下,单培养的WH8102和CC9311与混合培养时生理指标没有显著差异。因此,我们推测铁限制条件下混合培养的PCC7002比单培养时受到了更严重的铁限制。更多还原

【Abstract】 Iron supply limits primary production in the ocean, especially HNLC (high-nutrient low-chlorophyll) regions whose area is one-third of the world’s ocean. The important role of iron in oceanic productivity, biogeochemical cycle, and global climate can be seen from many iron enrichment experiments in HNLC regions since1988. Synechococcus species are ubiquitous and abundant in major oceanic regimes, underlying their ecological importance as significant contributors to the total photosynthetic biomass in the ocean. They also play a key role in pelagic food-web structure via energy transfer within the microbial loop, It has been estimated that35%-100%of the Synechococcus standing stock can be grazed per day. Picophytoplankton such as Synechococcus had previously been assumed not to be strongly limited by iron in HNLC regions because only the diatoms bloomed by escaping grazing pressure upon iron enrichment. But there were also some evidences indicated that Synechococcus also iron limited in the oceans. The physiolgical and biochemical response to iron limitation has been well studied in diatoms and other marine eukaryotic algae, but there are much less physiological information about ecological important Synechococcus.Iron plays a catalytic role in many biochemical reactions as a cofactor of enzymes and proteins involved in photosynthetic and respiratory electron transports, nitrogen assimilation and many other metabolic processes. But the majority of intracellular iron is required in the photosynthetic apparatus. Iron concentrations in coastal waters are higher than those in open ocean waters. Selection pressure imposed by iron limitation has resulted in oceanic eukaryotic diatoms less susceptible to iron limitation than coastal species. More iron was predicted to be required by using nitrate as the nitrogen source compared to ammonium. We did not know whether the oceanic Synechococcus strains were more tolerant to iron limitation than the coastal strains. Furthermore, there are conflicting data over whether ammonium, rather than nitrate, supports a higher growth rate under iron limited conditions. Iron concentrations are depleted in the surface ocean but light intensity decreased with depth in the ocean. According to the prediction as well as some data on diatoms, cellular iron demand enhanced under low irradiation but the physiological consequences of Fe limitation would increase susceptibility to PSII photoinhibition at high irradiance. Thus further studies need to be done about the interactive influences of iron and light on the growth and photosynthesis of green and red coastal synechococcus strains. Synechococcus sp. PCC7002has been proved to release extracellular Fe3+chelating agents (siderophore) to cope with iron limitation. It is worth us to explore whether PCC7002possessed a competitive advantage when they mixed-cultured with the strains that do not produce these ligands. In these studies, Synechococcus strains were grown in Aquil medium that was developed specifically for studying trace-metal physiology in algae, and the Metal Clean technique will be used to rigorously control the trace metal contaminations all through our experiments. The F0value measured by Water-PAM fluorometer was used to monitor the growth of Synechococcus. Water-PAM and FIRe fluorescence techniques as well as Flow cytometry technique were used to study the physiolgical response of different Synechococcus strains to iron limitation. The species-specific differences in physiology responses to iron limitation and the effects of iron in untilization of nitrogen and light in marine Synechococcus were explored. The results could help us to well evaluate the effects of iron limitation on primary productivity of the picophytoplankton, and to open out the effects of iron on the phytoplankton community composition of the ocean ecosystem. These results also provide us some bases to further research on biochemical and molecular mechanisms to cope with iron limitation in different strains. The mail results are as follows:1. The minimal fluorescence yield is a reliable and easy biomass measurement of picophytoplankton Synechococcus under semicontinuous batch culture. Two coastal Synechococcus strains (PCC7002and CC9311) and one oceanic strain (WH8102) were cultured with4-1000nM Fe in Aquil medium. The cell concentration and minimal fluorescence yield (F0) were measured daily by Flow cytometry and Water-PAM fluorometer in the exponential growth stage, and the growth rates obtained from these two methods showed little difference. Compared with those under iron-replete condition, their growth rates were significantly decreased by59%for WH8102at15nM Fe, by37%for CC9311at15nM Fe and by57%for PCC7002at4nM Fe. Among these three strains, PCC7002was the most tolerant to iron limitation while WH8102was the most sensitive to iron limitation. The linear correlation was established between F0value and cell concentration although Fo value per cell varied depending of the strains and iron levels. Under iron-replete condition, the minimal fluorescence yield per cell was100-fold higher for phycoerythrin-lacking strain PCC7002than two phycoerythrin-containing strains WH8102and CC9311. Under iron-deplete condition, it was increased respectively by128%and7%for WH8102and CC9311but was decreased by30%for PCC7002. This is mainly related to differences in the pigment composition of phycobilisomes of the various strains and to different effect of iron limitation on photosynthetic pigment contents. Furthermore, F0value per cell concentration for PCC7002and CC9311showed little difference throughout the light and dark diel cycle. However, it was significantly higher for WH8102in the daytime than in the dark. In a word, it is a reliable and easy method to assay the specific growth rate by measuring F0value at the steady state of cultures. 2. Different responses of photosynthesis and flow cytometric signals to iron limitation and nitrogen source in coastal and oceanic Synechococcus strains (Cyanophyceae). This study have been compared the photosynthesis and flow cytometric signals of four Synechococcus strains grown under different iron concentrations with either nitrate or ammonium as the sole nitrogen source. Two oceanic strains were much more sensitive to iron limitation than two coastal strains. The inhibition of iron limitation on the growth, maximal PSII photochemical yield, maximal rate of relative electron transport and photochemical quenching of the two oceanic strains was higher than for their coastal counterparts. Under iron limitation condition, the connectivity factor between individual photosynthetic units (p) increased for the two coastal strains while decreased for the two oceanic strains. Furthermore, iron limitation accelerated the QA re-oxidation of the two oceanic strains and the PQ pool re-oxidation of the two coastal strains. Under iron limitation condition, the cell size of the two coastal strains and intracellular pigment concentrations of the two oceanic strains decreased while the side light scatter/front light scatter (SS/FS) ratio of the two coastal strains increased. In contrast to iron limitation, nitrogen source only marginally affected the photosynthesis of the four Synechococcus strains. Ammonium enhanced the growth of the two coastal strains under iron-replete condition. For the two oceanic strains, ammonium increased their cell size and decreased their SS/FS ratio and intracellular pigment concentrations under iron-deplete and iron-replete conditions. Previously studies indicated that oceanic eukaryotic diatoms were less susceptible to iron limitation than coastal species. These were opposite to our results in Synechococcus. Further works need to be done to understand the biochemical mechanisms responsible for these physiological responses to iron limitation between oceanic and coastal Synechococcus strains.3. Different responses of growth, photosynthesis and flow cytometric pigments fluoresence to iron and light in one green and one red coastal synechococcus strains (Cyanophyceae). One green (PCC7002) and one red (CC9311) coastal synechococcus strains were cultured under different iron(10and1000nM) and light conditions (10and60μmol photons·m-1·s-1). The results indicated that PCC7002was more sensitive to iron limitation under low light while CC9311was more easily to suffer from photoinhibition under iron limitation. Anymore, CC9311was more sensitive to iron limitation than PCC7002both under high light and low light. The flow cytometric chl a, PE and PC fluorescence of the two strains decreased under iron limitation and high light conditions. Decrease in aPSII for PCC7002cultured under iron and light colimitation and for CC9311under iron limitation and photoinhibition conditions might resulted from decrease in the amount of pigments serving one RCII. Under low iron and low light conditions, the linear electron transport rate (rETRmax) decreased but PQ pool re-oxidation(1/τPQ) accelerated for PCC7002which might result from the stimulation of cyclic electron transport around PSI. Under low iron and high light conditions, the rETRmax decreased but Qa re-oxidation and PQ pool re-oxidation (1/τPQ) accelerated for CC93311which might result from the stimulation of cyclic electron transport around PSI and PSII respectively. Samples of PCC7002and CC9311cultured under different light and iron conditions were illuminated at300μmol photons-m·2-s·1for30min and then transferred to10μmol photons·m-2·s-1for60min for recovery. The Fv/Fm values were measured after high light treatment and low light recovery. The results indicated that CC9311was more susceptible to high light exposure and had lower capacity to recover from this photoinhibitory stress compared to PCC7002. Anymore, cells cultured at low light and low iron conditions were more easily to suffer from photoinhibition and had lower capacity to recover from photoinhibition. These might result from their lower NPQ (nonphotochemical quenching). Our results suggested that iron and light interaction might be important for vertical separation of red and green strains of synechococcus.4. Physiology response to iron limitation and effects of iron on the competition of PCC7002between WH8102or CC9311. PCC7002which could produce siderophore involved in iron sequestration under iron limitation conditions was predicted to own competitive advantage. In monoculture, the cell yieds and relative growth rate of WH8102and CC9311was inhibited more than PCC7002under iron limitation. Anymore, celluar ROS content and percentages of dead cells of WH8102and CC9311were also effected more by iron limitation. This results indicated that PCC7002was much more tolerant to iron limitation than WH8102and CC9311. Front light scatter (FS, related to cell size) of CC9311and WH8102increased significantly whereas the FS of PCC7002decreased significantly under iron limitation. Iron limitation effected cell cycle behavior and decreased DNA content of the three strains. The cell-cycle behavior of CC9311and WH8102were different from PCC7002, which corresponds to the slow and fast growth case of cell-cycle model respectively. Percentages of cells in G1phase decreased under low iron for WH8102and CC9311while PCC7002presented a broad and narrow unimodal under iron replete and deplete conditions respectively. Compared to monoculture, cell yieds, relative growth rate, Chl a fluorescence, FS and DNA content of PCC7002were decreased while celluar ROS content and percentages of dead cells increased in mixed-culture under iron limitation. However, there were no significant differences between the physiological characteristics for PCC7002in monoculture and mixed-cultured under iron replete conditions, and so is the physiological characteristics of WH8102or CC9311both under iron deplete and replete conditions. We concluded that cells of PCC7002in mixed-culture suffered from more heavily iron stress than it in monoculture under iron limitation conditions.更多还原