Comparative studies on photosynthesis and phosphate metabolism of Cylindrospermopsis raciborskii with Microcystis aeruginosa and Aphanizomenon flos-aquae

The physiological differences for three bloom-forming cyanobacteria (Cylindrospermopsis raciborskii, Microcystis aeruginosa, and Aphanizomenon flos-aquae) were investigated. In comparison with M. aeruginosa and A. flos-aquae, C. raciborskii exhibited a significantly higher concentration of carotenoids, higher values in maximum photosynthesis rate (P-m), apparent photosynthetic efficieny (a), and maximum electron transport rate (ETRmax) during the growth period. In addition, higher extracellular alkaline phosphatase activities and lower light compensation point (I-c) were also detected in C raciborskii (p < 0.05, ANOVA). Therefore, it is suggested that the higher photosynthetic activities, more effective uptake and utilization to phosphate, and low light requirements might play important roles in the occurrence and invasive behavior of C. raciborskii. Crown Copyright (C) 2009 Published by Elsevier B.V. All rights reserved.

Enhanced protection against oxidative stress in an astaxanthin-overproduction Haematococcus mutant (Chlorophyceae)

Many unicellular green algae can become yellow or red in various natural habitats due to mass accumulation of a secondary carotenoid, such as lutein, or astaxanthin. The accumulation of secondary carotenoids is generally thought to be a survival strategy of the algae under photo-oxidative stress or other adverse environmental conditions. The physiological role of the carotenoids in stress response is less well understood at the subcellular or molecular level. In this study, a stable astaxanthin overproduction mutant (MT 2877) was isolated by chemical mutagenesis of a wild type (WT) of the green microalga Haematococcus pluvialis Flotow NIES-144. MT 2877 was identical to the WT with respect to morphology, pigment composition, and growth kinetics during the early vegetative stage of the life cycle. However, it had the ability to synthesize and accumulate about twice the astaxanthin content of the WT under high light, or under high light in the presence of excess amounts of ferrous sulphate and sodium acetate. Under stress, the mutant exhibited higher photosynthetic activities than the WT, based on considerably higher chlorophyll fluorescence induction, chlorophyll autofluorescence intensities, and oxygen evolution rates. Cell mortality caused by stress was reduced by half in the mutant culture compared with the WT. Enhanced protection of the mutant against stress is attributed to its accelerated carotenogenesis and accumulation of astaxanthin. Our results suggest that MT 2877, or other astaxanthin overproduction Haematococcus mutants, may offer dual benefits, as compared with the wild type, by increasing cellular astaxanthin content while reducing cell mortality during stress-induced carotenogenesis.

The accumulation and metabolism of astaxanthin in Scenedesmus obliquus (chlorophyceae)

The biosynthesis and metabolism of astaxanthin in coenobium alga Scenedesmus obliquus were investigated using a two-stage culture. The first stage was for the analysis of biosynthesis and accumulation of astaxanthin in alga cells which were cultured under induction conditions (incubation at 30 degrees C and illumination of 180 mu mol m(-2) s(-1)) for 48 h. The composition of the secondary carotenoids in algal cells was analyzed and seven ketocarotenoids were identified. The results implied that S. obliquus synthesized astaxanthin from beta-carotene through three possible pathways. In the second stage, the cultures were transferred to normal conditions (incubation at 25 C and illumination of 80 mu mol m(-2) s(-1)) for 72 h. Algal cells accumulated more chlorophyll and biosynthesis of secondary carotenoids terminated, the content of secondary carotenoids decreased from 59.48 to 6.57%. The results inferred that accumulation and metabolism of astaxanthin could be controlled by cultivated conditions which also could lead the mobilization of secondary carotenoids to support the algal cell growth. The results also implied that presumed conversions from astaxanthin to lutein or antheraxanthin could be modulated by culturing conditions. (C) 2008 Published by Elsevier Ltd.

Photosynthesis and growth of Arthrospira (Spirulina) platensis (Cyanophyta) in response to solar UV radiation, with special reference to its minor variant

The minor variant of the economically important cyanobacterium, Arthrospira platensis, usually appears in commercial production ponds under solar radiation. However, how sensitive the minor variant to solar UVR and whether its occurrence relates to the solar exposures are not known. We investigated the photochemical efficiency of PSII and growth rate of D-0083 strain and its minor variant in semi-continuous cultures under PAR (400-700 nm) alone, PAR + UV-A (320-400 nm) and PAR + UV-A + UV-B (280-700 nm) of solar radiation. The effective quantum yield of D-0083 at 14:00 p.m. decreased by about 86% under PAR, 87% under PAR + UV-A and 92% under PAR + UV-A + UV-B (280-315 nm), respectively. That of the minor variant was reduced by 93% under PAR and to undetectable values in the presence of UV-A or UV-A + UV-B. Diurnal change of the yield showed constant pattern during long-term (10 days) exposures, high in the early morning and late afternoon but the lowest at noontime in both strains, with the UVR-related inhibition being always higher in the variant than D-0083. During the long-term exposures, cells of D-0083 acclimated faster to solar UV radiation and showed paralleled growth rates among the treatments with or without UVR at the end of the experiment; however, growth of the minor variant was significantly reduced by UV-A and UV-B throughout the period. Comparing to the major strain D-0083, the minor variant was more sensitive to UVR in terms of its growth, quantum yield and acclimation to solar radiation. (c) 2007 Elsevier B.V. All rights reserved.

Effects of solar ultraviolet radiation on the photochemical efficiency, photosynthetic pigments and biomass production of Spirulina platensis

Effects of solar ultraviolet radiation (UVR) on Spirulina platensis were studied by investigating its photochemical efficiency, photosynthetic pigments and biomass production while exposed to full spectrum solar radiation or depleted of UVR for understanding how and to what extent UVR influences its photosynthetic physiology and production. It was found that UVR brought about an extra inhibition of photochemical efficiency by 26%-30%. The greatest inhibition of photochemical efficiency in S. platensis was observed at noontime, and then recovered to some extent in late afternoon no matter which treatment they were exposed to. The contents of chlorophyll a, phycocyanin and carotenoids increased during initial stage of the exposure, but decreased with elongated exposure. UVR decreased the biomass yield by about 6%. It indicated that filtering out UVR of solar radiation would raise the productivity of S. platensis, which is an important factor that should be considered in the production.

Impacts of elevated CO2 concentration on biochemical composition, carbonic anhydrase, and nitrate reductase activity of freshwater green algae

To investigate the biochemical response of freshwater green algae to elevated CO2 concentrations, Chlorella pyrenoidosa Chick and Chlamydomonas reinhardtii Dang cells were cultured at different CO2 concentrations within the range 3-186 μ mol/L and the biochemical composition, carbonic anhydrase (CA), and nitrate reductase activities of the cells were investigated. Chlorophylls (Chl), carotenoids, carbonhydrate, and protein contents were enhanced to varying extents with increasing CO2 concentration from 3-186 μ mol/L. The CO2 enrichment significantly increased the Chl a/Chl b ratio in Chlorella pyrenoidosa, but not in Chlamydomonas reinhardtii. The CO2 concentration had significant effects on CA and nitrate reductase activity. Elevating CO2 concentration to 186 μ mol/L caused a decline in intracellular and extracellullar CA activity. Nitrate reductase activity, under either light or dark conditions, in C. reinhardtii and C. pyrenoidosa was also significantly decreased with CO2 enrichment. From this study, it can be concluded that CO2 enrichment can affect biochemical composition, CA, and nitrate reductase activity, and that the biochemical response was species dependent.

青杨组(Populus section Tacamahaca Spach)不同种对增强UV-B的反应差异

人类向大气中排放的大量氮氧化合物和氟氯烃类化合物(CFC’s)引起臭氧分子的分解，导致到达地球表面的紫外辐射增加，特别是UV-B辐射增强。本项目以青杨组杨树为模式植物，从形态和生理方面研究了来自不同UV-B背景下的康定杨与青杨在增强UV-B下的反应及其反应差异,并探讨了干旱、施肥对它们抗UV-B能力的影响。杨树具有分布广、适应性强、在生态环境治理和解决木材短缺方面均占有重要位置，研究成果可为生态系统的恢复与重建提供理论依据和科学指导。主要研究结果有以下： 1. 在温室中经过增强UV-B处理，杨树的外部形态及生理活动受到了一定程度的影响。增强UV-B导致康定杨、青杨的生物量、叶面积及节间长度降低，叶片增厚，SOD活性升高，膜伤害增加，而对叶片数目、R/S、叶绿素A、叶绿素B及整个叶绿素含量没有影响。两种杨树对UV-B胁迫的响应存在差异：在增强UV-B条件下，青杨的植株高度、生物量、叶面积、脯氨酸含量、长期用水效率受到的影响大于康定杨，相比而言，康定杨在比叶面积、叶片厚度、可溶性糖含量、UV-B吸收物质的含量及SOD和GPX活性方面增加的程度大于青杨。这些区别说明，来自于高海拔的康定杨比来自于低海拔的青杨对增强UV-B 具有更强的耐性。我们认为二者在叶片厚度、比叶面积、UV-B吸收物质含量及SOD、GPX活性差异是导致对增强UV-B耐性不同的原因。 2. 干旱与增强UV-B对杨树的生长和生理特性均产生了影响，而且两种胁迫共同作用时干旱表现减弱或加剧了UV-B对杨树某些形态和生理特性的影响。 据试验结果，干旱显著地降低了杨树的株高、叶片数目、叶面积，增加了叶片厚度，促进ABA的积累，提高了CAT活性。对于干旱，两种杨树之间也表现出了一定的差异性。可溶性蛋白质和脯氨酸在青杨叶片中得到显著积累，而在康定杨中没有变化。此外，CAT、长期用水效率在康定杨中受到的影响更加明显。长期用水效率的不同变化趋势说明两种杨树对水分胁迫采用了不同的用水策略，康定杨采用的是节水用水策略，提高用水效率，而青杨采用的是耗水的用水策略。根据干旱对叶面积、脯氨酸、ABA含量、CAT活性及长期用水效率等方面的影响，我们认为来自高海拔地区的康定杨比来自低海拔的青杨有更大的耐旱性，这是对生长环境长期适应的结果。在高海拔地区，因霜冻常带来土壤水分不可利用，降低了根系对水分的吸收，树木容易受到的生理性干旱。另外，高海拔的地区低的气温使植株对严寒有较强的耐性，减少了水分的需要。 生长于增强UV-B下的康定杨和青杨植株表现为高度降低，叶面积缩小，比叶面积增加；叶片栅栏组织、海绵组织均受到增强UV-B的影响，其厚度的增加导致整个叶片变厚。增强UV-B还显著提高了杨树的APX活性、UV-B吸收物质含量，而对叶片数目、ABA、可溶性蛋白质含量及CAT活性没有产生影响。试验中也观察到了两种杨树对增强UV-B响应的差异：与康定杨相比，在增强UV-B下青杨株高、叶面积降低的程度更大一些，SOD活性显著提高。另外UV-B吸收物质受到的影响不同。根据这些差别，高海拔的康定杨(3500 m)比来自低海拔的青杨(1500 m)增强UV-B有较强的耐性。 与水分充足情况下UV-B对植株的影响相比，干旱对杨树抗增强UV-B产生了一定的影响，表现为加剧或减弱UV-B对植物的影响，但这种影响与形态、生理指标有关。当干旱与增强UV-B共同作用时，杨树植株的株高、叶面积进一步降低、叶片进一步增厚。就脯氨酸的积累的而言，在没有水分胁迫时，增强UV-B促使它显著增加，而在干旱处理下这种效果变得不明显。干旱对增强UV-B的影响还与杨树的种类有一定的关系。在康定杨中，干旱减弱了增强UV-B对栅栏组织与海绵组织的影响，且在植株高度、叶面积上表现出累加效应，而在CAT上交互作用显著；但在青杨中干旱则加剧增强UV-B对栅栏组织与海绵组织的影响，在植株高度、叶面积及比叶面积上表现出显著的交互作用。据碳同素分析，在水分充足的条件下，无论是康定杨，还是青杨，增强UV-B均导致其长期用水效率的提高，然而当两种胁迫共同作用时，长期用水效率则表现出差异，在青杨中，长期用水效率得到进一步增高，而康定杨中干旱的效应被增强UV-B所减轻。 3. 田间试验表明，杨树的生长、生理特征都受到养分和增强UV-B的影响。施肥对杨树的影响表现为：提高了叶面积、生物量及SOD的活性，降低了抗坏血酸含量。对于施肥作用，两种杨树的反应也有区别：在康定杨中施肥显著增加了的叶片长度、宽度及光合色素的含量，降低了净光合速率、气孔导度及胞间CO2浓度；在青杨中，则SOD、GPX、APX活性表现增加。从试验看出，施肥对来自于高海拔地区的康定杨(3500 m)的影响较大，对来自低海拔的青杨(1500 m)影响较小，这与它们对原产地的生境适应有一定关系。在康定杨生长的高海拔地区，低温度和湿度不能为地上凋落物或土壤中的根分解提供理想的条件，造成当地土壤的低养分状况，所以当肥料施用以后，效果显著。 经过增强UV-B处理，杨树叶片中UV-B吸收物质含量、GPX的活性得到提高，而脯氨酸、丙二醛、可溶性蛋白质、叶绿素及类胡萝卜素含量没有受到影响。对于增强UV-B两种杨树受到的影响也有所不同：在青杨中增强UV-B导致叶面积缩小，生物量、净光合速率降低，APX的活性及长期用水效率的提高，而对康定杨的这些指标没有产生显著影响，相反抗氧化酶的活性明显高于青杨。这些差异性是由于两种杨树对原产地不同UV-B背景的长期适应结果。康定杨长期生长在较高UV-B环境中，对UV-B有较强的耐性。而青杨适应于较低的UV-B环境，对增强UV-B较为敏感。 试验中施肥也影响了植株对增强UV-B的反应，不过这种影响与杨树的种类及测定指标有一定的相关性。例如，在缺肥的情况下，青杨的长期用水效率和康定杨的叶绿素含量都受到增强UV-B的显著影响，而施肥以后这种影响变得不显著。在缺肥的条件下，GPX、APX在青杨中的活性、GPX在康定杨中的活性对增加UV-B反应不敏感；而施肥以后则变化显著，同样胞间CO2浓度在康定杨也有类似的变化。 For past decades, Ultraviolet radiation, especially UV-B reaching the Earth’s surface increased because of depletion of ozone layer resulted from emission of NxO and CFC’s from human activities. In this experiment, different species of Populus section Tacamahaca Spach from different UV-B background were selected as a model plant to assess the effects of enhanced UV-B radiation. Morphological and physiological traits induced by enhanced UV-B were observed and the different responses between P. kangdingensis and P. cathayana were discussed, furthermore the influences of drought and fertilizer on responses induced by enhanced UV-B were studied. Since poplars play an important role in lumber supply, and are important component of ecosystems due to their fast growth and wide adaptation, the study could provide a strong theoretical evidence and scientific direction for the afforestation, and rehabilitation of ecosystem. The results are as follows: 1. The experiment conducted in a greenhouse indicated that morphological and physiological traits of two poplars were affected by enhanced UV-B radiation. Enhanced UV-B radiation not only reduced biomass, leave area and internode length, but also increased leaf thickness and SOD activity as well as MDA concentration and electrolyte rate. However, no significant changes in leaf numbers, root shoot ratio, and total chlorophyll and chlorophyll component were observed. There were different responses to enhanced UV-B radiation between two species. Compared with P. kangdingensis, cuttings of P. cathayana, exhibited lower height increment and smaller leaf area. In addition, there were significant differences in free proline, soluble protein, and UV-B absorbing compounds, and the activity of SOD and GPX, long-term WUE between them. Differences in plant height, biomass, leaf area, free proline concentration, and long-termed WUE showed that P. cathayana were more affected by enhanced UV-B radiation than P. kangdingensis. In contrast, more increase of specific leaf mass, leaf thickness, and soluble sugar, and UV-B absorbing compounds, and activity of SOD and GPX were observed in P. kangdingensis. According to these results, we suggested that P. kangdingensis from high elevation, which adapted to higher UV-B environments, had more tolerance to enhanced UV-B than P. cathayana from low elevation, which adapted to lower UV-B environment. We believe it was the difference of leaf thickness, specific leaf mass, and UV-B absorbing compounds as well as the activity of SOD and GPX resulted in lower adaptation of P. cathayana to enhanced UV-B radiation. 2. Growth and physiological traits of two poplars were affected by both drought and enhanced UV-B radiation. Moreover, it was observed that when two stresses applied together drought could exacerbate UV-B effects or decrease sensitivity to UV-B. In the experiment, drought significantly decreased plant height, leaf numbers, leaf area, and increased leaf thickness, and ABA, and CAT activity of two poplars. There were significant interspecific differences to drought stress. Exposed to drought, soluble protein and proline concentration were increased in P. cathayana but not in P. kangdingensis. However, more changes in CAT and long-term WUE were observed in kangdingensis. Different change in long-term WUE suggests that two poplars adapted different water-use strategies. P. kangdingensis employ a conservative water-use strategy, whereas P. cathayana employ a prodigal water-use strategy. Based on the differences in leaf area, accumulation of free proline and ABA, CAT activity as well as long-term WUE, we believed that P. kangdingensis from high elevation had a greater tolerance to drought than P. cathayana from low elevation，which is the result of adaptation to local environment. In high elevation area, trees are prone to suffer from physiological drought because of un-movable water caused by frost. Besides lower temperature enable the plants had greater adaptability to frost as a results the requirement of water is reduced Enhanced UV-B radiation decreased shoots height, leaf area, and increased specific leaf mass and thickness of palisade and sponge layer as well as APX activity and UV-B absorbing compounds in both species. Whereas, leaf numbers, ABA content, soluble protein and CAT activity showed no differences to enhanced UV-B radiation. Interspecific differences were also observed. Compared with P. kangdingensis, P. cathayana showed lower shoot height and smaller leaf area, higher SOD activity. Besides, variation in UV-B absorbing compounds was found. These differences suggested that P. kangdingensis from high elevation (3500 m) was more tolerant to enhanced UV-B radiation than P. cathayana from low elevation (1500 m). Compared with morphological and physiological changes induced by enhanced UV-B radiation under well-watered conditions, drought exacerbated or decreased these changes. However, these effects vary with parameters measured. When two stresses applied together, shoot height and leaf area further decreased while leaf thickness further increased. Under well-watered conditions, enhanced UV-B radiation significantly increased proline content, but such effect was not observed under drought conditions. The effect of drought on enhanced UV-B radiation was related to species. For example, drought reduced the effects of enhanced UV-B radiation on palisade parenchyma and sponge mesophyll in P. kangdingensis, and additive effects in shoot height and leaf area and interactive effect CAT activity were observed. In contrast, for P. cathayana drought significantly exacerbated the effects of enhanced UV-B radiation on palisade parenchyma and sponge mesophyll; there were noticeable interaction in shoot height, leaf area and specific leaf mass. As far as long-term WUE is concerned, it was increased by enhanced UV-B radiation under well-watered conditions in both species. While different effect was observed between two species in combination of two stresses. Long-term water use efficiency was further increased in P. cathayana whereas the effect was less significant in P. kangdingensis. 3. The field experiment showed that growth and physiological traits of poplars were affected by nutrition and enhanced UV-B radiation. Fertilization significantly increased leaf area, biomass and SOD activity, reduced Ascorbic acid concentration. There was interspecific difference in response to fertilization. For P. kangdingensis, fertilization significantly increased leaf width, leaf length and photosynthetic pigments content while net photosynthetic rate and stomatal conductance, intercellular CO2 concentration were significantly decreased. However, for P. cathayana, these parameters were unaffected except the increase of SOD, GPX and APX activity. From above, it could concluded that P. kangdingensis from high elevation was more affected by fertilization than P. cathayana, This difference was due to adaptation to local environment., The low temperature and moisture where P. kangdingensis was collected can not provided optimum to decompose roots and litter fall as a result the nutrition in soil was poor. Exposed to enhanced UV-B radiation, for both species UV-B absorbing compounds and GPX activity were significantly increased while proline, MDA, soluble protein, chlorophyll, carotenoids were not affected. Different responses were also observed between the two species. Enhanced UV-B radiation caused significant decreases in leaf area, biomass, net photosynthetic rate and increase in APX activity and long-term WUE in P. cathayana but not in P. kangdingensis. In addition, activity in antioxidant enzymes was much higher in P. kangdingensis than in P. cathayana. In the experiment fertilization affected responses of cuttings to enhanced UV-B radiation, but it concern species and parameters measured. Long-term WUE in P. cathayana and chlorophyll in P. kangdingensis were significantly increased by enhanced UV-B radiation under non-fertilization treatments while the increase was not found under fertilization treatment. In contrast, under no fertilization treatment enhanced UV-B radiation did not affected GPX and APX activity in P. cathayana and GPX in P. kangdingensis while significant increase appeared after application of fertilization. Similar effect of enhanced UV-B radiation on intercellular CO2 concentration in P. kangdingensis was observed.

青杨(Populus cathayana Rehd.)雌雄植株对干旱胁迫的生理生态响应差异

随着全球气候变暖和温室效应加剧，干旱和荒漠化成为威胁人类生存和发展的主要 灾害，许多被子植物对干旱胁迫的生理、生态和生化响应已逐步得以报道，但很少有开 展干旱胁迫对雌雄异株植物的影响方面的研究。由于这类植物在长期进化过程中已经在 生长、性比、生殖格局、空间分布、资源配置和生物量分配等方面形成了明显的性别差 异，因此，干旱胁迫必将对其雌雄植株产生不同的生理生态影响。本研究以青杨为模式 植物，采用植物生态、生理及生物化学等研究方法，系统研究青杨雌雄植株在常温、增 温以及喷施外源脱落酸的条件下对干旱胁迫的响应，揭示其在生长形态、生物量分配、 光合作用、用水效率和生理生化等方面的性别间差异。主要研究结果如下： 1. 青杨雌雄植株对干旱胁迫的综合响应。 与较好水分条件相比，干旱胁迫显著降低了青杨雌雄植株的光合作用和生长发育， 影响了许多生理生化过程，并导致雌雄植株在生长发育、气体交换、用水效率、膜脂抗 氧化和抗氧化系统酶活性方面表现出显著的性别间差异。在较好水分条件下，雌雄植株 之间在株高、基径、生物量、净光合速率、蒸腾速率、用水效率以及丙二醛、脱落酸和 游离脯氨酸等生化物质含量方面均无显著差异。但在干旱胁迫下，雄株在生长发育、气 体交换、水分利用效率、膜脂过氧化保护和抗氧化系统酶活性方面均显著高于雌株，表 现出比雌株更高的株高、基径、叶面积、总叶片数、总生物量、总色素含量、类胡萝卜 素含量、净光合速率、蒸腾速率、羧化效率、光系统II最大光化学效率、内在水分利用 效率、碳同位素组分、过氧化氢酶和过氧化物酶活性等，而在CO2补偿点、比叶面积、 叶绿素a/b、丙二醛、脱落酸和超氧化物歧化酶活性等指标上显著低于雌株。与雌株相比， 雄株表现出更高的干旱胁迫适应能力，而雌株的生长发育和生理生化过程更易遭受干旱 胁迫的影响。 2. 干旱胁迫下的青杨雌雄植株对增温处理的综合响应 与环境温度相比，增温在干旱胁迫前后均显著促进了雌雄植株的生长发育、气体交 换，降低水分利用效率，影响生化物质含量，并促使青杨雌雄植株之间在干旱胁迫下表 现出显著的差异。在较好水分条件下，增温导致雌株的株高、基径、叶面积、总叶片数、 总生物量和超氧化物歧化酶活性显著高于雄株，而用水效率、丙二醛、脱落酸和游离脯 氨酸、抗坏血酸过氧化物酶和过氧化物酶活性低于雄株。在干旱胁迫下，增温将导致雄 株的株高、基径、叶面积、总生物量、净光合速率、蒸腾速率、气孔导度、总色素含量、 相对含水量、过氧化氢酶和抗坏血酸过氧化物酶活性等显著高于雌株，而光系统II 最大 光化学效率、内在水分利用效率、碳同位素组分、丙二醛、脱落酸、游离脯氨酸和超氧 化物歧化酶活性显著低于雌株。与雄株相比，水分较好条件下的增温有利于促进雌株的 生长发育，并在生理生态特征上优于雄株。而干旱胁迫下的增温则加剧了水分胁迫强度， 致使雌株的生长发育遭受比雄株更多的负面影响。 3. 干旱胁迫下的青杨雌雄植株对喷施外源脱落酸处理的综合响应 与对照相比，在干旱胁迫下喷施外源脱落酸可显著增加青杨雌雄植株的生长发育、 气体交换、降低水分利用效率，影响了生化物质含量，并导致青杨雌雄植株之间在干旱 胁迫下表现出显著的生理生态差异。在干旱胁迫下，喷施外源脱落酸致使雌株的株高、 叶面积、叶干重、细根干重、总生物量、净光合速率、蒸腾速率、气孔导度、光系统II 最大光化学效率、非光化学淬灭系数、相对含水量、总光合色素、类胡萝卜素、脱落酸、 超氧化物歧化酶和过氧化物酶活性的增加量显著高于雄株，而根重比、根冠比、细根/ 总根、比叶面积、内在水分利用效率、碳同位素组分、丙二醛、脯氨酸、过氧化氢酶和 抗坏血酸过氧化物酶活性等指标的减少量上显著低于雄株。与对照相比，干旱胁迫下的 喷施外源脱落酸则一定程度能减缓植株遭受胁迫的压力，促进植株生长和气体交换，减 少了植株体内的过剩自由基数量，并促使雌株的生长发育和光合能力显著提高，增强其 抗干旱胁迫能力。 With development of global warming and greenhouse effect, drought and desertification have been became main natural disasteres in resent years. Studies on ecophysiological responses of most angiosperm species to environmental stress have been reported, but little is known about dioecious plant responses to drought stress. Since significant differences on growth, survival, reproductive patterns, spatial distribution, as well as resource allocation between males and females of dioecious plant have been formed during evolutionary process, sexual different ecophysiological responses should be caused by drought stress. In this experiment, Populus cathayana Rehd. was used as model plant to study the sex-related responses to drought by using the ecological, physiological and biochemical methods under normal atmospheric temperature, elevated temperatures and exogenous abscisic acid (ABA) application treatment respectively, and to expose the sexual differences in growth, biomass allocation, photosynthesis, water use efficiency and some biochemical material contents in the males and females of dioecious plant. The results are follows: 1. A large set of parallel responses of males and females of P. cathayana to drought stress Compared with well-watered treatment, drought significantly decreased growth and photosynthesis of P. cathayana individuals, affected some physiological and biochemical processes, and induced males and females to exhibit obvious sexual differences in growth, gas exchange, water use efficiency, lipid peroxidation protection and antioxidant defenses enzyme system. Under well-watered treatment, there were no significant sexual differences in height growth (HG), basal diameter (BD), dry matter accumulation (DMA), net photosynthesis rate (A), transpiration (E), water use efficiency (WUE), and malondialdehyde (MDA), abscisic acid (ABA) and praline (Pro). However, under drought stress, males were found to exhibit higher HG, BD, leaf area (LA), total leaf number (TLA), DMA, total chlorophyll contents (TC), carotenoids content (Caro), A, E, carboxylation efficiency (CE), the maximum efficiency of PSII (Fv/Fm), intrinsic water use efficiency (WUE ), carbon isotope composition (δ13C), catalase (CAT), peroxidase (POD) and lower CO2 compensation point (Γ), specific leaf area (SLA), chlorophyll a/b ratio (Chla/Chlb), MDA, ABA and superoxide dismutase (SOD) than females. The results suggest that males possess greater drought resistance than do females and females suffer more negative effect on growth and development, physiological and biochemical processes than males under drought stress. 2. A large set of parallel responses of drought-stressed males and females of P. cathayana to elevated temperatures Compared with environmental temperature, elevated temperature treatment significant increased growth and gas exchange, decreased water use efficiency, changed some biochemical material contents of P. cathayana individuals, and induced males and females to exhibit obvious differences under drought stress. Under good water condition, elevated temperature treatment caused females to show significant higher HG, BD, LA, TLN, DMA, SOD activity, and great lower WUE, MDA, ABA, Pro, ascorbate peroxidase (APX) and POD than do males. On contrary, under drought condition, elevated temperature treatment induced males to exhibit higher HG, BD, LA, DMA, A, E, stomatal conductance (gs), relative water content (RWC), CAT, APX activity but lower Fv/Fm, WUE, δ13C, MDA, ABA, Pro, SOD activity than do females. The results suggest that females will benefit from elevating temperature under good water condition by possessing better ecophysiological processes than that of males, but will suffer from greater negative effects than do males when grown under drought stress with elevated temperature treatment. 3. A large set of parallel responses of drought-stressed males and females of P. cathayana to exogenous ABA application Compared with controls, exogenous ABA application under drought greatly increased growth and gas exchange, decreased water use efficiency, changed some biochemical material contents in P. cathayana individuals, and induced males and females to exhibit obvious sexual differences under drought. Under drought stress, exogenous ABA application induced females to exhibit more increases in HG, LA, leaf weight (LW), fine root weight (FRW), DMA, A, E, g, Fv/Fm, non-photochemical quenching coefficient (qN), RWC, TC, Caro, ABA, SOD, POD s activity than males, but to show lower decreases in root/weight ratio (RWR), root mass/foliage area ratio (RF), fine root/total root ratio (FT), SLA, WUE, δ13C, MDA, Pro, CAT, APX than males. The results suggest that exogenous ABA application under drought stress will eliminate negative damages caused by drought stress at a certain extent，promote the growth and gas exchange of plant and decrease the number of superfluous 1O2 in plant cells of males and females of P. cathayana. Furthermore, exogenous ABA application promoted more drought resistance in females than in males by increasing more growth and photosynthetic capacity in females under drought stress.

三孢布拉氏霉发酵产β-胡萝卜素的研究

本论文研究了利用三孢布拉氏霉(Blakeslea trispora)发酵产β-胡萝卜素的培养条件。主要包括：发酵培养基的确定，发酵条件的优化。还考察了发酵菌丝体中β-胡萝卜素的提取方法及薄层层析等。 首先研究了培养基成分对三孢布拉氏霉发酵产β-胡萝卜素的影响。确立了玉米淀粉作为碳源，黄豆粉（热榨）作为氮源，棉籽油作为植物油的发酵培养基配方，其成分为：玉米淀粉 3％，黄豆粉（热榨） 2％，棉籽油 3％，KH2PO4 0.2%，MgSO4·7H2O 0.2%，维生素B1 0.002％，pH值6.0。 其次，通过比较不同的发酵影响因子，分别得到最适的条件：如三孢布拉氏霉正负菌接种比例为1.3：0.7，培养基pH值为7.0（灭菌后），发酵促进因子为Triton X-100。并采用正交试验法，确定其最佳发酵条件为正负菌接种比例1.3/0.7，发酵培养基pH为7.0，在培养基中添加表面活性基Triton X-100 0.08％。使该菌株产β-胡萝卜素的量达到0.73g/L，较初始发酵条件提高了3.3倍。 研究中还找到一个简便有效的对β-胡萝卜素的提取方法，选用盐酸-热处理法进行细胞破壁，并选用沸程为60～90℃的石油醚进行萃取。 用三孢布拉霉菌丝体内类胡萝卜索的石油醚提取液点样于硅胶G板，以丙酮：石油醚（5：95）为展开剂能将β-胡萝卜素与其它类胡萝卜索分离。该方法简便快速，并有一定实用价值。 The fermentative conditions of β-carotene by Blakeslea trispora have been investigated. These conditions include fermentation medium, the optimization of some fermentation factor. The extracting methods and the TLC of carotenoids were also researched. Firstly, the effects of composition of fermentation medium on the yield of β-carotene were studied. the results showed that the best fermentation medium was corn starch 3％，soybean power 2％，cottonseed oil 3％，KH2PO4 0.2%，MgSO4·7H2O 0.2%，vitamin B1 0.002％，pH value 6.0. Secondly, through compared some factors, such as different proportion of plus and minus strains, pH value, nonionic surfactants, respective best values have been obtained. The best proportion of plus and minus strains is 1.3:0.7, pH value of fermentation medium (sterilized) is 7.0, fermentation accelerant which acts as surfactants is Triton x-100. Farther on, the fermentative conditions were optimized through orthogonal experiment, the optimization showed that proportion of plus and minus strains is 1.3:0.7，pH value is 7.0, content of Triton x-100 is 0.08％. And the yield of β-carotene reached 0.73g/L, which was up to 3.3 times through the fermentation. In the extracting study, it has showed hydrochloric acid-heat treatment is a simple, convenient and effective extracting methods is which was used to destroy the cell wall, and the extracting organic solvent is petroleum ether whose boiling range is 60~90 ℃. In the TLC experiments, extracting contents in the petroleum ether were spotted in the silicagel plate, and the mixed liquor of acetone and petroleum ether (5：95) is developping agent, which can distinguish β-carotene from other carotenoids. It is a simple and quick technique.

红发夫酵母发酵虾青素的研究

红发夫酵母分离于北美西部高山地区和日本一些岛屿上落叶树的渗出液中，因其所产主要色素为在水产养殖、食品和医药工业有广阔应用前景的虾青素而成为研究的热点。本论文对红发夫酵母Phaffia rhodozyma 的生长特性、培养参数与培养基组分对生长和虾青素积累的影响及其优化、虾青素合成的调节控制、虾青素的提取测定及红发夫酵母耐高温菌种的诱变进行了系统的研究。 虾青素是红发夫酵母的胞内色素，要对其进行分析首先要对红发夫酵母进行破壁处理，实验发现二甲亚砜是最有效的破壁溶剂，用氯仿和丙酮可以有效地把类胡萝卜素从二甲亚砜破壁后的红发夫酵母细胞中提取出来。 在固定摇床转速为200 rpm，温度为20 ℃的条件下，当种龄为36 h，以10％的接种量接入装液量为30 mL的250 mL三角瓶，初始pH为5.5时最有利于红发夫酵母的生长及类胡萝卜素的合成。 本实验中红发夫酵母最佳利用碳、氮源分别为蔗糖和蛋白胨，但蛋白胨价格昂贵，不适宜作单一氮源，因此使用硫酸铵和酵母膏作为复合氮源。 本论文采用了BP神经网络结合遗传算法的方法来优化红发夫酵母的发酵培养基，得到红发夫酵母发酵培养基的最佳配比为：蔗糖45.10 g/L、硫酸铵3.00 g/L、硫酸镁0.80 g/L、磷酸二氢钾1.40 g/L、酵母膏3.00 g/L、氯化钙0.50 g/L，使用优化后的培养基发酵类胡萝卜素产量达到8.20 mg/L，干重达到9.47 g/L，类胡萝卜素的产量比起始培养基提高了95.90%，干重提高了89.40%。 从代谢途径出发对红发夫酵母合成虾青素调控调控，选择谷氨酸、乙醇、VB1作为添加剂，通过正交试验设计得出三者添加水平分别为0.2 g/L，0.1% (V/V)，10 mg/L时，类胡萝卜素产量提高了25.73%，达到了10.31mg/L。 通过上述优化培养，本论文中红发夫酵母的虾青素产量从1.33 mg/L提高到9.12 mg/L，产量提高了6.86倍；总类胡萝卜素产量从4.23 mg/L提高到10.31 mg/L，产量提高了2.44倍；细胞干重从5.00 g/L提高到11.35 g/L，提高了2.27倍，总体提高效果显著。 红发夫酵母属于中低温菌，本论文采用紫外复合诱变的方式，通过高温筛选，得到一株能在35 ℃下能生长的突变株，但所产类胡萝卜素中虾青素所占比例很小，可能是诱变改变了红发夫酵母的代谢途径，阻断了虾青素的合成。 Phaffia rhodozyma is a heterobasidiomyceteous yeast that was originally isolated from the slime fluxes of brich tree wounds in mountain regions of northern Japan and southern Alaska. Phaffia rhodozyma produces astaxanthin as its principal carotenoid pigment, which has potential applications in acquaculture, food and pharmaceutical industry. This paper researched ways to break cell, analysis of astaxanthin, characteristics of growth, culture parameters and the effects of components of medium on growth and astaxanthin formation , optimization of culture medium, control of astaxanthin synthesis and mutagenesis of Phaffia rhodozyma. It is necessary to disrupt the yeast cell for extracting astaxanthin considering the yeast accumulating carotenoids in cell. Dimethyisulphoxide was the most effective solvent for breaking the yeast cell; acetone and chloroform were effective to extract carotenoids out of the disrupted cell. The optimum pH for growth and carotenoids synthesis is 5.5, the optimum medium volume is 30 mL (in 250 mL flask), the optimum culture time of inoculum is 36 h, the optimum inoculum concentration is 10%. The research on culture medium showed: sucrose is the best one of 6 carbon sources for growth and astaxanthin synthesis. Peptone is the best nitrogen source for growth and astaxanthin synthesis. Uniform Design was used for trial design of the formula medium components, then back-propagation neural network was established to modeling the relationships between the carotenoid yield and the concentration of medium components. Genetic algorithm (GA) was used for global optimization of the model. The optimum combination of the medium was obtained: sucrose 45.10 g/L, ammonium sulfate 3.00 g/L, magnesium sulfate 0.80 g/L, potassium dihydrogen phosphate 1.40 g/L, yeast extract 3.00 g/L, calcium chloride 0.50 g/L. The yield of carotenoid reached 8.20 mg/L, which was 95.90% higher than that of the original medium. Glu, VB1 and ethanol were selected as fermentation addictives, after Orthogonal Test, the carotenoid contents increased by 25.73% when adding 0.16 g/L Glu, VB1 10 mg/L and ethanol 0.1% (V/V). After the above optimization, the astaxanthin content increased 6.86 folds, which is 9.12 mg/L. The carotenoids content increased 2.44 folds, which is 10.31 mg/L. The biomass increased 2.27 folds, which is 11.35 g/L. Phaffia rhodozyma grows in the mild temperature range of 0 to 27 ℃, in this work, a thermotolerant mutant was selected through UV-irradiation. It can grows at 35 ℃, and showed increased carotenoid content. The optimal growth temperature for this mutant is 30 ℃. But the mutant can only produce carotenoids with little astaxanthin accumulation.

Functional characterization of sll0659 from Synechocystis sp PCC 6803

Synchocystis sp. PCC 6803 lacks a gene for the any known types of lycopene cyclase. Recently, we reported that sll0659 (unknown for its function) from Synechocystis sp. PCC6803 shows similarity in sequence to a lycopene cyclase gene-CruA from Chlorobium tepidum. To test, whether Sll0659 encoded protein serves as lycopene cyclase, in this study, we investigated the carotenoids of the wild types ans mutants, In the sll0659 deleted mutant, there is no blockage at the lycopene cyclization step. Our results demonstrate that sll0659 does not affect lycopene cyclization. However, the ultrastructure of mutants suggests the involvement or necessity of sll0659 in the cell division.

Molecular cloning, characterization and evolutionary analysis of phytoene desaturase (PDS) gene from Haematococcus pluvialis

Phytoene desaturase is one of the most important enzymes necessary for the biosynthesis of carotenoids in some cyanobacteria, green algae and plants. In this study, genomic DNA and cDNA of pds were cloned from unicellular green alga Haematococcus pluvialis strain323 using PCR and RT-PCR methods. The cDNA was cloned into plasmid pET-28a and efficiently expressed in Escherichia coli BL21. The complete genomic PDS gene of H. pluvialis, 3.3 kb in size, included eight exons and seven introns. To locate transcriptional regulation elements, an approximate 1 kb of 5'-flanking region was isolated by genome-walking method. Results of bioinformatic analysis showed several putative cis-elements e.g. the ABRE motif (abscisic acid responsive element), the C-repeat/DRE (dehydration responsive element) motif and the GCN4 motif were located in 5'-flanking region of pds. Results of phylogenetic analyses reveal that different sources of PDS genes form a separate clade, respectively, with 100% bootstrap support. Moreover, a maximum likelihood approach was employed to detect evidence of positive selection in the evolution of PDS genes. Results of branch-site model analysis suggest that 7.9% of sites along the green algal branch are under positive selection, and the PDS gene in green algae exhibits a different evolutionary pattern from its counterparts in cyanobacteria and plants.

Expression of beta-carotene hydroxylase gene (crtR-B) from the green alga Haematococcus pluvialis in chloroplasts of Chlamydomonas reinhardtii

A carotenoid gene (crtR-B) from the green alga Haematococcus pluvialis, encoding beta-carotene hydroxylase that was able to catalyze the conversion of beta-carotene to zeaxanthin and canthaxanthin to astaxanthin, was cloned into Chlamydomonas reinhardtii chloroplast expression vector p64D to yield plasmid p64DcrtR-B. The vector p64DcrtR-B was transferred to the chloroplast genome of C. reinhardtii using micro-particle bombardment. PCR and Southern blot analyses indicated that crtR-B was integrated into the chloroplast genome of the transformants. RTPCR assays showed that the H. pluvialis crt R-B gene was expressed in C. reinhardtii transformants. The transformants rapidly synthesized carotenoids in larger quantities than the wild-type upon being transferred from moderate to high-intensity white light. This research provides a foundation for further study to elucidate the possible mechanism of photo-protection by xanthophylls and other carotenoids in high light conditions or through exposure to UV radiation.

大西洋庸鲽Hippoglossus hippoglossus和大西洋鲑Salmo salar配子质量研究

种质问题是养殖健康发展的基础。在鱼类养殖中，卵子和精子的质量直接关系到受精、胚胎发育，仔稚鱼发育以及幼鱼生长等一系列过程。本论文针对大西洋庸鲽和大西洋鲑的配子质量进行研究。研究内容涉及大西洋庸鲽精子冷冻保存方法；促性腺激素释放激素类似物（GnRHa）使用对其精子冷冻保存效果、以及脂肪酸组成的影响；野生和驯养大西洋鲑卵子在脂肪酸、类胡萝卜素、矿物盐方面的差异比较。 精子冷冻保存通过提高对精子的利用效率，进而对于种质改良，推进鱼类养殖科研和生产具有重要意义。本实验建立了大西洋庸鲽精子大容量冷冻保存方法。八种抗冻剂冷冻保存实验结果表明：10% 及15% DMSO配以 HBSS 或KS 的抗冻剂组合冷冻保存效果最佳，4 mL体积冷冻保存可获得与1.6 mL同样的保存效果。 在繁殖季节后期注射GnRHa激素缓释剂，可获得质量稳定的大西洋庸鲽精液，将激素注射方法与精子冷冻保存方法相结合对于提高雄鱼利用率，扩大生产规模具有重要实用价值。本项研究分三个时间采集注射GnRHa激素后的雄鱼精子以及同期未注射激素的雄鱼精子，对所有精子样品使用同样的方法进行冷冻保存，检测冷冻保存后解冻精子的受精率与活力。结果表明，激素注射与否对于冷冻保存后精子的受精率和活力无显著影响，两类冷冻精液均达到鲜精水平。实验结果还表明，注射激素14天后的精子的密度显著的降低。说明GnRHa激素的使用可以显著降低精子密度，但不会影响精子的冷冻保存效果。 本相研究同时对注射GnRHa 缓释激素和未注射GnRHa 缓释激素的大西洋庸鲽精液脂肪酸成分进行分析，以检测该激素使用对精子生化组分的影响。结果表明激素的使用对在DHA (22:6n-3,二十二碳六烯酸)、EPA(20:5n-3,二十碳五烯酸)、AA(20:4n-6，花生四烯酸)等重要脂肪酸，不饱和脂肪酸、饱和脂肪酸以及n-3、n-6等重要种类的脂肪酸总量及其比例没有显著影响。精液脂肪酸中DHA含量最高，约占25%；PUFA约为44%。 作为世界性的重要养殖品种，野生和驯养大西洋鲑在形态、生化组成以及遗传 等方面表现出的差异被广泛关注。本论文，对野生和驯养大西洋鲑受精卵关键生化成分进行分析，通过与野生受精卵比较阐明驯养受精卵的质量状况，为亲鱼营养需求提供指导依据。本实验中野生配子和驯养配子的受精率没有显著差异，但重要脂肪酸组成、类胡萝卜素以及矿物盐含量都存在多方面显著差异。两类受精卵脂肪酸中含量最高的依次为18:1n-9（油酸）、DHA（二十二碳六烯酸）、16:0（棕榈酸）、EPA（二十碳五烯酸）。野生受精卵的单不饱和脂肪酸总量显著高于驯养受精卵，而多不饱和脂肪酸（PUFA）比例显著低于驯养的受精卵。在主要必需不饱和脂肪酸（EFA）中，DHA和EPA在野生受精卵中的比例高于驯养受精卵，AA（花生四烯酸）低于驯养受精卵。野生受精卵虾青素（Ax）的含量低于驯养受精卵而鸡油菌素（Cx）含量高于驯养受精卵。野生受精卵中多种矿物盐的含量（铝、铜、铁、硒和锌）含量显著高于驯养的受精卵。差别最大的为铜。诸多方面的差异表明，野生亲鱼与驯养亲鱼产出的卵子确实存在显著差异，因此关注亲鱼的营养极为重要。