花椒水浸液及酚酸类化感物质对作物种子萌发和幼苗生长的影响效应

花椒（Zanthoxylum piperitum）是川西干旱河谷地区的重要经济作物，化感作用是花椒连作障碍的原因之一。系统研究花椒化感作用有助于深入理解并最终解决花椒连作障碍。本文通过研究花椒叶、林下土壤浸提液及单一纯化感物质对花椒幼苗生长、苜蓿种子萌发及幼苗生长的影响作用，从生理生化角度揭示浸提液及纯化感物质的作用方式。通过室外和室内模拟实验，对浸提液及纯化感物质的化感效应进行比较，为花椒连作障碍的解决和化感作用机制的深入理解提供依据。主要结果如下： 1.花椒叶及林下土壤浸提液对地下生物量影响作用强与对地上生物量的化感效应，两种浸提液的化感效应强度不同，叶浸提液作用表现更显著。其中在Y6、Y8 、T6和T8处理时，花椒幼苗地下生物量分别降低了31.2%、32.1%、31.6%和31.7%。 2.两种浸提液均能显著影响花椒幼苗体内的保护性酶活性，总体说来，在高浓度下抑制各种抗氧化物酶活性，幼苗体内丙二醛含量增加，幼苗受害严重；在较低浓度下，各种保护性酶活性有所增加，丙二醛含量减少，幼苗伤害减轻。同时，不同月份里，各种酶的活性高低显著不同，9月份的活性显著低于7月份的酶活性。对于养分含量的影响，Y8、T8的影响强度最大，分别使碳元素含量降低了27.8%和30.8%，使钾元素含量降低了34.7%和25.6%。 3.花椒叶及林下土壤浸提液对苜蓿种子萌发及幼苗生长有化感作用，表现在最终萌发率、不同物质代谢及保护性酶活性的差异上。两种浸提液对苜蓿种子萌发过程中蛋白质的含量均无显著性影响，对淀粉和可溶性糖的影响作用类似，高浓度处理无明显化感效应，较低浓度处理显著降低二者在萌发苜蓿种子中的含量。Y2、Y4与T4处理分别使可溶性糖含量减少了32.3%、29.1%和18.8%，Y2与T2处理分别使淀粉含量降低了29.3%和26.8%。 4.苜蓿种子在4种单一化感物质最高浓度即10-3 mol•L-1处理下，萌发率显著降低，半数萌发时间推迟，随着处理浓度降低，抑制作用逐渐减弱，当降低到10-6 mol•L-1时，又能够表现出对苜蓿种子萌发的促进作用。 5.纯化感物质在10-6 mol•L-1时使苜蓿幼苗叶片的保护性酶活性显著升高，丙二醛含量显著降低；在10-3 mol•L-1时使苜蓿叶片中保护性酶活性显著降低，丙二醛含量增加，膜脂过氧化程度加重。 Zanthoxylum piperitum is one of the most important cash crops and has been extensively cultivated in Eastern Tibetan Plateau, especially in the fragile dry valley areas. Allelopathic effects could be a reason for Z. piperitum’s continuous cropping impediment. Systemmatically research of the effect of Z. piperitum allelopathy could help to comprehend the continuous cropping impediment. The allelopathic effects on seedlings growth and seed germination of aqueous extracts of Zanthoxylum piperitum and phenolic allelochemicals were studied, and the action mechanism of the two substances was also discussed from physiology. Indoor and outdoor experiments were set to compare the difference between aqueous extracts and pure allelochemicals. The main results showed that: 1. The aqueous extracts of leaf and soil had significant allelopathic effects on aboveground and underground biomass, but the effect on underground biomass was stronger than the effect on underground evidently. Treated with Y6、Y8 、T6 and T8, the underground biomass was reduced 31.2%、32.1%、31.6% and 31.7% respectively. 2. The activity of activities of superoxide dismutase, catalase, peroxidase and ascorbate peroxidase were significantly reduced, while the content of MDA was increased and the seedlings were suffered stronger, when treated by the high concentration; but at the low concentration, these were reversed. And then, at the different month, the activities of antioxidant enzyme were significantly distinct. As for the contents of nutrient element, Y8、T8 had the more intensive effects than other treatments. 3. The results showed that the two types of aqueous extracts had significant allelopathic effects on seed germination, substances metabolize and the activity of antioxidant enzyme. But the aqueous extracts had no effects on the content of protein, while had the similar effects on the content of starch and soluble sugar. At Y2、Y4 and T4, the content of soluble sugar decreased 32.3%、29.1% and 18.8% respectively. 4. Treated with 10-3 mol•L-1 of the four allelochemicals, the seed germination of alfalfa was significantly inhibited. Ferulic acid, coumarin and vanillic acid at 10-3 mol•L-1 significantly reduced the activities of antioxidant enzyme, while the content of MDA in alfalfa seedling was significantly increased. The restrain effects became weakened with the treat concentration falled. However, ferulic acid, coumarin and vanillic acid could increase the activities of antioxidant enzyme at 10-6mol•L-1.

遮荫与外源脱落酸喷施对粗枝云杉(Picea asperata Mast.)不同种群抗旱性的影响

本研究通过粗枝云杉不同种群进行的温室半控制试验，采用植物生态学、生理学和生物化学的研究方法，系统地研究了粗枝云杉不同种群抗旱性的生长、形态、生理和生化机理，并结合有关研究进行综合分析，得出主要研究结论如下： 1.粗枝云杉对干旱胁迫的综合反应 粗枝云杉在干旱胁迫下的适应机制为：（1）相对生长速率及植株结构的调整：干旱胁迫下虽然植株相对生长速率显著降低，且有相对较多的生物量向根部分配，但并未发现细根/总根比增加。（2）粗枝云杉对干旱胁迫的光合作用表现为：干旱胁迫显著地降低了控制的理想条件下的气体交换，但干旱胁迫对PSII最大光化学效率（Fv/Fm）没有影响，表明干旱并未影响到光合机构。（3）干旱还影响了很多生理生化过程，包括渗透调解物质（游离脯氨酸）、膜脂过氧化产物、脱落酸（ABA）含量的增加，以及保护酶活性的升高。这些结果证明植物遭受干旱胁迫后发生了一系列的形态、生理和生化响应，这些变化能提高干旱时期植物的存活和生长能力。 2.粗枝云杉不同种群对干旱胁迫反应的种群差异 粗枝云杉三个种群－干旱种群（四川丹巴和甘肃迭部）和湿润种群（四川黑水）对干旱适应不同，这种不同应归因于它们采用的用水策略不同：在水分良好和干旱胁迫条件下，受试种群在相对生长速率和水分利用效率（WUE）方面都表现出显著的种群间差异。与湿润种群相比，干旱种群在两种水分条件下有更高的WUE。粗枝云杉不同种群的碳同位素组分（δ13C）只在干旱胁迫下有显著差异，并且这种差异在水分良好时比干旱胁迫条件下小，说明生理响应和干旱适应性之间的关系受植物内部抗旱机制和外部环境条件（如水分可利用性）或两者互作效应的影响。这些结果说明干旱种群和湿润种群所采用的用水策略不同。干旱种群有更强的抗旱能力，采用的是节水型的用水策略，而湿润种群抗旱能力较弱，采用的是耗水型的用水策略。 3. 遮荫对粗枝云杉不同种群抗旱性影响 干旱胁迫显著降低了全光条件下叶相对含水量（RWC）、相对生长速率、气体交换参数、PSII的有效量子产量（Y），提高了非光化学猝灭效率（qN）、水分利用效率、脯氨酸（PRO）积累、脱落酸（ABA）含量及保护酶活性。然而这种变化在遮荫条件下不明显。我们得出结论适度遮荫降低了干旱对植物的胁迫作用。另一方面，在干旱条件下，与湿润种群相比，干旱种群抗旱性更强，表现在干旱种群净光合速率与单位重量上叶氮含量（Nmass）降低较少。另外，干旱种群表现出更为敏感的气孔导度，更高的热耗散能力（qN）能力、用水效率、ABA积累、保护酶活性，以及更低的总用水量、相对生长速率。这一结果表明这两种群采用不同的生理策略对干旱和遮荫做出反应。许多生长和生理反应差异与这两个种群原产地气候条件相适应。 4. 外源脱落酸(ABA)喷施对粗枝云杉不同种群抗旱性影响 外源ABA喷施在干旱和水分良好条件下均不同程度地提高了根/茎比，表明根和茎对ABA敏感程度不同。实验结果还表明，外源ABA喷施对这两个种群在干旱胁迫期间影响不同。干旱胁迫期间，伴随着ABA喷施，湿润种群净光合速率(A)显著降低，而干旱种群净光合速率变化不明显。另一方面，外源ABA喷施显著提高了干旱条件下干旱种群的单位叶面积重（LMA）、根/茎比、细根/总根（Ft）比、水分利用效率（WUE）、ABA含量, 以及保护酶活性。然而，外源ABA喷施对湿润种群的上述测定指标没有显著影响。这一结果表明干旱种群对外源ABA喷施更为敏感, 反应在更大的气孔导度降低，更高的生物量可塑性，及更高的水分利用效率、ABA含量和保护酶活性。综上所述，我们得出结论，粗枝云杉对外源ABA敏感性因种群的不同而不同。该研究结果可为两个明显不同种群在适应分化方面提供强有力的证据。 Arid or semi-arid land covers more than half of China's land territory. In arid systems, severe shortages of soil water often coincide with periods of high temperatures and high solar radiation, producing multiple stresses on plant performance. Protection from high radiation loads in shaded microenvironments during drought may compensate for a loss of productivity due to reduced irradiance when water is available. Additionally, ABA, a well-known stress-inducible plant hormone, has long been studied as a potential mediator for induction of drought tolerance in plants. Picea asperata Mast., which is one of the most important tree species used for the production of pulp wood and timber, is a prime reforestation species in western China. In this experiment, different population of P. asperata were used as experiment material to study the adaptability to drought stress and population differences in adaptabiliy, and the effects of shade and exogenous abscisic acid (ABA) application on the drought tolerance. Our results cold provide a strong theoretical evidence and scientific direction for the afforestation, and rehabilitation of ecosystem in the arid and semi-arid area, and provide a strong evidence for adaptive differentiation of different populations, and so may be used as criteria for species selection and tree improvement. The results are as follows: 1. A large set of parallel response to drought stress Drought stress caused pronounced inhibition of the growth and increased relatively dry matter allocation into the root; drought stress also caused pronounced inhibition of photosynthesis, while drought showed no effects on the maximal quantum yield of PSII photochemistry (Fv/Fm) in dark-adapted leaves, indicating that drought had no effects on the primary photochemistry of PSII. However, in light-adapted leaves, drought reduced the quantum yield of PSII electron transport (Y) and increased the non-photochemical quenching (qN). Drought also affected many physiological and biochemical processes, including increases in superoxide dismutase (SOD), ascorbate peroxidase (APX) activities, malondialdehyde and ABA content. These results demonstrate that there are a large set of parallel changes in the morphological, physiological and biochemical responses when plants are exposed to drought stress; these changes may enhance the capability of plants to survive and grow during drought periods. 2. Difference in adaptation to drought stress between contrasting populations of Picea asperata There were significant population differences in growth, dry matter allocation and water use efficiency. Compared with the wet climate population (Heishui), the dry climate population (Dan ba and Jiebu) showed higher LMA, fine root/total root ratio and water use efficiency under drought-stressed treatments. The results suggested that there were different water-use strategies between the dry population and the wet population. The dry climate population with higher drought tolerance may employ a conservative water-use strategy, whereas the wet climate population with lower drought tolerance may employ a prodigal water-use strategy. These variations in drought responses may be used as criteria for species selection and tree improvement. 3. The effects of shade on the drought tolerance For both populations tested, drought resulted in lower needle relative water content (RWC), relative growth rate (RGR), gas exchange parameters and effective PSII quantum yield (Y), and higher non-photochemical quenching (qN), water use efficiency (WUE), proline (PRO) and abscisic acid (ABA) accumulation, superoxide dismutase (SOD), ascorbate peroxidase (APX) activities as well as malondialdehyde (MDA) levels and electrolyte leakage in sun plants, whereas these changes were not significant in shade plants. Our study results implied that shade, applied together with drought, ameliorated the detrimental effects of drought. On the other hand, compared with the wet climate population, the dry climate population was more tolerant to drought in the sun treatment, as indicated by less decreases in A and mass-based leaf nitrogen content (Nmass), more responsive stomata, greater capacity for non-radiative dissipation of excitation energy as heat (analysed by qN), and higher WUE，higher level of antioxidant enzyme activities，higher ABA accumulation as well as lower MDA content and electrolyte leakage. Many of the differences in growth and physiological responses reported here are consistent with the climatic differences between the locations of the populations of P. asperata. 4. The effects of exogenous abscisic acid (ABA) application on the drought tolerance For both populations tested, exogenous ABA application increased root/shoot ratio (Rs) under well-watered and drought-stressed conditions, indicating that there was differential sensitivity to ABA in the roots and shoots. However, it appeared that ABA application affected the two P. asperata populations very differently during drought. CO2 assimilation rate (A) was significantly decreased in the wet climate population, but only to a minor extent in the dry climate population following ABA application during soil drying. On the other hand, ABA application significantly decreased stomatal conductance (gs), transpiration rate (E) and malondialdehyde (MDA) content, and significantly increased leaf mass per area (LMA), Rs, fine root/total root ratio (Ft), water use efficiency (WUE), ABA contents, superoxide dismutase (SOD), ascorbate peroxidase (APX) and catalase (CAT) activities under drought condition in the dry climate population, whereas ABA application did not significantly affect these parameters in the wet population plants. The results clearly demonstrated that the dry climate population was more responsive to ABA application than the wet climate population, as indicated by the strong stomata closure and by greater plasticity of LMA and biomass allocation, as well as by higher WUE, ABA content and anti-oxidative capacity to defense against oxidative stress, possibly predominantly by APX. We concluded that sensitivity to exogenous ABA application is population dependent in P. asperata. Our results provide strong evidence for adaptive differentiation between populations of P. asperata.

生姜化感作用机理及其栽培模式研究

近年来，随着对作物重茬（连年种植）障碍原因的深入研究，植物的化感作用越来越受到国内外众多学者的重视。而作为重要调料和药用植物的生姜，其连作障碍也备受关注，系统地研究生姜化感作用将有助于理解和最终解决生姜连作障碍问题。本文通过研究生姜不同部位、不同浓度的水浸液对与其间作的两个物种（大豆和四季葱）种子的萌发及幼苗生长的影响，从而证明生姜化感作用的存在；并通过温室盆栽实验研究了生姜的自毒作用（即研究生姜不同部位、不同浓度的水浸液对其幼苗的形态、生理生化、光合作用、土壤酶、土壤微生物多样性及土壤养分的影响），从而揭示生姜退化和衰老的机制，并为生姜筛选出合适的间作物种提供科学依据，对生姜连作障碍提出科学的解决方法。主要研究结果如下： 1. 与对照相比，生姜所有部位（根茎、茎、叶）、所有浓度（10、20、40、 80 g l-1）的水浸液均抑制了大豆种子和葱籽的萌发率、幼苗生长、水分吸收和脂肪酶活性，并且其抑制程度随着水浸液浓度的增加而增强，其生姜各部位水浸液抑制效应的强弱顺序为茎＞叶＞根茎。这一结果表明生姜根茎、茎、叶含有能够抑制大豆种子和葱籽种子萌发和幼苗生长的水溶性化感物质。根茎是生姜的主要收获部位，而生姜的残株（主要是茎和叶）应该从大田中处理掉以减轻其抑制效应。生姜水浸液中主要化感成分包括：根茎水浸液中主要是丁香酸和伞花内脂；茎水浸液中主要是阿魏酸，且其含量最高为73.4 ug/g；叶水浸液中除了阿魏酸，其他六种物质均检测出来，但含量较高的主要有丁香酸、伞花内脂和香豆酸。 2. 生姜茎和叶不同浓度的水浸液均显著抑制了生姜幼苗的株高、每株叶片数和叶面积，其抑制程度随着水浸液浓度的增加而有所增强，而生姜幼苗每株分枝数差异不显著；同时生姜水浸液也极大程度地影响了生姜幼苗的生物量（包括地下生物量、地上生物量和总生物量，均为鲜重）。在同一浓度下，茎水浸液对生姜幼苗形态指标及生物量指标均显示出最强的抑制作用，叶水浸液次之，根茎水浸液最弱。与对照相比，低浓度的生姜根茎水浸液提高了生姜幼苗叶片内四种抗氧化酶（SOD、POD、CAT、APX）活性，高浓度的根茎水浸液抑制了四种抗氧化酶活性，而茎和叶水浸液均随着浓度的增加而抑制了四种抗氧化酶活性，三种水浸液均随着浓度的增加降低了生姜幼苗叶片内叶绿素的含量，而增加了生姜幼苗叶片的相对电导率和丙二醛含量。同时，三种水浸液均随着浓度的增加降低了生姜幼苗的光合参数（包括胞间CO2浓度、气孔导度、蒸腾速率及净光合速率）。 3. 三种生姜水浸液对所测六种土壤酶活性均产生了不同程度的影响，其中影响最大的是酸性磷酸酶和蔗糖酶，在10 g l-1 时就达到了显著水平，并且所有酶均有随着水浸液浓度增加而增大的趋势；相同部位的水浸液随着浓度的增加，细菌和真菌的数量呈增加趋势，而放线菌的数量呈减少趋势；三种生姜水浸液均随着浓度的增加降低了土壤中有机质的含量，加剧了土壤中硝态氮含量的积累，根茎水浸液对土壤有效磷、速效钾和铵态氮均显示出低浓度提高其含量而高浓度降低其含量的趋势，而茎和叶水浸液则随着浓度的增加均降低了其含量。 4. 与生姜单作相比，所有间作系统均在旺盛生长期和收获期不同程度地提高了土壤酶活性，同时也增加了土壤细菌数量及土壤微生物总数但不显著；所有间作系统在旺盛生长期和收获期均不同程度地影响了土壤真菌及放线菌数量（增加或减少），所有间作系统间的多样性指数差异不显著，除了旺盛生长期四种作物（生姜-大豆-四季葱-大蒜）的间作模式显著降低了多样性指数，其值仅为生姜单作的33.18%；生姜与大豆间作不仅提高了19.6%的生姜产量而且获得了较好的经济效益，并且，所有间作系统均显著抑制了生姜姜瘟病的发生。 5. 不同栽培模式不同程度地影响了收获期生姜的株高、分枝数、根茎产量及内在品质。其中处理2显著地促进了生姜的分枝（10.5%），同时处理2、3和4也促进了生姜的生长（株高分别增加了15.0%、11.4%和14.0%），并且这三个处理提高了生姜的产量；处理2和3能有效提高生姜块茎中维生素C（分别较单作生姜显著提高了3.29%和4.05%）、处理3显著提高了可溶性糖（8.2%）、姜辣素（4.6%）和蛋白质等有益物质的含量，降低硝酸盐有害物质的含量（处理2显著降低了14.0%），改善了姜块的外观和内在品质。并且，生姜与大豆间作具有最高的纯收入和产投比，分别较生姜单作提高了24.80%和8.8%。Recently, allelopathy has been more and more paid attentions by national and foreign scholars with profound research on reasons of crop replanted (continuous planted) obstacle. Ginger rhizome is valuable all over the world either as a spice or herbal medicine and ginger replanted obstacle is also paid attentions. Systematic research on ginger allelopathy will contribute to understanding and ultimate solving problem of ginger replanted obstacle. The effects of ginger aqueous extracts with different parts and concentrations on seed germination and early seedling growth of soybean and chive were studied in this article to testify that ginger existed allelopathy. Furthermore, ginger autotoxicity was also studied by pot experiment in greenhouse (namely research on effects of ginger aqueous extracts with different parts and concentrations on morphological indexes, physiological and biochemical indexes, photosynthesis, soil enzymes, soil microbial diversity and soil nutrients) to reveal mechanism of ginger degeneration and senescence, provide scientific basis for selecting appropriate intercropping species and put forward scientific resolvent for ginger replanted obstacle. The main results were as follows: 1. All aqueous extracts at all concentrations inhibited seed germination, seedling growth, water uptake and lipase activity of soybean and chive compared with the control, and the degree of inhibition increased with the incremental extracts concentration. The degree of toxicity of different ginger plant parts can be classified in order of decreasing inhibition as stem＞leaf＞rhizome. The results of this study suggested that rhizome, stem and leaf of ginger contained water soluble allelochemicals which could inhibit seed germination and seedling growth of soybean and chive. The rhizome is the main harvested part of ginger. The residue (mainly stems and leaves) of the ginger plant should be removed from the field so as to diminish its inhibitory effect. The main allelopathic components of three kind of aqueous extracts were as follows: Rhizome extract chiefly contained syringic acid and vmbelliferone and stem extract mainly contained frulic acid whose content was the highest (73.4 ug/g). The other six substances were detected except of frulic acid, but only contents of syringic acid, vmbelliferone and p-coumaric acid were higher. 2. Stem and leaf aqueous extracts of ginger with different concentrations significantly inhibited plant height, leaf numbers per plant and leaf area, and the degree of inhibition increased with the incremental extracts concentration. However, tiller number per plant of ginger seedling showed no significant difference. At the same time, ginger aqueous extracts also influenced biomass including under-ground biomass, above-ground biomass and total biomass (fresh weight) to a large extent. Under the same concentration, stem aqueous extract showed the mostly inhibitory effect on morphological indexes and biomass indexes of ginger seedling. Rhizome aqueous extract showed the leastly inhibitory effect and leaf aqueous extract was intervenient. Enhanced concentration of ginger aqueous extracts significantly reduced total chlorophyll content, accompanying with increases in memberane permeability (REL) and lipid peroxidation (MDA). Compared with the control, rhizome ginger aqueous extract of lower concentration (10 g l-1) increased the activities of major antioxidant enzymes (superoxide dismutase, SOD; peroxidase, POD; catalase, CAT; ascorbate peroxidase, APX) of ginger leaf tissue and higher concentration inhibited the activities of four antioxidant enzymes. However, stem and leaf aqueous extract inhibited the activities of four antioxidant enzymes with increase in concentration. Meanwhile, enhanced concentration of ginger aqueous extracts significantly reduced photo-parameters of ginger seedling (including CO2 concentration, stoma conductivity, net photosynthesis rate and transpiration rate). 3. Rhizome, stem and leaf ginger aqueous extract showed different effect on six soil enzyme activities, and acid phosphatase and invertase showed significant effect when aqueous extract concentration got 10 g l-1. Furthermore, six soil enzyme activities increased with increase in aqueous extract concentration. Bcterial and fungi number tended to increase while antinomyces tented to decrease with the increase in aqueous extract concentration of identical part. Ginger aqueous extracts reduced soil organic matter content with increased concentration, accompanying with NO3-—N accumulation in soil. Rhizome aqueous extract showed the same tendency for available P, available K and NH4+—N, namely lower concentration increased their contents in soil and higher concentration reduced their contents. While stem and leaf aqueous extracts reduced their contents with the increamental concentration. 4. All intercropping systems increased soil enzyme activities to different extent both at VGS and at HS compared to solo ginger. All intercropping systems increased the colony numbers of soil bacteria and total of soil microbe but not significantly either at VGS or at HS. All intercropping systems increased the colony numbers of soil fungi and actinomytes to a different extent (increase or decrease) both at VGS and at HS. For DI, difference between all cultivation patterns and S-G was not significant either at VGS or at HS except that G-S-C-G whose value was only 33.18% of S-G at VGS significantly decreased. G-S not only increased ginger yield by 19.6% but also obtained better economic benefit. Furthermore, all intercropping systems significantly inhibited occurrence of bacterial wilt of ginger. 5. Different cultivated pattern influenced plant height, tiller numbers, rhizome yields and intrinsic quality of ginger. Treatment 2 significantly facilitated tiller occurring (10.5%). Treatment 2, 3 and 4 promoted ginger growth (plant height respectively increased 15.0%、11.4% and 14.0%) and enhanced rhizome yields. Treatment 2 and 3 effectively increased vitamin C content (significantly increased 3.29% and 4.05% compared to solo ginger). Treatment 3 significantly increased contents of beneficial substances such as soluble sugar (8.2%), gingerols (4.6%) and protein. Treatment 2 significantly decreased contents of deleterious substance namely nitrate (14.0%) and improved appearance and intrinsic quality of ginger rhizome. Furthermore, treatment 2 (ginger/soybean intercropping) could obtain better economic benefit and showed the highest net income and ratio of benefit and cost whose values respectively increased by 24.80% and 8.8% compared to solo ginger.

Effect of alginic acid decomposing bacterium on the growth of Laminaria japonica (Phaeophyceae)

We collected the diseased blades of Laminaria japonica from Yantai Sea Farm from October to December 2002, and the alginic acid decomposing bacterium on the diseased blade was isolated and purified, and was identified as Alterornonas espejiana. This bacterium was applied as the causative pathogen to infect the blades of L. japonica under laboratory conditions. The aim of the present study was to identify the effects of the bacterium on the growth of L. japonica, and to find the possibly effective mechanism. Results showed that: (1) The blades of L. japonica exhibited symptoms of lesion, bleaching and deterioration when infected by the bacterium, and their growth and photosynthesis were dramatically suppressed. At the same time, the reactive oxygen species (ROS) generation enhanced obviously, and the relative membrane permeability increased significantly. The contents of malonaldehyde (MDA) and free fatty acid in the microsomol membrane greatly elevated, but the phospholipid content decreased. Result suggested an obvious peroxidation and deesterrification in the blades of L. japonica when infected by the bacterium. (2) The simultaneous assay on the antioxidant enzyme activities demonstrated that superoxide dismutase (SOD) and catalase (CAT) increased greatly when infected by the bacterium, but glutathione peroxidase (Gpx) and ascorbate peroxidase (APX) did not exhibit active responses to the bacterium throughout the experiment. (3) The histomorphological observations gave a distinctive evidence of the severity of the lesions as well as the relative abundance in the bacterial population on the blades after infection. The bacterium firstly invaded into the endodermis of L. japonica and gathered around there, and then resulted in the membrane damage, cells corruption and ultimately, the death of L. japonica.

Efeitos da tetraciclina em Gambusia holbrooki

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Mammalian cell cultures as a model system for the determination of cytotoxicity induced by cholesterol oxidation products

Oxysterols are products of cholesterol oxidation, which may be produced endogenously or may be absorbed from the diet where they are commonly found in foods of animal origin. Oxysterols are known to be cyctotoxic to cells in culture and mode of toxicity has been identified as apoptosis in certain cell lines. The cytotoxicity of the oxysterols 25-hydroxycholesterol (25-OH) and 7β-hydroxycholesterol (7β-OH) was examined in two human cell lines, HepG2, a hepatoma cell line, and U937, a monocytic cell line. Both 25-OH and 7β-OH were cytotoxic to the HepG2 cell line but apoptotic cells were not detected and it was concluded that cells underwent necrosis. 25-OH was not cytotoxic to the U937 cell line but it was found to have a cytostatic effect. 7β-OH was shown to induce apoptosis in the U937 line. The mechanism of oxysterol-induced apoptosis has not yet been fully elucidated, however the generation of an oxidative stress and the depletion of glutathione have been associated with the initial stages of the apoptotic process. The concentration of cellular antioxidant enzyme, superoxide dismutase (SOD) was increased in association with 7β-OH induced apoptosis in the U937 cell line. There was no change in the glutathione concentration or the SOD activity of HepG2 cells, which underwent necrosis in the presence of 7β-OH. Many apoptotic pathways center on the activation of caspase-3, which is the key executioner protease of apoptosis. Caspase-3 activity was also shown to increase in association with 7β-OH-induced apoptosis in U937 cells but there was no significant increase in caspase-3 activity in HepG2 cells. DNA fragmentation is regarded as the biochemical hallmark of apoptosis, therefore the comet assay as a measure of DNA fragmentation was assessed as a measure of apoptosis. The level of DNA fragmentation induced by 7β-OH, as measured using the comet assay, was similar for both cell lines. Therefore, it was concluded that the comet assay could not be used to distinguish between 7β-OH-induced apoptosis in U937 cells and 7β-OH-induced necrosis in HepG2 cells. The cytotoxicity and apoptotic potency of oxysterols 25-OH, 7β-OH, cholesterol- 5a,6a-epoxide (a-epoxide), cholesterol-5β,6β-epoxide (β-epoxide), 19-hydroxy-cholesterol (19-OH), and 7-ketocholesterol (7-keto) was compared in the U937 cell line. 7 β-OH, β-epoxide and 7-keto were found to induce apoptosis in U937 cells. 7β-OH-induced apoptosis was associated with a decrease in the cellular glutathione concentration and an increase in SOD activity, 7-keto and β-epoxide did not affect the glutathione concentration or the SOD activity of the cells.a-Epoxide, 19-OH and 25-OH were not cytotoxic to the U937 cell line.

Fiber type-specific nitric oxide protects oxidative myofibers against cachectic stimuli.

Oxidative skeletal muscles are more resistant than glycolytic muscles to cachexia caused by chronic heart failure and other chronic diseases. The molecular mechanism for the protection associated with oxidative phenotype remains elusive. We hypothesized that differences in reactive oxygen species (ROS) and nitric oxide (NO) determine the fiber type susceptibility. Here, we show that intraperitoneal injection of endotoxin (lipopolysaccharide, LPS) in mice resulted in higher level of ROS and greater expression of muscle-specific E3 ubiqitin ligases, muscle atrophy F-box (MAFbx)/atrogin-1 and muscle RING finger-1 (MuRF1), in glycolytic white vastus lateralis muscle than in oxidative soleus muscle. By contrast, NO production, inducible NO synthase (iNos) and antioxidant gene expression were greatly enhanced in oxidative, but not in glycolytic muscles, suggesting that NO mediates protection against muscle wasting. NO donors enhanced iNos and antioxidant gene expression and blocked cytokine/endotoxin-induced MAFbx/atrogin-1 expression in cultured myoblasts and in skeletal muscle in vivo. Our studies reveal a novel protective mechanism in oxidative myofibers mediated by enhanced iNos and antioxidant gene expression and suggest a significant value of enhanced NO signaling as a new therapeutic strategy for cachexia.

A population-based association study of SNPs of GSTP1, MnSOD, GPX2 and Barrett's esophagus and esophageal adenocarcinoma

Oxidative stress appears to be important in the pathogenesis of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Single-nucleotide polymorphisms (SNPs) of antioxidant enzyme genes may play a part in determining individual susceptibility to these diseases. The Factors Influencing the Barrett's Adenocarcinoma Relationship (FINBAR) study is a population-based, case-control study of BE and EAC in Ireland. DNA from EAC (n = 207), BE (> or =3 cm BE at endoscopy with specialized intestinal metaplasia on biopsy, n = 189) and normal population controls (n = 223) were analyzed. Several SNPs spanning the genes for glutathione S-transferase P1 (GSTP1), manganese superoxide dismutase (MnSOD) and glutathione peroxidase 2 (GPX2) were genotyped using multiplex polymerase chain reaction and SNaPshottrade mark. The chi(2) test was used to compare genotype and allele frequencies between case and control subjects. Linkage disequilibrium between SNPs was quantified using Lewontin's D' value and haplotype frequency estimates obtained using Haploview. Eleven SNPs were genotyped (six for GSTP1, three for MnSOD and two for GPX2); all were in Hardy-Weinberg equilibrium. None was significantly associated with EAC or BE even before Bonferroni correction. Odds ratios for EAC for individual SNPs ranged from 0.68 [95% confidence interval (CI) 0.43-1.08] to 1.25 (95% CI 0.73-2.16), and for BE from 0.84 (95% CI 0.52-1.30) to 1.30 (95% CI 0.85-1.97). SNPs in all three genes were in strong linkage disequilibrium (D' > 0.887) but haplotype analysis did not show any significant association with EAC or BE. SNPs involving the GSTP1, MnSOD and GPX2 genes were not associated with BE or EAC. Further studies aimed at identifying susceptibility genes should focus on different antioxidant genes or different pathways.

Identification of serum stress biomarkers in pigs housed at different stocking densities

Eight Duroc × (Landrace × Large White) male pigs housed at a stocking rate of 0.50 m2/pig were subjected to a higher stocking rate of 0.25 m2/pig (higher density, HD) for two 4-day periods over 26 days. Using biochemical and proteomic techniques serum and plasma samples were examined to identify potential biomarkers for monitoring stress due to HD housing. HD housed pigs showed significant differences (P < 0.001) in total cholesterol and low density lipoprotein-associated cholesterol, as well as in concentrations of the pig-major acute phase protein (Pig-MAP) (P = 0.002). No differences were observed in serum cortisol or other acute phase proteins such as haptoglobin, C-reactive protein or apolipoprotein A–I. HD-individuals also showed an imbalance in redox homeostasis, detected as an increase in the level of oxidized proteins measured as the total plasma carbonyl protein content (P < 0.001) with a compensatory increase in the activity of the antioxidant enzyme glutathione peroxidase (P = 0.012). Comparison of the serum proteome yielded a new potential stress biomarker, identified as actin by mass spectrometry. Cluster analysis of the results indicated that individuals segregated into two groups, with different response patterns, suggesting that the stress response depended on individual susceptibility.

In utero exposure to cigarette chemicals induces sex-specific disruption of one-carbon metabolism and DNA methylation in the human fetal liver

Background: Maternal smoking is one of the most important modifiable risk factors for low birthweight, which is strongly associated with increased cardiometabolic disease risk in adulthood. Maternal smoking reduces the levels of the methyl donor vitamin B12 and is associated with altered DNA methylation at birth. Altered DNA methylation may be an important mechanism underlying increased disease susceptibility; however, the extent to which this can be induced in the developing fetus is unknown.

Methods: In this retrospective study, we measured concentrations of cobalt, vitamin B12, and mRNA transcripts encoding key enzymes in the 1-carbon cycle in 55 fetal human livers obtained from 11 to 21 weeks of gestation elective terminations and matched for gestation and maternal smoking. DNA methylation was measured at critical regions known to be susceptible to the in utero environment. Homocysteine concentrations were analyzed in plasma from 60 fetuses.

Results: In addition to identifying baseline sex differences, we found that maternal smoking was associated with sex-specific alterations of fetal liver vitamin B12, plasma homocysteine and expression of enzymes in the 1-carbon cycle in fetal liver. In the majority of the measured parameters which showed a sex difference, maternal smoking reduced the magnitude of that difference. Maternal smoking also altered DNA methylation at the imprinted gene IGF2 and the glucocorticoid receptor (GR/NR3C1).

Conclusions: Our unique data strengthen studies linking in utero exposures to altered DNA methylation by showing, for the first time, that such changes are present in fetal life and in a key metabolic target tissue, human fetal liver. Furthermore, these data propose a novel mechanism by which such changes are induced, namely through alterations in methyl donor availability and changes in 1-carbon metabolism.

Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood-brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen-glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection.

Lifespan, body size and oxygen : aerobic metabolism and oxidative stress in cultured fibroblasts /

The allometric scaling relationship observed between metabolic rate (MR) and species body mass can be partially explained by differences in cellular MR (Porter & Brand, 1995). Here, I studied cultured cell lines derived from ten mammalian species to determine whether cells propagated in an identical environment exhibited MR scaling. Oxidative and anaerobic metabolic parameters did not scale significantly with donor body mass in cultured cells, indicating the absence of an intrinsic MR setpoint. The rate of oxygen delivery has been proposed to limit cellular metabolic rates in larger organisms (West et al., 2002). As such cells were cultured under a variety of physiologically relevant oxygen tensions to investigate the effect of oxygen on cellular metabolic rates. Exposure to higher medium oxygen tensions resulted in increased metabolic rates in all cells. Higher MRs have the potential to produce more reactive oxygen species (ROS) which could cause genomic instability and thus reduced lifespan. Longer-lived species are more resistant to oxidative stress (Kapahi et al, 1999), which may be due to greater antioxidant and/or DNA repair capacities. This hypothesis was addressed by culturing primary dermal fibroblasts from eight mammalian species ranging in maximum lifespan from 5 to 120 years. Only the antioxidant manganese superoxide dismutases (MnSOD) positively scaled with species lifespan (p<0.01). Oxidative damage to DNA is primarily repaired by the base excision repair (BER) pathway. BER enzyme activities showed either no correlation or as in the case of polymerase p correlated, negatively with donor species (p<0.01 ). Typically, mammalian cells are cultured in a 20% O2 (atmospheric) environment, which is several-fold higher than cells experience in vivo. Therefore, the secondary aim of this study was to determine the effect of culturing mammalian cells at a more physiological oxygen tension (3%) on BER, and antioxidant, enzyme activities. Consistently, standard culture conditions induce higher antioxidant and DNA ba.se excision repair activities than are present under a more physiological oxygen concentration. Therefore, standard culture conditions are inappropriate for studies of oxidative stress-induced activities and species differences in fibroblast DNA BER repair capacities may represent differences in ability to respond to oxidative stress. An interesting outcome firom this study was that some inherent cellular properties are maintained in culture (i.e. stress responses) while others are not (i.e. MR).

Cellular Mechanisms of Resveratrol's Interaction with Mitochondrial Reactive Oxygen Species Metabolism

Resveratrol, a polyphenol found naturally in red wines, has attracted great interest in both the scientific community and the general public for its reported ability to protect against many of the diseases facing Western society today. While the purported health effects of resveratrol are well characterized, details of the cellular mechanisms that give rise to these observations are unclear. Here, the mitochondrial antioxidant enzyme Mn superoxide dismutase (MnSOD) was identified as a proximal target of resveratrol in vitro and in vivo. MnSOD protein and activity levels increase significantly in cultured cells treated with resveratrol, and in the brain tissue of mice given resveratrol in a high fat diet. Preventing the increase in MnSOD levels eliminates two of resveratrol’s more interesting effects in the context of human health: inhibition of proliferative cell growth and cytoprotection. Thus, the induction of MnSOD is a critical step in the molecular mechanism of resveratrol. Mitochondrial morphology is a malleable property that is capable of impeding cell cycle progression and conferring resistance against stress induced cell death. Using confocal microscopy and a novel ‘cell free’ fusion assay it was determined that concurrent with changes in MnSOD protein levels, resveratrol treatment leads to a more fused mitochondrial reticulum. This observation may be important to resveratrol’s ability to slow proliferative cell growth and confer cytoprotection. Resveratrol's biological activities, including the ability to increase MnSOD levels, are strikingly similar to what is observed with estrogen treatment. Resveratrol fails to increase MnSOD levels, slow proliferative cell growth and confer cytoprotection in the presence of an estrogen receptor antagonist. Resveratrol's effects can be replicated with the specific estrogen receptor beta agonist diarylpropionitrile, and are absent in myoblasts lacking estrogen receptor beta. Four compounds that are structurally similar to resveratrol and seven phytoestrogens predicted to bind to estrogen receptor beta were screened for their effects on MnSOD, proliferative growth rates and stress resistance in cultured mammalian cells. Several of these compounds were able to mimic the effects of resveratrol on MnSOD levels, proliferative cell growth and stress resistance in vitro. Thus, I hypothesize that resveratrol interacts with estrogen receptor beta to induce the upregulation of MnSOD, which in turn affects cell cycle progression and stress resistance. These results have important implications for the understanding of RES’s biological activities and potential applications to human health.

Rôle de la CuZn superoxyde dismutase dans la néovascularisation en réponse à l'ischémie

L’athérosclérose est à l’origine d’importantes obstructions vasculaires. La sévérité de l’ischémie tissulaire provoquée par l’athérosclérose dépend en partie de la capacité de l’organisme à former de nouveaux vaisseaux (néovascularisation). Les mécanismes de néovascularisation sont modulés par la balance oxydo-réductive. Une exacerbation du stress oxydant est retrouvée dans tous les facteurs de risque cardiovasculaire, et en particulier lors du vieillissement. Au niveau vasculaire, la CuZnSOD est la principale enzyme antioxydante. Cependant, son rôle spécifique dans le vieillissement vasculaire et dans le développement de nouveaux vaisseaux en réponse à l’ischémie n’est pas connu. Nos hypothèses de recherche sont: 1) qu’une absence de CuZnSOD diminue la néovascularisation réparatrice en réponse à l’ischémie 2) que cette diminution de la néovascularisation est dûe au vieillissement de la vasculature affectant à la fois les cellules endothéliales matures et les cellules progénitrices endothéliales. Nous avons démontré qu’une déficience en CuZnSOD diminue significativement la néovascularisation en réponse à l’ischémie. Cette diminution de néovascularisation est associée à une augmentation du stress oxydant et une réduction de la biodisponibilité du NO. La déficience en CuZnSOD réduit significativement le nombre de EPCs (moelle, rate). De plus, ces EPCs présentent une augmentation significative des niveaux de stress oxydant, une diminution de la production de NO et une capacité réduite à migrer et à s’intégrer à un réseau tubulaire. Fait important, il iv est possible d’améliorer la néovascularisation des souris déficientes en CuZnSOD par une supplémentation en EPCs provenant de souris contrôles. Nous avons également démontré que la récupération du flot sanguin suivant l’ischémie est significativement réduite par l’âge. À la fois chez les jeunes et les vieilles souris, la déficience en CuZnSOD mène à une réduction additionnelle de la néovascularisation. Fait intéressant, le potentiel néovasculaire des jeunes souris déficiente en CuZnSOD est similaire à celui des vieilles souris contrôles. Les niveaux de stress oxydant sont également augmentés de façon similaire dans ces deux groupes de souris. L’âge et la déficience en CuZnSOD sont tous deux associés à une réduction du nombre d’EPCs isolées de la moelle et de la rate. L’effet de l’âge seul sur la fonction des EPCs est modeste. Par contre, la déficience en CuZnSOD en condition de vieillissement est associée à d’importants effets délétères sur l’activité fonctionnelle des EPCs. En résumé, nos résultats suggèrent que la protection contre le stress oxydant par la CuZnSOD est essentielle pour préserver la fonction des EPCs et la néovascularisation réparatrice en réponse à l’ischémie. Le défaut de néovascularisation observé en absence de CuZnSOD est associé à un vieillissement vasculaire accéléré. Nos résultats suggèrent que dans le contexte du vieillissement, la CuZnSOD a un rôle encore plus important pour limiter les niveaux de stress oxydant, préserver la fonction des EPCs et maintenir l’intégrité des tissus ischémiques.

Le rôle et la régulation du pyroglutamylated RF-amide peptide dans le tissu adipeux lors de l’obésité

L’obésité est définie comme un surplus de masse adipeuse. Cette condition représente un problème de santé publique devenu pandémique dans les pays industrialisés. Elle prédispose à des maladies potentiellement mortelles comme le diabète de type 2, les maladies cardiovasculaires et la stéatose hépatique non-alcoolique. L’accumulation du tissu adipeux intra-abdominal, formé d’adipocytes, est corrélée avec la résistance à l’insuline. L’augmentation de la masse adipeuse se fait par l’hyperplasie des préadipocytes, la différenciation des préadipocytes en adipocytes et l’hypertrophie des adipocytes. La différenciation des préadipocytes se fait selon l’adipogenèse qui est régulée par une multitude de facteurs, mais qui est inhibée pas les stimuli inflammatoires qui sont aussi responsables de la résistance à l’insuline et de l’apparition des problèmes de santé liés à l’obésité. Nous avons identifié un nouveau système de régulation autocrine/paracrine de l’adipogenèse dans les cellules du tissu adipeux. Le pyroglutamylated RF-amide peptide (QRFP), qui était connu pour son rôle dans la régulation de l’appétit, est un activateur de l’adipogenèse par l’activation de son récepteur, le G protein-coupled receptor 103 (GPR103). Le QRFP est exprimé dans les macrophages et les adipocytes alors que le GPR103 de sous-type b est exprimé dans les adipocytes seulement. Un traitement des adipocytes avec le QRFP augmente le captage des acides gras, l’accumulation de lipides ainsi que l’expression et l’activité de l’enzyme LPL. Le QRFP augmente aussi l’expression des gènes des transporteurs d’acides gras CD36 et FATP1, de l’enzyme activatrice d’acides gras ACSL1 et des facteurs de transcription PPAR-γ et C/EBP-α, qui sont tous impliqués dans l’adipogenèse. En plus de ses effets sur l’adipogenèse, le QRFP possède aussi un effet inhibiteur sur l’activité lipolytique induite par les catécholamines. Nous avons montré que l’expression du QRFP est diminuée dans le tissu adipeux des souris obèses. Selon nos résultats, cette diminution pourrait être expliquée par une augmentation des endotoxines circulantes chez les obèses, appelée endotoxémie métabolique, qui agirait, entre autres, par l’induction des interférons dans les macrophages. Les voies de signalisation de ces effets ont aussi été identifiées. Nous avons montré un autre exemple de stimulus inflammatoire qui régule les signaux adipogènes à la baisse.