桃和苹果库源关系中源叶光合作用，光抑制及其光保护机制的研究

以‘早久保’（Prunus persica (L.) Batch.）为试材，在果实最后迅速生长期，通过去果处理降低库力，同时设留果对照，并通过环剥和保留相同数量叶片严格控制库源关系，进行了源叶净光合速率（Pn）、叶绿素荧光、叶黄素循环、抗氧化酶及抗氧化同化物日变化的研究。结果表明，和留果对照相比，去果处理显著降低了源叶Pn、气孔导度（gs）和蒸腾速率（E），但显著增加了胞间二氧化碳浓度（Ci）、叶面饱和蒸汽压亏缺（VPDl）和叶片温度（Tl）。光系统II光化学效率（ΦPSII）以及羧化速率（CE）与Pn平行降低。中午去果降低Pn主要归因于非气孔限制。在低库需条件下，开放的PSII反应中心捕获能量的降低以及关闭的PSII反应中心的增加导致了ΦPSII的降低。去果处理叶片中依赖于叶黄素循环的热耗散以及抗氧化系统的上调保护叶片免受光氧化破坏。和留果对照相比，去果处理的叶片有更大的叶黄素循环库，更高的脱环氧化状态以及更高的抗氧化酶活性，包括超氧化物歧化酶（SOD）、抗坏血酸过氧化物酶（APX）、单脱氢抗坏血酸还原酶（MDAR）和脱氢抗坏血酸还原酶（DHAR）的活性以及更高的还原型抗坏血酸（AsA）和还原型谷胱甘肽（GSH）的含量。但与此同时，去果显著增加了过氧化氢（H2O2）以及丙二醛（MDA）的含量，这意味着在去果处理的叶片中可能会发生光氧化破坏。 以一年生‘皇家嘎拉’苹果（Malus domestica Borkh.）组培苗为试材，通过环剥降低库力，进行了源叶Pn、叶绿素荧光、核酮糖-1,5-二磷酸羧化酶/氧化酶（Rubisco）以及光系统II（PSII）复合体关键蛋白PsbA和PsbO含量日变化的研究。和对照相比，环剥显著降低了源叶Pn、gs和E，但是却显著增加了Ci、Tl和淀粉的含量。在低库需下，开放的PSII反应中心捕获能量的降低以及关闭的PSII反应中心的增加导致了ΦPSII的降低。另一方面，环剥降低了光合作用关键酶Rubisco以及PSII复合体PsbA和放氧复合体PsbO的含量。以上结果表明，环剥降低Pn主要归因于非气孔限制。

玉米种子脱水耐性与其线粒体完整性及抗氧化系统的关系

本文以正常性玉米种子‘农大108’(Zea Mays L. ‘Nongda 108’) 种胚为实验材料，研究了玉米种子发育过程中脱水耐性的变化规律，细胞匀浆以及线粒体水平上活性氧清除酶活性与种子脱水耐性/敏感性的关系，以及线粒体结构和功能完整性在发育过程中不同阶段对脱水的应答，以期在亚细胞分区水平上，针对活性氧产生的源头位点 (线粒体) 探明种子细胞脱水耐性/敏感性与抗氧化系统运转的关系。结果表明： 玉米种子在发育过程中先获得萌发能力后获得脱水耐性，并且脱水耐性的获得是一个渐进的过程。人工授粉后26天 (Days after pollination, DAP) 之前的种胚不具有脱水耐性，26 DAP时开始获得脱水耐性，到34 DAP后种胚完全获得脱水耐性。 在发育过程中，种胚线粒体的呼吸速率逐渐降低，并且对脱水的敏感性也逐渐下降。脱水会降低脱水敏感性种胚线粒体的结构完整性；脱水同时会降低线粒体功能的完整性，包括线粒体能量产生的速率和效率，以及三羧酸循环关键酶的活性。但当种胚获得脱水耐性后，脱水将不再影响种胚线粒体结构和功能的完整性。 玉米种胚发育过程中脱水耐性的变化与细胞中的抗氧化系统有关。在细胞匀浆水平上，脱水过程中脂质过氧化产物的积累与细胞脱水耐性的关系不明显；但是在线粒体水平上脱水会明显导致脱水敏感性种胚线粒体膜质过氧化程度的升高。脱水导致脱水敏感种胚细胞中几个重要的抗氧化酶活性的下降，但是与细胞匀浆水平相比，在线粒体水平上抗氧化酶系统对脱水更加敏感。 总之，发育早期玉米胚对脱水之所以敏感有两方面的原因，一方面是发育早期线粒体具有较高的代谢速率因而产生过多的活性氧，另一方面是由于脱水导致各抗氧化酶活性的显著降低，失去了抗氧化保护功能。而在发育晚期，早期本来很活跃的许多代谢随之关闭，呼吸速率降到很低，因而产生的活性氧减少，同时由于抗氧化系统对脱水的耐受性，所以脱水不会对线粒体的结构和功能造成伤害。与细胞匀浆水平相比，线粒体水平上抗氧化系统的运转与种胚在发育过程中脱水耐性的获得的关系更加密切。

Ultraviolet-B exposure induces photo-oxidative damage and subsequent repair strategies in a desert cyanobacterium Microcoleus vaginatus Gom.

Biological soil crusts are important in reversing desertification. Ultraviolet radiation, however, may be detrimental for the development of soil crusts. The cyanobacterium Microcoleus vaginatus can be a dominant species occurring in desert soil crusts all over the world. To investigate the physico-chemical consequences of ultraviolet-B radiation on M. vaginatus, eight parameters including the contents of chlorophyll a, reactive oxygen species, malondialdehyde and proline, as well as the activities of photosynthesis, superoxide dismutase (EC 1.15.1.1), peroxiclase (EC 1.11.1.7) and catalase (EC 1.11.1.6) were determined. As shown by the results of determinations, ultraviolet-B radiation caused decreases both in contents of chlorophyll a and in ratios of variable fluorescence over maximum fluorescence that indicate the growth and photosynthesis of M. vaginatus, besides, increases both in levels of reactive oxygen species and in contents of malondialdehyde and proline, while intensified activities of superoxide dismutase, peroxiclase and catalase reflecting the abilities of enzymatic preventive substances to oxidative stress of the treated cells. Therefore, ultraviolet-B radiation affects the growth of M. vaginatus and leads to oxidative stress in cells. Under ultraviolet-B radiation, the treated cells can improve their antioxidant abilities to alleviate oxidative injury. The change trends of reactive oxygen species, superoxide dismutase, peroxiclase and catalase are synchronous. These results suggest that a balance between the antioxidant system and the reactive oxygen species content may be one part of a complex stress response pathway in which multiple environmental factors including ultraviolet-B radiation affect the Survival of M. vaginatus. (C) 2009 Elsevier Masson SAS. All rights reserved.

Trends of Superoxide Dismutase and Soluble Protein of Aquatic Plants in Lakes of Different Trophic Levels in the Middle and Lower Reaches of the Yangtze River, China

A limnological study was carried out to determine the responses of superoxide dismutase (SOD) activities and soluble protein (SP) contents of 11 common aquatic plants to eutrophication stress. Field investigation in 12 lakes in the middle and lower reaches of the Yangtze River was carried out from March to September 2004. Our results indicated that non-submersed (emergent and floating-leafed) plants and submersed plants showed different responses to eutrophication stress. Both SOD activities of the non-submersed and submersed plants were negatively correlated with their SP contents (P < 0.000 1). SP contents of non-submersed plants were significantly correlated with all nitrogen variables in the water (P < 0.05), whereas SP contents of submersed plants were only significantly correlated with carbon variables as well as ammonium and Secchi depth (SD) in water (P < 0.05). Only SOD activities of submersed plants were decreased with decline of SD in water (P < 0.001). Our results indicate that the decline of SOD activities of submersed plants were mainly caused by light limitation, this showed a coincidence with the decline of macrophytes in eutrophic lakes, which might imply that the antioxidant system of the submersed plants were impaired under eutrophication stress.

Molecular cloning and characterization of the copper/zinc and manganese superoxide dismutase genes from the human parasite Clonorchis sinensis

A copper/zinc superoxide dismutase (Cu/ZnSOD) gene and a manganese superoxide dismutase (MnSOD) gene of the human parasite Clonorchis sinensis have been cloned and their gene products functionally characterized. Genes Cu/ZnSOD and MnSOD encode proteins of 16 kDa and 25.4 kDa, respectively. The deduced amino acid sequences of the two genes contained highly conserved residues required for activity and secondary structure formation of Cu/ZnSOD and MnSOD, respectively, and show up to 73.7% and 75.4% identities with their counterparts in other animals. The genomic DNA sequence analysis of Cu/ZnSOD gene revealed this as an intronless gene. Inhibitor studies with purified recombinant Cu/ ZnSOD and MnSOD, both of which were functionally expressed in Escherichia coli, confirmed that they are copper/zinc and manganese-containing SOD, respectively. Immunoblots showed that both C. sinensis Cu/ZnSOD and MnSOD should be antigenic for humans, and both, especially the C. sinensis MnSOD, exhibit extensive cross-reactions with sera of patients infected by other trematodes or cestodes. RT-PCR and SOD activity staining of parasite lysates indicate that there are no significant differences in mRNA level or SOD activity for both species of SOD, indicating cytosolic Cu/ZnSOD and MnSOD might play a comparatively important role in the C. sinensis antioxidant system.

苜蓿幼苗芽、根器官对盐胁迫的生理生化响应

探讨盐胁迫对苜蓿幼苗不同器官生长及抗氧化酶系统的影响,为苜蓿植被恢复技术的研发提供理论参考。【方法】以新牧一号(盐忍耐品种)和北极星(盐敏感品种)2个苜蓿品种为材料,以0mmol/L NaCl处理为对照,用200mmol/L NaCl胁迫处理,测定萌发7d苜蓿幼苗芽、根生长及其H2O2、丙二醛(MDA)含量,相对质膜透性、抗氧化保护酶活性及其同功酶的变化。【结果】200mmol/L NaCl胁迫抑制了苜蓿幼苗的生长,并导致芽、根H2O2、MDA含量及相对质膜透性升高,但新牧一号芽、根器官均表现出比北极星较低程度的生长抑制或质膜损伤。盐胁迫后超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)、过氧化氢酶(CAT)、过氧化物酶(POD)的活性均明显增加。CAT和POD对活性氧的清除具有器官差异性,CAT活性在芽中较强,POD则与之相反。SOD-Ⅱ、APX-Ⅰ、POD-Ⅲ及POD-Ⅳ为芽、根中盐胁迫敏感的同功酶谱带。【结论】苜蓿盐忍耐品种通过更强的抗氧化保护能力,降低了盐胁迫对其幼苗的损伤。

水分亏缺和凋落物水浸液对云杉幼苗生长和种子萌发的影响

在人类活动导致全球变暖的前提下，由于全球气温的升高，地表水分加速向空中蒸发。从20世纪70年代至今，地球上严重干旱地区的面积几乎扩大了一倍。这一增长的一半可归因于气温升高而不是降雨量下降，因为实际上同期全球平均降水量还略有增长。干旱对陆地植物和农林生态系统产生深远影响，并已成为全球变化研究的一个重要方面。位于青藏高原东部的川西亚高山针叶林是研究气候变暖对陆地生态系统影响的重要森林类型。森林采伐迹地、人工林下和林窗环境作为目前该区人工造林和森林更新的重要生境，其截然不同的光环境对亚高山针叶林更新和森林动态有非常重要的影响。凋落物产生的化感物质可通过影响种子萌发和早期幼苗的定居而影响种群的建立和更新，而人工林和自然林物种以及更新速度的差异性也都受凋落物的影响。 云杉是川西亚高山针叶林群落的重要树种之一,在维持亚高山森林的景观格局和区域生态安全方面具有十分重要的作用，其自然更新能力及其影响机制一直是研究的热点问题。本试验以云杉种子和2年生幼苗为研究对象，从萌发、根尖形态、幼苗生长、光合作用、渗透调节和抗氧化能力等方面研究了不同光环境下水分亏缺和凋落物水浸液对云杉种子和幼苗生长的影响。旨在从更新的角度探讨亚高山针叶林自然更新的过程，其研究成果可在一定程度上为川西亚高山针叶林更新提供科学依据，同时也可为林业生产管理提供科学指导。主要研究结论如下： 水分亏缺在生长形态、光合作用、抗氧化能力、活性氧化对云杉幼苗都有显著影响。总体表现为，水分亏缺导致了云杉幼苗的高度、地径、单株总生物量降低，增加了地下部分的生长；水分亏缺显著降低了云杉叶片中相对含水量、光合色素、叶氮含量，净光合速率和最大量子产量(Fv/Fm)，提高了幼苗叶片中膜脂过氧化产物（MDA）的含量；水分亏缺提高了幼苗叶片中过氧化氢(H2O2)含量，超氧荫离子（O2-）生成速率以及脯氨酸和抗氧化系统的活性（ASA, SOD, CAT, POD, APX和GR）。从这些结果可知，植物在遭受水分亏缺导致的伤害时，其自身会形成防御策略，并通过改变形态和生理方面的特性以减轻害。但是，这种自我保护机制依然不能抵抗严重水分亏缺对植物的伤害。 模拟林下低光照条件显著增加单株植物的地上部分生长，尤其是其叶片的比叶面积（叶面积/叶干重），同时其光合色素含量和叶片相对含水量也显著增加，这些改变直接导致植株光合速率和生物量的增加。同时，与高光照水平相比，低光照幼苗的膜脂过氧化产物（MDA）和活性氧物质均较低，显示出低光照比高光照水平对植物的更低的氧化伤害。尽管低光照也导致大部分抗氧化酶活性降低，但这正显示出植物遭受低的氧化伤害，更印证了前面的结论。 凋落物水浸液影响了云杉种子的萌发和根系的生长，更在形态、光合作用、抗氧化能力、活性氧物质以及叶氮水平上显著影响了云杉幼苗，其中，以人工纯林凋落物的影响更有强烈。具体表现在，种子萌发速率和萌发种子幼根的长度表现为对照>自然林处理>人工纯林；凋落物水浸液抑制种子分生区和伸长区的生长，人工林处理更降低了根毛区的生长，使根吸水分和养分困难。对2年生幼苗的影响主要表现在叶绿素含量、光合速率以及叶氮含量的降低；膜脂过氧化产物、活性氧物质和抗氧化酶系统的显著增加。同样的，人工纯林处理对云杉幼苗的影响显著于自然林处理。 在自然生态系统中，由于全球变暖气温升高导致的水分亏缺和森林凋落物都存在森林的砍伐迹地，林窗和林下环境中。我们的研究表明，与迹地或林窗强光照比较，林下的低光照环境由于为植物的生长营造了较为湿润的微环境，因此水分亏缺在林下对云杉幼苗造成的影响微弱。这可以从植物的形态、光合速率以及生物量积累，过氧化伤害和抗氧化酶系统表现出来。另一方面，凋落物水浸液在模拟林下低光照环境对植物的伤害也微弱于强光照环境，这与强光照环境高的水分散失导致环境水分亏缺有关；而人工纯林处理对云杉幼苗的伤害比对照和自然林处理显示出强烈的抑制作用。 Under the pre-condition of global warming resulted from intensive human activities, water in the earth’s surface rapidly evaporates due to the increase of global air temperature. From 1970s up to now, the area of serious drought in the world is almost twice as ever. This increase might be due to the increasing air temperature and not decreasing rainfall because global average rainfall in the corresponding period slightly is incremental. Drought will have profound impacts on terrestrial and agriculture-forest system and has also become the important issue of global change research. The subalpine coniferous forests in the eastern Qinghai-Tibet Plateau provide a natural laboratory for the studying the effects of global warming on terrestrial ecosystems. The light environment significantly differs among cutting blanks, forest gap and understory, which is particularly important for plant regeneration and forest dynamics in the subalpine coniferous forests. Picea asperata is one of the keystone species of subalpine coniferouis forests in western China, and it is very important in preserving landscape structure and regional ecological security of subalpine forests. The natural regeneration capacities and influence mechanism of Picea asperata are always the hot topics. In the present study, the short-term effects of two light levels (100% of full sunlight and 15% of full sunlight), two watering regimes (100% of field capacity and 30% of field capacity), two litter aqueous extracts (primitive forest and plantation aqueous extracts) on the seed germination, early growth and physiological traits of Picea asperata were determined in the laboratory and natural greenhouse. The present study was undertaken so as to give a better understanding of the regeneration progress affected by water deficit, low light and litter aqueous extracts. Our results could provide insights into the effects of climate warming on community composition and regeneration behavior for the subalpine coniferous forest ecosystem processes, and provide scientific direction for the forest production and management. Water deficit had significant effects on growth, morphological, physiological and biochemical traits of Picea asperata seedlings. Water deficit resulted in the decrease in height, basal diameter, total biomass and increase in under-ground development; water deficit significantly reduced the needle relative water content, photosynthetic pigments, needle nitrogen concentration, net photosynthetic rate and the maximum potential quantum yield of photosynthesis (Fv/Fm), and increased the degree of lipid peroxidation (MDA) in Picea asperata seedlings; water deficit also increased the rate of superoxide radical (O2-) production, hydrogen peroxide (H2O2) content, free proline content and the activities of antioxidant systems (ASA, SOD, POD, CAT, APX and GR) in Picea asperata seedlings. These results indicated that some protective mechanism was formed when plants suffered from drought stress, but the protection could not counteract the harm resulting from the serious drought stress on them. Low light in the understory significantly increased seedling above-ground development, especially the species leaf area (SLA), and photosynthetic pigments and relative needle content. These changes resulted in the increase in net photosynthetic rate and total biomass. Moreover, the lower MDA content and active oxygen species (AOS) (H2O2 and O2-) in low light seedlings suggested that low light had weaker oxidative damage as compared to high light. Lower antioxidant enzymes activities in low light seedlings indicated the weaker oxidative damage on Picea asperata seedlings than high light seedlings, which was correlative with the changes in MDA and AOS. Litter aqueous extracts affected seed germination and root system of Picea asperata seedlings. Significant changes in growth, photosynthesis, antioxidant activities, active oxygen species and leaf nitrogen concentration were also found in Picea asperata seedlings, and plantation treatment showed the stronger effects on these traits than those in control and primitive forest treatment. The present results indicated that seed germination and radicle length parameters in control were superior to those in primitive forest treatment, and those of primitive forest treatment were superior to plantation treatment; litter aqueous extracts inhibited the meristematic and elongation zone, and plantation treatment caused a decrease in root hairs so as to be difficult in absorbing water and nutrient in root system. On the other hand, litter aqueous extracts significantly decreased chlorophyll content, net photosynthetic rate and leaf nitrogen concentration of Picea asperata seedlings; MDA, AOS and antioxidant system activities were significantly increased in Picea asperata seedlings. Similarly, plantation treatment had more significant effect on Picea asperata seedlings as compared to primitive forest treatment. In the nature ecosystem, water deficit resulted from elevating air temperature and litter aqueous extract may probably coexist in the cutting blank, forest gap and understory. Our present study showed that water deficit had weaker effects on low light seedlings in the understory as compared to high light seedlings in the cutting blank and forest gap. The fact was confirmed from seedlings growth, gas exchange and biomass accumulation, peroxidation and antioxidant systems. This might be due to that low light-reduced leaf and air temperatures, vapour-pressure deficit, and the oxidative stresses can aggravate the impact of drought under higher light. On the other hand, litter aqueous extracts in the low light had weaker effects on the Picea asperata seedlings than those at high light level, which might be correlative to the water evapotranspiration under high light. Moreover, plantation litter aqueous extracts showed stronger inhibition for seed germination and seedling growth than control and primitive forest treatments.

增强UV-B辐射对粗枝云杉(Picea asperata Mast.)和青杨(Populus cathayana Rehd.)的影响

光是植物赖以生存的重要环境因子，但是植物在获得光的同时不可避免的会受到紫外辐射的伤害。尤其是近年来，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。而另一方面，植物对UV-B辐射反应的敏感性在种间和品种间存在差异，主要受植物基因型，生态型和生活型的控制。本项目分别以粗枝云杉和青杨组杨树为模式植物，从形态和生理生化方面分别研究了来自不同水分背景下的粗枝云杉种群和来自不同UV-B背景下的青杨种群在增强UV-B下的反应及其反应差异,并探讨了干旱、喷施外源脱落酸(ABA)对它们抗UV-B能力的影响。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。主要研究结果如下： 1. 粗枝云杉的两个种群，湿润种群(来自四川黑水)和干旱种群(来自甘肃迭部)在水分良好和干旱状况下表现出对增强UV-B的不同响应。同时，干旱对粗枝云杉抗UV-B能力的影响也得到研究：两种胁迫共同作用时，干旱表现出在一定程度上减弱了增强UV-B对粗枝云杉的生理特性的影响。 干旱胁迫显著降低了两个粗枝云杉种群的光合同化速率(A), 气孔导度(gs)和PSII的有效光量子产量(Y), 同时，提高了非光化学猝灭效率(qN)和超氧化物歧化酶(SOD)的活性。与湿润种群相比，干旱种群抗旱性更强，表现为干旱种群拥有更高的SOD和干旱进一步加剧了UV-B的胁迫效应。 本研究中，干旱胁迫单独作用时，显著降低了青杨两个种群的生物量积累和气体交换，具体包括A、gs、蒸腾速率(E)和光合氮利用效率(PNUE)，提高了两个种群的瞬时水分利用效率(WUEi)、长期水分利用效率(WUET)、碳同位素组分(δ13C)和氮含量(N)。同时，UV吸收物质和ABA含量也得到积累。另一方面，增强UV-B对青杨两个种群各个指标的影响，同干旱所引起的效应有着相似的趋势。同低海拔种群相比，高海拔种群有着更强的抗旱和抗UV-B能力，具体表现在高海拔种群有着更多的生物量积累，更强的气体交换和水分利用效率及更高水平的ABA和UV吸收物质含量。相比干旱诱导的生物量积累和气体交换的降低，在干旱和增强UV-B两个胁迫同时作用于青杨时，这种降低表现的更为明显。显著的干旱和UV-B的交互作用还表现在WUEi, WUET, δ13C, 可溶性蛋白含量, UV吸收物质含量, ABA, 叶片和茎中的N含量以及C/N比中。 3. 经过一个生长季的试验观察，增强UV-B、外源ABA及两因子共同作用对青杨的生物量积累、气体交换、内源ABA和UV吸收物质含量、抗氧化系统以及碳、氮含量和碳/氮比均产生显著影响。本试验中，青杨的两个种群分别来自中国西南部的不同海拔地区，高海拔种群来自青海大通而低海拔种群来自四川九寨。外源ABA的胁迫为直接喷施ABA到青杨叶片，而增强UV-B胁迫是利用平方波系统分别保证青杨苗暴露于外界UV-B强度和两倍于外界UV-B强度下。 研究结果显示，增强UV-B显著的降低了两个青杨种群的株高、基茎、总叶面积和总生物量等生长指标，同时也导致其A、gs、E和叶片中碳含量的减少。而显著增加了SOD和过氧化物酶(GPx)活性水平，诱导了过氧化氢(H2O2)和MDA的显著增加，促进了UV吸收物质和不同器官中内源ABA含量的显著积累。另一方面，外源ABA引起了青杨光合同化速率的下降，SOD和GPx酶活性的增强，H2O2 和 MDA含量也表现出显著增加，同时，内源ABA含量得到显著累积。同低海拔种群相比，高海拔种群具有更加抗UV-B和外源ABA的特性。显著的UV-B和ABA的交互作用表现在A, E, SOD和GPx活性，以及叶片和根部的内源ABA等一系列指标中。在所有胁迫下，叶片中的碳和氮含量同其在茎和根中的含量显著相关，另外，叶片和茎中的氮含量同茎中的碳含量显著相关。 Sunlight is an indispensable environment factor for plants survival and development. Meanwhile, photosynthetic organisms need sunlight and are thus, inevitably, exposed to UV radiation. Especially for recent years, 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. On the other hand, the sensitivity of plants to UV-B radiation depends on the species, developmental stage and experimental conditions. In this experiment, two populations of Picea asperata Mast from different water background and two populations of Populus cathayana Rehder from different altitude background were selected as model plants to assess the effects of enhanced UV-B radiation. Morphological and physiological traits induced by enhanced UV-B in each plant species were observed and the different responses were discussed, furthermore the influences of drought and exogenous ABA on responses induced by enhanced UV-B were studied. The study could provide a strong theoretical evidence and scientific direction for the afforestation and rehabilitation of ecosystem. The results are as follows: 1. Different responses of two contrasting Picea asperata Mast. populations to enhanced ultraviolet-B (UV-B) radiation under well-watered and drought conditions were investigated. And the effects of enhanced UV-B on tolerance of drought were also observed in our study that the UV-B exposure may have alleviated some of the damage induced by drought. Two contrasting populations, originating from a wet and dry climate region in China, respectively, were employed in our study. Drought significantly decreased CO2 assimilation rate (A), stomatal conductance (gs) and effective PSII quantum yield (Y), while it significantly increased non-photochemical quenching (qN) and the activity of superoxide dismutase (SOD) in both populations. Compared with the wet climate population, the dry climate population was more acclimated to drought stress and showed much higher activities of SOD and ascorbate peroxidase (APX), and much lower levels of malondialdehyde (MDA) and electrolyte leakage. On the other hand, enhanced UV-B radiation also induced a significant decrease in the chlorophyll (Chl) content in both populations under well-watered conditions, and a significant increase in UV-absorbing compounds in the wet climate population. After one growing season of exposure to different UV-B levels and watering regimes, the increases in MDA and electrolyte leakage, as induced by drought, were less pronounced under the combination of UV-B and drought. In addition, an additive effect of drought and UV-B on A and gs was observed in the wet climate population, and on the activity of APX and qN in the dry climate population. 2. The significant effects of drought, enhanced UV-B radiation and their combination on Populus cathayana Rehd. growth and physiological traits were investigated in two populations, originating from high and low altitudes in south-west China. Our results showed that UV-B acts as an important signal allowing P. cathayana seedlings to respond to drought and that the combination of drought and UV-B may cause synergistically detrimental effects on plant growth in both populations. In both populations, drought significantly decreased biomass accumulation and gas exchange parameters, including A, gs, E and photosynthetic nitrogen use efficiency (PNUE). However, instantaneous water use efficiency (WUEi), transpiration efficiency (WUET), carbon isotope composition (δ13C) and nitrogen (N) content, as well as the accumulation of soluble protein, UV-absorbing compounds and abscisic acid (ABA) were significantly increased by drought. On the other hand, cuttings from both populations, when kept under enhanced UV-B radiation conditions, showed very similar changes in all above-mentioned parameters, as induced by drought. Compared with the low altitude population, the high altitude population was more tolerant to drought and enhanced UV-B, as indicated by the higher level of biomass accumulation, gas exchange, water-use efficiency, ABA concentration and UV-absorbing compounds. After one growing season of exposure to different UV-B levels and watering regimes, the decrease in biomass accumulation and gas exchange, induced by drought, was more pronounced under the combination of UV-B and drought. Significant interactions between drought and UV-B were observed in WUEi, WUET, δ13C, soluble protein, UV-absorbing compounds, ABA and in the leaf and stem N, as well as in the leaf and stem C/N ratio. 3. During one growing season, significant effects induced by enhanced UV-B radiation, exogenous ABA and their combination on biomass accumulation, gas exchange, endogenous ABA and UV-absorbing compounds concentrations, antioxidant system as well as carbon (C) content, nitrogen (N) content and C/N ratio were investigated in two contrasting Populus cathayana populations, originating from high and low altitudes in south-west China. Exogenous ABA was sprayed to the leaves and enhanced UV-B treatment was using a square-wave system to make the seedlings under ambient (1×) or twice ambient (2×) doses of biologically effective UV-B radiation (UV-BBE). Enhanced UV-B radiation significantly decreased height, basal diameter, total leaf area, total biomass, A, gs, E and carbon (C) content in leaves, and significantly increased activities of SOD and guaiacol peroxidase (GPx), hydrogen peroxide (H2O2) and malonaldehyde (MDA) content as well as the accumulation of UV-absorbing compounds and endogenous ABA concentrations among different organs in both populations. In contrast, exogenous ABA showed significant decrease in A and significant increases in activities of SOD and GPx, H2O2, MDA content and the endogenous ABA concentrations. Compared with the low altitude population, the high altitude population was more tolerant to enhanced UV-B and exogenous ABA. Significant interactions between UV-B and ABA were observed in A, E, activities of SOD and GPx, as well as in endogenous ABA in leaves and roots of both populations. Across all treatments, C and N content in leaves was strongly correlated with those were in stems and roots, respectively. Additionally, leaf and stem N content were significant correlated with stem C content.

青杨(Populus cathayana Rehd)雌雄植株对锈病和干旱胁迫的生理响应及差异蛋白质组比较

雌雄异株植物对环境的不同响应一直是一个有趣而新颖的研究领域，由于雌雄个体不同的繁殖成本及不同的生存策略，使得雌雄植株在生长、存活、生殖格局、空间分布、资源配置等方面已经表现出明显的不同，在生理和分子水平上也表现出明显的性别间差异。干旱是制约农林业发展的环境因子之一，叶锈病是对杨树危害最严重的病害之一，由于长期进化的结果，不同性别的植物必然对生物和非生物胁迫有着不同的响应。本文以雌雄异株的青杨为模式植物，研究雌雄间在生理、生化、亚细胞结构和蛋白质水平上对生物和非生物胁迫的差异响应。主要研究结果如下： （1） 青杨雌雄植株对锈病胁迫的生理生化差异响应 在正常的对照组中，雄株叶片比雌株叶片有着较高的活性氧自由基产生速率、较高的SOD、POD、PPO 和较低的CAT 活性；在锈病感染的早期阶段， SOD、POD、CAT 活性、活性氧自由基产生速率、H2O2 含量、膜脂过氧化程度和细胞膜的电渗率在雌雄株中都增加，而PPO 仅在雄株中增加明显，APX 仅在雌株中增加明显，并且雌株比雄株有着更严重的锈病感染程度、细胞膜的伤害程度和光合系统II 的破坏程度，雌株有更多的净光合速率、气孔导度和叶绿素a 含量的降低，在同工酶变化上，雌雄间对锈病也显示出不同的表达模式。结果显示，雄株比雌株对锈病有着更好的抗性和更有效的ROS 清除系统。 （2） 青杨雌雄植株对干旱胁迫的生理生化及亚细胞结构的差异响应 与较好水分条件相比，干旱下雄株比雌株有着更高的A-Ci 响应参数，如Rubisco 最大羧化速率、光呼吸速率、暗呼吸速率和最大电子传递速率等。干旱显著地增加了膜脂过氧化程度和游离脯氨酸含量，并且雄株比雌株表现出较低的膜脂过氧化程度，较高的总蛋白和游离脯氨酸含量。无论是中度干旱还是极度干旱，除了CAT 外，雄株比雌株表现为较强的抗氧化酶活性，在同工酶谱带上，雌雄间表现出不同的变化模式，并且有些条带是干旱影响应的，而有些条带是性别特异性的，这些性别特异性条带能够作为鉴定性别快速而准确的标记。干旱显著地影响了线粒体、叶绿体和细胞壁的结构，尤其在中度干旱胁迫下，雄株线粒体和叶绿体比雌株呈现出较好的完整性，并且雄株细胞壁要比雌株更厚。因此， 雄株比雌株表现出更强的干旱忍耐性和更高效的抗氧化酶系统。 （3） 青杨雌雄植株对干旱胁迫的蛋白质组差异响应 用双相电泳检测到雌雄间近1000 个蛋白点，通过对比发现对照组雌雄间有54 个差异蛋白点，干旱下雌雄间有108 个差异点，其中102 个被质谱成功鉴定。对照组雌雄间的差异蛋白主要集中在与光合作用相关蛋白、抗氧化酶、胁迫防御蛋白和一些调节基因表达的蛋白；干旱胁迫下雌雄间差异蛋白明显增多，主要有参与信号转导、调节基因表达、蛋白质加工、转录产物的转录翻译后修饰的调节性蛋白蛋白和参与氧化还原平衡、抗胁迫、细胞壁合成、光合作用、能量代谢、氨基酸代谢和脂肪酸代谢等的功能性蛋白。干旱下这些蛋白的表达量在雌雄中有的表现出相同的表达模式，如干旱下雌雄株中Rubisco 激活酶、小热激蛋白等表达都增加，而有的表现出相反的表达模式，如Rubisco 大亚基的降解片段、羰酸酯酶等在雄株中表达量上调而在雌株中却是下调。因此，雌雄间在蛋白质水平上对干旱胁迫响应的差异是显著的，也是复杂的。 It is an interesting and novel topic that dioecious plants possess different responses to environmental stress. As for the different productive cost and different survive strategy, different sexual plants have shown obviously morphological, physiological and molecular differences. Drought is one of the most worldwidely important environmental stress factors that limit plant growth and ecosystem productivity. Rust disease is one of the economically important diseases in many trees. As a result of the long evolutionary process, male and female plants should show different responses to abiotic and biotic stress. In this paper, using a dioeious tree of Populus cathayana Rehd as a model, we study the sexual differences to drought and rust disease stress in physiological, biochemical, sub-cellular and proteomics levels. The main results are follows: (1) The sexual differences in physiology and biochemistry of poplar to rust disease In controls, males showed higher production of superoxide radicals, higher activities of SOD, POD, PPO and lower CAT activity. Under rust disease, the activities of antioxidant, the content of ROS and the degree of cellular member destroyed were increased in both sexes, except for PPO in diseased males and APX in diseased females. However, females showed more seriously disease severity and cellular member and PS II destroyed degrees. Net photosynthesis rate, transpiration rate and chlorophyll a content were decreased more in diseased females than in males. There were also some different changes inantioxidant isozymes under rust disease. The results suggested that male poplar possessed a more effectively antioxidant system and were more resistant to rut disease than females. (2) The sexual differences in physiology and biochemistry of poplar to drought stress Under drought stress, there were higher rates of RuBP-saturated CO2 assimilation, dark respiration, photorespiratory release of oxygen, the max electron transportrate in CO2-saturated and carboxylation efficiency in males than in females. And males showed lower TBARS and higher proline content. Except for CAT, the activities of other antioxidants were higher in males than in females. Meanwhile, there were obviously differences in isozyme changes between teo sexes. Drought stress obviously destroyed the integralities of chloroplasts and mitochondria and the sexual differences in sub-cellular level were obviously under the moderate water stress. Male cell walls were more sensitive to drought stress than did female. The results suggested males were more resistant to drought stress. (3) The sexual differences in proteomics of poplar to drought stress By 2-D and MS analysis, we identified 102 different protein spots between males and females. Under control conditions, the different proteins were mainly in photosynthesis related proteins, antioxidants, stress response proteins and some gene expression related proteins. Under drought stress, the different proteins were focused on (i) regulated proteins such as signaling conduction, kinase, HSP, gene expressional regulation and protein modification, (ii) functional proteins such as photosynthesis, energy metabolism, antioxidant, redox, stress response, lipid metabolism and amino acid metabolism. Some protein showed the same expressional pattern, while some showed contrary expressional pattern. Thus, the results suggested that sexual differences in proteomics were significant and complex.

Response of integrated biomarkers of fish (Lateolabrax japonicus) exposed to benzo[a]pyrene and sodium dodecylbenzene sulfonate

Fish Lateolabrax japonicus were exposed to 0.1 and 1 mg/L of anion surfactant sodium dodecylbenzene sulfonate (SDBS) and to 2 and 20 mu g/L of benzo[a]pyrene (B[a]P) for 6, 12, and 18 days, with control and solvent control groups. Liver antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), reduced glutathione (GSH), and glutathione S-transferase (GST), were determined; brain acetyleholinesterase (AChE) and liver inducible nitric oxide synthase (iNOS) activities were also measured. The results indicated that (1) L. japonicus avoided oxidative damage through antioxidant systems; (2) SOD, GPx, and GSH were induced, and GST was inhibited and then induced by B[a]P exposure; and (3) CAT, GPx, and AChE were induced while NOS was inhibited, and GST was induced and then inhibited by SDBS stress in experimental period. (c) 2005 Elsevier Inc. All rights reserved.

Trace elements as predictors of preeclampsia in type 1 diabetic pregnancy

Preeclampsia (PE) affects approximately 5% of all pregnancies, but is increased several-fold in women with pre-gestational type 1 diabetes mellitus (T1DM). Increased oxidative stress and altered maternal plasma trace elements that modulate the antioxidant system have been implicated in PE. In non-diabetic women, increased plasma copper and iron and decreased manganese, selenium, and zinc have been associated with PE in cross-sectional studies. In a longitudinal study, we hypothesized that plasma levels of trace elements differ between T1DM women with vs. without subsequent PE. Samples were collected during the first (gestation 12.2 ± 1.9 weeks, [mean ± SD]), second (21.6 ± 1.5 weeks), and third (31.5 ± 1.7 weeks) trimesters of pregnancy, all before the onset of PE. We compared 23 T1DM women who subsequently developed PE with 24 T1DM women who remained normotensive; and we included 19 non-diabetic (non-DM) normotensive pregnant women as reference controls. Trace elements were measured using inductively coupled plasma mass spectroscopy. In T1DM women with subsequent PE vs normotensive, only plasma zinc was significantly higher at the first trimester, while copper:zinc and copper:high-density lipoprotein cholesterol ratios were higher throughout gestation (all P < .05). These findings persisted after adjustment for covariates. Higher copper:zinc ratios may contribute to oxidative stress in T1DM women who develop PE. Ratios of pro- to anti-oxidant factors may predict risk for PE in diabetic pregnancies more effectively than individual trace element levels.

Toxicity assessment of essential metals in aquatic detritivores

Os contaminantes provenientes quer de fontes naturais quer como consequência da atividade humana, têm contribuído para a degradação dos ecossistemas aquáticos. Entre estes encontram-se os metais que podem, ou não ser essenciais mediante o papel que desempenham no metabolismo dos organismos. O cobre e o zinco são exemplos de metais essenciais, contudo quando atingem concentrações elevadas podem tornar-se tóxicos. Os detritívoros aquáticos desempenham um papel fundamental na decomposição da matéria orgânica, alimentando-se de carcaças e partes de plantas que caem nos cursos de água. Assim, estes organismos permitem que o ciclo dos nutrientes se complete e servem como elo de ligação entre todos os grupos funcionais do ecossistema mantendo o seu equilíbrio estrutural e funcional. Sendo a matéria orgânica a sua principal fonte de energia estão sujeitos à contaminação existente no meio, pelo que é de todo o interesse proceder-se à avaliação dos efeitos da toxicidade de metais nestes organismos. Uma vez que as diferenças comportamentais consequentes desta exposição podem originar variações na densidade e diversidade, o que se refletirá a nível das comunidades, originando alterações na estrutura e funcionamento do ecossistema. Tendo em vista a avaliação dos efeitos da contaminação por metais em detritívoros, o principal objetivo deste trabalho foi comparar a sensibilidade a metais essenciais de dois detritivoros aquáticos, o camarão Atyaephyra desmarestii e o anfípode Echinogammarus meridionalis. Para tal, avaliaram-se os efeitos do cobre e do zinco a diferentes níveis de organização biológica. Primeiro, foram determinadas as preferências alimentares de A. desmarestii e E. meridionalis considerando tanto a área das folhas como a contaminação por metais das folhas. Em seguida, avaliaram-se os efeitos do cobre e do zinco na sobrevivência e inibição alimentar de ambas as espécies. Finalmente, avaliaram-se os efeitos destes mesmos metais a nível bioquímico utilizando uma bateria de biomarcadores que incluiu enzimas de stresse oxidativo, o sistema de defesa antioxidante e as colinesterases. Ambos os organismos não mostraram preferência em relação a folhas de área diferente. A presença de uma maior ou menor concentração de metais essenciais no alimento não teve qualquer influência na sua escolha pelo alimento (contaminado ou não). Os ensaios agudos de cobre e zinco mostraram que o cobre é mais tóxico para ambas as espécies do que o zinco. O camarão demonstrou ser mais sensível ao zinco que o anfípode, tendo este sido mais sensível ao cobre ( CL50 do cobre para A. desmarestii foi de 0,128 mg.l-1 e o de E. meridionalis foi de 0,050 mg.l-1; os valores correspondentes para o zinco foram 7,951 e 11,860 mg.l-1, respectivamente. Em relação aos efeitos subletais, o cobre teve efeitos notórios na taxa de alimentação de E. meridionalis, mas não afectou a de A. desmarestii. No que diz respeito à exposição ao zinco, ambas as espécies parecem apresentar tendência para inibir a alimentação. A caracterização das colinesterases revelou que a principal forma presente em ambas as espécies é a acetilcolinesterase, a qual que não foi afetada pela presença dos metais, no caso do camarão, mas parece ser inibida pelo zinco no caso do anfípode. O cobre inibiu o sistema de defesa enzimático de ambas as espécies, sem sinais de danos lipídicos. Para além disto, inibiu uma das enzimas antioxidantes (GPx) do anfípode. Apesar de não ter ocorrido dano lipídico após exposição ao cobre, observou-se um ligeiro aumento dos níveis das LPO, o que pode ser indicativo de uma potencial existência de dano oxidativo, como resultado da falha do sistema de defesa antioxidante. Por outro lado, o zinco induziu o sistema de defesa em E. meriodionalis prevenindo o dano lipídico. Enquanto em A. desmarestii o sistema enzimático antioxidante não respondeu, tendo ocorrido dano celular oxidativo considerando-se, assim, que o sistema de defesa antioxidante do camarão pode ser comprometido por exposição a metais. Ainda que os danos celulares oxidativos tivessem ocorrido a baixas concentrações de zinco. A exposição a este metal também induziu a actividade da GST de E. meriodionalis. Considerando que a taxa de alimentação foi severamente reduzida no caso deste organismo, o zinco parece ser o metal cuja concentração no ecossistema requer maior atenção. Integrando as respostas dos biomarcadores parece também evidente que A. desmarestii responde de uma maneira geral a maiores concentrações dos dois metais, enquanto a resposta de E. meridionalis ocorre a concentrações inferiores. Pelo que, E. meridionalis parece ser mais sensível ao nível bioquímico. Neste trabalho, os dois detritívoros, com ligeiras diferenças no modo como utilizam a matéria orgânica disponível, apresentam diferenças na sensibilidade aos metais essenciais a vários níveis de organização biológica, sendo o zinco o metal que poderá causar maior preocupação a nível bioquímico, enquanto o cobre parece ser o mais tóxico ao nível do organismo, causando mortalidade a concentrações mais baixas.

Genotoxic risk of herbicides to Anguilla anguilla L.

No contexto dos contaminantes aquáticos, os herbicidas são considerados como um dos grupos mais perigosos. Uma vez aplicados, estes são facilmente transportados para cursos de água, quer devido a uma pulverização pouco cuidada ou devido a fenómenos de escorrência superficial e/ou subterrânea. A presença destes agroquímicos no ambiente tem vindo a ser associada a efeitos nefastos em organismos não-alvo, como é o caso dos peixes. Contudo, existe ainda uma grande lacuna no que diz respeito à informação científica relacionada com o seu impacto genotóxico. Deste modo, a presente tese foi delineada com o intuito de avaliar o risco genotóxico em peixes de duas formulações de herbicidas: o Roundup®, que tem como princípio activo o glifosato, e o Garlon®, que apresenta o triclopir na base da sua constituição, produtos estes largamente utilizados na limpeza de campos agrícolas, assim como em florestas. Foi ainda planeado desenvolver uma base de conhecimento no que diz respeito aos mecanismos de dano do ADN. Como último objectivo, pretendeu-se contribuir para a mitigação dos efeitos dos agroquímicos no biota aquático, nomeadamente em peixes, fornecendo dados científicos no sentido de melhorar as práticas agrícolas e florestais. Este estudo foi realizado adoptando a enguia europeia (Anguilla anguilla L.) como organismo-teste, e submetendo-a a exposições de curta duração (1 e 3 dias) dos produtos comerciais mencionados, em concentrações consideradas ambientalmente realistas. Para a avaliação da genotoxicidade foram aplicadas duas metodologias: o ensaio do cometa e o teste das anomalias nucleares eritrocíticas (ANE). Enquanto o ensaio do cometa detecta quebras na cadeia do ADN, um dano passível de ser reparado, o aparecimento das ANE revela lesões cromossomais, sinalizando um tipo de dano de difícil reparação. O ensaio do cometa foi ainda melhorado com uma nova etapa que incluiu a incubação com enzimas de reparação (FPG e EndoIII), permitindo perceber a ocorrência de dano oxidativo no ADN. No que diz respeito ao Roundup®, o envolvimento do sistema antioxidante como indicador de um estado próoxidante foi também alvo de estudo. Uma vez que as referidas formulações se apresentam sob a forma de misturas, o potencial genotóxico dos seus princípios activos foi também avaliado individualmente. No caso particular do Roundup®, também foram estudados o seu surfactante (amina polietoxilada; POEA) e o principal metabolito ambiental (ácido aminometilfosfórico; AMPA). Os resultados obtidos mostraram a capacidade do Roundup® em induzir tanto dano no ADN (em células de sangue, guelras e fígado) como dano cromossómico (em células de sangue). A investigação sobre o possível envolvimento do stresse oxidativo demonstrou que o tipo de dano no ADN varia com as concentrações testadas e com a duração da exposição. Deste modo, com o aumento do tempo de exposição, os processos relacionados com o envolvimento de espécies reactivas de oxigénio (ERO) ganharam preponderância como mecanismo de dano no ADN, facto que é corroborado pela activação do sistema antioxidante observado nas guelras, assim como pelo aumento dos sítios sensíveis a FPG em hepatócitos. O glifosato e o POEA foram também considerados genotóxicos. O POEA mostrou induzir uma maior extensão de dano no ADN, tanto comparado com o glifosato como com a mistura comercial. Apesar de ambos os componentes contribuirem para a genotoxicidade da formulação, a soma dos seus efeitos individuais nunca foi observada, apontando para um antagonismo entre eles e indicando que o POEA não aumenta o risco associado ao princípio activo. Deste modo, realça-se a necessidade de regulamentar limiares de segurança para todos os componentes da formulação, recomendando, em particular, a revisão da classificação do risco do POEA (actualmente classificado com “inerte”). Uma vez confirmada a capacidade do principal metabolito do glifosato – AMPA – em exercer dano no ADN assim como dano cromossómico, os produtos da degradação ambiental dos princípios activos assumem-se como um problema silencioso, realçando assim a importância de incluir o AMPA na avaliação do risco relacionado com herbicidas com base no glifosato. A formulação Garlon® e o seu princípio activo triclopir mostraram um claro potencial genotóxico. Adicionalmente, o Garlon® mostrou possuir um potencial genotóxico mais elevado do que o seu princípio activo. No entanto, a capacidade de infligir dano oxidativo no ADN não foi demonstrada para nenhum dos agentes. No que concerne à avaliação da progressão do dano após a remoção da fonte de contaminação, nem os peixes expostos a Roundup® nem os expostos a Garlon® conseguiram restaurar completamente a integridade do seu ADN ao fim de 14 dias. No que concerne ao Roundup®, o uso de enzimas de reparação de lesões específicas do ADN associado ao teste do cometa permitiu detectar um aparecimento tardio de dano oxidativo, indicando deste modo um decaimento progressivo da protecção antioxidante e ainda uma incapacidade de reparar este tipo de dano. O período de pós-exposição correspondente ao Garlon® revelou uma tendência de diminuição dos níveis de dano, apesar de nunca se observar uma completa recuperação. Ainda assim, foi evidente uma intervenção eficiente das enzimas de reparação do ADN, mais concretamente as direccionadas às purinas oxidadas. A avaliação das metodologias adoptadas tornou evidente que o procedimento base do ensaio do cometa, que detecta apenas o dano nãoespecífico no ADN, possui algumas limitações quando comparado com a metodologia que incluiu a incubação com as enzimas de reparação, uma vez que a última mostrou reduzir a possibilidade de ocorrência de resultados falsos negativos. Os dois parâmetros adoptados (ensaio do cometa e teste das ANE) demonstraram possuir aptidões complementares, sendo assim recomendado a sua utilização conjunta com vista a efectuar uma avaliação mais adequada do risco genotóxico. Globalmente, os resultados obtidos forneceram indicações de grande utilidade para as entidades reguladoras, contribuindo ainda para a (re)formulação de medidas de conservação do ambiente aquático. Neste sentido, os dados obtidos apontam para a importância da avaliação de risco dos herbicidas incluir testes de genotoxicidade. A magnitude de risco detectada para ambas as formulações adverte para a necessidade de adopção de medidas restritivas em relação à sua aplicação na proximidade de cursos de água. Como medidas mitigadoras de impactos ambientais, aponta-se o desenvolvimento de formulações que incorporem adjuvantes selecionados com base na sua baixa toxicidade.

Effect of high CO2 and ocean acidification on photosynthesis and response to oxidative stress in seagrasses

Climate change scenarios comprise significant modifications of the marine realm, notably ocean acidification and temperature increase, both direct consequences of the rising atmospheric CO2 concentration. These changes are likely to impact marine organisms and ecosystems, namely the valuable seagrass-dominated coastal habitats. The main objective of this thesis was to evaluate the photosynthetic and antioxidant responses of seagrasses to climate change, considering CO2, temperature and light as key drivers of these processes. The methodologies used to determine global antioxidant capacity and antioxidant enzymatic activity in seagrasses were optimized for the species Cymodocea nodosa and Posidonia oceanica, revealing identical defence mechanisms to those found in terrestrial plants. The detailed analysis and identification of photosynthetic pigments in Halophila ovalis, H.stipulacea, Zostera noltii, Z marina, Z. capricorni, Cymodocea nodosa and Posidonia oceanica, sampled across different climatic zones and depths, also revealed a similarity with terrestrial plants, both in carotenoid composition and in the pigment-based photoprotection mechanisms. Cymodocea nodosa plants from Ria Formosa were submitted to the combined effect of potentially stressful light and temperature ranges and showed considerable physiological tolerance, due to the combination of changes in the antioxidant system, activation of the VAZ cycle and accumulation of leaf soluble sugars, thus preventing the onset of oxidative stress. Cymodocea nodosa plants living in a naturally acidified environment near submarine volcanic vents in Vulcano Island (Italy) showed to be under oxidative stress despite the enhancement of the antioxidant capacity, phenolics concentration and carotenoids. Posidonia oceanica leaves loaded with epiphytes showed a significant increase in oxidative stress, despite the increase of antioxidant responses and the allocation of energetic resources to these protection mechanisms. Globally, the results show that seagrasses are physiologically able to deal with potentially stressful conditions from different origins, being plastic enough to avoid stress in many situations and to actively promote ulterior defence and repair mechanisms when under effective oxidative stress.

Le diabète maternel influence la morphogenèse rénale et la programmation périnatale

Le diabète maternel est un facteur de risque majeur pour le développement de malformations congénitales. Dans le syndrome de l’embryopathie diabétique, l’exposition prolongée du fœtus à de hautes concentrations ambientes de glucose induit des dommages qui peuvent affecter plusieurs organes, dont les reins. Les malformations rénales sont la cause de près de 40 pourcent des cas d’insuffisance rénale infantile. L’hyperglycémie constitue un environnement utérin adverse qui nuit à la néphrogenèse et peut causer l’agenèse, la dysplasie (aplasie) ou l’hypoplasie rénale. Les mécanismes moléculaires par lesquels les hautes concentrations ambientes de glucose mènent à la dysmorphogenèse et aux malformations demeurent toutefois mal définis. Le diabète maternel prédispose aussi la progéniture au développement d’autres problèmes à l’âge adulte, tels l’hypertension, l’obésité et le diabète de type 2. Ce phénomène appelé ‘programmation périnatale’ a suscité l’intérêt au cours des dernières décennies, mais les mécanismes responsables demeurent mal compris. Mes études doctorales visaient à élucider les mécanismes moléculaires par lesquels le diabète maternel ou un environnement in utero hyperglycémique affecte la néphrogenèse et programme par la suite la progéniture a développer de l’hypertension par des observations in vitro, ex vivo et in vivo. Nous avons utilisé les cellules MK4, des cellules embryonnaires du mésenchyme métanéphrique de souris, pour nos études in vitro et deux lignées de souris transgéniques (Tg) pour nos études ex vivo et in vivo, soient les souris HoxB7-GFP-Tg et Nephrin-CFP-Tg. Les souris HoxB7-GFP-Tg expriment la protéine fluorescente verte (GFP) dans le bourgeon urétérique (UB), sous le contrôle du promoteur HoxB7. Les souris Nephrin-CFP expriment la protéine fluorescente cyan (CFP) dans les glomérules, sous le contrôle du promoteur nephrin spécifique aux podocytes. Nos études in vitro visaient à déterminer si les hautes concentrations de glucose modulent l’expression du gène Pax2 dans les cellules MK4. Les cellules MK4 ont été traitées pendant 24h avec du milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants ou inhibiteurs de p38 MAPK, p44/42 MAPK, PKC et NF-kB. Nos résultats ont démontré que le D-glucose élevé (25mM) augmente la génération des espèces réactives de l’oxygène (ROS) dans les cellules MK4 et induit spécifiquement l’expression du gène Pax2. Des analogues du glucose tels le D-mannitol, L-glucose ou le 2-Deoxy-D-glucose n’induisent pas cette augmentation dans les cellules MK4. La stimulation de l’expression du gène Pax2 par le D-glucose dans les cellules MK4 peut être bloquée par des inhibiteurs des ROS et de NF-kB, mais pas par des inhibiteurs de p38 MAPK, p44/42 MAPK ou PKC. Ces résultats indiquent que la stimulation de l’expression du gène Pax2 par les concentrations élevées de glucose est due, au moins en partie, à la génération des ROS et l’activation de la voie de signalisation NF-kB, et non pas via les voies PKC, p38 MAPK et p44/42 MAPK. Nos études ex vivo s’intéressaient aux effets d’un milieu hyperglycémique sur la morphogenèse de la ramification du bourgeon urétérique (UB). Des explants de reins embryonnaires (E12 à E18) ont été prélevés par micro-dissection de femelles HoxB7-GFP gestantes. Les explants ont ensuite été cultivés dans un milieu contenant soit 5mM D-glucose et 20mM D-mannitol ou 25mM D-glucose et avec ou sans antioxydants, catalase ou inhibiteur de PI3K/AKT pour diverses durées. Nos résultats ont démontré que le D-glucose stimule la ramification du UB de manière spécifique, et ce via l’expression du gène Pax2. Cette augmentation de la ramification et de l’expression du gène Pax2 peut être bloquée par des inhibiteurs des ROS et de PI3K/AKT. Ces études ont démontré que les hautes concentrations de glucose altèrent la morphogenèse de la ramification du UB via l’expression de Pax2. L’effet stimulant du glucose semble s’effectuer via la génération des ROS et l’activation de la voie de signalisation Akt. Nos études in vivo visaient à déterminer le rôle fondamental du diabète maternel sur les défauts de morphogenèse rénale chez la progéniture. Dans notre modèle animal, le diabète maternel est induit par le streptozotocin (STZ) chez des femelles HoxB7-GFP gestantes (E13). Les souriceaux ont été étudiés à différents âges (naissants et âgés de une, deux ou trois semaines). Nous avons examiné leurs morphologie rénale, nombre de néphrons, expression génique et les événements apoptotiques lors de cette étude à court terme. La progéniture des mères diabétiques avait un plus faible poids, taille et poids des reins, et possédait des glomérules plus petits et moins de néphrons par rapport à la progéniture des mères contrôles. La dysmorphogenèse rénale observée est peut-être causée par l’augmentation de l’apoptose des cellules dans la région du glomérule. Nos résultats ont montré que les souriceaux nés de mères diabétiques possèdent plus de podocytes apoptotiques et plus de marquage contre la caspase-3 active dans leurs tubules rénaux que la progéniture des mères contrôles. Les souriceaux des mères diabétiques montrent une augmentation de l’expression des composants du système rénine angiotensine (RAS) intrarénal comme l’angiotensinogène et la rénine, ainsi qu’une augmentation des isoformes p50 et p65 de NF-kB. Ces résultats indiquent que le diabète maternel active le RAS intrarénal et induit l’apoptose des glomérules, menant à une altération de la morphogenèse rénale de la progéniture. En conclusion, nos études ont permis de démontrer que le glucose élevé ou l’environnement in utero diabétique altère la morphogenèse du UB, qui résulte en un retard dans la néphrogenèse et produit des reins plus petits. Cet effet est dû, au moins en partie, à la génération des ROS, à l’activation du RAS intrarénal et à la voie NF-kB. Nos études futures se concentreront sur les mécanismes par lesquels le diabète maternel induit la programmation périnatale de l’hypertension chez la progéniture adulte. Cette étude à long terme porte sur trois types de progénitures : adultes nés de mères contrôles, diabétiques ou diabétiques traitées avec insuline pendant la gestation. Nous observerons la pression systolique, la morphologie rénale et l’expression de divers gènes et protéines. Nous voulons de plus déterminer si la présence d’un système antioxydant (catalase) peut protéger la progéniture des effets néfastes des ROS causés par l’environnement in utero hyperglycémique. Les souris Catalase-Tg expriment la catalase spécifiquement dans les tubules proximaux et nous permettrons d’explorer notre hypothèse sur le rôle des ROS dans notre modèle expérimental de diabète maternel.