【作者】 冯人伟；

【导师】 涂书新； 韦朝阳；

【作者基本信息】 华中农业大学 ， 植物营养， 2009， 博士

【摘要】 随着工业和农业的发展,重金属不可避免的大量进入环境,导致环境污染问题日益严重。超富集植物以其对重金属的耐性、富集性等特性展示了它在重金属污染修复方面的巨大潜力。近年来,利用超富集植物修复环境污染的研究获得了学术界和公众的普遍关注。砷、硒、锑三种元素的污染问题在世界范围内广泛存在。研究这三种元素在植物体内的吸收、富集机理及相互作用过程,对于认识植物耐性机理以及植物修复技术的实践均具有理论与实际意义。本文通过一系列的水培与土培试验,以蕨类植物为主要材料,应用生理生化测定技术、氢化物原子荧光光谱法、ICP-OES测定技术以及亚细胞分离技术等手段,研究了植物对硒、锑的吸收、富集机理以及砷、硒、锑在植物体内的两两相互作用,探索研究了利用硒缓解砷、锑毒性的策略。研究成果不仅有助于了解环境有害元素在植物体内的行为特征,对于降低有害元素的环境以及健康风险也提出了可行的技术手段。取得的主要结果有:（1）研究了蜈蚣草富集硒的能力及硒胁迫条件下的抗氧化反应。结果表明:蜈蚣草是一种硒的耐性植物,其地下部硒的最大富集量为1573.3 mg kg^-1,远高于地上部硒的富集量263.6 mg kg^-1。0-2 mg L^-1硒处理浓度对蜈蚣草有益,显著降低蜈蚣草叶片中MDA的含量;但是≥5 mg L^-1硒处理浓度对蜈蚣草产生损伤,显著增加植物叶片中MDA的含量,特别是在硒的最高处理浓度20 mg L^-1时。0-5 mg L^-1的硒处理显著诱导了酶POD,APX和CAT的活性,而＞5 mg L^-1的硒处理浓度则降低了这三种酶的活性。＞5 mg L^-1的硒处理浓度显著增加了蜈蚣草叶片中GSH的含量以及GR酶的活性,20 mg L^-1的硒处理浓度显著增强蜈蚣草叶片中SOD酶。以上结果说明,GSH、GR酶以及SOD酶可能与蜈蚣草的耐硒机理有关,起到调控超氧阴离子自由基（O₂^-）的作用;而酶POD,APX和CAT仅在低浓度硒处理条件下起到清除H₂O₂的作用。（2）利用水培和土培试验研究了硒对蜈蚣草体内必需营养元素含量及分布的影响。结果表明:水培条件下蜈蚣草硒富集量高于土培条件下蜈蚣草的富硒量。在土培条件下,蜈蚣草同样也富集了大量的硒,其地上部和地下部硒的最大富集量分别为81 mg kg^-1和233 mg kg^-1。在土培试验中,硒抑制了几乎所有测定元素的吸收,包括镁（Mg）、钾（K）、磷（P）、铁（Fe）、铜（Cu）和锌（zn）。在水培试验条件下,当蜈蚣草体内硒含量相对较低时,硒同样也抑制了几乎所有被测元素的吸收。然而,当蜈蚣草体内硒含量增加时,硒则促进了蜈蚣草对Ca,Mg,K的吸收。在水培条件下,低剂量硒（或低硒含量）抑制、而高剂量硒（或高硒含量）促进了蜈蚣草对Fe的吸收。以上结果说明Ca,Mg,K可能与蜈蚣草的耐硒机理有关,同时我们推测硒对Fe的吸收调控可能与硒在蜈蚣草体内的双面作用有关。（3）采用正交旋转回归设计,利用水培试验研究了蜈蚣草体内砷硒间的交互作用。结果表明:当硒处理水平小于2.5 mg L^-1时,增加砷处理浓度促进蜈蚣草根部硒的吸收,这一促进作用可能与硒对植物的有益作用有关。而当硒处理浓度高于2.5 mgL^-1时,砷抑制了蜈蚣草根部硒的吸收。蜈蚣草地上部和地下部砷的吸收均被硒抑制,表明硒对砷的拮抗作用。另外,当硒的处理浓度小于2.5 mg L^-1时,砷的加入促进了硒向蜈蚣草地上部的转移;硒却导致砷向蜈蚣草地上部转移能力的降低。（4）利用水培试验比较研究了四种蕨类植物对锑的吸收富集能力以及相应的耐性机理。结果表明:锑的加入没有显著影响砷超富集植物白玉凤尾蕨的生物量,却抑制了贯众、鳞盖蕨和齿牙毛蕨的生物量。与各自对照相比,随着锑的加入,贯众、鳞盖蕨和齿牙毛蕨的生物量降低幅度分别达到12.5%,35.0%和38.3%。表明四种植物由高到低的耐锑能力。四种植物吸收的锑主要富集在根部,表明四种植物转移锑的能力较低。四种植物中,白玉凤尾蕨根部锑最大平均富集量为358 mgkg^-1,贯众为224 mg kg^-1,齿牙毛蕨为124 mg kg^-1,鳞盖蕨为123 mg kg^-1。在20 mg L^-1锑处理条件下,与各自对照相比,鳞盖蕨和齿牙毛蕨叶片中MDA的含量分别增加41.3%和171.6%,而白玉凤尾蕨和贯众叶片中MDA的含量没有显著性变化,表明这两种蕨类植物中脂质过氧化反应较低。在5 mg L^-1锑处理条件下,白玉凤尾蕨叶片中抗坏血酸过氧化物酶（APX）、过氧化氢酶（POD）以及过氧化物酶（CAT）活性均显著高于其他三种蕨类植物,表明这三种酶类在抵御锑毒性的过程中起到重要作用。在锑胁迫下,白玉凤尾蕨不变的生物量、较高的根部锑富集量、较低的叶片MDA含量以及较高的叶片抗氧化酶活性均表明白玉凤尾蕨耐锑能力高于其他三种蕨类植物。结果表明,抗氧化物酶类可能与白玉凤尾蕨较高的耐锑能力有关。（5）利用水培试验研究了砷、锑在砷超富集植物大叶井口边草体内的相互作用以及亚细胞分布特征。结果表明:大叶井口边草是一种潜在的锑超富集植物。在不加砷、锑处理条件下,大叶井口边草叶片和茎部胞液组份聚集了绝大部分砷和锑,而细胞壁和细胞器中砷、锑含量相对较少;在单独加入锑处理时,随着锑处理浓度的增加,大叶井口边草体内锑的含量逐渐增加,叶片、茎和根部中的各个亚细胞组份中锑的含量也逐渐增加,胞液中锑含量的相对比例却降低,而细胞壁组份中锑含量的比例随着锑处理浓度的增加而增加。当砷、锑联合处理时,砷的加入显著促进了植物对锑的吸收,并且低剂量砷的促进作用要高于高剂量砷的促进作用。增加的砷处理浓度诱导更多的锑被转移富集于植物胞液组份中,而这一过程伴随着砷在胞液中比例的降低。在低砷处理条件下,锑的加入轻微促进了植物对砷的吸收,然而这种促进作用伴随着植物富集砷潜力的降低,表现为大叶井口边草胞液组份中砷含量以及相对比例的降低;在高砷处理下,锑的加入抑制了植物对砷的吸收,显著降低了茎部砷的含量,同时也降低了叶片和茎不胞液组份中砷含量的比例。表明锑对水稻具有很强的毒性。当不加锑时,0.1 mg L^-1硒的加入未显著影响水稻的生物量,却显著抑制了叶片MDA;≥1 mg L^-1硒的加入则显著降低了水稻的生物量并增加了叶片MDA的含量,表现出硒对水稻的两面性作用。硒的加入显著增加了水稻叶片中蛋白质的含量,而5 mg L^-1锑的加入却逆转了这一趋势,蛋白质含量随着硒处理浓度的增加而降低。当硒、锑联合处理时,增加的硒处理浓度降低了水稻各个部位锑的含量,表现出硒对锑的拮抗作用。另外,与5 mg L^-1单独锑处理浓度相比,1 mg L^-1硒的添加降低了5 mg L^-1单独锑处理下的叶片MDA含量,并且显著增加了该锑处理下的水稻生物量,此时水稻的生物量甚至要高于不加硒、锑的对照处理下水稻的生物量,以上结果表明硒缓解了锑对水稻的毒性。5 mg L^-1锑的加入也能缓解1 mg L^-1或者5 mg L^-1硒对水稻的毒害,表现为水稻生物量的增加、降低的叶片MDA含量以及降低的地上部硒含量。意外的是,当硒处理浓度为1 mg L^-1或者5 mg L^-1时,随着锑处理浓度的增加,硒被优先富集在水稻根部。以上结果反映了水稻体内硒、锑互相解毒的过程,而硒对锑的解毒功能可能主要与硒对锑的拮抗作用有关,而部分与硒的抗氧化功能有关。更多还原

【Abstract】 With the development of industry and agriculture,continuous deposition of heavy metals in the environment is unavoidable,which may result in the aggravation of environmental pollution.Resently,increasing concerns from the scientists and public have been paid for the technology using hyperaccumulating plants to phytoremediate the comtaminated environment.Investigations on the mechanisms for uptake,accumulation and interactions among deleterious elements in plants will contribute to the understandings of tolerance mechanisms,and also benefit to provide more theoretical knowledge in phytoremediation process.In this study,a series of experiments in nutrition culture or in soil culture have been conducted to investigate:1) the mechanisms on the uptake and accumulation of selenium and antimony in plants;2) the interactions among arsenic,selenium and antimony in plants;3) the strategies to alleviate the toxicity of arsenic and antimony to plants using the extra supplementation of selenium to the substrate.In this study,most employed plants were fern plants and at the same time, physiological and chemical determination methods,hydride generation atomic fluorescence spectrometer test technology,inductively coupled plasma optical emission spectrometry test technology and the subcellular fractionation technology were also used.The main results were as follows:1.We examined the selenium（Se） accumulation as well as its related antioxidant responses in Chinese brake fern（Pteris vittata L.）,an arsenic hyperaccumulator.The results showed that Se was accumulated more in roots than in fronds,with the highest Se concentration of 1573.3 mg kg^-1.Addtion of Se in the range of 0-2 mg L^-1 exerted a beneficial effect that was indicated by a significantly decreased content of malondialdehyde（MDA） in the fronds of Chinese brake fern.However,the toxicity of Se in Chinese brake fern occurred with Se addition of greater than 5 mg L^-1,which was shown by significant increases in MDA contents,especially at the highest Se addition rate of 20 mg L^-1.The enzymes catalase（CAT）,ascorbate peroxidase（APX） and peroxidase （POD） in the fronds of Chinese brake fern were largely induced at low Se addition rates from 0 to 5 mg L^-1 while Se addtion of greater than 5 mg L^-1 reduced their activities.The contents of glutathione（GSH） and activities of GR increased when Se addition was greater than 5 mg L^-1,and a significant increase of superoxide dismutase（SOD） activities was observed at the highest addition rate of Se at 20 mg L^-1.The results suggested that participation of APX and POD in the destruction of H₂O₂ might be promoted by low addition of Se,while SOD,GSH and GR contributed to high Se tolerance through the reduction of superoxide radicals（O₂^-） accumulation. 2.Hydroponic（nutrient solution culture） and pot（soil culture） experiments were simultaneously conducted to investigate the effects of Se on the uptake and distribution of essential elements in Chinese brake fern.Chinese brake fern took up much more Se in nutrient solution culture than that in soil culture.In soil culture,Chinese brake fern also accumulated high contents of Se,with the highest contents of 81 mg kg^-1 and 233 mg kg^-1 in the fronds and roots,respectively.In soil culture,the addition of Se suppressed the uptake of most measured elements,including magnesium（Mg）,potassium（K）, phosphorus（P）,iron（Fe）,copper（Cu） and zinc（Zn）.In nutrient solution culture,when the Se contents in the tissues of Chinese brake fern were relatively low,the supplementation of Se suppressed the uptake of most essential elements;however,with the increase of Se contents,stimulation effects of Se on the uptake of Ca,Mg,K were observed.An initial decrease followed by a rapid increase of Fe contents in the fronds of Chinese brake fern were found with Se addition and tissue Se contents increasing in nutrient solution culture,suggesting antagonistic and synergic roles of Se on these elements under low to high Se exposure,respectively.The results indicated that Ca,Mg, K might be involved in the tolerance mechanism of Se,and that the regulation of Fe accumulation by Se in the fronds might be partially due to the dual effects of Se on Chinese brake fern.3.The interactive effects of As and Se on their uptake by Chinese brake fern were explored in two hydroponic experiments based on a two-factor,five-level central composite design.At Se levels of less than 2.5 mg L^-1,increasing amounts of As stimulated the uptake of Se in Chinese brake fern roots,possibly because of the beneficial effects of Se.In contrast,at Se concentrations greater than 2.5 mg L^-1,As suppressed the uptake of Se in Chinese brake fern roots.Uptake of As by both fronds and roots of Chinese brake fern was suppressed by the addition of Se,indicating the antagonistic effects of Se on As.In addition,at Se concentrations of less than 2.5 mg L^-1,As stimulated the translocation of Se from roots to fronds;meanwhile,the addition of Se resulted in reduced translocation of As from roots to fronds.These findings demonstrate the interactive effects of As and Se on their uptake by Chinese brake fern.4 Investigation of the potential of antimony（Sb） tolerance and accumulation by plants as well as the antioxidative responses to Sb in four fern plants were carried out.The biomass of fern PCA（Pteris Cretica ’Albo-lineata’） remained constant with Sb addition, whereas the biomass of ferns CYF（Cyrtomium Fortunei）,MH（Microlepia Hancei） and CYD（Cyclosorus Dentatus） at the high Sb rate exposure decreased by 12.5%,35.0%and 38.3%,respectively as compared with their controls.This suggested a high to low Sb tolerance order for these four fern plants.For all of these fern plants,more Sb was accumulated in the roots than in the fronds.Antimony concentration in the roots at the high rate of Sb addition was recorded,on average,as 358 mg kg^-1 for fern PCA,224 mg kg^-1 for fern CYF,124 mg kg^-1 for fern CYD and 123 mg kg^-1 for fern MH.A high rate of addition of Sb increased the contents of malondialdehyde（MDA） by 41.3%and 171.6% for ferns MH and CYD,respectively,as compared with their controls.No changes for MDA contents were observed in ferns PCA and CYF with Sb addition,indicating no lipid peroxidation reaction in these two plants.At a medium rate of Sb addition,the activities of peroxidase,catalase and ascorbate peroxidase in fern PCA were much higher than those in ferns CYF,CYD and MH,demonstrating the important role of these three enzymes in resisting Sb toxicity.The consistency in unchanged biomass,high accumulation of Sb in roots,lower MDA contents,as well as high enzyme production in fronds,indicated that fem PCA was more tolerant to Sb than the other three fern plants. Antioxidative enzymes（peroxidase catalase and ascorbate peroxidase） might be involved in Sb toxicity resistance of fern PCA.5 Pteris Cretica might be a potential antimony hyperaccumulator.Without As and Sb addition,most As and Sb absorbed by this plant were stored in the cytoplasmic supematant fraction in the fronds and stems,while the cellwall and cytoplastic organelles only sequestrated relatively few of As and Sb.In the presence of antimony alone in the solution,with Sb treatment concentrations increasing,the contents of Sb in all tissues of Pteris Cretica and in all subcellular fractions from leaves,stems and roots were all enhanced;However,the percentages of Sb distribution in the cytoplastic supernatant fraction in all tissues dereased,accompanied with the increases of that in the fraction of cellwall.When both As and Sb were present in the solution,the uptake of Sb by this fern plant were stimulated by the increasing As treatment concentrations,however,more efficient stimulation of Sb uptake by relatively low As treatment concentrations than high As treatment concentrations was also observed.The increased As treatment concentrations induced the more translocation of Sb to the cytoplastic supernatant fraction, along with the decrease in the percentage of As distribution in this fraction.At low As treatment concentrations,the supply of Sb slightly enhanced the uptake of As,but this operation simultaneously reduced the accumulation potential of As in Pteris cretica, reflecting on the decreased percentage of As in the fraction of cytoplastic suprnatant. However,at high As treatment concentrations,the uptake of As by this plant was suppressed by the addition of Sb.Significant decreases in the stem As contents and in the percentages of As in the cytoplastic supematant fractions of both leaves and stems were observed along with the supply of Sb.6.The effects of Sb on the growth of paddy-rice（WeiYouⅡ416） and the supposition of detoxifying Sb by the addition of selenium to plants were explored.Aider 14 days exposure,the single addition of 5 mg L^-1 Sb slightly reduced the biomass of paddy-rice and increased the leaf malondialdehyde（MDA） contents,and higher Sb addition as 50-100 mg L^-1 Sb totally resulted in the death of paddy-rice,suggesting the toxicity of Sb to paddy-rice.Without Sb addition,0.1 mg L^-1 Se remarkably subdued the formation of MDA in the leaves accompanied with an unaffected biomass,but≥1 mg L^-1 Se evidently decreased the biomass and enhanced the leaf MDA contents,suggesting antioxidant and pro-oxidant roles of Se to paddy-rice under different dosages of Se.The increasing Se treatment concentrations enhanced the synthesis of soluble protein in the leaves of paddy-rice,however,with 5 mg L^-1 Sb in the solution,increasing Se treatment concentrations reversely suppressed it.With both Se and Sb in the solution,the increasing Se levels depressed the uptake of Sb in all tissues of paddy-rice,indicating an antagonistic effect of Se on the uptake of Sb.Moreover,as compared with the single Sb level of 5 mg L^-1,1 mg L^-1 Se addition reversely decreased the leaf MDA contents and remarkably increased the biomass even being higher than that of the control,suggesting an alleviated process of Sb toxicity by Se.Interestingly,the addition of 5 mg L^-1 Sb also ameliorate the toxicity of 1 or 5mg L^-1 Se,reflecting on more or less increased biomass,decreased leaf MDA contents,and reduced above ground Se contents.Unexpected,when Se was imposed at the concentrations of 1 or 5 mg L^-1,more Se were accumulated in the root of paddy-rice with increasing Sb treatment concentrations.The results of this study suggested a mutual detoxification process between Se and Sb in paddy-rice,and the detoxification of Sb by Se might be related with the antagonistic effects of Se on the uptake of Sb,partially with the antioxidant role of Se.更多还原