苎麻镉耐性机制及应用研究
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摘要
由人类工业和农业生产活动所引起的重金属镉(Cd)污染正在成为一个世界性的环境问题。由于Cd具有极高的生物毒性以及从土壤向植物体的高度迁移和富集能力,因此环境Cd污染不仅对生态系统产生了极大的破坏作用,而且威胁粮食生产安全,直接危害人类生命健康。目前,我国受到Cd、砷(As)、铅(Pb)等重金属／类金属污染的耕地面积近2000万公顷,约占总耕地面积的1/5,其中Cd污染耕地面积近1.33万公顷,污染范围涉及11个省25个地区,而且污染程度有逐年加重的趋势,因此Cd污染土壤治理及安全高效利用是目前生态恢复及可持续发展中所面临的一个重要课题。
苎麻是我国所特有的一种耐Cd能力强、经济价值高的麻类作物,同时具有很强的耐旱、耐贫瘠能力,在土壤和水体Cd污染修复及矿山废弃地生态恢复中具有较高的应用价值和开发潜力。本研究以Cd耐性植物苎麻为研究对象,重金属Cd为目标污染物,旨在探明苎麻对重金属Cd的积累模式及生理机制,明确苎麻在我国南方,特别是湖南省Cd污染农田高效利用及矿区生态恢复中的作用。主要研究内容及结论包括：
水培条件下,苎麻对Cd的积累随胁迫浓度的升高而增加,苎麻体内的Cd浓度以根部为最高,茎部次之,叶片最低,在0.1-10mg L-1Cd浓度范围内,麻根部Cd浓度为地上部分的4-60倍,且地上部分Cd浓度<100mg kg-1dw,因此苎麻不是Cd的超积累植物。
随着Cd胁迫浓度从0.1mg L-1增加至10mg L-1,苎麻对Cd的转运系数由0.29降至0.04,说明苎麻根部对Cd在体内的分布具有严格的调控作用,高浓度胁迫条件下,减少Cd向地上部分的转运比例是苎麻耐受Cd胁迫的一道重要生理防线。苎麻根部对Cd的吸收符合Michaelis-Menten方程(p<0.0001),其中的动力学参数值分别为亲和系数Km=5.4mg L-1,最大吸收速率Vmax=99.3mg kg-1dw h-1,反映出苎麻根系对Cd的吸收是一个由转运载体所介导的逐渐趋于饱和的过程。重金属Cd胁迫直接影响苎麻对大量元素和微量元素的吸收：Cd胁迫使苎麻叶片中Mg和Ca含量显著降低,低浓度Cd(1mg L-1)促进叶片对S、P、K的吸收,而高浓度Cd(10mg L-1)则明显抑制了叶片对S、 P、 K的积累,叶片对微量元素B、 Mn、 Fe、Cu的积累随Cd胁迫浓度的增加逐渐降低,而Zn含量基本不受Cd胁迫的影响；在苎麻茎部,低浓度Cd(1mg L-1)处理使苎麻对Mg和Ca的积累高于对照样,而P和K含量随Cd浓度的增加逐渐降低,茎部对微量元素Zn、Mn、 Fe、 Cu的积累随Cd胁迫浓度的增加均有所下降,而B含量基本不受Cd胁迫影响；在根部,低浓度Cd(1mg L一)使苎麻对所测定的所有大量及微量元素的吸收均显著增加,而Ca是唯一受到吸收抑制的元素,当Cd胁迫浓度升高至10mg L-1时,苎麻对各种元素吸收的增加量(Ca除外)开始下降。在Cd胁迫浓度≤7mg L-1条件下,苎麻的细胞膜透性增加并不显著,而当Cd胁迫浓度升高至10mg L-1时,细胞膜透性增加至对照样的1.6倍,说明Cd胁迫对细胞膜产生了生理结构损伤。在Cd胁迫下苎麻叶片中富马酸含量与茎中富马酸、苹果酸和柠檬酸含量随着Cd处理浓度的增加逐渐减少,说明以上几种有机酸并未参与苎麻体内Cd的络合与解毒机制。Cd胁迫同时引起了细胞对脯氨酸和亚精胺的积累,作为一种有效的细胞渗透调节剂,脯氨酸的积累反映了Cd对苎麻体内水分平衡的干扰。作为对Cd胁迫的响应,亚精胺的迅速积累表明其参与了苎麻的耐Cd防御机制。
Cd在亚细胞水平上的分布模式及化学形态是苎麻Cd耐性和解毒机制中一个重要的组成部分。在苎麻叶片和根细胞中约48.2%—61.9%的Cd与细胞壁相结合,细胞质中Cd含量次之,约占30.2%—38.1%,而细胞器中Cd含量最少。化根中,NaCl提取态Cd约占总量的50%,其次为醋酸和去离子水提取态,分别约占总量的23%和15%；在茎叶中, NaCl和醋酸提取态占绝对优势,两者总和分别约占茎和叶中Cd总量的80%和65%,说明与蛋白质或果胶酸相结合以及形成磷酸盐沉淀是Cd在苎麻细胞中存在的主要化学形态及解毒机制。Cd在苎麻细胞内的富集直接影响了叶片硝酸还原酶活性和根系活力,其中,1、3mg L-1Cd胁迫显著刺激了硝酸还原酶活性和根系活力,而7mg L-1Cd则使叶片硝酸还原酶的活性及根系活力分别降低至对照样的72%和84%,说明苎麻的解毒及防御机制在超过其生理耐受阈值的Cd浓度胁迫下对细胞生理代谢的保护作用非常有限。
抗坏血酸-谷胱甘肽循环是植物体清除活性氧自由基的核心抗氧化机制。在低浓度Cd(1、3mg L-1)处理下,苎麻叶绿素含量显著增加,而在7mg L-1Cd长期胁迫下,叶绿素含量开始降低,并伴随着丙二醛(MDA)含量的显著增加。苎麻对Cd的氧化胁迫反应与不同Cd浓度条件下抗坏血酸-谷胱甘肽循环的运行效率密切相关：1、3mg L-1Cd胁迫明显激活了抗坏血酸-谷胱甘肽循环,该循环中两种关键的小分子抗氧化剂即还原性抗坏血酸(AsA)和还原性谷胱甘肽(GSH)的含量以及两种关键抗氧化酶即抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)的活性均显著升高；而在7mg L-1Cd胁迫下.随着处理时间的延长,苎麻根部GSH含量及GR活性低于对照,成为整个循环高效清除氧自由基的主要限制因素。
以上通过水培实验了解到苎麻幼苗(营养生长期)对Cd的耐受闽值约为7mgL-1,远远高于Cd在水体中产生毒性效应的浓度范围(0.001-0.01mg L-1),说明苎麻具有较强的耐Cd性。为了进一步了解苎麻对土壤Cd污染胁迫的生理反应及耐性机制,根据湖南省典型矿区及农田Cd污染水平,模拟设置土壤中6个Cd污染浓度：O、10、25、50、80、150mgkg-1。实验结果表明土培条件下,Cd在苎麻体内的分布规律为根>茎>叶,Cd转运系数约为0.3,这与水培条件下苎麻对Cd的分布规律相一致。营养生长期苎麻对土壤Cd污染的耐受阈值约为80mgkg-1左右,当土壤Cd浓度在此范围内时,苎麻通过调节体内叶绿素、类胡萝卜素和可溶性蛋白质含量,维持光合作用的正常进行,保持各种代谢酶的数量和活性,虽然MDA含量随Cd浓度的增加有所升高,但苎麻通过加速AsA-GSH循环有效清除活性氧自由基,调节细胞的氧化还原势,因此Cd对苎麻的生理毒害并不明显；而当土壤Cd浓度高于80mg kg-1时,苎麻体内AsA-GSH循环中的四个关键指标值(AsA,GSH,GR,APX)开始降低,膜脂过氧化加剧,叶绿素、类胡萝卜素和可溶性蛋白质含量显著下降,生理代谢平衡遭到破坏,说明其自身防御系统的保护功能是有限度的,高于忍耐阈值的Cd胁迫将导致苎麻各项生理活动受到严重影响。外源喷施精胺(spmm)和微量元素Se4+可以显著改善不同Cd浓度胁迫下苎麻的生理代谢状况,两种外源物质最适宜的浓度分别为0.5×10-4molL-1和1.26×10-6mol L-1。
通过分析湖南省Cd污染农田及矿业废弃地尾渣的典型代表即七宝山矿区周边农田土壤和湘潭Mn精炼尾渣的理化特性与重金属含量,发现七宝山矿区周边农田土壤含有Cd、Cu、Zn等重金属污染物,其含量已接近或超出国家土壤质量三级标准,其中Cd含量是国家土壤质量三级标准所允许值的8倍。湘潭小浒尾渣库中的Mn精炼尾渣富含氮、磷、钾等植物生长所必需的营养元素,重金属Mn、Cd、Cu、Zn含量分别为对照土样的239倍、11900倍、5.94倍和58.9倍,但与尾渣库边缘有大量植被生长的根际土相比,Mn、Cd、Cu、Zn含量的差异并不显著。尾渣中含有大量细小颗粒,易于板结,空气通透性差,这是与边缘根际土的最主要的差异,也是尾渣库实现植被恢复的关键性限制性因素。通过将Mn精炼尾渣与砂子(1：1)混合增加空气通透性,实现尾渣物理结构的初步改良。
苎麻幼苗在Cd污染农田土壤及Mn精炼尾渣改良基质中的成活率均达到100%,且苎麻的生长显著增加了根际土壤中的活菌数量。在Mn精炼尾渣改良基质中生长的苎麻,其地上部分生物量与对照样相比降低了约30%,但在Cd污染农田土壤中生长的苎麻,其地上部分生物量没有受到显著影响,说明苎麻作为非食用性经济作物,可以考虑在Cd污染农田系统中推广种植,是目前高效利用Cd污染土壤的一种理想作物。由于苎麻地上部分对Cd的积累浓度较低,苎麻不符合植物提取的要求,而更适用于植物固定,通过根系的吸收富集作用,避免Cd向地下水渗透或向地上部分迁移而进入食物链,在矿区植被恢复和水上保持中具有很大的应用潜力。
Worldwide contamination of Cd as a consequence of industrial and agricultural activities is becoming a major environmental problem due to the great toxicity of Cd and its high mobility from soil to plants and further to the food chain. In China, presently, total area contaminated by heavy metals like Cd, As, Pb are approaching to2×107hm2, accounting for c.20%of the total cultivated area in our country. Particularly, the cultivated land contaminated by Cd is almost1.33×104hm2distributed in25districts in11provinces. Furthermore, Cd contamination is becoming heavier year by year. Therefore, the remediation and efficient use of Cd-contaminated soil in a safe way are of great importance for ecological restoration and sustainable development.
Bechmeria nivea (L.) Gaud (ramie), indigenous to China, is an important fiber crop used for textile with a high tolerance to Cd stress as well as to drought and poor nutrition. It displays a high potential in the remediation of Cd-contaminated soil and water and ecological restoration of mine tailings. The main goal of this study was to investigate the accumulation of Cd and tolerance mechanisms in ramie. Furthermore, based on hydroponic and soil culture, we aimed to find out the role of this species in the efficient use of Cd-contaminated agriculture soils and re-vegetation of mine tailings. The main content and conclusions are as follows:
In hydroponics, Cd accumulation in ramie was increased with external Cd concentrations. The highest Cd level occurred in root followed by shoot and the leaves had the least Cd concentrations. With Cd concentration in uptake media ranging from0.1to10mg L-1, Cd concentrations in root were4-60times higher than the above ground part where Cd concentrations were lower than100mg kg-1dw, indicating that ramie is not a Cd-hyperaccumulator. Cd translocation factor in ramie was decreased from0.29to0.04with external Cd increasing from0.1to10mg L-suggesting that tight control of Cd in sensitive tissues may act as an important defense line to counteract Cd toxicity in ramie. Cd uptake by the root of ramie followed Michaelis-Menten kinetics, with Km value of5.4mg L-1and Vmax value of99.3mg kg-1dw h-1, indicating that Cd uptake was a transporter-mediated process and gradually saturated. The accumulation of macro-and micro-nutrients by ramie were apparently affected by Cd stress:the level of Mg and Ca were significantly reduced by Cd in leaves. Low Cd (1mg L-1) stimulated the uptake of S, P and K, while with higher Cd (10mg L-1), the accumulation of S, P and K in leaves were markedly decreased. The levels of B、Mn、Fe、Cu in leaves were decreased at higher external Cd with Zn kept unaffected. In shoots, the concentrations of Mg and Ca were higher than the controls at1mg L-1Cd, while P and K were gradually decreased in a Cd concentration-dependent manner. This held true for the miicronutrients in shoot except B which kept stable at varying Cd concentrations. In roots, almost all the macro-and micro-nutrients uptakes were significantly increased at1mg L-1Cd except Ca which was reduced upon Cd exposure. At10mg L-1Cd, the increasing degree of each element was smaller except Ca. The permeability of cell membrane in ramie changed little at lower Cd concentration. While the maximum cell membrane permeability up to1.6times higher than the control was observed at10mg L-1Cd. Fumarate in leaves and roots as well as citric and malic acid in roots decreased with Cd in a concentration-dependent manner, suggesting that they were not likely to be involved in Cd detoxification mechanisms in ramie under Cd stress. Cellular accumulation of proline and spermidine were rapidly induced by Cd. As an effective buffer for osmotic stress, the increase of proline indicated the disturbance of water balance by Cd. As a quick response to Cd stress, the accumualtion of spermidine in ramie reflected its role in Cd tolerance.
Determination of Cd distribution and chemical speciation in ramie is essential for understanding the mechanisms involved in Cd accumulation, transportation and detoxification. To assess the effect of Cd uptake on plant performance, nitrate reductase activity in leaves androot activity were analyzed during the entire experimental period. Subcellular fractionation of Cd-containing tissues indicated that about48.2-61.9%of the element was localized in cell walls and30.2-38.1%in soluble fraction, and the lowest in cellular organelles. Cd taken up by ramie rapidly equilibrated among different chemical forms. Results showed that the greatest amount of Cd was found in the extraction of1M NaCl and2%HAC, and the least in residues in all test tissues, suggesting that the majority of Cd was detoxified by integrating with pectates and protein followed by forming Cd-phosphate complexes in cells. In roots, the subdominant amount of Cd was extracted by d-H2O and80%ethanol, followed by0.6M HCl. While in stems and leaves, the amount of0.6M HCl-extractable Cd was comparable with that extracted by80%ethanol or d-F2O.1mg L-1Cd stimulated nitrate reductase activity in leaves and root activity, while a concentration-dependent inhibitory effect was observed with increasing Cd concentration, particularly at7mg L-1Cd. It could be suggested that the protective mechanisms evolved by ramie play an important role in Cd detoxification at relatively low Cd concentrations (below3mg L-1Cd) but become restricted to maintain internal homeostasis with higher Cd stress.
Among the antioxidant mechanisms the ascorbate-glutathione cycle has been shown to play a central role in removing reactive oxygen species (ROS) and maintaining the cellular redox status.Ramie under Cd stress exhibited increased level of lipid peroxidation, as was evidenced by the increased malondialdehyde content (MDA, an index of lipid peroxidation) in leaves and roots.Shorter exposure to lower Cd concentrations (1and3mg L-') led to a stimulation of chlorophyll synthesis, while longer exposure and higher Cd concentration (7mg L-1) led to a remarkable breakdown of chlorophyll, suggesting that Cd caused oxidative stress in ramie. The antioxidant system as represented by the ascorbate-glutathione pathway was clearly activated following Cd exposure. An overall increase in the metabolite levels and major constituting enzyme activities (ascorbate peroxidase APX and glutathione reductase GR) was observed in ramie treated with1and3mg L-1Cd throughout the entir experimental period, while prolongation of exposure to7mg L-1Cd resulted in decrease of GR activity and GSH pool in roots which may limit the operation of the whole cycle. Results suggest that Cd-induced oxidative damage in ramie is closely associated with the efficiency of its intrinsic antioxidant mechanisms and the accelerated operation of ascorbate-glutathione cycle provides ramie with enhanced Cd-stress tolerance.
By hydroponics, it is understood that the threshold of ramie seedlings are7mg L-1, which is well above the concentrations of Cd (0.001-0.01mg L-1) exerting toxic effect to most organisms in aquatic system. It is concluded that ramie is a remarkable Cd-tolerant species. To further investigate the physiological response and tolerant mechanism of ramie to Cd in soil system, a series of soil Cd concentrations were set up according the typical Cd contamination degree in agriculture soils and mine tailings in Hunan province. As a result, Cd distribution pattern in ramie followed roots>shoots> leaves and the translocation factor was0.3, consistent with the results from the hydroponics. Ramie seedling in vegetative growth phase can tolerant up to80mg kg-1in soils. Within this range, the chlorophyll, carotenoid and soluble protein were all stimulated, and four key indices involving in AsA-GSH cycle including ascorbic acid (AsA、glutathione (GSH)、ascorbate peroxidation (APX)、glutathione reductase (GR) were all enhanced, which indicates that reactive oxygen species induced by Cd could be eliminated as much as possible by this acceleratory cycle. While the defending system of ramie suffered at higher Cd concentrations. It was proved that the optimum exogenous spm and Se4+concentrations for ramie was0.5×10-4mol L-1and1.26×10-6mol L-1, which could promote the content of chlorophyll, carotenoid and soluble protein significantly and inhibit the cell membrane-lipid peroxidation effectively. At the same time, the content of GSH, AsA and the activity of APX and GR were also increased markedly with exogenous spm. As a result, the whole cycle could operate at higher speed and this means a lot to strengthen the resistance of ramie to Cd.
By analyzing the physiochemical characteristics and heavy metal concentrations in the agriculture soils around Qibaoshan mine and Mn tailings from Xiangtan Mn mine, it was found that elevated Cd, Cu and Zn concentration were approaching to or exceeded Chinese Environmental Quality Standards for soils. Particularly, Cd concentrations were up to8-fold higher than the third grade permitted by the soil standard. The Mn tailings contained rich N, P and K, which would benefit the survival of plants during re-vegetation. Total Mn (31903mg kg-1), Cd (119mg kg-1), Cu (126mg kg-1) and Zn (2490mg kg-1) in Mn tailings were all at phytotoxic levels, but did not differ significantly from those in rhizosphere soils. Mn tailings were clay textured, while rhizosphere soils were silty loam or clay loam. The compaction and anoxic nature of Mn tailings were considered to be the major constraints for plant establishment. By mixing the tailings with sand at1:1ratio, we tried to improve the aeration of the tailings, making it suitable for plant growth. The survival rate of ramie seedlings in the contaminated soils and improved tailings were100%and the growth of ramie greatly increased the bacteria amount in the rhizosphere. As compared to the control plants, the aboveground biomass of ramies grown in the tailings was reduced by30%while those grown in contaminated soils were unaffected. Presently, large scale of cultivated lands has been polluted by relatively low Cd concentrations. As a non-food crop, ramie is recommended to be cultivated in Cd-polluted land before food crops and vegetables are grown, which is considered as an efficient and safe way to use these types of soils. With low Cd concentration in leaves, ramie exhibits great potential for Cd phytostabilization rather than phytoextraction. Taking into account its rich root system, ramies can serves as good candidates for re-vegetation of degraded mining aera and soil and water conservation.
苎麻是我国所特有的一种耐Cd能力强、经济价值高的麻类作物,同时具有很强的耐旱、耐贫瘠能力,在土壤和水体Cd污染修复及矿山废弃地生态恢复中具有较高的应用价值和开发潜力。本研究以Cd耐性植物苎麻为研究对象,重金属Cd为目标污染物,旨在探明苎麻对重金属Cd的积累模式及生理机制,明确苎麻在我国南方,特别是湖南省Cd污染农田高效利用及矿区生态恢复中的作用。主要研究内容及结论包括：
水培条件下,苎麻对Cd的积累随胁迫浓度的升高而增加,苎麻体内的Cd浓度以根部为最高,茎部次之,叶片最低,在0.1-10mg L-1Cd浓度范围内,麻根部Cd浓度为地上部分的4-60倍,且地上部分Cd浓度<100mg kg-1dw,因此苎麻不是Cd的超积累植物。
随着Cd胁迫浓度从0.1mg L-1增加至10mg L-1,苎麻对Cd的转运系数由0.29降至0.04,说明苎麻根部对Cd在体内的分布具有严格的调控作用,高浓度胁迫条件下,减少Cd向地上部分的转运比例是苎麻耐受Cd胁迫的一道重要生理防线。苎麻根部对Cd的吸收符合Michaelis-Menten方程(p<0.0001),其中的动力学参数值分别为亲和系数Km=5.4mg L-1,最大吸收速率Vmax=99.3mg kg-1dw h-1,反映出苎麻根系对Cd的吸收是一个由转运载体所介导的逐渐趋于饱和的过程。重金属Cd胁迫直接影响苎麻对大量元素和微量元素的吸收：Cd胁迫使苎麻叶片中Mg和Ca含量显著降低,低浓度Cd(1mg L-1)促进叶片对S、P、K的吸收,而高浓度Cd(10mg L-1)则明显抑制了叶片对S、 P、 K的积累,叶片对微量元素B、 Mn、 Fe、Cu的积累随Cd胁迫浓度的增加逐渐降低,而Zn含量基本不受Cd胁迫的影响；在苎麻茎部,低浓度Cd(1mg L-1)处理使苎麻对Mg和Ca的积累高于对照样,而P和K含量随Cd浓度的增加逐渐降低,茎部对微量元素Zn、Mn、 Fe、 Cu的积累随Cd胁迫浓度的增加均有所下降,而B含量基本不受Cd胁迫影响；在根部,低浓度Cd(1mg L一)使苎麻对所测定的所有大量及微量元素的吸收均显著增加,而Ca是唯一受到吸收抑制的元素,当Cd胁迫浓度升高至10mg L-1时,苎麻对各种元素吸收的增加量(Ca除外)开始下降。在Cd胁迫浓度≤7mg L-1条件下,苎麻的细胞膜透性增加并不显著,而当Cd胁迫浓度升高至10mg L-1时,细胞膜透性增加至对照样的1.6倍,说明Cd胁迫对细胞膜产生了生理结构损伤。在Cd胁迫下苎麻叶片中富马酸含量与茎中富马酸、苹果酸和柠檬酸含量随着Cd处理浓度的增加逐渐减少,说明以上几种有机酸并未参与苎麻体内Cd的络合与解毒机制。Cd胁迫同时引起了细胞对脯氨酸和亚精胺的积累,作为一种有效的细胞渗透调节剂,脯氨酸的积累反映了Cd对苎麻体内水分平衡的干扰。作为对Cd胁迫的响应,亚精胺的迅速积累表明其参与了苎麻的耐Cd防御机制。
Cd在亚细胞水平上的分布模式及化学形态是苎麻Cd耐性和解毒机制中一个重要的组成部分。在苎麻叶片和根细胞中约48.2%—61.9%的Cd与细胞壁相结合,细胞质中Cd含量次之,约占30.2%—38.1%,而细胞器中Cd含量最少。化根中,NaCl提取态Cd约占总量的50%,其次为醋酸和去离子水提取态,分别约占总量的23%和15%；在茎叶中, NaCl和醋酸提取态占绝对优势,两者总和分别约占茎和叶中Cd总量的80%和65%,说明与蛋白质或果胶酸相结合以及形成磷酸盐沉淀是Cd在苎麻细胞中存在的主要化学形态及解毒机制。Cd在苎麻细胞内的富集直接影响了叶片硝酸还原酶活性和根系活力,其中,1、3mg L-1Cd胁迫显著刺激了硝酸还原酶活性和根系活力,而7mg L-1Cd则使叶片硝酸还原酶的活性及根系活力分别降低至对照样的72%和84%,说明苎麻的解毒及防御机制在超过其生理耐受阈值的Cd浓度胁迫下对细胞生理代谢的保护作用非常有限。
抗坏血酸-谷胱甘肽循环是植物体清除活性氧自由基的核心抗氧化机制。在低浓度Cd(1、3mg L-1)处理下,苎麻叶绿素含量显著增加,而在7mg L-1Cd长期胁迫下,叶绿素含量开始降低,并伴随着丙二醛(MDA)含量的显著增加。苎麻对Cd的氧化胁迫反应与不同Cd浓度条件下抗坏血酸-谷胱甘肽循环的运行效率密切相关：1、3mg L-1Cd胁迫明显激活了抗坏血酸-谷胱甘肽循环,该循环中两种关键的小分子抗氧化剂即还原性抗坏血酸(AsA)和还原性谷胱甘肽(GSH)的含量以及两种关键抗氧化酶即抗坏血酸过氧化物酶(APX)和谷胱甘肽还原酶(GR)的活性均显著升高；而在7mg L-1Cd胁迫下.随着处理时间的延长,苎麻根部GSH含量及GR活性低于对照,成为整个循环高效清除氧自由基的主要限制因素。
以上通过水培实验了解到苎麻幼苗(营养生长期)对Cd的耐受闽值约为7mgL-1,远远高于Cd在水体中产生毒性效应的浓度范围(0.001-0.01mg L-1),说明苎麻具有较强的耐Cd性。为了进一步了解苎麻对土壤Cd污染胁迫的生理反应及耐性机制,根据湖南省典型矿区及农田Cd污染水平,模拟设置土壤中6个Cd污染浓度：O、10、25、50、80、150mgkg-1。实验结果表明土培条件下,Cd在苎麻体内的分布规律为根>茎>叶,Cd转运系数约为0.3,这与水培条件下苎麻对Cd的分布规律相一致。营养生长期苎麻对土壤Cd污染的耐受阈值约为80mgkg-1左右,当土壤Cd浓度在此范围内时,苎麻通过调节体内叶绿素、类胡萝卜素和可溶性蛋白质含量,维持光合作用的正常进行,保持各种代谢酶的数量和活性,虽然MDA含量随Cd浓度的增加有所升高,但苎麻通过加速AsA-GSH循环有效清除活性氧自由基,调节细胞的氧化还原势,因此Cd对苎麻的生理毒害并不明显；而当土壤Cd浓度高于80mg kg-1时,苎麻体内AsA-GSH循环中的四个关键指标值(AsA,GSH,GR,APX)开始降低,膜脂过氧化加剧,叶绿素、类胡萝卜素和可溶性蛋白质含量显著下降,生理代谢平衡遭到破坏,说明其自身防御系统的保护功能是有限度的,高于忍耐阈值的Cd胁迫将导致苎麻各项生理活动受到严重影响。外源喷施精胺(spmm)和微量元素Se4+可以显著改善不同Cd浓度胁迫下苎麻的生理代谢状况,两种外源物质最适宜的浓度分别为0.5×10-4molL-1和1.26×10-6mol L-1。
通过分析湖南省Cd污染农田及矿业废弃地尾渣的典型代表即七宝山矿区周边农田土壤和湘潭Mn精炼尾渣的理化特性与重金属含量,发现七宝山矿区周边农田土壤含有Cd、Cu、Zn等重金属污染物,其含量已接近或超出国家土壤质量三级标准,其中Cd含量是国家土壤质量三级标准所允许值的8倍。湘潭小浒尾渣库中的Mn精炼尾渣富含氮、磷、钾等植物生长所必需的营养元素,重金属Mn、Cd、Cu、Zn含量分别为对照土样的239倍、11900倍、5.94倍和58.9倍,但与尾渣库边缘有大量植被生长的根际土相比,Mn、Cd、Cu、Zn含量的差异并不显著。尾渣中含有大量细小颗粒,易于板结,空气通透性差,这是与边缘根际土的最主要的差异,也是尾渣库实现植被恢复的关键性限制性因素。通过将Mn精炼尾渣与砂子(1：1)混合增加空气通透性,实现尾渣物理结构的初步改良。
苎麻幼苗在Cd污染农田土壤及Mn精炼尾渣改良基质中的成活率均达到100%,且苎麻的生长显著增加了根际土壤中的活菌数量。在Mn精炼尾渣改良基质中生长的苎麻,其地上部分生物量与对照样相比降低了约30%,但在Cd污染农田土壤中生长的苎麻,其地上部分生物量没有受到显著影响,说明苎麻作为非食用性经济作物,可以考虑在Cd污染农田系统中推广种植,是目前高效利用Cd污染土壤的一种理想作物。由于苎麻地上部分对Cd的积累浓度较低,苎麻不符合植物提取的要求,而更适用于植物固定,通过根系的吸收富集作用,避免Cd向地下水渗透或向地上部分迁移而进入食物链,在矿区植被恢复和水上保持中具有很大的应用潜力。
Worldwide contamination of Cd as a consequence of industrial and agricultural activities is becoming a major environmental problem due to the great toxicity of Cd and its high mobility from soil to plants and further to the food chain. In China, presently, total area contaminated by heavy metals like Cd, As, Pb are approaching to2×107hm2, accounting for c.20%of the total cultivated area in our country. Particularly, the cultivated land contaminated by Cd is almost1.33×104hm2distributed in25districts in11provinces. Furthermore, Cd contamination is becoming heavier year by year. Therefore, the remediation and efficient use of Cd-contaminated soil in a safe way are of great importance for ecological restoration and sustainable development.
Bechmeria nivea (L.) Gaud (ramie), indigenous to China, is an important fiber crop used for textile with a high tolerance to Cd stress as well as to drought and poor nutrition. It displays a high potential in the remediation of Cd-contaminated soil and water and ecological restoration of mine tailings. The main goal of this study was to investigate the accumulation of Cd and tolerance mechanisms in ramie. Furthermore, based on hydroponic and soil culture, we aimed to find out the role of this species in the efficient use of Cd-contaminated agriculture soils and re-vegetation of mine tailings. The main content and conclusions are as follows:
In hydroponics, Cd accumulation in ramie was increased with external Cd concentrations. The highest Cd level occurred in root followed by shoot and the leaves had the least Cd concentrations. With Cd concentration in uptake media ranging from0.1to10mg L-1, Cd concentrations in root were4-60times higher than the above ground part where Cd concentrations were lower than100mg kg-1dw, indicating that ramie is not a Cd-hyperaccumulator. Cd translocation factor in ramie was decreased from0.29to0.04with external Cd increasing from0.1to10mg L-suggesting that tight control of Cd in sensitive tissues may act as an important defense line to counteract Cd toxicity in ramie. Cd uptake by the root of ramie followed Michaelis-Menten kinetics, with Km value of5.4mg L-1and Vmax value of99.3mg kg-1dw h-1, indicating that Cd uptake was a transporter-mediated process and gradually saturated. The accumulation of macro-and micro-nutrients by ramie were apparently affected by Cd stress:the level of Mg and Ca were significantly reduced by Cd in leaves. Low Cd (1mg L-1) stimulated the uptake of S, P and K, while with higher Cd (10mg L-1), the accumulation of S, P and K in leaves were markedly decreased. The levels of B、Mn、Fe、Cu in leaves were decreased at higher external Cd with Zn kept unaffected. In shoots, the concentrations of Mg and Ca were higher than the controls at1mg L-1Cd, while P and K were gradually decreased in a Cd concentration-dependent manner. This held true for the miicronutrients in shoot except B which kept stable at varying Cd concentrations. In roots, almost all the macro-and micro-nutrients uptakes were significantly increased at1mg L-1Cd except Ca which was reduced upon Cd exposure. At10mg L-1Cd, the increasing degree of each element was smaller except Ca. The permeability of cell membrane in ramie changed little at lower Cd concentration. While the maximum cell membrane permeability up to1.6times higher than the control was observed at10mg L-1Cd. Fumarate in leaves and roots as well as citric and malic acid in roots decreased with Cd in a concentration-dependent manner, suggesting that they were not likely to be involved in Cd detoxification mechanisms in ramie under Cd stress. Cellular accumulation of proline and spermidine were rapidly induced by Cd. As an effective buffer for osmotic stress, the increase of proline indicated the disturbance of water balance by Cd. As a quick response to Cd stress, the accumualtion of spermidine in ramie reflected its role in Cd tolerance.
Determination of Cd distribution and chemical speciation in ramie is essential for understanding the mechanisms involved in Cd accumulation, transportation and detoxification. To assess the effect of Cd uptake on plant performance, nitrate reductase activity in leaves androot activity were analyzed during the entire experimental period. Subcellular fractionation of Cd-containing tissues indicated that about48.2-61.9%of the element was localized in cell walls and30.2-38.1%in soluble fraction, and the lowest in cellular organelles. Cd taken up by ramie rapidly equilibrated among different chemical forms. Results showed that the greatest amount of Cd was found in the extraction of1M NaCl and2%HAC, and the least in residues in all test tissues, suggesting that the majority of Cd was detoxified by integrating with pectates and protein followed by forming Cd-phosphate complexes in cells. In roots, the subdominant amount of Cd was extracted by d-H2O and80%ethanol, followed by0.6M HCl. While in stems and leaves, the amount of0.6M HCl-extractable Cd was comparable with that extracted by80%ethanol or d-F2O.1mg L-1Cd stimulated nitrate reductase activity in leaves and root activity, while a concentration-dependent inhibitory effect was observed with increasing Cd concentration, particularly at7mg L-1Cd. It could be suggested that the protective mechanisms evolved by ramie play an important role in Cd detoxification at relatively low Cd concentrations (below3mg L-1Cd) but become restricted to maintain internal homeostasis with higher Cd stress.
Among the antioxidant mechanisms the ascorbate-glutathione cycle has been shown to play a central role in removing reactive oxygen species (ROS) and maintaining the cellular redox status.Ramie under Cd stress exhibited increased level of lipid peroxidation, as was evidenced by the increased malondialdehyde content (MDA, an index of lipid peroxidation) in leaves and roots.Shorter exposure to lower Cd concentrations (1and3mg L-') led to a stimulation of chlorophyll synthesis, while longer exposure and higher Cd concentration (7mg L-1) led to a remarkable breakdown of chlorophyll, suggesting that Cd caused oxidative stress in ramie. The antioxidant system as represented by the ascorbate-glutathione pathway was clearly activated following Cd exposure. An overall increase in the metabolite levels and major constituting enzyme activities (ascorbate peroxidase APX and glutathione reductase GR) was observed in ramie treated with1and3mg L-1Cd throughout the entir experimental period, while prolongation of exposure to7mg L-1Cd resulted in decrease of GR activity and GSH pool in roots which may limit the operation of the whole cycle. Results suggest that Cd-induced oxidative damage in ramie is closely associated with the efficiency of its intrinsic antioxidant mechanisms and the accelerated operation of ascorbate-glutathione cycle provides ramie with enhanced Cd-stress tolerance.
By hydroponics, it is understood that the threshold of ramie seedlings are7mg L-1, which is well above the concentrations of Cd (0.001-0.01mg L-1) exerting toxic effect to most organisms in aquatic system. It is concluded that ramie is a remarkable Cd-tolerant species. To further investigate the physiological response and tolerant mechanism of ramie to Cd in soil system, a series of soil Cd concentrations were set up according the typical Cd contamination degree in agriculture soils and mine tailings in Hunan province. As a result, Cd distribution pattern in ramie followed roots>shoots> leaves and the translocation factor was0.3, consistent with the results from the hydroponics. Ramie seedling in vegetative growth phase can tolerant up to80mg kg-1in soils. Within this range, the chlorophyll, carotenoid and soluble protein were all stimulated, and four key indices involving in AsA-GSH cycle including ascorbic acid (AsA、glutathione (GSH)、ascorbate peroxidation (APX)、glutathione reductase (GR) were all enhanced, which indicates that reactive oxygen species induced by Cd could be eliminated as much as possible by this acceleratory cycle. While the defending system of ramie suffered at higher Cd concentrations. It was proved that the optimum exogenous spm and Se4+concentrations for ramie was0.5×10-4mol L-1and1.26×10-6mol L-1, which could promote the content of chlorophyll, carotenoid and soluble protein significantly and inhibit the cell membrane-lipid peroxidation effectively. At the same time, the content of GSH, AsA and the activity of APX and GR were also increased markedly with exogenous spm. As a result, the whole cycle could operate at higher speed and this means a lot to strengthen the resistance of ramie to Cd.
By analyzing the physiochemical characteristics and heavy metal concentrations in the agriculture soils around Qibaoshan mine and Mn tailings from Xiangtan Mn mine, it was found that elevated Cd, Cu and Zn concentration were approaching to or exceeded Chinese Environmental Quality Standards for soils. Particularly, Cd concentrations were up to8-fold higher than the third grade permitted by the soil standard. The Mn tailings contained rich N, P and K, which would benefit the survival of plants during re-vegetation. Total Mn (31903mg kg-1), Cd (119mg kg-1), Cu (126mg kg-1) and Zn (2490mg kg-1) in Mn tailings were all at phytotoxic levels, but did not differ significantly from those in rhizosphere soils. Mn tailings were clay textured, while rhizosphere soils were silty loam or clay loam. The compaction and anoxic nature of Mn tailings were considered to be the major constraints for plant establishment. By mixing the tailings with sand at1:1ratio, we tried to improve the aeration of the tailings, making it suitable for plant growth. The survival rate of ramie seedlings in the contaminated soils and improved tailings were100%and the growth of ramie greatly increased the bacteria amount in the rhizosphere. As compared to the control plants, the aboveground biomass of ramies grown in the tailings was reduced by30%while those grown in contaminated soils were unaffected. Presently, large scale of cultivated lands has been polluted by relatively low Cd concentrations. As a non-food crop, ramie is recommended to be cultivated in Cd-polluted land before food crops and vegetables are grown, which is considered as an efficient and safe way to use these types of soils. With low Cd concentration in leaves, ramie exhibits great potential for Cd phytostabilization rather than phytoextraction. Taking into account its rich root system, ramies can serves as good candidates for re-vegetation of degraded mining aera and soil and water conservation.
引文
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