【作者】 司江英；

【导师】 封克；

【作者基本信息】 扬州大学 ， 植物学， 2007， 博士

【摘要】 铜是植物生长发育所必需的微量营养元素,但过量的铜也会对植物产生毒害作用。近年来,由于铜矿的开采,含铜农药的大量使用,以及生活污水的排放,造成土壤中铜的浓度大幅度增加,铜已成为土壤中主要重金属污染物之一,并已引起国内外研究者们的极大关注。然而,有关铜对植物的毒害机理和植物对铜的耐性机理还有待深入地研究。本论文采用水培试验,选择玉米（Zea mays L.,品种为苏玉19）作为供试材料,对铜胁迫条件下植物根系形态结构进行分析;通过透射电子显微镜技术观察了不同浓度铜对玉米细胞超微结构的影响及铜在玉米细胞中的分布位点;通过木质部汁液的搜集研究了铜胁迫条件下玉米体内各种养分及铜的转运情况;采用差速离心法和逐步梯度提取法研究了铜在玉米体内的存在形态及亚细胞水平分布情况;研究了不同铜水平下玉米生长及体内铜和养分吸收累积的差异。旨在阐明铜对植物的毒害机理及植物对铜的耐性机理,为铜污染土壤的植物修复提供理论基础。通过研究得到了以下结果:（1）研究不同铜水平对玉米幼苗生长和根系形态几种指标的影响表明,1μmol·L^-1Cu处理时,玉米的生长并不受影响,根系长度和根表面积明显增加,根体积和根平均直径与对照相比并未表现出显著的差异。随着铜浓度的增加,幼苗的生长受到不同程度的抑制,根系长度、根表面积、根体积和根平均直径均显著减小。在各类分级中,20μmol·L^-1Cu条件下, 0.4 mm<直径≤0.8mm时的根系长度、根表面积和根体积均未受到明显抑制,甚至在0.6mm<直径≤0.8mm时三者的值极显著地大于对照。（2）研究不同铜水平对玉米根尖细胞结构和细胞发育的影响表明,1μmol·L^-1Cu处理时,玉米根尖细胞结构及根尖伸长区单位长度细胞数和细胞长度与对照间均未表现出明显差异。随着铜浓度的增加,根冠细胞开始破碎脱落,根冠明显变短;皮层薄壁细胞和导管的破坏逐渐加重,至80μmol·L^-1Cu处理时,薄壁细胞发生崩溃,导管壁断裂破碎;根尖伸长区细胞的长度增加,单位长度的细胞个数逐渐减少,可见,过量的铜对根尖细胞伸长的影响并不明显,很可能抑制根尖细胞的分裂。（3）研究不同浓度铜对玉米根细胞超微结构的影响及铜在根细胞中的分布位点表明,1μmol·L^-1 Cu处理时,玉米根皮层细胞胞质浓,质膜紧贴于细胞壁,含有丰富的细胞器,有较小的细胞核和核仁,核质均匀,核膜皱缩,根维管束细胞中质壁发生分离,原生质膜皱缩。随着外源铜浓度的增加,根皮层细胞中质壁分离和原生质膜破坏加重,细胞器从较少直至完全溶解,细胞核变得更小,核仁分散,核膜断裂,染色质凝集,直至核膜完全溶解,核质和游离的核仁分散在细胞质中,20μmol·L^-1 Cu处理时液泡中有少量颗粒状物质,50μmol·L^-1 Cu处理时,核膜上出现黑色沉积,80μmol·L^-1 Cu处理时原生质膜上出现黑色沉积物;根维管束细胞中,原生质膜和细胞器结构破坏加重, 80μmol·L^-1 Cu处理时细胞质、原生质膜及导管侧壁上均有有黑色沉积。（4）研究不同浓度铜对玉米叶细胞超微结构的影响及铜在叶细胞中的分布位点表明,1μmol·L^-1 Cu处理时,叶表皮细胞中原生质膜模糊,叶肉细胞的超微结构无明显变化。随着外源铜浓度的增加,叶表皮细胞中原生质膜受损和质壁分离更加严重,细胞质和细胞壁中出现黑色沉积物;叶肉细胞中原生质膜破损,至80μmol·L^-1 Cu处理时膜上出现黑色沉淀,叶绿体膜破损,片层结构紊乱,20μmol·L^-1 Cu处理时形成了大的脂质颗粒,50μmol·L^-1 Cu处理时叶绿体上有很多大小不同的空泡,线粒体脊突数目减少,被膜断裂,直至线粒体膜和脊突完全消失。（5）研究不同铜水平对玉米植株内铜积累和养分吸收的影响表明,1μmol·L^-1Cu处理时,玉米地上部中铜的含量和累积量与对照相比均未表现出显著差异,随着外源铜浓度的增加,地上部中的铜含量显著的高于对照,但玉米根系中铜的含量和累积量在4种铜浓度处理时均显著的高于对照,且二者最大值均出现在20μmol·L^-1Cu处理时;不同铜浓度处理对玉米吸收P素的影响很小,而在80μmol·L^-1Cu处理时玉米根部吸收N素及N素在地上部的分配明显受到抑制,在50和80μmol·L^-1Cu处理时玉米根部吸收K素及K在素在地上部的分配都明显受抑制;4种铜浓度处理均不利于Ca在玉米地上部的分配,而1μmol·L^-1Cu处理却有助于玉米根吸收Ca,在50μmol·L^-1Cu处理时玉米根对Mg的吸收及Mg在地上部的分配同时受到抑制;在80μmol·L^-1Cu处理时玉米根部吸收Zn及Zn在地上部的分配同时受到抑制,1μmol·L^-1Cu处理有利于Fe在地上部的分配,随着铜浓度的增加玉米根部对Fe和Mn的吸收及其在地上部的分配均受到抑制。（6）研究不同铜水平下玉米木质部汁液转运速率的差异及木质部伤流液中N、P、K和Cu的浓度和转运速率的差异表明,不同铜浓度处理对玉米木质部汁液转运速率的影响是不同的,与对照相比,1和20μmol·L^-1Cu处理时木质部伤流液的转运速率较快,而50和80μmol·L^-1Cu处理时转运速率减慢。20μmol·L^-1Cu处理时,玉米木质部汁液中Cu的浓度较高,转运速率也较快。对于木质部伤流液中不同形态氮而言,NH4+-N的运输对于铜胁迫最为敏感,仅在很低的铜浓度条件下就受到了抑制,有机氮的转运在铜胁迫条件下不受抑制,随着营养液中铜浓度的升高,其转运速率加快,有机氮/无机氮也逐渐升高。在几种铜浓度处理中,1μmol·L^-1Cu处理时有利于玉米木质部汁液中养分NO3--N、氨基酸、P和K的转运。（7）研究铜在玉米细胞内的分布和存在的主要化学形态表明,细胞壁和细胞溶质部分是铜在玉米细胞内分布的主要位点,细胞核、叶绿体及线粒体等细胞器中铜的含量较低。在对照和1μmol·L^-1Cu处理下根部铜主要分布在细胞壁部分,其次为细胞溶质部分,随着铜浓度的升高,铜向细胞壁部分的分配减少,而向细胞溶质部分的分配增加;茎中,在不同铜浓度处理中亚细胞组分中铜的含量均以细胞溶质部分中的值最高,其次为细胞壁部分;而叶中,其均以细胞壁部分中的值最高,其次为细胞溶质部分。在各种形态铜中,对照中根部主要以HCl提取态铜为主,茎和叶中铜以多种化学形态存在,1μmol·L^-1Cu处理时,根部仍主要以HCl提取态铜为主,而茎和叶中主要以NaCl提取态铜占优势,随着铜浓度的升高,根部和叶部主要以乙醇提取态铜占优势,而茎中20和50μmol·L^-1铜浓度时主要以NaCl提取态铜为主,在80μmol·L^-1 Cu处理时主要以去离子水提取态铜和NaCl提取态铜为主。更多还原

【Abstract】 Copper is an essential nutrient for the growth and development of plants. It is a constituent micronutrient of protein components of several enzymes, mainly of those participating in electron flow, catalyzing the redox reaction in mitochondria and chloroplasts in the cytoplasm of plant cells. When absorbed in excess amounts, copper can cause damage leading to total inhibition of plant growth. Recently, the increasing application of fertilizer and agrochemical, the mining and smelting of metal and the irrigation of wastewater result in the progressive accumulation of toxic copper in the soil. Copper, one of main toxic metals in the soil, has attracted considerable attention. However, the mechanisms of copper toxicity and tolerance need for the further investigation. In this paper, solution culture experiments were conducted using maize and rice as experimental materials. The effects of different copper levels on morphology and structure of maize roots were investigated. Transmission electron microscopy was used to observe effects of different copper concentrations on cell ultrastructure in maize roots and leaves as well as distribution of copper. Xylem sap was collected to study the effects of different copper concentrations on translocation of nutrients and copper in maize. The differential centrifugation technique and sequential chemical extraction method were used to study the subcellular distribution and the chemical forms of copper in the roots, leaves and stems of maize. Growth, copper accumulation and nutrient uptake of maize were studied. The main results were as follows:（1） Under the condition of 1μmol·L-1Cu treatment, growth, root length, root surface area, root volume and average diameter of maize seedlings were not inhibited. With elevation of copper concentration, growth of maize was inhibited. All the parameters of root morphology markedly decreased under high copper concentrations. According to classification, the growth of 0.6mm<D≤0.8mm roots was significantly improved in length, surface area and volume at 20μmol·L^-1Cu level.（2） For root tip of maize, cell structure, cell number unit length and cell length in zone of elongation were no obvious difference between the 1μmol·L^-1Cu level and the control treatment. With the increase of copper concentration, calyptrogens became obviously short, cells of which began to destroy and shell; in root cortex the destruction of parenchymatous cells and vessels was more serious, and parenchymatous cells collapsed and vessels broke at 80μmol·L^-1Cu level; cell length increased, and cell number unit length decreased in zone of elongation. Excess copper may cause reduction in cell division instead of reduction in cell elongation.（3） As for the ultrastructure of maize root cells, dense cytoplasm, plasmalemma sticking cell walls, abundance of cell organelles, small nuclei and nucleolus, homogenous nucleoplasm and folded nuclei membrane were observed in cortical cells of 1μmol·L^-1Cu-treated roots; plasmolysis and folded plasmalemma were found in vascular bundle cells. With the increase of copper concentration, in cells of root cortex plasmolysis and the destruction of plasmalemma became more serious, cell organelles disintegrated completely, nucleus with disintegrated nucleolus became smaller, nucleus membrane bursted, chromatin agglomerated, nucleus membrane finally disappeared leading to nucleolus and nucleoplasm distributing in cytoplasm, and dense and compact materials deposited in vacuole at 20μmol·L^-1 Cu level, in nuclei membrane at 50μmol·L^-1 Cu level and in plasmalemma at 80μmol·L^-1 Cu level; in vascular bundle cells plasmalemma and structure of cell organells were badly destroyed, and dark sediment distributed in cytoplasm, plasmalemma and vessel walls.（4） Compared with the control treatment, blurred plasmalemma was found in epidermal cells of maize leaves while no obvious significance was appeared in mesophyllic cells cells under 1μmol·L^-1 Cu treatment. With the increase of copper concentration, the damage of plasmalemma and plasmolysis became more serious in epidermal cells of maize leaves, and dark granular materials deposited in cytoplasm and cell walls; damaged plasmalemma and disintegrated membrane and disorderly lamella of chloroplasts were detected in mesophyllic cells; dark materials deposited in plasmalemma under 80μmol·L^-1 Cu-treated condition; under 20μmol·L^-1 Cu and 50μmol·L^-1 Cu treatments large lipids and many variform vesicles were formed in chloroplasts; the decrease number of cristae and disintegrated membrane were found in mitochondria, and finally cristae and membrane of mitochondria disappeared.（5） The content and accumulation of copper in maize shoots had no obvious difference between 1μmol·L^-1 Cu treatment and the control. With the elevation of copper supply, compared with the control, the copper content in shoots markedly increased. The content and accumulation of copper in maize roots increased, which in the 20μmol·L^-1Cu-treated roots were the largest among the five Cu levels. Excess copper had little influence on the uptake of P by maize. Both N uptake by maize roots and N allotment in shoots were inhibited under the 80μmol·L^-1Cu treatment. K uptake by maize roots and K distribution in shoots were obviously retarded. All 4 copper levels inhibited Ca allotment in maize shoots, whereas 1μmol·L^-1Cu treatmentimproved Ca uptake by roots. Mg uptake by roots and Mg allotment in maize shoots were limited under 50μmol·L^-1Cu treatment. Zn uptake by roots and Zn allotment in maize shoots were limited under 80μmol·L^-1Cu treatment. Fe distribution in maize shoots was accelerated at 1μmol·L^-1Cu level, while uptake and allotment of Fe and Mn in maize plants were obviously inhibited with the elevation of copper supply.（6） The effects of different copper levels on translocation rates of maize xylem sap were different. Compared with the control, transport rates of xylem sap increased in 1 and 20μmol·L^-1Cu-treated maize whereas just the contrary in 50 and 80μmol·L^-1 Cu-treated plants. Under 20μmol·L^-1Cu treatment, copper concentration in maize xylem sap was higher, and copper transport rates increased. For different forms N in xylem sap, NH4+-N was sensitive to copper stress, translocation of which was retarded simply under the low copper concentration. Excess copper had no inhibition on transport of organic nitrogen. Its translocation rates and organic N/inorganic N increased with the elevation of copper supply. Among 5 copper levels, 1μmol·L^-1Cu treatment stimulated the translocation of NO3--N, organic N, P and K synchronously.（7） Copper was mainly bound to cell walls and soluble fraction, and little was found in the cell organelle fraction in maize cells. Copper content in cell walls was more than in the soluble fraction in the control and 1μmol·L^-1Cu-treatment roots. With the elevation of copper concentration in solution, distribution of copper was reduced in cell wall fraction and increased in the soluble fraction. Copper content in soluble fraction was highest in subcellular parts of maize stems, whereas that in cell wall fraction came next under different copper concentrations. On the contrary, copper mainly distributed in cell wall fraction in leaves under different copper concentrations. Chemical forms of copper in roots, stems and leaves of maize were significantly different at different copper levels. HCl extractable copper was the main form in roots while several copper forms were observed in shoots under the control condition. HCl extractable copper was still superior to other forms in roots and NaCl extractable copper was advantage in shoots under 1μmol·L^-1Cu concentration condition. Ethanol extractable copper became dominant in roots and leaves with the increase of copper supply. In stems, NaCl extractable copper was mainly existed in the 20 and 50μmol·L^-1 Cu treatments while water and NaCl extractable copper most in 80μmol·L^-1 Cu treatment.更多还原