【作者】 郭智；

【导师】 奥岩松；

【作者基本信息】 上海交通大学 ， 蔬菜学， 2009， 博士

【摘要】 镉作为一种毒性较强的重金属污染物,由于长期施用磷肥、金属电镀和塑料工业发展等原因而在农田长时间残留,对人类健康造成潜在的威胁。针对目前日益严重的镉污染,植物修复是一种绿色廉价、被人们广泛认可的有效的原位治理措施,其核心技术在于利用极少数镉超富集植物来去除土壤中的镉以达到修复和治理环境的目的。本文以新型镉超富集植物龙葵为研究对象,通过营养液培养方法深入研究了系列镉处理浓度下苗期龙葵植株生长、抗氧化系统、氮代谢、光合作用参数等生理特性和镉吸收积累特性,探讨镉胁迫下超富集植物龙葵的生理响应机制。在此基础上,研究了镉在龙葵体内的移动性及其在龙葵不同叶位叶片间亚细胞分布、化学赋存形态的差异性,部分揭示龙葵的镉耐性机制。并且,对镉、锌单一和复合污染条件下龙葵和茄子对锌、镉的吸收和积累特性的差异性进行了研究,同时通过人工模拟镉污染土壤研究了镉对大量元素及微量营养元素吸收的影响,以探讨元素互作的内在机制。另外,本研究通过外源添加水杨酸考察其对龙葵幼苗叶片镉毒害的缓解效应,通过外源添加壳聚糖考察其对土壤镉的钝化效应。主要研究内容和结果如下:①镉胁迫对龙葵幼苗生长、部分生理特性及镉积累的影响镉处理使龙葵和茄子幼苗生长受到不同程度影响,并且具有明显的浓度效应和时间效应。龙葵在镉浓度低于50μM条件下,地上部干物质量仅下降15.48%,根系则有所增加,低浓度镉(25μM)处理显著促进龙葵幼苗根系活力,而高浓度(150μM)镉处理10 d后达到峰值;茄子地上部干物质量和根系活力在所有镉处理下均呈下降趋势。镉胁迫还导致龙葵和茄子幼苗叶片色素含量降低。龙葵体内的SOD、POD、CAT对镉胁迫起到较强的抗氧化保护作用。随着镉浓度的升高和胁迫时间的延长,龙葵幼苗叶片相对电导率、MDA含量均显著上升,而可溶性糖和脯氨酸含量也相应上升,起到一定的渗透调节作用。②镉胁迫对龙葵幼苗叶片抗坏血酸-谷胱甘肽循环的影响及外源水杨酸对镉毒害的缓解效应通过营养液培养方法,研究龙葵叶片GSH-AsA循环及其相关酶对Cd胁迫的响应,并且通过外源添加水杨酸(SA)考察其对龙葵幼苗叶片镉毒害的缓解效应。结果表明:镉处理显著影响龙葵幼苗叶片还原型谷胱甘肽(GSH)、氧化型谷胱甘肽(GSSG)、还原型抗坏血酸(AsA)、脱氢抗坏血酸(DHA)含量和谷胱甘肽还原酶(GR)和抗坏血酸过氧化物酶(APX)活性,且具有浓度效应和时间效应。镉处理3 d后,GSH和GSSG含量首先小幅下降。镉处理9 d后GSH含量达到峰值。GSSG含量在25μM镉处理下表现为“降-升-降”的变化规律,12 d后含量最低,而150μM镉处理下则表现为“升-降-升”,6 d后达到峰值。25μM镉处理下,AsA含量随时间延长先升后降,平均含量较CK均值提高48.58%,而150μM镉处理下则呈现“降-升-降”的变化规律,6 d后含量最低,但平均含量较CK均值提高40.88%。DHA含量随镉浓度提高和处理时间的延长逐渐增加,25μM镉处理9 d和150μM镉处理12 d后均达最大值。GR活性随镉处理时间的延长先升后降,9 d时达到峰值。同时,25μM和150μM镉处理下平均GR活性分别较CK提高30.90%和51.67%。APX活性变化规律与GR有所不同。25μM镉处理下,APX随处理时间的延长呈现“升-降-升”的变化规律,平均活性较CK升高47.37%,而150μM镉处理下表现为“降-升-降”,3 d时达到峰值,较对照增加37.81% (P<0.05)。外源SA的添加可以显著提高龙葵叶片色素、GSH、AsA和DHA含量,降低MDA含量,也能显著提高GR和APX活性,SA处理3 d和6 d后,GR活性分别较未添加SA的T2 (150μM)镉处理增加27.50%和32.08% (P<0.05),APX增加23.74%和22.48% (P<0.05)。③镉胁迫对龙葵幼苗光合特性、氮代谢的影响采用模拟镉污染土壤培养法研究了镉对龙葵光合特性及营养元素吸收的影响。结果表明:随镉处理浓度的增加,龙葵幼苗叶片净光合速率(Pn)、气孔导度(Gs)和蒸腾速率(Tr)均呈下降趋势,而胞间CO2浓度(Ci)则增加。同时,可变荧光和最大荧光之比(Fv/ Fm)显著降低。采用营养液培养方法,研究了系列镉浓度处理对龙葵幼苗氮代谢的影响。结果表明:龙葵根系可溶性蛋白质较叶片受Cd影响更大。龙葵植株叶片和根系NO3--N含量、硝酸还原酶(NR)、谷氨酰胺合成酶(GS)活性均随镉浓度提高而先增后降,且随处理时间的延长而逐渐下降。叶片NO3--N含量和NR活性均在25μM镉处理8 d后达到峰值,而根系NO3--N则在50μM镉处理8 d后最高。叶片NO3--N含量受镉处理影响较根系稍大。然而,龙葵叶片中NH4+-N含量随镉浓度升高和时间延长逐渐升高,100μM镉处理16 d后最高,表明镉胁迫下龙葵叶片铵态氮富集效应明显。龙葵叶片和根系中谷氨酸脱氢酶(GDH)活性随镉处理浓度提高和处理时间延长而逐渐升高,100μM镉处理16 d后达到峰值。④镉在超富集植物龙葵体内的移动性及其在龙葵不同叶位叶片中的亚细胞分布及赋存形态研究从积累部位来看,镉主要积累在龙葵幼苗地上部,镉含量由高到低依次为叶片>茎>根系。同时,龙葵幼苗叶片镉含量依叶位不同呈相应变化,本试验结果表明第7片叶镉含量最高,且叶脉部分镉含量较叶片边缘和叶脉间隙部分高。限制蒸腾处理24 h使第7片叶镉含量下降76.73%,而未影响其他叶片镉含量分布情况。将龙葵植株移至去镉营养液中继续处理后发现新展开2片新叶镉含量极低。通过比较加镉-去镉两种处理下龙葵植株各部位镉含量和镉积累量差异,用以考察镉在龙葵体内的移动性。去镉处理后,各老叶的镉含量均较对照下降,而镉积累量却有所上升。然而,加镉-去镉两种处理下植株总体镉积累量保持不变,去镉处理并未改变镉在龙葵体内的分布模式。去镉处理使龙葵根系中约37.78%的镉转运至地上部。因此,可以推断在龙葵体内镉离子随蒸腾流较易转运至地上部,一旦进入叶片,镉离子很难通过韧皮部转运至其他组织器官。镉在龙葵不同叶位叶片中的亚细胞分布模式基本相同,主要分布在细胞壁部分,占62%～66%,其次是细胞质可溶部分,而在细胞器组分的分布较少,仅占5%~7%。提高镉处理水平,镉向叶片细胞壁部分和细胞器部分的分配比例增加,而向细胞质可溶部分的分配比例相对减少。同时,镉向下位叶细胞器的分配比例增加幅度较上位叶更大。龙葵叶片中镉主要以氯化钠提取态和醋酸提取态存在,尤其是氯化钠提取态,占40.09%~56.09%。即主要是果胶酸、蛋白质结合态或吸附态及难溶于水的磷酸盐类镉,进一步表明龙葵叶片中镉主要分布在细胞壁。⑤镉、锌单一或复合胁迫对龙葵和茄子幼苗生长及镉、锌积累的影响镉、锌单一和复合污染条件下龙葵和茄子幼苗生长反应及其对锌、镉的吸收和积累特性的研究结果表明:高浓度镉锌使苗期龙葵和茄子植株生长受到不同程度的抑制,而低浓度镉处理和部分低浓度镉锌复合处理却具有促进作用。不同锌浓度处理均能大幅度提高苗期龙葵根、叶的镉含量,并随锌浓度的提高先升后降,均在100μM时达到峰值。龙葵和茄子各器官镉含量高低主要由溶液中的镉浓度决定,镉锌互作的影响有限,但相同镉浓度下龙葵均以100μM锌处理组合最高,而茄子以50μM锌处理组合最高。从植株整体的镉积累量来看,龙葵地上部镉积累量是根系的7~16倍多,而茄子主要积累在根系。龙葵各器官的平均锌含量均以50μM镉浓度时最高,尤其当Cd/ Zn为50/ 500μM时,地上部锌积累量高达445.9μg·株-1,而茄子地上部锌积累量在Cd/ Zn为25/ 500μM时达到最大值,仅为48.3μg·株-1。龙葵和茄子各器官锌含量高低主要由溶液中的锌浓度决定,镉锌互作对龙葵作用有限,而对茄子具有一定的互作效应。从植株整体的锌积累量来看,锌主要积累在龙葵幼苗地上部,叶片锌积累量是根系的7~17倍多,而对于茄子而言,锌在叶片中积累量较根系稍高。镉处理还导致龙葵植株营养元素吸收紊乱。镉促进龙葵叶片和根系K吸收,对Na吸收影响不显著。同时,镉促进根系Mg吸收,但抑制其向地上部转运。低浓度镉处理促进叶片Ca吸收。龙葵根、茎、叶Zn含量随镉处理浓度的提高均表现为低促高抑。根系Cu吸收随镉浓度提高而增加,叶片先增后降,各器官Fe含量随镉浓度提高逐渐降低,而根系Mn含量受镉抑制。⑥壳聚糖对土壤镉离子的钝化效应外源壳聚糖对镉污染土壤中有效态镉含量及其垂直分布的研究结果表明,壳聚糖对土壤中Cd2+含量有较大的影响。施入壳聚糖后,土壤中的有效Cd2+含量明显降低,并且随壳聚糖施用量的增加而进一步降低,0.9 g kg-1 DW壳聚糖施用量钝化效应最佳。壳聚糖对土壤中重金属离子的作用主要是发生在处理后的7d内。同时,壳聚糖还影响镉离子在土壤中的垂直分布。更多还原

【Abstract】 Cadmium (Cd), accumulated in soils as a consequence of agricultural activities (excessive phosphatic fertilizers application) and industrial activities (mining and smelting of metalliferous ores, electroplating, etc.) is becoming a major problem due to the great toxicity, high mobility from soil to plant, long half-life of Cd and further entry into food chain. Phytoremediation of Cd polluted soils has attracted world-wide attention and research, and in which the hyperaccumulation of Cd in the shoots by plants is the main technique to remove Cd in the contaminated soils. In the present study, the newly discovered Cd hyperaccumulator plant Solanum nigrum L. was grown in hydroponics with increasing Cd concentrations to study the growth, antioxidant system, Nitrogen metabolism, photosynthetic parameters, and Cd accumulation characteristics. Based these, distribution and mobility of Cd in the S. nigrum plant, and subcellular distribution and chemical forms of Cd in the leaves of different position were investigated. Further, the difference of Cd, Zn accumulation between S. nigrum and S. melongena plants subjected to Cd, Zn, and Cd-Zn complex stress was studied by hydroponic culture method. Moreover, the interactions between Cd and macro- and micro-nutrient elements were investigated through a pot experiment. In addition, exogenous SA was applied in the hydroponic system to study the alleviating effects to Cd toxicity in the leaves of Solanum nigrum L.; exogenous chitosan was applied in different textural soils to study the effects on the available contents and vertical distribution of Cd2+. The main results were indicated as follows:①The growth status, physiological characteristics, and Cd accumulation in the hyperaccumulator plant Solanum nigrum L seedlings subjected to Cd stress.Physiological responses of S. nigrum and S. melongena seedlings to Cd stress were investigated by nutrient solution culture method. The results showed that Cd could affect the growth of S. nigrum and S. melongena seedlings, with concentration-dependent and time-dependent manners. Dry shoot biomass of S. nigrum decreased 15.48% when Cd concentration was below 50μM (P > 0.05), but dry root biomass increased 111.11% with the application of Cd (≤100μM, P < 0.05). Root activities of S. nigrum increased at low Cd concentrations (≤50μM) and then decreased. However, dry shoot biomass and root activities of S. melongena decreased significantly in all Cd treatments. The leaves pigment contents of S. nigrum and S. melongena seedlings decreased with the increase of Cd concentration. SOD, POD, and CAT in the leaves of S. nigrum played an important role against Cd stress. With increasing Cd concentration in the solution and along with treatment time, the relative elective conductivities, malondialdehyde (MDA) contents increased, soluble sugar and proline contents also increased, which played an important role of osmotic adjustment.②Response of the ascorbate-glutathione cycle and the alleviating effects of exogenous SA to Cd toxicity in the hyperaccumulator plant Solanum nigrum L.Response of the ascorbate-glutathione cycle and the alleviating effects of exogenous SA to Cd toxicity in the hyperaccumulator plant S. nigrum seedlings under Cd stress were investigated by nutrient solution culture method. The results showed that Cd stress significantly influenced the contents of GSH, GSSG, AsA, DHA and the activities of GR and APX in the leaves of S. nigrum, and have concentration-dependent and time-dependent effects. After 3 d of Cd exposure, the contents of GSH and GSSG decreased with a little extent firstly, and reached maximum after 9 d of Cd exposure. GSSG content presented a rule of“decrease-increase-decrease”subjected to 25μM of Cd, and reached minimum after 12 d of Cd exposure, but when treated with 150μM of Cd, GSSG content presented a reverse rule-“increase-decrease-increase”, and reached maximum after 6 d of Cd exposure. And, AsA content increased firstly and decreased along with treatment time subjected to 25μM of Cd, average content increased 48.58% compared to the control. However, AsA content presented a rule of“decrease-increase-decrease”subjected to 150μM of Cd, and reached minimum after 6 d of Cd exposure, but the average content increased 40.88% than the control. DHA content increased with increasing Cd concentration and along with treatment time, and reached maximum after 9 d of 25μM Cd and 12 d of 150μM Cd exposures. GR activity increased firstly and decreased along with exposure time, and reached peak values after 9 d of Cd exposure, further, average GR activities increased 30.90% and 51.67% subjected to 25 and 150μM of Cd, respectively. APX activity presented a different rule with GR activity, and a rule of“increase-decrease-increase”was found when the seedlings subjected to 25μM of Cd, average activity increased 47.37% compared to the control, however, APX activity presented the rule of“decrease-increase-decrease”, and reached maximum after 3 d of 150μM Cd exposure, increased 37.81% compared to the control (P < 0.05).Application of exogenous SA significantly increased the contents of leaf pigments, GSH, AsA, and DHA, decreased the MDA contents. Furthermore, increased the activities of GR and APX, after 3 d and 6 d of SA application, GR activities increased 27.50% and 32.08% compared to that of T2 (no SA application, 150μM Cd, P < 0.05), and APX activities increased 23.74% and 22.48%, respectively (P < 0.05).③Effects of Cd stress on the leaf photosynthesis, Nitrogen metabolism, and uptake of nutrient elements in the hyperaccumulator plant Solanum nigrum L.Effects of Cd stress on the leaf photosynthesis and macro- and micro-nutrient elements uptake in the hyperaccumulator plant S. nigrum were investigated through a soil pot experiment. The results showed that net photosynthesis rate (Pn), stomatal conductance (Gs), and transpiration rate (Tr) decreased with increasing Cd concentrations, but intercellular CO2 concentration (Ci) increased. And, Fv/ Fm ratios decreased significantly.Nitrogen metabolism in S. nigrum seedlings under Cd stress were investigated by nutrient solution culture method. The results showed that NO3--N and soluble protein contents, NR and GS activities in the leaves and roots increased firstly and decreased with increasing Cd concentrations in the solutions, and decreased along with treatment time. And, soluble protein contents in the roots decreased with more extent. After 8 d of Cd exposure (25μM), NO3--N content and NR activity in the leaves of S. nigrum reached peak values, but root NO3--N contents reached maximum after 8 d of 50μM Cd exposure, and leaf NO3--N contents were more sensitive to root. However, NH4+-N contents in the leaves of S. nigrum increased with increasing Cd concentrations and along with treatment time, and reached maximum after 16 d of 100μM Cd exposure, which suggested significant NH4+-N accumulation in the leaves of S. nigrum subjected to Cd stress. Furthermore, GDH activities in the leaves and roots increased with increasing Cd concentrations and along with exposure time, and reached peak values after 16 d of 100μM Cd exposure.④Subcellular distribution, chemical forms, and mobility of Cadmium in the hyperaccumulator plant Solanum nigrum L.Cd mainly accumulated in the shoot of S. nigrum seedlings, and Cd concentrations in the plant tissues could be drawn as followed: leaf﹥stem﹥root. The Cd concentrations in leaves of S. nigrum varied with leaf position, the highest concentration was found in the 7th leaf (2373.51μg Cd g-1 DW). And, an increase in Cd concentration from the marginal leaf to the midrib part was observed. When the 7th leaf was wrapped in a transparent plastic bag for 24 h, the Cd concentration of this leaf decreased 76.73% than that in the corresponding leaf of no wrapping. And, the treatment did not affect the distribution of Cd in other leaves. Cd concentrations in the two new leaves (11th and 12th leaves), which had appeared after the cessation of Cd treatment, were extremely low. The mobility of Cd in S. nigrum was investigated in this study by firstly growing seedlings in one-half-strength Hoagland nutrient solution containing Cd, followed by a further growth period in a nutrient solution without Cd. After the plant has been transplanted into Cd-free nutrient solution, Cd concentrations in the older leaves decreased compared to the control, but Cd contents increased. And, the total Cd contents of seedlings remained stable after the two treatments. The distribution pattern of Cd in the S. nigrum did not change, and approximately 37.78% of the Cd content in the roots was translocated to shoots after the external Cd source was removed. Therefore, we suggest that Cd could be readily translocated from roots to shoots along with transpiration stream; however, once it reached the leaves, it could not be remobilized through the phloem to other organs.Subcellular fractionation of Cd-containing tissues indicated that about 62%~66% of Cd was localized in cell walls and 26%~32% in soluble fraction, and the lowest in cellular organelles. The distribution rates of Cd in the cell walls and cellular organelles increased with increasing external Cd levels, and larger distribution rates in the leaves of lower position than upper leaves, but decreased in the soluble fraction. And, results showed that the greatest amount of Cd was found in the extraction of 1 M NaCl and 2% HAC, particularly 1 M NaCl (40.09%~56.09%), which suggested that Cd in the leaves of S. nigrum mainly detoxified as the combination of protein and phosphates, further suggested that Cd mainly localized in cell walls. ⑤Cadmium uptake and interactions with Zinc in two species of Solanum --Solanum nigrum L and Solanum melongena L: A short-time hydroponic study.In the present study, the growth status and metal accumulation of S. nigrum and S. melongena were examined under Cd, Zn, and Cd-Zn complex polluted conditions using nutrient solution culture method. The results showed that high concentrations of Cd (200μM Cd) and Zn (500μM Zn) greatly affected the growth of S. nigrum and S. melongena seedlings, but dry biomass of part Cd-Zn treated plants were higher compared with the control plants under low concentrations of Cd-Zn. With the application of Zn in the nutrient solution, Cd concentration in the roots and leaves of S. nigrum and S. melongena seedlings increased firstly, then decreased, and reached maximum when 100μM Zn and 50μM Zn were applied, respectively (P < 0.05). Cd concentrations in the roots and shoots of S. nigrum and S. melongena seedlings were mainly determined by the Cd concentrations in the Hoagland solution, the effect of Cd-Zn interaction was limited. From the view of total heavy metal contents, shoot Cd contents of S. nigrum were 7~16-fold of roots, but Cd mainly accumulated in the roots of S. melongena seedlings.Cd application significantly influenced the shoots and roots Zn contents of S. nigrum and S. melongena seedlings, shoot Zn contents reached maximum when applied 50μM Cd, especially reached peak value 445.9μg·Plant-1 when Cd/ Zn ratio was 50/ 500μM, however, shoot Zn contents reached peak value 48.3μg·Plant-1 when Cd/ Zn ratio was 25/ 500μM. Zn concentrations in the roots and shoots of S. nigrum and S. melongena seedlings were mainly determined by the Zn concentrations in the Hoagland solution, the effect of Cd-Zn interaction on the Zn concentration of S. nigrum was limited, but significant interaction was found in the S. melongena seedlings. From the view of total heavy metal contents, shoots Zn contents of S. nigrum were 7~17-fold of roots, but shoots Zn contents were slightly higher than roots in the S. melongena seedlings.Moreover, Cd application led to a disturbance of macro- and micro-nutrient elements uptake. Cd increased K uptake in the root and shoot of S. nigrum, but had no significant effect on Na. And, Cd increased root Mg uptake, but decreased the transport of Mg from root to shoot. Moreover, low concentration of Cd increased Ca uptake in the leaves of S. nigrum. Low Cd increased Zn concentrations in the roots, stems, and leaves, but high Cd decreased them. Cu concentration in the root increased, but leaf Cu concentration increased firstly, then decreased. Furthermore, Fe concentrations in the root, stem, and leaf decreased with increasing Cd concentration in the soils; however, Cd decreased Mn concentration in the root of S. nigrum significantly.⑥Effect of Chitosan on the available contents and vertical distribution of Cd2+ in different textural soilsIn this study, an environment-friendly biopolymer- chitosan, has been adopted to remedy contaminated soils by heavy metals of Cd2+. Experimental results demonstrated that, within the first 7 d, available Cd2+ contents in three textural soils (clay, loam, and sandy soil) decreased significantly after chitosan application. Moreover, the available Cd2+ contents in soil layers of 14-16 cm and 24-26 cm were significantly reduced than that in 4-6 cm after 7 d of chitosan application. Our investigation suggested that application of 0.9 g chitosan kg-1 DW soil for 7 d could be perfect for the remediation of the soil contaminated by Cd2+.更多还原