【作者】 杨卫东；

【导师】 陈益泰；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2008， 博士

【摘要】 植物修复是用绿色植物及相联微生物清洁环境污染,作为非破坏性技术近10年已经获得很大进展。柳树用于原位修复土壤中Cd、Zn污染。旱柳、杞柳、垂柳是我国重要乡土树种,它们有高生物量,生长快,适应水湿环境等特点。本论文先对我国广泛栽培的灌木柳——杞柳对Cd和Zn积累的特点以及重要绿化树种——垂柳对Cd积累特点作了研究,最后以我国重要的乡土树种—旱柳为材料,探讨Cd对柳树光合作用、矿质元素影响、Cd-Zn交互作用以及柳树对Cd忍耐和解毒机制。本论文研究内容共有8部分组成。1.杞柳3品种微山湖、大红头、一枝笔,通过水培方法,比较杞柳品种对Cd的积累与忍耐差异。Cd处理浓度为0、10、50μmol·L^-1,处理6周。结果表明:3种品种在植物枝、根生物量以及对Cd忍耐水平和积累存在差异. Cd不同程度降低了3种枝和根生物量;Cd在枝中浓度与根中浓度比值低于1,并且随介质中Cd剂量提高,其比值降低,但Cd在枝中积累总量与Cd在根中积累总量高于1,但随介质浓度升高而降低;微山湖品种忍耐系数TIroot（以根干重表示）为0.61⁰.82;大红头品种TIroot为0.88,一枝笔品种TIroot为0.92⁰.93。添加0、100、1000μmol·L^-1 Zn,调果Zn对3杞柳品种生长参数影响、吸收和忍耐情况。结果表明:依赖Zn剂量,这些生长参数在品种之间有明显差异,在高浓度(1000μmol·L^-1)时,Zn降低杞柳3品种茎高、根长、枝和根生物量。在Zn 100μmol·L^-1时,3种杞柳枝吸收与积累Zn量最高,而根中积累量随培养介质中Zn浓度增加而升高,枝中浓度与根中浓度比值接近1。忍耐系数随介质Zn含量升高而降低。Cd处理浓度为0、10、50μmol·L^-1,处理时间为30d,评价Cd对垂柳3无性系生长参数的影响、吸收和忍耐情况。结果表明:Cd改变垂柳3无性系生长参数;在枝中Cd积累量无性系差别不明显,在10μmol·L^-1 Cd,3种无性系枝积累量>100μg?g-1,Cd主要积累于根部,并且积累量增加随介质浓度增加,在根部,无性系之间Cd吸收量明显不同,并且枝吸收Cd总量与根吸收Cd总量大于1;3种垂柳无性系对Cd忍耐系数与处理介质浓度有关,在10μmol·L^-1,忍耐系数为0.80⁰.93。2.调查Cd( 5和25μmol·L^-1)胁迫对叶相对电导率、根K+渗透、脂过氧化及抗氧化酶的变化影响。叶相对电导率和根K+渗透以及根和叶丙二醛量（MDA）没有发生显著变化;高浓度Cd诱导根超氧歧化酶（SOD）活性增强,不同浓度Cd促进叶SOD活性升高;Cd使根抗坏血酸过氧化物酶（APX）活性增加,但对叶APX活性并没有显著影响,Cd显著促进叶愈创木酚过氧化酶（POD）活性,但对根POD活性并没有显著改变,Cd并没有改变根谷胱甘肽过氧化物酶（GPX）,但显著增进叶GPX活性,根和叶GST活性随Cd浓度增加而增加。这些结果说明,叶和根细胞质膜保持较大稳定性,抗氧化酶不同程度改变,在旱柳对Cd忍耐中起重要作用。3.柳树作为植物修复Cd污染的木本植物,这主要依赖柳树生物量大,Cd的植物毒性会抑制植物光合作用,降低植物生物量,会影响提取效率。添加Cd(0、5、25、50μmol·L^-1),培养14d,分析叶绿素含量、Rubisco和PEPC活性以及游离氨基酸含量及内肽酶活性。结果发现:受Cd影响减少了总叶绿素含量及叶绿素a和叶绿素b含量,但是处理间叶绿素降低量差异不大。随着介质中Cd剂量增加,Rubisco活性逐渐降低。同时Cd也抑制根和叶PEPC活性。根中游离氨基酸含量没有变化,而叶中游离氨基酸含量增加,这种增加依赖Cd浓度;Cd不同处理根的内肽酶活性降低,但在高浓度使叶内肽酶活力增加。这些结果表明Cd干旱柳光合作用、蛋白质降解和CO2同化。4. Cd毒性抑制植物生长,干扰矿质营养代谢。本研究调查Cd对旱柳N、P、K等矿质元素吸收和利用影响。由于K、P为大量元素,它们在旱柳中的吸收与器官和Cd剂量有关。Cd胁迫并没有显著影响根对K吸收,只在低浓度(Cd=5μmol·L^-1)增加叶中K含量,在25⁵0μmol·L^-1 Cd叶中K含量没有明显变化;5⁵0μmol·L^-1 Cd抑制根对P吸收,在25-50μmol·L^-1 Cd降低叶中P含量;受Cd胁迫根中NH4+含量增加,叶NH4+含量降低;根和叶中NO-13含量都轻微上升;在5μmol·L^-1 Cd,根NR活性显著上升,其它剂量Cd没有明显改变NR活性,Cd降低叶NR活性。本研究为通过营养管理策略提高修复对Cd效率,提供理论借鉴。5.柳树已经用于植物提取Cd,这需要忍耐一定剂量Cd,抗坏血酸—谷胱甘肽循环（ascorbate-glutathione cycle, AsA-GSH cycle）在植物忍耐Cd胁迫中起重要作用。分析根和叶还原型抗坏血酸（ascorbate, AsA）、氧化型抗坏血酸（脱氢抗坏血酸, dehydroascorbate, DHA）,还原型谷胱甘肽（reduced glutathione, GSH）,氧化型谷胱甘肽（oxidized glutathione, GSSG）及相关酶变化。Cd显著降低根AsA含量,增进叶AsA积累;5μmol·L^-1Cd促使根DHA含量升高,25μmol·L^-1Cd降低根和叶DHA含量,并且使根和叶AsA/DHA比率上升。5μmol·L^-1Cd促进根和叶GSH积累,25μmol·L^-1抑制根GSH含量积累,Cd使根和叶GSSG含量降低,在5μmol·L^-1时根和叶GSH/GSSG比率高于对照。在Cd处理下,Cd促进叶抗坏血酸过氧化物酶（ascorbate peroxidase, APX）不同程度增强,根APX活性没有显著变化,根中单脱氢抗坏血酸还原酶（monodehydroascorbate reductase, MDHAR）在5μmol·L^-1活性最高,叶MDHAR活性随介质Cd浓度增加显著增加,根和叶的脱氢抗坏血酸还原酶（dehydroascorbate reductase, DHAR）活性显著高于对照,根GR仅在5μmol·L^-1Cd活性显著增加,叶GR活性高于对照;同对照相比,谷胱甘肽过氧化酶（glutathione peroxidase, GPX）和谷胱甘肽转硫酶（glutathione-S-transferase, GST）活性也不同程度上升。以上结果表明在Cd胁迫下抗坏血酸—谷胱甘肽循环参与旱柳对Cd忍耐。6. Cd在旱柳叶和根亚细胞积累及存在化学形式。随着培养介质Cd浓度增加叶和根各亚细胞组份Cd含量增加。Cd主要积累于叶细胞壁、叶绿体和可溶性部分,叶中FI（细胞壁）占65⁶9%,而FII（叶绿体）为14²2%,FIV（可溶性部分）为6.8⁷.7%,仅少量Cd发现于FIII（细胞器）中,Cd在叶亚细胞组份中积累顺序为FI>FII>FIV>FIII;而根中Cd主要积累于细胞壁和可溶性部分,其中Cd在根细胞壁中含量为59⁶6%,可溶性部分为14²5%,而Cd在根亚细胞组份中积累顺序为FI（细胞壁）>FIV（可溶性部分）>FII（质体）>FIII（细胞器）。Cd在旱柳以不同的化学形态存在,大部分为HCl、NaCl、HAC提取态,极少部分为乙醇和水,其中叶和根中Cd FHCl和FNaCl提取态>30%,Cd在叶和根中5种溶剂提取态顺序为FHCl>FNaCl>FHAC>Fwater>Fethanol。这些结果与旱柳对Cd脱毒与忍耐相关。7.柳树是具有修复潜力的木本植物,尤其适合于植物提取Cd、Zn,重金属往往是多种元素组成复合污染。调查了Cd―Zn交互作用对金属在枝根积累与分配影响。Zn、Cd在根部表现为互相竞争,即Zn抑制Cd吸收,Zn吸收也受Cd抑制;而在枝中,Cd抑制Zn积累,但是Zn对Cd吸收积累的影响依赖培养介质中Cd、Zn浓度。Zn、Cd交互作用改变它们在枝根中分配,这种改变仍与它们在介质中浓度相关,Cd、Zn主要积累于根部。在交互作用中,Cd-Zn单一或结合处理,干扰了Mn、Cu、Fe、Mg、Ca等矿质元素在枝根中含量,对K没有明显改变。这些结果表明Cd-Zn对旱柳有显著交互作用,影响二者在植物中积累、分配和对矿质元素吸收。8.低分子量含巯基醇化合物在植物解毒和积累中起重要作用。测定根和叶Cys、非蛋白巯基（Non-protein thiols, NPTs）、蛋白质巯基（Protein thiols, PTs）以及总巯基（Total thiols, TTs）含量,以及γ-GCS活力,了解低分子量巯基醇在旱柳对Cd忍耐和解毒中作用。发现不同剂量Cd处理下诱导NPTs、PTs和TTs产生,这表明低分子量巯基参与旱柳对Cd解毒与忍耐,有助于旱柳对Cd植物提取。由于Cd抑制旱柳根和叶γ-GCS活性,旱柳脱毒并不是由GSH生物合成引起的。更多还原

【Abstract】 Phytoremediation, the use of plants and their associated microbe for environmental cleanup, has gained advance in the past 10 years as cost-effective, noninvasive alternative technology. Willows were used for the in situ decontamination of soils polluted with Cd, Zn. Salix matsudana, S. integra and S. babylonica are native to china, and they hold high biomass, fast growth and good tolerance of wet soils. S. integra and S. babylonica for Cd, S.integra for Zn metal resistance and accumulation were studies in this paper. S. matsudana was used as material, effects on photosynthesis and mineral nutrition under Cd stress, tolerance and detoxification mechanisms to Cd were investigated. This paper was consisted of 8 parts.1. Three varieties of S. integra in hydroponic experiments for their metal resistance and accumulation were investigated. Plants were exposed to Cd (0, 10, 50μmol·L^-1) for 6 weeks. Plant biomass, Cd tolerance and accumulation pattern in shoots and roots varied between varieties. 10μmol·L^-1 and 50μmol·L^-1 Cd treatments reduced the dry mass of shoots and roots, the leaf: root ratios for Cd in all varieties were <1, But total amount of Cd in shoot: in root were highly significantly >1, ratios of total amount of Cd decreased with Cd level in the medium. Tolerance index （TIroot） of Weshanhu variety was 0.62⁰.82, Dahongtou variety had a TIroot of 0.88, TIroot of Yizhibi was about 0.92⁰.93.Varieties of S. integra on zinc were evaluated with three Zn levels (0, 100, 1000μmol·L^-1) for 41d. Stem heighten, root length, biomass of shoots and roots of three varieties decreased significantly with high Zn concentration (1000μmol·L^-1). At 100μmol?L Zn, uptake and accumulation of three varieties shoots on Zn is highest. Zinc concentration in roots was elevated with Zn level in the medium, ratio of shoots to roots Zn concentration was near to 1 at 100μmol·L^-1, Index tolerance of three warieties reduced with Zn concentration.Cadmium uptake and tolerance potential of three weeping willow （Salix babylonica） clones were evaluated with three Cd level (0, 10, 50μmo·L^-1). There were not significantly difference on Cd concentration of shoots among clones, at 10μmol·L^-1, Cd concentration in shoots of three clones was >100μg?g-1, and total amount of Cd in shoots to Cd in roots ratio was >1. Three clones tolerance index were related to Cd concentration in medium. 2. Salix matsudana exposed to 5 and 25μmol·L^-1 of Cd for 21d in hydroponic culture were analyzed with level of lipid peroxide, ion leakage and antioxidative enzymes in roots and leaves. Electrical conductivity of leaves, K+ efflux of roots and level of lipid peroxidation of roots and leaves were not significantly influenced under Cd treatment. Changes in antioxidant enzyme levels in roots and leaves were examined, high levels of Cd(25μmol·L^-1) enhanced significantly the activity of SOD in roots, Cd-induced SOD in leaves were showed more at 25μmol·L^-1 than 5μmol·L^-1; the activity of APX in roots was observed raised compared with controls, but APX of leaves was not significantly changed; POD in leaves increased but in roots remained almost unmodified; GPX in roots was not significantly altered, but in leaves induced markedly increase with Cd levels in the medium; upon exposure to 5μmol·L^-1 and 25μmol·L^-1 Cd, GST prominently increased in roots and leaves. These results suggested that cell membranes in roots and leaves had considerably stability, and changed activity of antioxidative enzymes in Cd-treated had antioxidative function in Salix matsudana to cadmium stress.3. Willows have been shown to be suited for phytoextaction of cadmium, which is depend on large biomass, Cd is phototoxic to plants, Cd depresses photosynthesis, effects plants productivity, reduces phytoextraction rate of Cd. Salix matsudana was hydroponic culture for 14d with CdCl2 (0, 5, 25, 50μmol·L^-1), Chlorophyll, Rubisco, PEPC, free amino acid and endopetidase were analyzed. Total Chlorophyll, Chlorophyll a and Chlorophyll b were decreased in Cd-treated plant, Rubisco activity reduced with Cd concentration in the medium, Cd decreased PEPC activity in roots and leaves,Free amino acids of roots remained no change, but free amino acids of leaves increased depending on Cd concentration; endopeptidase of roots were hindered at different dose of Cd, endopetidase of leaves increased at higher dose of Cd. The result showed that photosynthesis of Salix matsudana was disturbed under Cd stress.4. Cd toxicity is associated with growth inhibition and imbalances in many macro- and micronutrient levels. Effect on uptake of mineral nutrient in S. matsudana under cadmium stress was studied at four Cd levels (0, 5, 25, 50μmol·L^-1) for 14d. K content of roots remained no change in Cd-treated plant, Except that only at 5μmol·L^-1 Cd-treated plant K content of leaves was increased, no significant effect on K content of leaves was found at 25⁵0μmol·L^-1 Cd. P uptake in roots was inhibited at all Cd levels, at 25⁵0μmol·L^-1 Cd treatment decreased P content in leaves. Ammonium content of roots was elevated, but of leaves was decreased. Nitrate Content of roots and leaves showed a slight increase compared with the control. Only at lowest level (5μmol·L^-1) Cd NR activity of roots was increased, NR activity of roots didn’t change at 25⁵0μmol·L^-1, compared to the control, NR activity of leaves was reduced in all Cd levels. These results suggested that the possibility to improve nutrient enhance to phytoextract Cd in willows.5. Willows have been shown to be promising for Cd phytoextraction, which requires tolerance to Cd stress, the ascorbate-glutathione cycle has been shown to be of great importance in Cd stress, an operation of ascorbate-glutathione cycle enzymes was investigated in roots and leaves of Salix matsudana grown on hydroponics containing 0, 5, 25μmol·L^-1 CdCl2 for 28d. AsA content of roots decreased but AsA in leaves increased with increase in Cd level, DHA content of roots increased at 5μmol·L^-1 Cd, however, DHA content of leaves decreased at 25μmol·L^-1 Cd, significant enhancement of AsA /DHA ratio in roots and leaves was observed at 25μmol·L^-1. GSH content of roots and leaves was increased at 5μmol·L^-1 Cd, 25μmol·L^-1Cd inhibited GSH of roots, GSSG content decreased in roots and leaves compared to control, but at 25μmol·L^-1 the ratios of GSH to GSSG was higher in roots and leaves as compared to the control. APX increased in leaves but didn’t change significantly in roots respect to the controls, MDHAR of roots was elevated at 5μmol·L^-1, and MDHAR of leaves was markedly increased at all concentrations of Cd, DHAR in roots and leaves significantly increased compared with the controls, The results suggested that the ability of Salix matsudana to regulate the enzyme antioxidant system during different conditions might be an important attribute linked to cadmium tolerance.6. The subcellular distribution and chemical of cadmium in Salix matsudana grown in nutrient solution containing 10 and 30μmol·L^-1 were investigated. Increased Cd levels in the medium caused a significant increase of Cd concentration in all fractions of leaves and roots, most of the accumulated Cd was isolated to the cell wall of leaves （65⁶9%）,high Cd levels were also found in the crude chloroplastic fraction （14²2%）, the cytosolic fraction accumulated 6.8⁷.7% of Cd, the fraction containing the lowest level of Cd was the organelle fraction. The largest proportion of Cd was located in the walls of roots （59⁶6%）,about 14²5% of the total Cd contained in the root cell were accumulated in the soluble fraction, Cd concentration of the fractions of roots decreased in the order: FI （cell walls）>FIV （soluble）>FII （trophoplast）>FIII （organelles）. The Cd concentrantion was bound to the different chemical forms, chemical forms of Cd （>30%） were extracted with 0.6 mol·L^-1 HCl and 1 mol·L^-1 NaCl, The leaves and roots, the greatest amount of Cd was found in extraction solution of 0.6 mol·L^-1 HCl and 1 mol·L^-1 NaCl, followed by 2% HAC, and lowest in extraction of 80% ethanol or d H2O. There was a distinct difference among clones in Cd concentration in subcellular and chemical forms. These results would suggest that Salix matsudana has metabolic mechanism related to cadmium detoxification and tolerance.7. Willows were suitable for Cd and Zn in phytoextraction, contamination contained multiple heavy metals rather than simple. Salix matsudana were grown 30 days in nutrient solution at two concentrations of cadmium （0, 5, 10 and 25μmol·L-1） and zinc （0, 25 and 50μmol·L-1） singly and in factorial combination, Effects of Cd?Zn interactions on metals accumulation and distribution were investigated. Cd uptake was inhibited by Zn and Zn uptake was inhibited by Cd in roots, the results revealed a competitive interaction between Cd and Zn in roots; Zn accumulation was hindered by Cd in shoots, influence of Zn on Cd contents in shoots were dependent on concentrations of those metals in the medium. Cd-Zn interactions altered their accumulation and distribution in shoots and roots, these changes were related to Cd and Zn level in the medium, cadmium and zinc concentrated mainly in the roots. At Cd-Zn interactions, Cd, Zn single or combination disturbed Mn, Cu, Fe, Mg and Ca contents in shoots and roots; K was not significantly altered in treatments. The results showed interactions of Zn and Cd affected those metals uptake and distribution in shoots and roots of S. matsudana.8. Low molecular weight thiol-containing compounds have been reported to play an important role in metal detoxification and accumulation in some higher plants. Relationships between detoxification response to Cd and low molecular weight thiols in S. matsudana were investigated exposed to Cd (5, 25, 50, 100μmol·L^-1) for 14d. At 25μmol·L^-1 Cys content increased significantly, Cys content of leaves didn’t significantly changed. The concentration of NPTs, PTs and TTs showed a very strong increase with Cd level in the mediums. Only 50μmol·L^-1γ-GCS activity of leaves was markedly elevated compared to the control,γ-GCS of roots was reduced. These results showed that low macular weight thiols contributed to detoxification in S. matsudana, but didn’t depend on GSH biosynthesis.更多还原