铅在茶树体内的分布及化学形态特征

采用水培试验,结合亚细胞组分分离和化学形态提取技术,研究了茶树品种龙井43和迎霜不同器官的Pb含量,及其在根系亚细胞中的分布和化学结合形态.结果表明： 在Pb胁迫下,两个品种茶树根系的形态特征不同;龙井43新叶中Pb含量随营养液Pb浓度的提高显著增加,而迎霜的变化不显著.两个品种茶树根系亚细胞中Pb的含量分布及结合形态均存在差异,低浓度Pb处理下, 龙井43根亚细胞组分（除可溶物质外）中Pb含量均低于迎霜;而高浓度Pb处理下,龙井43根亚细胞组分（除细胞壁外）中Pb含量均高于迎霜;龙井43根中醋酸(HAc)提取态的Pb含量比例最高,其他化学形态的高低顺序为NaCl提取态>HCl提取态/H₂O提取态>乙醇提取态;而迎霜根中NaCl提取态Pb成分比例最高,其他化学形态的高低顺序为HAc提取态>HCl提取态/H₂O提取态>乙醇提取态.迎霜对Pb毒害的耐受能力强于龙井43.     

霞多丽苗木中镉的积累、亚细胞分布及化学存在形态

以一年生盆栽葡萄品种霞多丽(Vitis vinifera L.cv.Chardonnay)自根苗及SO4砧嫁接苗为试材,采用CdCl2和CaCl2根部灌入的方法,研究了镉在霞多丽植株内的亚细胞分布和存在形态,以及外源添加氯化钙对植株镉吸收的影响.结果表明:大部分镉积累在霞多丽自根苗及嫁接苗的地下部器官;4 mmol·L-1浓度的CdCl2处理下,镉在自根苗根及根颈部分的积累量占77.1％,在叶片中的积累量占1.4％,而嫁接苗中镉在嫁接口以下部分的积累量高达93.9％,叶片中的积累量仅占0.1％;5 mmol·L-1外源钙缓解了植株对镉的吸收积累,而10mmol ·L-1外源钙则显著增加了植株对镉的吸收积累.镉在根系和叶片中的亚细胞分布规律为细胞壁＞可溶性部分＞细胞器,且在细胞壁中积累50％以上;镉在根系中主要以氯化钠提取态存在,其次为乙酸提取态,去离子水提取态含量最少.随着镉处理浓度的增加,各提取态含量有所变化.     

Identification of rice varieties with high tolerance or sensitivity to lead and characterization of the mechanism of tolerance

Pb inhibits plant growth. To study Pb tolerance in rice (Oryza sativa), we screened 229 varieties for Pb tolerance or sensitivity. Three-day-old seedlings were treated for 12 d with 20 microM Pb solution. Based on the dry weight of the root, three Pb-tolerant (var CH-55, var KH-2J, var Kumnung) and three Pb-sensitive (var Aixueru, var C-9491, var Milyang23) rice varieties were selected. The root biomasses of the tolerant varieties were approximately 10-fold higher than those of the sensitive ones. The greatest morphological difference between the two groups was in the growth of the adventitious roots, as tolerant lines were able to develop adventitious roots after 6 d of Pb treatment, whereas sensitive ones did not develop any even after 15 d. The growth of adventitious roots in the tolerant varieties was dependent on a mechanism, whereby Pb was altered to a form that cannot be taken up by the tissue, because (a) the solution in which the tolerant varieties of rice had grown still contained Pb but nevertheless did not affect the root growth of new rice seedlings, and (b) the adventitious roots of tolerant seedlings developed in Pb solution contained little Pb. The oxalate content in the root and root exudate increased upon Pb treatment in the tolerant varieties, whereas the opposite was observed for the sensitive ones. Oxalate added to the growth solution ameliorated the inhibition of root growth by Pb. These results suggest that compounds such as oxalate secreted from the root may reduce the bio-availability of Pb, and that this may constitute an important Pb tolerance mechanism in the tolerant rice varieties studied here.     

不同类型镉积累水稻细胞镉化学形态及亚细胞和分子分布

利用水培试验结合亚细胞组分分级分离和凝胶过滤等技术,研究了水稻根和叶中镉的化学结合形态及其亚细胞和分子分布,比较了低镉积累品种“广源占No.3”和高镉积累品种 “珍桂矮”的差异.结果表明：随着营养液中镉浓度的升高,根和叶亚细胞镉含量显著上升,大部分镉积累在细胞壁（F_Ⅰ）和细胞可溶部分（F_Ⅲ）.高镉积累品种“珍桂矮”根和叶中可溶部分镉含量显著高于低镉积累品种“广源占No.3”.根和叶各种形态镉中,以氯化钠提取态占优势,其次是醋酸提取态,盐酸提取态镉含量最低.与“广源占No.3”相比,“珍桂矮”中迁移性较强的去离子水和乙醇提取态镉比例较高.凝胶过滤结果表明,两种类型的水稻可溶部分镉的出峰位置与样品流份中可溶性蛋白的出峰位置大致相同.可溶部分中的镉大多与分子量为3kD的物质结合,属于植物鳌合肽（PCs）或低分子量物质.“广源占No.3” 根系中镉与PCs配合的组分（Cd-PCs）含量远小于“珍桂矮”.“广源占No.3”细胞可溶部分较低的镉含量以及根系中较少的Cd-PCs形成量,降低了镉的移动及其向地上部转运的可能性.     

褪黑素对水稻镉积累及其化学结合形态的影响

研究了添加外源褪黑素对水稻幼苗中Cd积累以及水稻体内Cd化学形态分布的影响.结果表明: Cd胁迫显著降低水稻幼苗地上部和根部的生物量,并且显著降低水稻叶片的叶绿素含量.适宜的外源褪黑素添加能明显提高Cd胁迫下水稻的地上部和根部生物量,降低水稻地上部和根部Cd含量.当Cd胁迫浓度为5 μmol·L^-1时,添加20 μmol·L^-1褪黑素使水稻地上部和根部Cd含量分别比对照处理降低48.4%和16.9%,添加100 μmol·L^-1褪黑素水稻地上部和根部Cd含量分别降低67.5%和47.9%.添加外源褪黑素也显著降低了水稻体内Cd的转运效率.当Cd胁迫浓度为20 μmol·L^-1时,添加20 和100 μmol·L^-1褪黑素使水稻Cd的转运效率分别比对照降低24.4%和46.8%.通过逐步提取法对水稻幼苗Cd的化学结合形态进行分析发现,添加外源褪黑素使水稻体内氯化钠提取态Cd的比例提高,而水溶态Cd、乙醇提取态Cd的比例明显降低,说明添加褪黑素可促进水稻体内移动性较强的Cd形态向移动性较弱的Cd形态转移,从而降低水稻对Cd的吸收和转运.     

低温对茶树叶片膜脂脂肪酸和蛋白质的影响

本文研究低温胁迫过程中龙井43和大叶云峰叶片膜脂脂肪酸的变化。结果发现,低温下不同品种茶树叶片膜脂脂肪酸配比变化趋势不同,较抗寒的龙井43,不饱和脂肪酸指数(IUFA)和亚麻酸(183)比例随低温期间不同阶段呈现出"低-高-低"的变化趋势;而不抗寒的大叶云峰的变化趋势无明显规律性。此外,还研究了越冬过程中龙井43叶片可溶性蛋白和膜蛋白的变化,发现低温期间龙井43叶片可溶性蛋白含量和组分基本稳定,而膜蛋白含量在低温胁迫时大幅度上升,且经低温诱导出现了46KD、38KD两种新的蛋白组分,并在温度升高后消失。     

Zinc distribution and zinc-binding forms in Phragmites australis under zinc pollution

The influence of zinc (Zn) on physiological and biochemical parameters was studied to elucidate the mechanism of Zn resistance in Phragmites australis. Zn concentrations in roots, stems and leaves increased with exogenous Zn concentration, while Zn content in roots was much higher than in shoots. X-ray microanalysis was used to reveal compartments in which Zn accumulated in root cortex. Zinc concentrations followed a gradient with the sequence: intercellular space>cell wall >vacuole >cytoplasm, indicating that most Zn was immobilized in the apoplast or sequestered into the vacuolar lumen. Sequential extraction of various Zn chelates revealed that the ratio of Zn extracted with different extraction media was markedly different. Ethanol, HAc (acetic acid) and NaCl-extractable Zn were dominant in both roots and leaves of P. australis. Zn-binding protein fractions were found in the roots and leaves after gel filtration chromatography, among which a polypeptide with an apparent molecular mass of 14kDa bound Zn most effectively. Two newly synthesized polypeptides of 58 and 45kDa appeared under Zn pollution, whereas a prominent fraction of 72kDa disappeared. The involvement of Zn distribution in plant tissues, subcellular compartments and chelates and Zn-inducing proteins in the acclimation mechanism of P. australis to Zn pollution is discussed.     

铅污染下芦苇体内铅的分布和铅胁迫相关蛋白

用Pb(NO3 ) 2 10mmol/L处理芦苇幼苗 7d后 ,检测铅在其不同器官、细胞不同区隔和不同化学状态之间的分布以及铅胁迫相关蛋白。结果显示 ,受铅污染后芦苇幼苗根、地下茎、茎和叶片的铅含量增加 ,它们的大小顺序为根 >地下茎 >茎 >叶片。细胞不同部分铅含量的大小顺序为细胞间隙 >细胞壁 >液泡 >细胞质。在根部和叶片中 ,均以活性较低的醋酸可提取态铅和盐酸可提取态铅占优势。在铅胁迫下 ,从芦苇根和叶片中检测出一些铅结合蛋白。另外 ,铅除诱导芦苇合成一种新的不结合铅的蛋白质外 ,还导致一种分子量大约为 72 0 0 0的蛋白质消失。芦苇抗铅胁迫有以下几种机制 :根部比地上部分积累较多的铅 ;铅在体内形成难溶性化合物 ;铅沉淀在质外体内 ;形成铅结合蛋白质和诱导蛋白     

Lead bioaccumulation,subcellular distribution and chemical form in sugarcane and its potential for phytoremediation of lead-contaminated soil

Abstract

Sugarcane is a promising species for lead (Pb) phytoextraction due to its large biomass and high tolerance toward Pb content. To understand the mechanisms involved in Pb tolerance and detoxification and its potential for phytoremediation in sugarcane, the bioaccumulation, subcellular distribution, and chemical forms of Pb in different tissues were investigated through pot cultivation sugarcane with increasing Pb concentrations in the present study. Results showed that sugarcane could tolerate high concentrations of Pb (up to 1250.0–1750.0?mg/kg); the Pb content in roots and shoots increased with increasing Pb concentration. A large amount of Pb content was stored in roots. The subcellular distribution of Pb in sugarcane revealed that the majority of Pb was bound to the cell wall. Meanwhile, the greatest amount of Pb was extracted by 2.0% acetic acid and 0.6?mol/L HCl, which indicated that most of Pb was combined with undissolved phosphate and oxalate. These results implied that the Pb formation of undissolved salts and compartmentalization in the cell wall may be a key strategy for Pb detoxicity and tolerance in sugarcane.     

茶树叶片蛋白双向电泳体系建立与应用

为开展茶树Camellia sinensis 低温和干旱胁迫下差异蛋白的分离和鉴定,以抗逆性较强的茶树品种‘迎霜’为试材,通过对提取方法、IPG 胶条pH 范围、上样量、分离胶浓度、染色方法的比较,筛选适用于茶树叶片的蛋白质双向电泳体系。结果表明,采用TCA-丙酮法或Tris-HCl 法提取叶片总蛋白,选用17 cm pH 4~7IPG 胶条用于等电聚焦,选择1.6~2.2 mg 上样量、13.5%聚丙烯酰胺凝胶进行分离,随后通过高敏考马斯亮蓝R-250 法染色;最终,叶片各分子量的蛋白充分分离,获得的双向电泳图谱分辨率高、背景清晰、重复性好,适用于‘迎霜’低温和干旱胁迫下叶片差异蛋白分析。     

铜尾矿库区土壤与植物中重金属形态分析

对铜陵铜尾矿区土壤和植物中重金属形态进行了研究.结果表明,尾矿库区种植地极端贫瘠,有机质含量仅2.6～.8 g·kg^-1,而土壤Cu、Cd、Pb、Zn含量皆高于对照土壤,其中Cu含量达809.30～1 39.4 mg·kg^-1,Cd含量达3.2～6.3 mg·kg^-1,达到对照土壤30～60倍.结缕草和三叶草体内重金属含量与土壤重金属交换态及有机结合态含量成正相关,与碳酸盐结合态、铁锰氧化物结合态成显著或极显著负相关,与矿物态含量相关性不显著.在两种优势植物中,Cu、Zn、Pb均以活性较低的醋酸提取态、盐酸提取态和残渣态为主;Zn在根系和茎叶中,NaCl提取态占有较大比例,而Cd均以NaCl提取态为主.     

Antioxidative defense to lead stress in subcellular compartments of pea root cells

Lead, similar to other heavy metals and abiotic factors, causes many unfavorable changes at the subcellular and molecular levels in plant cells. An increased level of superoxide anion in Pisum sativum root cells treated with 1 mM Pb(NO3)2 evidenced oxidative stress conditions. We found increased activities of enzymatic components of the antioxidative system (catalase and superoxide dismutase) in the cytosol, mitochondrial and peroxisomal fractions isolated from root cells of Pisum sativum grown in modified Hoagland medium in the presence of lead ions (0.5 or 1 mM). Two isoenzyme forms of superoxide dismutase (Cu,Zn-SOD and Mn-SOD) found in different subcellular compartments of pea roots were more active in Pb-treated plants than in control. Increased amount of alternative oxidase accompanied by an increased activity of this enzyme was found in mitochondria isolated from lead-treated roots. These results show that plants storing excessive amounts of lead in roots defend themselves against the harmful oxidative stress caused by this heavy metal.     

复合胁迫下茭白体内镉、铅的亚细胞分布和植物络合素的合成

以茭白（Zizania latifoliaTurcz.）的单季茭品种‘蒋墅茭’和双季茭品种‘葑红早’为试材,进行Cd2＋、Pb2＋的单一及复合胁迫处理,测定了茭白根系和叶片中的非蛋白巯基（NPT）、谷胱甘肽（GSH）、植物络合素（PCs）的含量,同时测定了茭白植株各亚细胞组分中Cd2＋、Pb2＋的积累量,以探讨茭白对重金属镉、铅胁迫的耐性机理。结果表明：Cd2＋、Pb2＋的单一及其复合胁迫均能促进两茭白品种根系和叶片中NPT、GSH、PCs的含量及茭白各亚细胞组分中Cd2＋、Pb2＋积累量的显著增加;复合胁迫时两茭白品种的NPT、GSH、PCs含量及各亚细胞组分中Cd2＋、Pb2＋的积累量均高于单一胁迫,茭白的不同部位间,以根系中的NPT、GSH、PCs含量显著高于叶片;茭白各亚细胞组分中Cd2＋、Pb2＋的积累量表现为：细胞壁高于原生质体,而可溶性部分高于细胞器。     

Characteristics of Cd accumulation and distribution in two sweet potato cultivars

In this study, we compared the chemical forms and subcellular distribution of Cd in high-Cd (X16) and low-Cd (N88) sweet potato cultivars through hydroponic experiments and examined the Cd distribution in their roots by histochemical staining. The results showed that inorganic and pectate/protein-integrated Cd predominated in the leaves, and Cd concentrations were significantly higher in X16 than in N88. However, in the roots, Cd was mostly integrated with pectate and protein, and Cd concentration was higher in N88 than in X16. It was mainly stored through vacuolar sequestration and cell wall binding. In the leaves and stems, Cd concentrations in all subcellular fractions were higher in X16 than in N88; the opposite was observed in the roots. In X16, Cd was mostly accumulated in the root stele, and its Cd translocation factor was higher than that of N88. Overall, the subcellular fractions of X16 roots retained less Cd than N88 roots, and more Cd entered the root stele of X16 and subsequently moved to the shoots. The higher amounts of inorganic, water-soluble, and pectate/protein-integrated Cd with high mobility in the shoots of X16 than in N88 might facilitate Cd remobilization to other tissues, but this needs to be further studied.     

Variations in Metal Tolerance and Accumulation in Three Hydroponically Cultivated Varieties of Salix integra Treated with Lead

Willow species have been suggested for use in the remediation of contaminated soils due to their high biomass production, fast growth, and high accumulation of heavy metals. The tolerance and accumulation of metals may vary among willow species and varieties, and the assessment of this variability is vital for selecting willow species/varieties for phytoremediation applications. Here, we examined the variations in lead (Pb) tolerance and accumulation of three cultivated varieties of Salix integra (Weishanhu, Yizhibi and Dahongtou), a shrub willow native to northeastern China, using hydroponic culture in a greenhouse. In general, the tolerance and accumulation of Pb varied among the three willow varieties depending on the Pb concentration. All three varieties had a high tolerance index (TI) and EC50 value (the effective concentration of Pb in the nutrient solution that caused a 50% inhibition on biomass production), but a low translocation factor (TF), indicating that Pb sequestration is mainly restricted in the roots of S. integra. Among the three varieties, Dahogntou was more sensitive to the increased Pb concentration than the other two varieties, with the lowest EC50 and TI for root and above-ground tissues. In this respect, Weishanhu and Yizhibi were more suitable for phytostabilization of Pb-contaminated soils. However, our findings also indicated the importance of considering the toxicity symptoms when selecting willow varieties for the use of phytoremediation, since we also found that the three varieties revealed various toxicity symptoms of leaf wilting, chlorosis and inhibition of shoot and root growth under the higher Pb concentrations. Such symptoms could be considered as a supplementary index in screening tests.     

茶树脂氢过氧化物裂解酶基因CsiHPL1的克隆及表达

根据茶树基因CsiHPL1的cDNA序列设计引物,采用RT-PCR方法从茶树品种龙井43′中克隆了CsiHPL1序列,并分析了CsiHPL1在生物和非生物胁迫下的诱导表达情况及亚细胞定位。结果表明,CsiHPL1包含一个1 476 bp的最大开放阅读框,编码491个氨基酸,预测为13-HPL基因。Real-Time PCR分析结果表明,CsiHPL1的表达受到茶尺蠖取食、机械损伤和茉莉酸的诱导;构建了CsiHPL1与绿色荧光蛋白（GFP）基因的重组表达载体,经农杆菌瞬时转化烟草叶片,利用激光共聚焦显微镜在叶绿体中观察到GFP荧光,表明CsiHPL1编码的蛋白质为叶绿体蛋白质。     

香蒲对不同浓度Pb~(2+)胁迫的生理应答及其细胞超微结构变化

通过室内水培试验,研究了不同浓度Pb2+(0、0.25、0.50、1.00和2.00mmol·L-1)胁迫对东方香蒲根和叶中Pb含量、叶绿素含量、丙二醛(MDA)含量、抗氧化酶(SOD、CAT和POD)活性以及亚细胞结构的影响。结果显示:(1)随着外源Pb2+浓度的增加,Pb在香蒲根和叶中的积累量均显著高于对照,且Pb在根中的含量明显高于叶中,并与外源Pb2+浓度呈显著正相关关系。(2)香蒲叶片中的叶绿素a和叶绿素b含量随着外源Pb2+浓度的增加呈先升后降趋势,均在处理浓度为0.50mmol·L-1时达到峰值。(3)胁迫处理叶片的MDA含量与对照相比变化不显著,但根中MDA含量呈显著下降趋势。(4)叶片中SOD活性在1.00mmol·L-1 Pb2+处理时达到峰值,然后下降,但始终高于对照,CAT和POD活性则均低于对照组;根中SOD活性除1.00mmol·L-1 Pb2+处理组外均显著低于对照组,CAT和POD活性分别在0.25和0.50mmol·L-1 Pb2+处理时达到峰值,然后随处理Pb2+浓度升高而下降。(5)电镜观察发现,Pb2+胁迫使香蒲叶细胞中叶绿体被膜破裂,类囊体膨胀、破损;根和叶细胞中的线粒体被膜均破裂、内腔空泡化,细胞核核膜破损、核仁消失、染色质凝集。研究表明,Pb2+胁迫致使东方香蒲根、叶生理代谢失衡,亚细胞结构出现不可逆的损伤,这为从分子水平研究Pb2+作用的具体机理以及香蒲在重金属污染修复中的应用提供了依据。     

茶树CsPLK基因的鉴定与表达分析

茶树中富含茶氨酸、儿茶素和咖啡碱等重要功能成分,具有较高的价值功效,茶树在生命周期中经常遭受逆境胁迫,维生素B6(VB6)在植物体内参与逆境应答,吡哆醛激酶(pyridoxal kinase,PLK)是VB6补救途径中的关键酶。为进一步了解PLK在茶树生物合成中的功能和作用机理,该研究基于茶树基因组数据库,以龙井43为材料,采用逆转录PCR(RT-PCR)的方法从茶树中克隆出CsPLK的基因。结果表明:该基因序列长为1 179 bp,编码393个氨基酸; CsPLK蛋白和已知物种中PLK蛋白具有较高的同源性,都是核糖激酶超家族成员;通过构建pET-CsPLK载体进行原核表达,并鉴定出重组蛋白有很强的催化活性;组织表达特异性分析表明,叶中的表达量比茎、根的高,在根中最低;荧光定量PCR表示,低温诱导CsPLK上调表达,干旱诱导CsPLK下调表达,发现该基因在茶树中有明显的逆境应答,推测CsPLK在茶树的生长发育、逆境胁迫发挥重要作用。     

Distribution of polycyclic aromatic hydrocarbons in subcellular root tissues of ryegrass (Lolium multiflorum Lam.)

Background

Because of the increasing quantity and high toxicity to humans of polycyclic aromatic hydrocarbons (PAHs) in the environment, several bioremediation mechanisms and protocols have been investigated to restore PAH-contaminated sites. The transport of organic contaminants among plant cells via tissues and their partition in roots, stalks, and leaves resulting from transpiration and lipid content have been extensively investigated. However, information about PAH distributions in intracellular tissues is lacking, thus limiting the further development of a mechanism-based phytoremediation strategy to improve treatment efficiency.

Results

Pyrene exhibited higher uptake and was more recalcitrant to metabolism in ryegrass roots than was phenanthrene. The kinetic processes of uptake from ryegrass culture medium revealed that these two PAHs were first adsorbed onto root cell walls, and they then penetrated cell membranes and were distributed in intracellular organelle fractions. At the beginning of uptake (< 50 h), adsorption to cell walls dominated the subcellular partitioning of the PAHs. After 96 h of uptake, the subcellular partition of PAHs approached a stable state in the plant water system, with the proportion of PAH distributed in subcellular fractions being controlled by the lipid contents of each component. Phenanthrene and pyrene primarily accumulated in plant root cell walls and organelles, with about 45% of PAHs in each of these two fractions, and the remainder was retained in the dissolved fraction of the cells. Because of its higher lipophilicity, pyrene displayed greater accumulation factors in subcellular walls and organelle fractions than did phenanthrene.

Conclusions

Transpiration and the lipid content of root cell fractions are the main drivers of the subcellular partition of PAHs in roots. Initially, PAHs adsorb to plant cell walls, and they then gradually diffuse into subcellular fractions of tissues. The lipid content of intracellular components determines the accumulation of lipophilic compounds, and the diffusion rate is related to the concentration gradient established between cell walls and cell organelles. Our results offer insights into the transport mechanisms of PAHs in ryegrass roots and their diffusion in root cells.     

茶树原位转化方法的建立

解决茶树缺乏高效、稳定的遗传转化体系问题,建立了一种基于根癌农杆菌介导的茶树原位转化转基因方法,为茶树基因功能研究和种质创新奠定坚实的基础。以茶树品种‘铁观音’、‘白叶1号’和‘龙井43’的实生幼苗为受体材料,进行去顶芽和腋芽处理。利用根癌农杆菌菌液侵染实生苗伤口,通过抗性筛选获得再生芽,经分子生物学鉴定后,采用短穗扦插法获得茶树转基因植株。结果表明,再生芽中GUS(β-glucuronidase)和HYG(hygromycin)标记基因经多次PCR检测均为阳性,经测序验证PCR产物序列与标记基因序列一致。‘铁观音’、‘白叶1号’和‘龙井43’的转化率分别为8.14%、2.99%和2.53%。建立了不依赖茶树组织培养的原位转化转基因体系,具有操作简便、转化率高、成本低、试验周期短的特点。