摘要
采用水培试验,利用电感耦合等离子体、透射电镜、扫描电镜等技术研究了镉污染对烟草(N icotiana tabacum)叶绿素含量及叶绿素a/b值、叶下表皮气孔器密度、腺毛密度、叶片细胞超微结构和P、K、C a等元素含量的影响,以及叶片细胞、腺毛对镉污染的反应。结果表明随着营养液中镉浓度的增高,烟草叶绿素含量及叶绿素a/b值降低;叶下表皮气孔器的密度及腺毛的密度增加;叶绿体中组成基粒的类囊体层数减少、分布不均、或粘连成索状,叶绿体膜系统崩溃,内外膜均解体,类囊体消失;但烟草可通过将镉隔离于细胞壁中或排出表皮细胞或通过腺毛分泌作用来减少其毒害。随着营养液中镉浓度的增高,腺毛分泌物中S i、K、A l、C a、M g、F e的含量增加;浓度为3m g/L的镉污染可增加叶片中P、C a、M g、F e、Cu、Zn、A l元素的含量,但浓度为30m g/L的镉污染造成叶片中P、C a、M g、F e、Cu、Zn、A l元素含量的减少;镉污染可引起叶片中N a含量增加,且随着营养液中镉浓度的增高N a含量增加;镉污染造成K、M n在叶片中的含量下降,而且随着营养液中镉浓度的增高,K、M n含量下降幅度增加。
To determinate the effects of cadmium （Cd） toxicity on tobaccoes （Nicotiana tabacum L. ）, chlorophyll content and the value of chlorophyll a/b, density of stomatas and trichomes on abaxial epidermis of leaves, ultramicroscopic structure of the leaves,content of some elements in leaves and secreting materials by trichomes were studied under Cd stress. Tobacco seedlings with same number of leaves （six leaves） and same height were selected. After roots were washed with deionized water, the seedlings were transferred and cultivated hydroponically in pots containing nutrient solution （according to method of Hoagland）. After 20d cultivated in nutrient solution, the cadmium was added to nutrient solution at the following concentrations （calculated with pure Cd）： 0 mg/L, 3 mg/L, 30 mg/L, 100 mg/L in Cd （CH3COO）2. Each treatment was replicated three times. The nutrient solution with different concentrations of Cd was renewed every 5d. The seedlings were aerated 3 h everyday. After 5d the seedlings cultivated in Cdl00 mg/L died. The other samples were collected after 40 day＇ cultivation.
The assay of chlorophyll content and the value of chlorophyll a/b in leaves were according the method of Zhu Guanglian.
To determine the mineral elements content in leaves treated with different concentrations of Cd, the samples were selected at the sixth leaf from the base to top in each treatment. 0. 1g dried leaf samples at same location of the leaf （removed main veins） were ground and the powder was digested with a mixture of HNO3＋HCl＋HClO4（in the proportion of 3 ： 2 ： 1.5）. The concentration of HNO3, HCl and HClO4 were 1.40g/ml, 1.19g/ml, and 1.67g/ml respectively. Samples were evaporated to dry on a plank heated by electricity at 300℃. The elements were extracted with 1 ml 10% HCl, Then the HCl was added to 10 ml. The samples were put in ICP （IRIS Intrepid） to determine content Of the elements.
To observe ultramicroscopic structure of chloroplasts and cell walls, the samples were selected at the fifth leaf from the base to top in each treatment at the same location of the leaf. Leaf segments were fixed in 3% glutaraldehyde （PH7.2） for 4 h at 4℃ and post fixed in 1% OsO4 over a night. The segments were dehydrated with ethyl alcohol and embedded in Spurr＇resin at 60℃ for 8 h. Sections （60-90nm） were made with LKB-8800 ultramicrotome, stained with 1% uranyl acetate and observed by JEM-100SX.
To observe secreted materials by trichomes in leaves, leaves were initially examined using a multi-material microscope （AXIOPLAN2, ZEISS, German）. Then leaf segments were attached to aluminum stubs glued on both sides. The aluminum stubs with specimens were put on the stage of chamber. Samples were analyzed using environmental scanning electronic microscope （QUANTA 600F, FEI, Holland） with an energy-dispersive X-ray analysis system （INCA x-sight, OXFORD INSTRUMENTS, Britain）.
After 40 days cultivated hydroponically in nutrient solution with different Cd concentrations, the chlorophyll contents of leaves and their a/b value were separately 7.8, 3.9, 1.9, and 3. 36, 2.83, 2.31 respectively under 0 mg/L, 3mg/L and 30 mg/L of Cd treatments. The density of stomatas and trichomes on abaxial epidermis of leaves increased with the Cd concentration in nutrient solution.
For the chloroplast under the Cd 3mg/L stress, both the numbers of granas and the thylakoids sacked to granas were reduced. Under the Cd 30 mg/L stress, the membrane system of chloroplast collapsed. The outer membrane edge of chloroplast became vague, and granas disappeared. There were some cavities in the thylakoids system. In some chloroplasts there were no obvious membrane systems. It suggested that Cd had obvious effects on chloroplasts and Cd might have some effects on photosynthesis, and eventually affect on the growth and development of tobacco.
The content of Cd in leaves increased with Cd concentration in nutrient solution. The contents of P,Ca,Mg,Fe,Cu,Zn,Al in leaves increased in Cd 3mg/L treatment, but decreased sharply in Cd30 mg/L treatment. The content of Na in leaves increased along with the rise of Cd concentration in nutrient solution, but the content of Mn decreased along with the rise of Cd concentration in nutrient solution.
By observation of the leaf ultramicroscopic structure with transmission electronic microscope, it was found that there were some globular osmiopohilic materials appearing in cell wall, intercellular layer, cavity among adjacent cell and on the outer surface of the cell walls of epidermis under Cd stress, but there were no globular osmiopohilic materials in controlled tobacco leaves. By analysis of globular osmiopohilic materials with an energy-dispersive X-ray analysis system fitted with ESEM, it revealed that globular osmiopohilic materials contained Cd. It suggested that tobacco leaves could reduce Cd ions by segregating Cd in cell wall or exuded Cd out of the cells to diminish Cd toxicity.
In tobacco there are mainly two kinds of trichomes in morphologically. One is long, the other is short. Using environmental scanning electronic microscope it was found that both long and short trichomes could secrete crystals. An energy-dispersive X-ray analysis system fitted with ESEM revealed that the crystals had Cd and other elements after Cd treatments, but there was no Cd element in controlled one. In Cd 3mg/L treatment the content of Cd was 0. 11% of crystals weight, and in Cd 30 mg/L treatment the content of Cd was 0. 25% of crystals weight. It suggested that the content of Cd in crystals increased with the concentration of Cd in nutrient solution. The content of elements of Cd, Si, K, Al, Ca, Mg and Fe in crystals secreted by Cd treated tobaccoes increased with the rise of concentration of Cd in nutrient solution.
The results of this study demonstrated that Cd greatly inhibited the growth and development of tobacco leaves. On one hand Cd stress resulted in the leaf chlorosis, because Cd inhibited synthesis of chlorophyll and replication of chloroplasts, reduced chlorophyll contents, and changed a/b value. On the other hand Cd resulted in ultramicroscopic structure changes of chloroplasts such as the numbers of granas reduction, some cavities appearance in thylakoids system, even collapse of membrane systems. The content of Cd in treated leaves increased with the rise of Cd concentration in nutrient solution. The study revealed that under Cd stress tobaccoes in some degree could segregate Cd in cell walls, intercellular layers, cavities among adjacent cells and on the outer surface of the cell walls of epidermis, and secrete Cd by trichomes to reduce Cd toxicity. The content of Cd, Si, A1, K, Ca, Fe in crystals secreted by trichomes of treated tobacco increased with the rise of Cd concentration in nutrient solution.
出处
《生态学报》
CAS
CSCD
北大核心
2005年第11期2919-2927,共9页
Acta Ecologica Sinica
基金
国家烟草专卖局重大招标资助项目(110200201005)~~
关键词
烟草
镉污染
叶片
超微结构
元素含量
Tobacco leaves
Cd contamination
ultramicroscopic structure
content of elements
