| The quality safety of tea is a hot issue in current researches of tea science, however the heavy metal ions contents of tea are too high or "over standards" have been important hazard factors.Most of relevant researches in tea heavy metals pollution were about searching causes in whole process, analyzing source and investageting background values of garden on application techniques. some were about absorption and accumulation status of tea plant, content of tea and its influential factors were investigated on application basis. in this thesis, accumulation, distribution status were researched connected with relevant physiological characteristics of tea plant on theory, the characteristics of lead, cuprum, chromium, and cadmium four heavy metal ions accumulation, allocation and distribution of fractions in tea plant was systematically studied. and tolerance, resistance of tea plant was evaluated in this thesis. Characterization of ultra-structure localization was conducted at the subcellular level, the stress physiology and biochemical mechanisms of detoxification in tea plant were in-depth explored.This research is an important practical issue in relevant field, which is directly related to quality safety of tea, and will provide a theoretical basis for "the source elimination" and "whole course control" etc. techniques.The heavy metal ions contents and distribution rates in fractions, tissues of tea plant were determined by atomic absorption spectroscopy firstly. on this basis, characteristics of tolerance, resistance were investigated by a system hydroponic test. furthermore, density gradient centrifugation, TEM-EDS and SEM-EDS etc. advanced techniques were used to analyze lead ultra-structure localization and characterization in tea tissues, cells, and subcellular fractions. DDPT-PCR were tentatively used to explore mechanisms of heavy metals tolerance and the molecular defense in tea plant. The main results are as follows:1.The lead, cuprum were trend to accumulate in eugonic early shoots of field planting tea. however the chromium, cadmium were trend to accumulate in steady mature shoots. The contents of four heavy metal ions in early shoots were 2.72±0.42mg/kg (lead, r=0.97**),12.04±0.58mg/kg (cuprum,r=0.84**),0.23±0.01mg/kg (chromium,r=0.97**) and 0.29±0.02mg/kg (cadmium,r=0.97**), respectively, the heavy metal ion accumulation of tea plant would change obviously or be significantly different with the variation of growth periods, tenderness, seasons and varieties, it would provide an available technological path to control the heavy metal residues.2. There were significant differences in accumulation capacity to different heavy metal ions of various organs, the accumulation of which were ranged from 3.93% to 16.45%(lead),5.23% to 13.09%(cuprum),0.37% to 25.17%(chromium) and 1.58% to 33.01%(cadmium). heavy metal ions of tea plant would be transported, redistributed among various organs to some extent, which was reflected by the correlation of accumulation in organs, the concrete correlations:the accumulations in leaves were significantly related to which in whole plant (rpt,=0.71**, rcu-0.93**, rcr=0.61*), lateral roots (rcu=0.79**,rcr=0.79**), and the cuprum accumulations in main roots were significantly related to which in lateral roots. The bioaccumulation factors order of heavy metal ions in tea organs was roots>leaves>stems, and ranged from 0.13 to 1.42 (lead), 0.18 to 3.07(cuprum),0.08 to 1.59(chromium)and 0.32 to 3.59(cadmium). The research suggested that the heavy metals accumulated by absorbing roots which had a function of accumulation were the highest, then which accumulated by lateral roots, older leaves and mature leaves which had a function of storage were at the middle, which accumulated by the trunks and scaffold branches which had a function of transport were relatively lower.3. The tea plant absorbed heavy metal ions mainly from soil, correlation between accumulation of fractions and total (effective) content were studied, the correlation coefficients of cuprum accumulation and effective soil content were productive branches(-0.78**), lateral roots (r=0.69**), while the correlation coefficients of cadmium accumulation were whole plant(r=0.57**), leaves(r=0.62**) and productive branches(r=-0.78**), respectively. The research suggested effective heavy metal was the main form absorbed by tea plant, the lower PH was more conducive to the release of lead ions, cuprum ions and chromium ions and improvement of activities, but passivation of cadmium ions and reduction of activities.4. There were differences in characteristics of lead, cuprum, chromium and cadmium uptake, accumulation in hydroponic tea plant, four typical correlation models were built between accumulation and variational concentration:Hill model, Linear model, Gaussian model and Boltzmann model, which were cuprum(74.21%), chromium(70.11%) and cadmium(85.89%) absorbed by hydroponic tea roots were mainly cumulated in roots, distribution of leaves were lead(0.57%), cuprum(24.20%), chromium(1.03%) and cadmium(3.01%) approximately. However when absorbed by hydroponic tea leaves, distribution rate of leaves were 27.27%(Pb),31.98% (Cu),82.98%(Cr) and 41.31%(Cd), and distribution rate of roots were 21.59%(Pb), 17.22%(Cu),2.36%(Cr) and 13.54%(Cd). Which showed that the heavy metal ions accaccumulated mainly distributed in the absorbing organs, only a few transported, allocated and redistributed to other organs.5. When the hydroponic tea plant had absorbed lead, cuprum, chromium and cadmium for 35 days, there were great differences in undamaged critical concentrations including Pb2+400mg/L, Cu2+200mg/L, Cr3+ 20mg/L and Cd2+2 mg/L, which were relative values determined by cultured concentration and time. And activities of SOD, POD and CAT had been increased under the concentration, it showed that the antioxidant enzyme activities had been induced increasing and the plant exhibited a certain adaptation and resistance to heavy metals, the tea plant exhibited poisoning symptoms at different degrees when had been cultured in hydroponics of Pb2+≥800 mg/L,Cu2+≥400mg/L,Cr3+≥30mg/L,Cd2+≥3 mg/L for 35 days, SOD, POD and CAT activities of leaves had been decreased, MDA content and CMP had been increased, leaf membrane lipid peroxidation had been changed because of heavy metal ions, structures and functions of cell membrane might been partial injuried, leaf photosynthesis had been restrained, the leaf net photosynthetic rate had decreased by 58.01 (Pb),32.92%(Cu),54.09%(Cr) and 58.72%(Cd)6. The research showed that differences in lead uptake and distribution were related to the structure function and composition of various subcellular fractions. The order of subcellular distribution rate in roots, stems and leaves was cell wall and residue (70.43%) >soluble component and cell membrane (17.93%)>organelle (14.26%)>cell nucleus (1.37%). It showed that there were strong filtration and sedimentary function in complex structures and components of cell membranes, where were the most distributed, however the nucleus was the main place where information carrier RNA stored, it was protected and the least distributed.7. The research suggested that the separate distributions of accaccumulated lead in tissues were influenced by organizational structures and functions. The results showed that: lead accaccumulation werexylem (15.78%)>epidermis (12.44%)>cortex (3.51%) in roots; which were phloem (25.82%)>cambium (18.08%)>epidermis (16.59%) >xylem (12.76%)>palisade tissue (11.21%)>spongy tissue (5.43%) in leaves.8. The research suggested that subcellular location and characterization of lead could been conduct by SEM-EDS and TEM-EDS accurately,a method of heavy metals subcellular characterization was constructed in this thesis, there were method innovativeness in the research. The TEM-EDS location results showed that lead of roots mainly distributed in cell walls, plasma membrane and tonoplast. lead contents of stems were obvious lower than roots and mainly distributed in cell walls, chloroplast, plasma membrane and tonoplast. lead of leaves mainly distributed in plasma membrane and cytoplasm, and lead in cell walls and other parts were so few that was not observed.9. The protein contents and free amino acids of hydroponic tea leaves cultured in lead solution were compared, it was presumed that the free amino acids contents varied because of the variation of nitrogen metabolism, amino acids and amino acid synthesis in tea plant could be promoted by the low concentration of lead, and then the tolerance was improved, however it would be restrained when the concentration over a certain standard, it showed that the tea plant would start its physiologic emergency response under the lead stress.10. The DTRT-PCR was adopted to filtrate, clone, transfer and sequencing, gene fragments which controlled serine-threonine kinase family genes differentlly expressed had been aligned in a part of shoots, and had provided a certain theory reference and evidence for revealing the mechanisms of Pb2+ tolerance and resistance in tea plant on the molecular view. |
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