【作者】 李春雷；

【导师】 倪德江；

【作者基本信息】 华中农业大学 ， 茶学， 2011， 博士

【摘要】 茶树是自然界中一类富集氟能力较强的植物,能从土壤、大气及水中吸收大量的氟,比一般植物高出2-3个数量级,但氟对茶树生理生化方面的影响的研究还较少。本试验以中国主栽品种福鼎大白茶为材料,研究了氟对茶树品质成分、超微结构、生理指标方面的影响；分析了抗氧化系统及抗坏血酸-谷胱甘肽循环在茶树耐氟性方面的作用及机理,解析了氟在亚细胞中的分布及与氟铝钙之间在茶树中的相互作用。本研究主要结果如下：1氟对茶叶品质成分的影响采用水培法,研究了不同浓度的氟对茶叶品质成分的影响。结果表明：随着氟浓度的增加,茶多酚、蛋白质、总儿茶素及其单体含量降低；氨基酸和可溶性糖含量显著升高；氨基酸组分大部分先升高后降低；金属元素Ca, K、Cu、Zn的含量显著下降,Mn的含量显著升高,Fe的含量先升高后降低,Mg先降后升,但各处理间差异不显著；各类香气成分的相对含量变化趋势不尽相同,但香气成分总量降低。以上结果表明氟对茶叶品质成分的影响与氟水平有关,随着氟浓度的升高,茶叶品质下降。2氟对茶树叶片光合特性及细胞超微结构的影响采用水培法,研究了不同浓度的氟对茶苗光合特性及细胞超微结构的影响,结果表明：在低氟浓度(<2 mg/L)范围,叶绿素含量和光合速率轻微增加,但无显著差异；超过2 mg/L,随氟浓度的增加,叶绿素含量和光合速率均显著下降。通过透射电镜发现,在低浓度氟(<4 mg/L)处理下细胞超微结构有轻微损伤,如质壁分离现象、类囊体轻微膨胀等,当氟浓度达到6 mg/L时,细胞超微结构遭到严重破坏,且随着氟浓度的增加破坏越来越重,如叶绿体降解、线粒体空化等。以上结果说明,氟对茶叶细胞超微结构的破坏导致了叶绿素含量和光合作用的下降。3氟对茶树体内活性氧代谢的影响3.1不同氟浓度对茶树活性氧代谢的影响采用水培法,茶苗在不同氟浓度下培养,发现低浓度氟(2 mg/L)处理的茶苗叶片中的MDA和H202含量与对照比均无显著差异,但随着氟浓度的升高,两者含量显著升高；总SOD及分型SOD活性显著下降,这对清除ROS是不利的,POD及CAT活性均是先升高后降低,最大值分别在6 mg/L和4 mg/L,脯氨酸含量显著增加,表明了抗氧化系统在低浓度范围内对氟胁迫作出了积极响应；在ASA-GSH循环系统中,随着氟浓度的增加,APX、GR、MDHAR均是先升高后降低,分别在4,2,2 mg/L达到最大值,之后活性下降,DHAR活性显著下降；抗氧化剂ASA先升高后降低,这些均表明了在低浓度氟处理下大部分的抗氧化酶及抗氧化物质对氟胁迫做出了抵御响应,保护茶树不受ROS的破坏,但随着氟浓度的增加,超出了这些酶及抗氧化物质的清除能力,导致过量的ROS积累。GSH显著下降,ASA/DHA和GSH/GSSG的比值均显著下降,也说明了茶苗遭到了过量积累的ROS的破坏。以上结果表明了,通过抗氧化系统及ASA-GSH循环只能在一定程度上(低氟范围)清除过量的ROS,随着氟浓度的升高,这两大系统的防御机能达到最大后下降。3.2茶树体内活性氧代谢对氟胁迫的动态响应采用水培法,一年生扦插苗在氟浓度为16 mg/L的条件下培养0,6,12,24,48,72 h(不加氟为对照),结果表明：随着处理时间的延长,MDA和H202与对照均无显著差异,SOD活性显著下降,POD、CAT、APX、GR等酶活性先升高后降低,在48h达到最大值；MDHAR、DHAR的活性及抗氧化剂ASA均显著高于对照,GSH与对照差别不明显；ASA/DHA及GSH/GSSG比值升高,这些结果均说明茶树在短期内完全可以通过抗氧化酶及ASA-GSH循环系统清除过多的ROS,从而保护茶树不受ROS伤害,这也正是茶树耐氟的机制之一。4钙减轻茶苗氟铝毒害的原因及茶树耐氟的机制不论是氟铝单独处理还是氟铝交互处理对茶树叶片及根尖超微结构的破坏均非常严重,而增施钙后明显减轻了叶片及根尖超微结构的破坏程度,说明钙能有效的减轻氟铝对茶树的伤害。铝处理和氟铝交互处理的茶苗根细胞壁中的果胶含量均高于对照,增施钙后即铝钙处理的茶苗根细胞壁中果胶含量比铝单独处理显著下降,氟铝钙处理的茶苗根细胞壁中果胶含量比氟铝交互处理显著下降。这说明钙减轻氟铝对茶树的伤害是因为钙改变了细胞壁的组分。通过不同比例的氟铝处理,结果表明在所设比例范围内铝均未减轻氟对茶苗的伤害程度,反而加重了对茶苗的伤害。因此,氟和铝以形成氟铝络合物而相互解毒的观点还有待于进一步的验证。在本试验中还发现,无论是大田茶园的福安大白、乌牛早品种还是营养液培养的福鼎大白品种,氟在亚细胞中的分布均是细胞壁(F1)>细胞核和叶绿体(F2)>含核糖体的可溶性组分(F4)>线粒体(F3)。大部分的氟(60%)被固定在细胞壁中,因此,细胞壁阻止了氟进一步进入细胞正是茶树耐氟的重要机制,结合大田中的茶树含有大量氟茶树并未出现中毒现象,而水溶液培养的茶苗却出现了中毒现象,这说明了茶树耐氟机制除与叶片细胞壁有关外,还与土壤中的某种成分关系密切。因此,氟-土壤中的某种成分-细胞壁正是茶树耐氟的模式。更多还原

【Abstract】 Camellia sinensis (L.) is a kind of plant that can concentrate fluoride in nature; it can absorb a large amount of fluorine from the soil, air and water, higher than other plants of 2 to 3 orders-of-magnitude. However, research of fluoride on the physiological and biochemical of tea plant are less. In this experiment Chinese cultivars Fu ding da bai was used as the material to made a study of fluoride effect on the tea quality components, cell ultrastructure, physiological indicators of tea, analysed the the role and mechanism of antioxidant system and ascorbic acid-glutathione cycle in fluoride resistance of tea, analysis, investigated the fluoride distribution in sub-cellular and the effects on the interaction among fluoride, aluminum and calcium in tea. The results of this study are as follows:1. Effect of F on the chemical composition in tea leaves.Seedlings of the tea plant, Camellia sinensis (L.), were grown hydroponically for 30 days to study the effect of fluoride (F) on the chemical composition in the leaves. Polyphenols, protein, total catechins and monomeric catechins decreased significantly with increasing F concentration. On the other hand, the content of amino acids and soluble-sugars increased significantly, but the differences in caffeine and water-soluble extracts were not statistically significant, in the same time, most of the amino acid composition are first increased and then decreased. Metallic elements content of Ca, K, Cu, Zn decreased significantly, except for content of Mn increased significantly yet Fe first increased and then decreased and Mg appeared in contradiction to Fe, However, differences among treatments were not significant. The changing trend of the relative content of aroma components was different; however, the relative content of total aroma components was decreased. These results suggestted that quality of tea was related to fluoride level and the main chemical constituents of tea leaves decreased with F increasing.2. Effect of F on tea leaves photosynthesis feature and ultrastructureSeedlings of Camellia sinensis were grown hydroponically for 30 d in order to study the effect of fluoride (F) on photosynthesis and leaf ultrastructure. Chl content and PN increased slightly at low F concentration (2 mg/L) but not significantly. However, these two parameters decreased significantly (P<0.05) with increasing F. concentration. The cell ultrastructure had little change under 0-4 mg/L F, e.g. plastoglobulus and thylakoid expanded slightly, however, an irreversible destruction under the concentration of F higher than 6 mg/L was observed, such as membrane rupture, thylakoid expantion and even disintegration was found. These results were interpreted that the destruction of F to tea leaves ultrastructure leaded the decreased of Chl content and photosynthesis.3. Effect of F on metabolism of active oxygen in tea plant.3.1 Effect on metabolism of active oxygen in tea plant under different fluoride concentrationsSeedlings of Camellia sinensis were grown hydroponically for 30 d in order to study the effect of fluorine (F) on antioxidant defence and ascorbate-glutathione cycle system. MDA and H2O2 content did not have a significant difference compared to control at low F concentration (2 mg/L), however, these two parameters increased significantly with increasing F concentration. SOD activity was significantly decreased, which was detrimental to remove ROS, POD and CAT activity that was first increased then decreased, and the maximum at 6 and 4 mg/L, proline increased significantly, indicating that at low F concentration the antioxidant system had made a positive action to fluoride stress. In the ASA-GSH cycle system, APX, GR, MDHAR activities increased first and decreased afterwards, reached maximun under 4,2,2 mg/L, respectively, DHAR activity was significantly decreased; ASA increased first and then decreased, which showed that the antioxidant enzymes had made a positive action to fluoride stress in a degree and protected cells from ROS. However, the balance between formation and detoxification of ROS was lost with increasing of F concentrations. GSH content, ASA/DHA and GSH/GSSG were significantly decreased, and also showed an excessive accumulation of ROS resulting in the destruction of tea seedlings. Therefore, our results suggestted that at low F concentrations, the leaf antioxidant defence system and ascorbate-glutathione cycle system can scavenge reactive oxygen species and sufficiently protect cells from free radical injury. However, antioxidant defence system and ascorbate-glutathione cycle system couldn’t scavenge excessive reactive oxygen species to protect the tissue from free radical injury under the stress of higher F. 3.2 Dynamic response of activity oxygen metabolism in tea plant under fluoride stressThrough nutrient fluid culturing, annual cuttings were exposed to 16 mg/L fluoride concentrations and cultivited for 0,6,12,24,48,72 h (without fluoride as control), the results indicated that with the time increasing, MDA and H2O2 had no significant difference, the activities of SOD decreased significantly, activities of POD, CAT, APX, GR increased first and then decreased, reached the highest at 48 h, the activities of antioxidases above MDHAR、DHAR and antioxidant ASA increased significantly, GSH had no obvious difference, ASA/DHA and GSH/GSSG increased, all these suggested that to protect tea from injury, tea could eliminate redundant ROS in the short term through antioxidases and AsA-GSH cycle, this is one of the mechanisms of fluoride-resistant of tea.4. Cause of Ca can alleviate the toxicity of Al-F to tea seedlings and fluoride-resistant mechanisms of teaUltrastructure of tea leaves and roots were severely damaged no matter under the separated treating of Al and For or the interaction of them, yet after adding Ca, it can significantly reduced the damage degree of the ultrastructure of leaves and roots, which explained that Ca could effectively reduce the damage of Al-F to tea cell. Under the processing of Al or interaction treatment of F-Al, content of pectin in root cell wall were both higher than the control. After adding Ca, when tea under the treatment of Ca-Al, the content of pectin in root cell wall decreased significantly than Al treatment alone, whereas under the treatment of F-Al-Ca, pectin content was significantly decreased than under interaction treatment of Al-F. This showed that Ca could reduce the damage of F-Al to tea may be due to Ca could change the composition of tea cell wall.Through the treating of F-Al of different proportions, the results showed that during the range, Al did not reduce the damage of F to tea seedlings, actually the damage was worsened. Therefore, the argument of F-Al compounds that could detoxify the damage needed further verification. In this study also found that, no matter cultivars Fu an da bai and Wu niu zao cultivated in field, or Fu ding da bai cultured in nutrient solution, Fluoride distribution in the sub-cells were Cell wall (F1)>Nuclear and chloroplast (F2) > Soluble fraction containing ribosomal (F4)>Mitochondria (F3). Most of the fluoride (60%) was fixed in the cell wall, so that cell wall could prevent F get into the cell which was an important mechanism of fluoride-resistant of tea. Combining with the phenomenon that field-cultivated tea bush containing amount of F but did not appear F-poisoning, while solution-cultured tea was poisoned by F, we inferred that the mechanism of fluoride-resistant in tea besides the protection of cell wall of tea leaves, also had relationship with some other components existing in soil, so that F-poisoning occurred in solution-cultured tea just because of lacking this composition in the solution. Therefore, we believed that fluoride-some components in the soil-the cell wall may be the fluoride-resistant mode of tea.更多还原