长春花幼苗生理代谢对锌胁迫的响应特点及其受乙烯调控的研究
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摘要
长春花(Calharanlhus roseus (L.) G. Don),夹竹桃科(Apocynaceae),长春花属(Calharanthus)我国西南、中南及华东等地栽培品种,是一种防治癌症的良药,长春花茎、叶中提取出来的抗癌成分有长春碱、长春新碱等,是目前国际上应用最多的抗癌植物药源。锌(Zn)作为植物生长所必需的元素,同时也是一种具有毒性的重金属元素。重金属污染一般导致植物生理代谢紊乱,加速植物的衰老和死亡
本论文以药用植物长春花为实验材料,采用珍珠岩为培养基质的营养液方式培养,设置不同浓度的外源锌、乙烯条件,从长春花在锌胁迫的逆境条件下培养入手,并且辅助以外源乙烯调控,对其初生代谢、Zn在植株内部的富集和转运、抗氧化系统以及次生代谢等方面进行初步研究,为进一步研究长春花中各项生理指标响应外源锌胁迫条件下的变化以及在此基础上外源乙烯的调控作用提供参考依据,并且验证长春花是否可以作为一种新的Zn超富集植物。
研究结果如下：
1.低浓度的外源锌可以促进长春花植株的生长,而高浓度的外源锌则抑制长春花植株的生长。长春花植株根系表现出的耐受性较强,说明长春花对Zn有着较强的耐受性和适应性,有超富集的潜质。叶绿素和植株光合作用在锌胁迫条件下表现出低促高抑的现象,可能是较高浓度Zn加速了叶绿素的降解,从而影响了植物的光合作用。在锌胁迫的基础上加入外源乙烯缓解了Zn胁迫对长春花植株的伤害作用。锌胁迫条件下长春花有一定的乙烯气体释放,随着时间的增加释放量都有小幅度的增加。而在锌胁迫的基础上加入外源乙烯后乙烯气体的释放量大幅度增加,最高可增加17.8倍。而在相同乙烯处理条件下随着时间的增加长春花乙烯气体释放量逐渐减少,说明随着时间的延长,乙烯气体释放量逐渐降低。
2.外源锌胁迫条件下长春花植株根部对Zn的富集能力较强,因此Zn对根部的伤害也较大。长春花响应锌胁迫条件下根部对Zn的吸收富集能力随着Zn处理浓度的增高表现出低促高抑的现象,茎、叶片中Zn含量随着Zn处理浓度的增加逐渐升高,说明长春花对于Zn的富集能力较强,且有一定的超富集潜力。而转运能力主要表现为Zn由成熟组织向幼嫩组织转运率较强。在锌胁迫基础上加入外源乙烯后,降低了植株各组织部位对Zn的富集能力,降低了锌胁迫对长春花植株的胁迫伤害。促进了Zn由成熟叶片向幼嫩组织的转运率,但是抑制了Zn在长春花植株体内其余组织间的的运输。
3.在外源锌胁迫条件下长春花植株细胞脂膜过氧化作用增强,POD、CAT、SOD活性升高,有利于活性氧物质的清除,使细胞膜免受伤害。外源锌胁迫刺激导致细胞内H202的积累对生物大分子产生细胞毒害的能力。并且使MDA、可溶性糖含量增加,在一定程度上减弱了重金属对植物体的毒害效应。Zn对植物伤害的重要机制之一是ROS的过量积累导致质膜过氧化,并通过SOD、CAT、POD等来消除或减少ROS带来的伤害。在锌胁迫的基础上加入外源乙烯后缓解了Zn对长春花植株的伤害。
4.锌胁迫条件下药用植物长春花叶片中吲哚生物碱：文多灵、长春碱、长春质碱,以及根中含有的长春质碱具有组织器官和生长发育的特异性。不同叶位的叶片中文多灵、长春质碱含量呈现规律性变化,在上部幼嫩叶片中含量最高,而下部成熟叶片中含量最低。这一特点符合一般生物碱的变化规律：幼嫩组织中含量较高,随着组织逐渐成熟,含量逐渐降低,以最大效率维持植物正常的发育和生长。在此基础上加入外源乙烯则促进长春花吲哚生物碱含量的提高。
Catharanihus roseus (L.) is widely cultivated in south, east and southwest of China. Catharanthus roseus is a kind of medicine on prevention and treatment of cancer, the anticancer ingredient of vinblastine and vincristine that extracted from stems and leaves, is currently a critical source of most anti-cancer medicine. As an essential micro element for plant, Zinc (Zn) is also a kind of heavy metal that threatens plant growth. In general, heavy metal disorders of physiological metabolism and accelerates plant senescence and death.
In this paper, to investigate the primary metabolism in plants and Zn ion transformation as well as its accumulation combined with the secondary metabolism response to Zn stress in plant, Catharanthus roseus was selected as experiment material. A cultivate system that nutrient solution irrigated to perlite was sued. Moreover, exogenous ethylene was applied to Zn stressed seedlings to study the effects of ethylene in regulating Catharanihus roseus response to Zn stress.
The results are as follows:
1. The growth of Catharanthus roseus was promoted under low concentrations of Zn presence while it was inhibited under high levels Zn condition. The root system of Catharanthus roseus was more tolerant to excess Zn, indicating the tolerance of Catharanthus roseus to Zn was high. Under Zn stress, the chlorophyll and photosynthesis was prompted and depressed under low or high levels of Zn. respectively, suggesting that high levels of Zn results in the degradation of chlorophyll by which decreased its photosynthesis ability. Under Zn stress, application of exogenous ethylene alleviated excess Zn induced damage to plants. Excess Zn treatment increased ethylene production and the production will increased slightly. On contrary, application of exogenous ethylene under Zn stress increased ethylene production dramatically,17.8times higher than that of control at its peak value. However, as the treatment time prolonged, the production of ethylene decreased under ethylene sole treatment condition, suggesting that ethylene production will decrease as the time past.
2. As the high ability in absorbing and accumulating Zn ion, the root system of Catharanthus roseus was hypersensitive to Zn stress. The ability of absorbing and accumulating of Zn ion in Catharanthus roseus root was increased under low Zn condition while depressed under high Zn condition. The accumulation of Zn ion in plant stem and root was increased as the Zn treatment concentration increased, suggesting the higher ability of Catharanthus roseus in absorbing and accumulating Zn ion. Specifically, the high transformation ability was mainly due to the transformation of Zn ion from mature tissue to younger tissue. Under Zn stress, application of exogenous ethylene decreased the accumulation of Zn ion in many tissues, and alleviated the damage induced by excess Zn ion to plant. Furthermore, its application also promotes transformation of Zn ion from mature tissue to younger tissue and inhibited its transformation between other tissues.
3. Under Zn stress, cells was severe damaged by stress induced oxidative stress, however. the activities of POD. CAT and SOD was increased that was considered to be ROS scavengers and protect cell membranes from oxidative damage. Also, the concentrations of MDA and soluble sugars were also increased which then alleviated the damage induced by-excess Zn to plants. One of the important mechanisms that Zn induced damage to plants was the oxidative stress that caused by ROS. However, application of exogenous ethylene alleviated Zn stress induced damage to plants.
4. Vindoline, vinblastine and catharanthine, three forms of indole alkaloids in Catharanthus roseus, combined the vinblastine in roots displayed characteristics of tissue specific. The concentrations of vindoline and vinblastine varies depends on the location of tissue, it was higher in upper younger leaves but lower in bottom mature ones. This phenomenon was consist with the classical theory that there was more secondary metabolites in younger tissue while less in mature ones, by which to guarantee the organism develop and growth ordinarily. Application of exogenous ethylene stimulates biosynthesis of indole alkaloid in Catharanthus roseus under excess Zn stress.
本论文以药用植物长春花为实验材料,采用珍珠岩为培养基质的营养液方式培养,设置不同浓度的外源锌、乙烯条件,从长春花在锌胁迫的逆境条件下培养入手,并且辅助以外源乙烯调控,对其初生代谢、Zn在植株内部的富集和转运、抗氧化系统以及次生代谢等方面进行初步研究,为进一步研究长春花中各项生理指标响应外源锌胁迫条件下的变化以及在此基础上外源乙烯的调控作用提供参考依据,并且验证长春花是否可以作为一种新的Zn超富集植物。
研究结果如下：
1.低浓度的外源锌可以促进长春花植株的生长,而高浓度的外源锌则抑制长春花植株的生长。长春花植株根系表现出的耐受性较强,说明长春花对Zn有着较强的耐受性和适应性,有超富集的潜质。叶绿素和植株光合作用在锌胁迫条件下表现出低促高抑的现象,可能是较高浓度Zn加速了叶绿素的降解,从而影响了植物的光合作用。在锌胁迫的基础上加入外源乙烯缓解了Zn胁迫对长春花植株的伤害作用。锌胁迫条件下长春花有一定的乙烯气体释放,随着时间的增加释放量都有小幅度的增加。而在锌胁迫的基础上加入外源乙烯后乙烯气体的释放量大幅度增加,最高可增加17.8倍。而在相同乙烯处理条件下随着时间的增加长春花乙烯气体释放量逐渐减少,说明随着时间的延长,乙烯气体释放量逐渐降低。
2.外源锌胁迫条件下长春花植株根部对Zn的富集能力较强,因此Zn对根部的伤害也较大。长春花响应锌胁迫条件下根部对Zn的吸收富集能力随着Zn处理浓度的增高表现出低促高抑的现象,茎、叶片中Zn含量随着Zn处理浓度的增加逐渐升高,说明长春花对于Zn的富集能力较强,且有一定的超富集潜力。而转运能力主要表现为Zn由成熟组织向幼嫩组织转运率较强。在锌胁迫基础上加入外源乙烯后,降低了植株各组织部位对Zn的富集能力,降低了锌胁迫对长春花植株的胁迫伤害。促进了Zn由成熟叶片向幼嫩组织的转运率,但是抑制了Zn在长春花植株体内其余组织间的的运输。
3.在外源锌胁迫条件下长春花植株细胞脂膜过氧化作用增强,POD、CAT、SOD活性升高,有利于活性氧物质的清除,使细胞膜免受伤害。外源锌胁迫刺激导致细胞内H202的积累对生物大分子产生细胞毒害的能力。并且使MDA、可溶性糖含量增加,在一定程度上减弱了重金属对植物体的毒害效应。Zn对植物伤害的重要机制之一是ROS的过量积累导致质膜过氧化,并通过SOD、CAT、POD等来消除或减少ROS带来的伤害。在锌胁迫的基础上加入外源乙烯后缓解了Zn对长春花植株的伤害。
4.锌胁迫条件下药用植物长春花叶片中吲哚生物碱：文多灵、长春碱、长春质碱,以及根中含有的长春质碱具有组织器官和生长发育的特异性。不同叶位的叶片中文多灵、长春质碱含量呈现规律性变化,在上部幼嫩叶片中含量最高,而下部成熟叶片中含量最低。这一特点符合一般生物碱的变化规律：幼嫩组织中含量较高,随着组织逐渐成熟,含量逐渐降低,以最大效率维持植物正常的发育和生长。在此基础上加入外源乙烯则促进长春花吲哚生物碱含量的提高。
Catharanihus roseus (L.) is widely cultivated in south, east and southwest of China. Catharanthus roseus is a kind of medicine on prevention and treatment of cancer, the anticancer ingredient of vinblastine and vincristine that extracted from stems and leaves, is currently a critical source of most anti-cancer medicine. As an essential micro element for plant, Zinc (Zn) is also a kind of heavy metal that threatens plant growth. In general, heavy metal disorders of physiological metabolism and accelerates plant senescence and death.
In this paper, to investigate the primary metabolism in plants and Zn ion transformation as well as its accumulation combined with the secondary metabolism response to Zn stress in plant, Catharanthus roseus was selected as experiment material. A cultivate system that nutrient solution irrigated to perlite was sued. Moreover, exogenous ethylene was applied to Zn stressed seedlings to study the effects of ethylene in regulating Catharanihus roseus response to Zn stress.
The results are as follows:
1. The growth of Catharanthus roseus was promoted under low concentrations of Zn presence while it was inhibited under high levels Zn condition. The root system of Catharanthus roseus was more tolerant to excess Zn, indicating the tolerance of Catharanthus roseus to Zn was high. Under Zn stress, the chlorophyll and photosynthesis was prompted and depressed under low or high levels of Zn. respectively, suggesting that high levels of Zn results in the degradation of chlorophyll by which decreased its photosynthesis ability. Under Zn stress, application of exogenous ethylene alleviated excess Zn induced damage to plants. Excess Zn treatment increased ethylene production and the production will increased slightly. On contrary, application of exogenous ethylene under Zn stress increased ethylene production dramatically,17.8times higher than that of control at its peak value. However, as the treatment time prolonged, the production of ethylene decreased under ethylene sole treatment condition, suggesting that ethylene production will decrease as the time past.
2. As the high ability in absorbing and accumulating Zn ion, the root system of Catharanthus roseus was hypersensitive to Zn stress. The ability of absorbing and accumulating of Zn ion in Catharanthus roseus root was increased under low Zn condition while depressed under high Zn condition. The accumulation of Zn ion in plant stem and root was increased as the Zn treatment concentration increased, suggesting the higher ability of Catharanthus roseus in absorbing and accumulating Zn ion. Specifically, the high transformation ability was mainly due to the transformation of Zn ion from mature tissue to younger tissue. Under Zn stress, application of exogenous ethylene decreased the accumulation of Zn ion in many tissues, and alleviated the damage induced by excess Zn ion to plant. Furthermore, its application also promotes transformation of Zn ion from mature tissue to younger tissue and inhibited its transformation between other tissues.
3. Under Zn stress, cells was severe damaged by stress induced oxidative stress, however. the activities of POD. CAT and SOD was increased that was considered to be ROS scavengers and protect cell membranes from oxidative damage. Also, the concentrations of MDA and soluble sugars were also increased which then alleviated the damage induced by-excess Zn to plants. One of the important mechanisms that Zn induced damage to plants was the oxidative stress that caused by ROS. However, application of exogenous ethylene alleviated Zn stress induced damage to plants.
4. Vindoline, vinblastine and catharanthine, three forms of indole alkaloids in Catharanthus roseus, combined the vinblastine in roots displayed characteristics of tissue specific. The concentrations of vindoline and vinblastine varies depends on the location of tissue, it was higher in upper younger leaves but lower in bottom mature ones. This phenomenon was consist with the classical theory that there was more secondary metabolites in younger tissue while less in mature ones, by which to guarantee the organism develop and growth ordinarily. Application of exogenous ethylene stimulates biosynthesis of indole alkaloid in Catharanthus roseus under excess Zn stress.
引文
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