【作者】 贾永霞；

【导师】 郭世荣；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2009， 博士

【摘要】 无土栽培中水培营养液供氧不足或基质培根垫的形成均会造成低氧逆境,影响植株正常生长发育,成为困扰无土栽培技术大规模应用于生产的限制性因子。因此,提高植株在低氧胁迫下的耐性和抗性对农业生产具有重要的意义。多胺(Polyamines, PAs)是生物代谢过程中产生的一类具有生物活性的低分子量脂肪族含氮碱,主要包括腐胺(Put)、亚精胺(Spd)和精胺(Spm)。研究表明,多胺广泛作用于植物生长、形态建成、衰老和对环境胁迫的反应,然而,关于多胺种类和形态变化与植物低氧胁迫耐性的关系以及多胺的生理调节功能尚不清楚。本文以耐低氧能力较弱的‘中农八号’黄瓜(Cucumis sativus L.)品种为试材,采用营养液栽培的方法,通过营养液中添加外源多胺和多胺代谢抑制剂,研究了低氧胁迫下黄瓜幼苗体内多胺种类和形态的变化与碳代谢、光合作用之间的关系,探讨了多胺在黄瓜植株适应低氧胁迫中的生理调节功能。主要研究结果如下：低氧胁迫下,营养液中添加适宜浓度的Put、Spd和Spm均可缓解低氧胁迫对黄瓜幼苗生长的抑制作用,其中以0.05 mmol·L-1 Spd的缓解效果最佳。低氧胁迫下,黄瓜幼苗根系和叶片中游离态、结合态、束缚态Put、Spd和Spm含量均显著提高；营养液中添加外源Spd后进一步提高了低氧胁迫下黄瓜幼苗根系和叶片中游离态Spd、Spm含量,却显著降低了游离态Put含量；同时进一步提高了结合态、束缚态多胺含量,尤其是束缚态多胺含量,并且促进了黄瓜幼苗生长。Put合成抑制剂D-精氨酸(D-Arg),不仅显著降低了黄瓜幼苗体内游离态Put、Spd和Spm的含量,而且降低了抗氧化酶活性,导致植株体内活性氧(ROS)大量积累,加重了低氧胁迫的伤害。Put转化抑制剂甲基乙二醛双(脒基腙)(MGBG)和降解抑制剂氨基胍(AG)混合施用造成游离态Put和ROS的大量积累,并且Spd、Spm含量和抗氧化酶活性均显著降低,进一步加重了低氧胁迫对植株的伤害。低氧胁迫下,游离态多胺向束缚态多胺转化的抑制剂邻菲咯啉(o-phen)处理后,显著降低了幼苗体内束缚态多胺含量,并加重了低氧胁迫对幼苗生长的抑制作用。这些结果表明,黄瓜幼苗体内多胺的种类和形态与低氧胁迫耐性密切相关：从多胺的种类来看,根系和叶片中游离态Put迅速向游离态Spd和Spm转化,有利于提高黄瓜幼苗的低氧胁迫耐性；从多胺的形态来看,游离态Put迅速向结合态和束缚态多胺转化,尤其是向束缚态多胺的转化,有利于增强黄瓜幼苗的低氧逆境适应能力。低氧胁迫下,黄瓜植株的净光合速率(Pn)显著降低,并且饱和光强下的Pn、表观量子效率(AQY)和PSⅡ的最大光化学效率(Fv/Fm)均显著下降,天线耗散能量(D)的比率、非光化学猝灭系数(NPQ)和光抑制程度(1-qP/qN)显著升高,说明低氧胁迫下黄瓜植株受到了光合作用的光抑制。低氧胁迫下,NPQ与叶黄素脱环氧化状态(DEPS)变化趋势相一致,且两者均被抗坏血酸(AsA)所促进,被二硫苏糖醇(DTT)所抑制,说明黄瓜植株通过叶黄素循环耗散过量光能,以适应低氧胁迫。外源Spd降低了低氧胁迫下黄瓜植株的1-qP/qN,同时也降低了NPQ和DEPS,说明Spd并不是通过增强叶黄素循环来耗散过剩光能从而缓解光抑制。低氧胁迫下,外源Spd提高了植株的Pn、Fv/Fm和实际光化学效率(ΦPSⅡ)以及光化学反应的能量部分(P),降低了天线耗散的能量部分(D)和PSⅡ反应中心过剩的光能(Ex)。说明,低氧胁迫下,外源Spd通过增强光能利用效率来缓解黄瓜植株的光抑制,从而保持较高的PSⅡ光化学效率,防御光破坏。低氧胁迫下,黄瓜叶片中淀粉和葡萄糖含量显著升高,而根系中淀粉和葡萄糖含量显著降低,表明可溶性糖从叶片向根系的转运受到抑制,根系通过消耗自身储存的淀粉和葡萄糖来维持生理代谢,以增强低氧适应性。另外,黄瓜根系三羧酸循环过程中的关键酶琥珀酸脱氢酶(SDH)和异柠檬酸脱氢酶(IDH)活性显著降低,三羧酸循环(TCA)严重受阻,ATP含量显著降低；同时,黄瓜根系无氧呼吸代谢能力增强,乳酸脱氢酶(LDH)、丙酮酸脱羧酶(PDC)、乙醇脱氢酶(ADH)和丙氨酸氨基转移酶(AlaAT)活性显著升高,造成有毒产物乳酸和乙醛大量积累,因此抑制了黄瓜幼苗的生长。外源Spd通过调节低氧胁迫下黄瓜幼苗体内多胺种类与形态的变化,提高了根系中糖含量以及糖酵解代谢酶和SDH、IDH活性,有利于维持较长时间的糖酵解,促进TCA循环,刺激ATP的合成,同时抑制无氧呼吸代谢和乳酸、乙醛积累,从而提高黄瓜幼苗的低氧胁迫耐性。黄瓜幼苗根系细胞器上束缚态多胺含量变化与植株低氧胁迫耐性密切相关：低氧胁迫下,黄瓜根系线粒体膜上束缚态多胺含量显著增加,添加外源Spd后进一步提高了线粒体膜上束缚态多胺含量,同时提高了线粒体膜上H+-ATPase、H+-PPase、ca2+-ATPase和Mg2+-ATPase活性,降低呼吸4态无效耗氧,提高呼吸3态、呼吸控制率和氧化磷酸化效率。表明,低氧胁迫下,外源多胺通过促进黄瓜根系游离态多胺以自身的多聚阳离子特性与线粒体膜上带负电荷的磷脂、膜蛋白结合后,有助于稳定线粒体膜、调节膜蛋白的构象,提高线粒体膜上ATPase活性,增强线粒体的呼吸功能,促进线粒体的电子传递和氧化磷酸化偶联作用,从而部分恢复有氧呼吸,提高根系呼吸速率,增强黄瓜植株对低氧胁迫的耐性。低氧胁迫下,黄瓜根系质膜束缚态多胺含量显著增加,而液泡膜束缚态多胺含量显著降低,添加外源Spd后显著提高了质膜和液泡膜束缚态多胺含量以及质膜H+-ATPase、液泡膜H+-ATPase和H+-PPase活性。表明,低氧胁迫下,外源多胺通过促进黄瓜根系游离态多胺与根系质膜H+-ATPase、液泡膜H+-ATPase和H+-PPase蛋白结合后,可以防止膜蛋白在逆境下变性,稳定膜蛋白的天然构象以及调节膜蛋白的生物功能,从而提高黄瓜幼苗对低氧胁迫的耐性。更多还原

【Abstract】 When grown in hydroponic and substrate cultivation, it was very often that the plant would undergo hypoxia. Now the levels of O2 has been becoming a determined factor in popularization of soilless cultivation. Therefore, improving the tolerance of crops to hypoxia is very important in agriculture. Polyamines (PAs), mainly including putrescine (Put), spermidine (Spd) and spermine (Spm), are kinds of aliphatic amine alkaloid produced by biosynthetic pathway. Many evidences have showed that polyamines were associated with plants growth, differentiation, senescence and responses to environmental stress. However, little is known about their physiological function in plants subjected to hypoxia stress. Cucumber cultivar (Cucumis sativus L.) cv. Zhongnong No.8 (sensitive to hypoxia stress) was used as a material in this study and the seedlings were grown hydroponically. They were treated with aeration and hypoxia with nutrient solutions add or not add PAs and metabolic inhibitor of PAs. The study aimed to investigate the relationship between kinds and formations of PAs and plant respiratory metabolism, photosynthesis, et al., to elucidate the physiological regulation function of polyamines played in cucumber seedlings that adapted to hypoxia stress. Main results were as follows:Suitable concentration of exogenous Put, Spd and Spm could alleviate the inhibition of cucumber seedlings growth caused by hypoxia stress, and the most effective application was 0.05 mmol·L-1 Spd.Under the hypoxia stress, the contents of free, soluble conjugated and insoluble bound Put, Spd and Spm in roots and leaves of cucumber seedlings were significantly increased. Application of exogenous Spd markedly suppressed the accumulation of free Put but promoted further the content of free Spd and Spm, soluble conjugated and insoluble bound Put in roots and leaves of cucumber seedlings, and the growth of the plant was improved. Application of D-arginine (D-Arg), an inhibitor of Put biosynthesis, not only significantly decreased accumulation of free Put, Spd and Spm, but also suppressed the activities of antioxidant enzymes, which lead to a mass accumulation of reactive oxygen species (ROS) in cucumber seedlings and aggravate the injury caused by hypoxia to seedlings. Application with mixed methylglyoxyl-bis (guanylhydrazone) (MGBG), an inhibitor of Put transform to Spd or Spm, and aminogunidine (AG), an inhibitor of Put degradation, enhanced accumulation of free-Put but suppressed accumulation of free-Spd, free-Spm. The activity of antioxidant enzymes was inhibited and resulted in ROS accumulation in roots and leaves of cucumber seedlings. All of these led to aggravate the seedlings injury caused by hypoxia. Compared to the hypoxia stress, exogenous o-phen, an inhibitor of transform of free PAs into insoluble bound PAs, decreased contents of insoluble bound PAs in seedlings and aggravated the hypoxia stress-induced plant growth inhibition. These results suggested that there were a close relationship between the kinds and formations of PAs in cucumber seedlings and the tolerance of hypoxia stress. Considered from the view of the kinds and formations of PAs, both rapid conversion of free Put into free Spd and Spm, and the conversion of free Put into soluble conjugated and insoluble bound polyamines, especially bound polyamines in roots and leaves could be beneficial to hypoxia tolerance of cucumber seedlings.When exposed to hypoxia stress, net photosynthetic rate (Pn), apparent quanta yield (AQY) and maximal photochemical efficiency (Fv/Fm) in leaves of cucumber seedlings were significantly decreased, but the allocation of dissipation energy (D) by antenna, non-fluorescence quenching coefficient (NPQ) and photo-inhibition degree (1-qP/qN) were significantly increased, which indicated that hypoxia caused photo-inhibition to cucumber seedlings. The daily movements and dynamics of NPQ in leaves of cucumber seedlings were consistent with that of xanthophyll de-epoxidation state (DEPS), and both of which were promoted by ascorbic acid (AsA) but inhibited by 1,4-dithiothreitol (DTT), showing that excess energy dissipated by xanthophyll cycle under the hypoxia stress. Exogenous Spd decreased 1-qP/qN, NPQ and DEPS in leaves of cucumber seedlings during hypoxia stress, showing that exogenous Spd did not alleviate photo-inhibition of cucumber seedlings by xanthophyll cycle through dissipation excess energy under hypoxia stress. However, exogenous Spd enhanced Fv/Fm, PSⅡphotochemisty rate (OPSⅡ) and photochemistry energy (P), but suppressed D and excessive energy in PSⅡ(Ex) in leaves of cucumber seedlings, these results suggested that exogenous Spd enhanced light transform efficiency to alleviate photo-inhibition in leaves of cucumber seedlings, and kept higher photochemical efficiency with lower photo-destroy under hypoxia stress. In the condition of the hypoxia stress, content of starch and glucose in leaves of cucumber seedlings was significant enhanced but that of which in roots was suppressed, indicating that transportation of soluble sugar from leaves to roots was inhibited. Therefore, starch and glucose stored in roots were consumed to maintain physiological metabolism in order to survive from the hypoxia stress. Besides, activities of succinic dehydrogenase (SDH) and isocitric dehydrogenase (IDH), some key enzymes in tricarboxylic acid cycle (TCA), were significantly decreased by hypoxia stress, which resulted in severe declination of TCA and a significant decrease in ATP content. At the same time, the fermentation metabolism in roots of seedlings was triggered due to enhancement of activities of lactate dehydrogenase (LDH), pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), alanine aminotransferase (AlaAT) and resulted in accumulation of lactate and aladehyde which is harmful to plants. As a result, the plant growth was severely inhibited by hypoxia stress. Exogenous Spd increased the concentrations of fructose, sucrose and glucose, and promoted the activities of enzymes such as SDH and IDH in roots of seedlings, which was beneficial to maintain glycolysis metabolism, promote TCA, stimulate synthesis of ATP, suppress anaerobic respiration and inhibit the accumulations of lactate and aladehyde, subsequently, hypoxia-induced inhibition of plant growth was alleviated.Contents of insoluble bound polyamines in organelles of cucumber seedlings roots played an important role in improving hypoxia tolerance. During exposure to hypoxia stress, contents of insoluble bound PAs in mitochondria of cucumber roots increased significantly. Comparing to treatment of hypoxia, application of exogenous Spd to hypoxic nutrient solution promoted insoluble bound polyamines contents further and increased the activity of H+-PPase, Ca2+-ATPase, Mg2+-ATPase and R3 (oxygen consumption rate with ADP and substrate present), RCR (respiratory control ratio), P/O (oxidative phosphorilation ratio) in mitochondria of cucumber roots, but suppressed activity of R4 (oxygen consumption rate with substrate). All of these would be advantage to stabilize functions of mitochondria respiratory. The results indicated that exogenous PAs promoted free PAs bound to phospholipid and proteins in mitochondria membrane, stabilized the structure of mitochondria, regulated conformation of membrane protein, promoted ATPase activity in mitochondria membrane, enhanced respiration of mitochondria and promoted the coupling of electron and oxidative-phosphorylation. Therefore, aerobic respiration in mitochondria of seedling was resumed in some degree and root respiration rate was improved which resulted in enhancement of tolerance of cucumber plants to root-zone hypoxia. Content of insoluble bound PAs in plasma membranes was significantly increased, while insoluble bound PAs in tonoplasts of seedling roots was remarkably decreased by hypoxia stress. Application of exogenous Spd to hypoxic nutrient solution significantly increased content of insoluble bound PAs in plasma membranes and tonoplasts, and promoted activities of plasma membrane H+-ATPase, tonoplast H+-ATPase and tonoplast H+-PPase of seedling roots compared to that of hypoxia treatment. These results suggested that exogenous PAs could enhance combination of free PAs with plasma membrane H+-ATPase, tonoplast H+-ATPase and tonoplast H+-PPase in roots, and this combination would prevent denaturalization of membrane proteins, stabilize the natural conformation of membrane proteins and regulate its biological function. Therefore, tolerance of cucumber plants to root-zone hypoxia was enhanced.更多还原