| 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. |
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