| Zizania latifolia Turcz., a gramineae species native to China, is a perennial aquatic plant. The swollen stem of the plant is an important vegetable in China since ancient times. The enlargement of the succulent stem was accompanied by proliferation of smut fungus(Ustilago esculenta P. Henn.) and hyperplastic growth of the first 3 to 5 nodes beneath the apical meristem. Besides a few histopathological and phytohormonal reports related to the process, however, little is known about the mechanism of enlargement. The present study examines the oxidative stress during stem enlargement of Z. latifolia. Major results are presented as follows:1. Spores of U. esculenta were able to germinate at 75μΗM of H2O2 and their germination was promoted at 15μM of H2O2. Further experiments showed that the smut fungus spores enhanced degradation of H2O2 with an equivalece of 5μM Fe2+ per 2.0 x 106/ml spores.2. The stem enlargement process was ushered by rapid increases in contents of H2O2 and MDA, which reached maxima at 6- and 8-d, respectively, and followed by decline to their initial levels. Ascorbate (AsA) content was raised before enlargement and dropped quickly to a lower level afterwards, whereas the ratio of dehydroascorbate in total ascorbate maintained at a high level except at 0-d, at which the lowest ratio was observed. GSH content took a similar time-course as that of AsA. The uppermost part of non-enlarging stems of the same plant species contained similar or lower levels of MDA, H2O2, AsA and GSH, as compared to those in the pre-enlargement state of succulent stem. Similar to the non-enlarging stems, mature leaves of plant with enlarging stems were also not infected by U. esculenta and relatively small fluctuations occurred in MDA and GSH levels during period of stem enlargement taking place in the same tiller. These results showed that the progressive oxidative stress in the succulent stem of Z. latifolia during enlargement was caused by activities of the systematic smut fungus. Antioxidants such as ascorbate and glutathione probably contribute little to the scavenging of ROS during stem enlargement.3. Distinct temporal patterns of activities of antioxidative enzymes including catalase (CAT), ascorbate peroxidase (APX), guaiacol-peroxidase (POD) and superoxide dismutase (SOD), were found during the stem enlargement. Both of the activities of CAT and APX displayed a rapid "rise-and-fall" pattern with a peak at 8-d, while that of SOD rose to maximum at 2-d and then declined to levels lower than that of pre-enlargement state. In contrast, the activity of POD rose slowly before 8-d but drastically thereafter. Similar patterns of MDA/H2O2 levels and APX/CAT activities during stem enlargement imply that CAT and APX are the principal ROS-scavengers. By analogy, SOD probably plays the role of scavenging ROS mainly in the initial stage of stem enlargement. POD, on the other hand, is likely to be involved in the cellular expansion at later stages of stem development. Comparison between changes in levels of antioxidants and activities of antioxidative enzymes during the stem enlargement also revealed that induction of antioxidants preceded that of antioxidative enzymes in response to oxidative stress.4. TiIv-PAR method was used to estimate the H2O2 amount in the present study. Major limitations of the assay system were examplified as follows, (1) metaphosphoric acid, often used in extracting ascorbate from plant tissues, interfered severely with the TiIV-PAR assay; and (2) the pH range usable for ammonia-neutralized system was much narrower than reported. Tris-buffered system was proposed to replace the ammonia-neutralized one. It was quite simple to bring the pH of reaction mixture to the suitable range with the buffering system. Compared to the ammonia-neutralized system, Tris-buffered system was also found to have wider pH range (pH7.6-9.0) and shorter assay time (10 min at 3℃). |
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