摘要
茭白(Zizania latifolia Turcz.)是原产于中国大陆的一种重要的水生蔬菜,属于禾本科多年生水生植物。茭白的茎端数节能够在短期内迅速膨大形成肥大的肉质茎,其膨大机制一般认为与茭白植株中寄生的一种称为菰黑粉菌(Ustilago esculenta P. Henn.)的真菌大量繁殖有关。
本文旨在从氧化胁迫的角度,研究茭白肉质茎膨大期间氧化胁迫程度的变化情况,为进一步研究茭白肉质茎膨大机理提供科学依据。主要研究结果如下：
1、以“灰茭”中的黑粉菌孢子为材料,研究了H2O2对菰黑粉菌孢子萌发的效应。结果显示,75μM的H2O2下仍可观察到孢子萌发,而低浓度H2O2(15μM)不仅不会抑制黑粉菌孢子的萌发,反而可以起到促进萌发的作用。进一步研究表明,黑粉菌孢子具有促进H202降解的能力,2.0×106/ml孢子降解H2O2速率与5μMFe2+相当。
2、以“雄茭”为参照研究茭白肉质茎膨大期间的氧化胁迫状况。结果显示,膨大开始后,茎中H2O2和丙二醛(MDA)含量快速上升,并分别在膨大6和8d达到最大,此后降至膨大初期水平。抗坏血酸(AsA)在即将膨大前小幅上升,随后快速下降并处于较低水平,氧化型抗坏血酸比例在膨大开始时较低,随后则处于较高水平。谷胱甘肽(GSH)含量变化与AsA类似,也呈现先升后降的趋势。“雄茭”茎顶端组织的MDA、H2O2、AsA及GSH含量与膨大前的肉质茎相近或更低些,而同样没有黑粉菌侵入的成熟叶片中,MDA和GSH水平在植株孕茭期间的相对变化幅度都较小。可见,肉质茎膨大期间氧化胁迫逐渐加重是菰黑粉菌活动引起的,抗坏血酸和谷胱甘肽等抗氧化剂在清除膨大肉质茎活性氧的过程中贡献不大。
3、肉质茎膨大期间,过氧化氢酶(CAT)和抗坏血酸过氧化物酶(APX)的活性都呈现先升后降的趋势,且都在8d达到峰值。超氧化物歧化酶(SOD)活性在膨大前比较稳定,膨大开始后活性升高,至2d达到最大,随后降低,至6d后又缓慢回升,但直至12d仍低于膨大前期水平。过氧化物酶(POD)活性在膨大期间呈持续升高趋势,尤其后期(8d后)上升迅速。“雄茭”茎顶端组织中的CAT活性接近正常茭未膨大时的活性水平,而APX、POD和SOD活性则均高于正常茭。从抗氧化酶活力在肉质茎膨大期间的变化趋势分析,CAT和APX可能在清除ROS过程中起着主导作用,SOD主要在膨大初期发挥作用,而POD则有可能在肉质茎发育后期的细胞体积迅速膨大过程中发挥作用。对照抗氧化剂水平的变化趋势可见,对氧化胁迫最先响应的是AsA和GSH等抗氧化剂,随后才是SOD等抗氧化酶的响应。
4、H2O2含量是氧化胁迫水平的重要特征,其测定的准确性尤为重要。本文研究了Ti(Ⅳ)-PAR法测定H2O2含量需要注意的一些影响因素。主要内容包括：(1)论证了偏磷酸(HPO3)会严重干扰TiIV-PAR-H2O2复合体的形成,不适宜作为H2O2测定的提取液；(2)研究了pH对H2O2测定的影响,指出了能准确测定H2O2的pH范围；(3)提出了采用Tris作为缓冲液的H202测定系统,该系统能有效缓冲TCA提取液酸性,操作简便,适合测定的pH范围较宽(7.6～9.0)、反应时间缩短(10min,37℃)。
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℃).
引文
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