Oung Hui-Min, Lin Ke-Chun, Wu Tsung-Meng, Chandrika Nulu Naga Prafulla, Hong Chwan-Yang
Department of Agricultural Chemistry, College of Bioresources and Agriculture, National Taiwan University, Taipei, 10617, Taiwan.
Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, 91201, Taiwan.
Plant Mol Biol. 2015 Dec;89(6):577-88. doi: 10.1007/s11103-015-0380-4. Epub 2015 Sep 28.
The aminoglycoside antibiotic hygromycin B (Hyg) inhibits prokaryotic, chloroplast and mitochondrial protein synthesis. Because of the toxic effect of Hyg on plant cells, the HPT gene, encoding hygromycin phosphotransferase, has become one of the most widely used selectable markers in plant transformation. Yet the mechanism behind Hyg-induced cell lethality in plants is not clearly understood. In this study, we aimed to decipher this mechanism. With Hyg treatment, rice calli exhibited cell death, and rice seedlings showed severe growth defects, leaf chlorosis and leaf shrinkage. Rice seedlings also exhibited severe lipid peroxidation and protein carbonylation, for oxidative stress damage at the cellular level. The production of reactive oxygen species such as O2(·-), H2O2 and OH(·) was greatly induced in rice seedlings under Hyg stress, and pre-treatment with ascorbate increased resistance to Hyg-induced toxicity indicating the existence of oxidative stress. Overexpression of mitochondrial Alternative oxidase1a gene without HPT selection marker in rice enhanced tolerance to Hyg and attenuated the degradation of protein content, whereas the rice plastidial glutathione reductase 3 mutant showed increased sensitivity to Hyg. These results demonstrate that Hyg-induced cell lethality in rice is not only due to the inhibition of protein synthesis but also mediated by oxidative stress.
氨基糖苷类抗生素潮霉素B（Hyg）可抑制原核生物、叶绿体和线粒体的蛋白质合成。由于Hyg对植物细胞具有毒性作用，编码潮霉素磷酸转移酶的HPT基因已成为植物转化中使用最广泛的选择标记之一。然而，Hyg诱导植物细胞致死的机制尚不清楚。在本研究中，我们旨在破解这一机制。经Hyg处理后，水稻愈伤组织出现细胞死亡，水稻幼苗表现出严重的生长缺陷、叶片黄化和叶片皱缩。水稻幼苗还表现出严重的脂质过氧化和蛋白质羰基化，这是细胞水平上的氧化应激损伤。在Hyg胁迫下，水稻幼苗中诸如超氧阴离子（O2(·-)）、过氧化氢（H2O2）和羟基自由基（OH(·)）等活性氧的产生被极大诱导，用抗坏血酸预处理可增强对Hyg诱导毒性的抗性，表明存在氧化应激。在水稻中过表达无HPT选择标记的线粒体交替氧化酶1a基因可增强对Hyg的耐受性并减弱蛋白质含量的降解，而水稻质体谷胱甘肽还原酶3突变体对Hyg的敏感性增加。这些结果表明，Hyg诱导水稻细胞致死不仅是由于蛋白质合成受到抑制，还由氧化应激介导。
