ROS resistance in Pisum sativum cv. Alaska: the involvement of nucleoside diphosphate kinase in oxidative stress responses via the regulation of antioxidants
ROS resistance in Pisum sativum cv. Alaska: the involvement of nucleoside diphosphate kinase in oxidative stress responses via the regulation of antioxidants
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DOI：
10.1007/s00425-010-1173-2
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发表时间：
2010-07-01
期刊：
影响因子：
4.3
通讯作者：
Hasunuma, Kohji
中科院分区：
文献类型：
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作者：
Haque, Md. Emdadul;Yoshida, Yusuke;Hasunuma, Kohji
以抗百草枯的豌豆品系R3-1为材料，以野生型为对照，研究了抗百草枯豌豆品系R3-1的活性氧耐受机制。生理生化分析表明，在表型，如延迟的叶片和花的发展，上级分支，更大的生物量和产量的R3-1线，以及抗氧化色素的水平增加和细胞脂质过氧化反应的速率较低的抗性R3-1。此外，粗蛋白的磷酸化显示出R3-1和WT植物之间的条带迁移率和强度的可区分的差异。NDPKs的cDNA克隆和序列分析表明，NDPK 2中的两个氨基酸（IL 12 L和Glu 205 Lys）和NDPK 3中的一个氨基酸（P45 S）在R3-1中发生了突变。使用谷胱甘肽S-转移酶-NDPK融合构建体，我们发现前体重组R3-1 NDPK 2在R3-1植物中与WT植物相比显示出增加的活性和自磷酸化水平。对粗蛋白的非变性聚丙烯酰胺凝胶电泳分析表明，NDPK和过氧化氢酶（CAT）活性共存于凝胶的同一区域。在酵母双杂交试验中，NDPK 2的N-末端区域显示与全长CAT 1蛋白的相互作用。此外，我们发现WT在光照下和/或在含有ROS释放试剂的培养基上显示出与R3-1相比降低的CAT活性水平。综上所述，这些结果表明，在R3-1植物中NDPK 2和CAT 1之间存在强烈的相互作用，这可能在抗氧化防御ROS中起着重要作用。
This study investigated the reactive oxygen species (ROS) tolerance mechanism of a paraquat-resistant Pisum sativum line (R3-1) compared with the wild type (WT). Physiological and biochemical analyses showed significant differences in the phenotypes, such as delayed leaf and floral development, superior branching, and greater biomass and yields in the R3-1 line, as well as an increased level of antioxidant pigments and a lower rate of cellular lipid peroxidation in the resistant R3-1. Additionally, the phosphorylation of crude proteins showed distinguishable differences in band mobility and intensity between the R3-1 and WT plants. cDNA cloning and sequence analysis of NDPKs, which were candidate phosphorylated proteins, revealed that two of the deduced amino acids in NDPK2 (IL12L and Glu205Lys) and one in NDPK3 (P45S) were mutated in R3-1. Using glutathione S-transferase-NDPK fusion constructs, we found that the precursor recombinant R3-1 NDPK2 showed an increased level of activity and autophosphorylation in R3-1 plants compared to WT plants. Native PAGE analysis of the crude proteins revealed that NDPK and catalase (CAT) activity co-existed in the same area of the gel. In a yeast two-hybrid assay, the N-terminal region of NDPK2 showed an interaction with the full-length CAT1 protein. Furthermore, we found that WT showed a decreased level of CAT activity compared with R3-1 under illumination and/or on media containing ROS-releasing reagents. Taken together, these results suggest that there is a strong interaction between NDPK2 and CAT1 in R3-1 plants, which possibly plays a vital role in the antioxidant defense against ROS.