ROS resistance in Pisum sativum cv. Alaska: the involvement of nucleoside diphosphate kinase in oxidative stress responses via the regulation of antioxidants.
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.
Haque
Md Emdadul
ME
Kihara Institute for Biological Research, Yokohama City University, Totsuka-ku, Yokohama, Japan. mehaque@affrc.go.jp
Yoshida
Yusuke
Y
Hasunuma
Kohji
K
D000975:Antioxidants / Q000378:metabolism*; D002374:Catalase / Q000378:metabolism; D015227:Lipid Peroxidation / Q000235:genetics / Q000502:physiology; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000235:genetics / Q000502:physiology*; D018532:Pisum sativum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020798:Two-Hybrid System Techniques
D000975:Antioxidants; D010940:Plant Proteins; D017382:Reactive Oxygen Species; D002374:Catalase; D009701:Nucleoside-Diphosphate Kinase
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