Skip to main content
SpringerLink
Log in

ROS resistance in Pisum sativum cv. Alaska: the involvement of nucleoside diphosphate kinase in oxidative stress responses via the regulation of antioxidants

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Abbreviations

WT:

Wild type

3Chl:

Chlorophyll triplet state

3O2:

Ground state oxygen

PQ:

Paraquat

ROS:

Reactive oxygen species

1O2:

Singlet oxygen

O ·−2 :

Superoxide

H2O2 :

Hydrogen peroxide

RF:

Riboflavin

MB:

Methylene blue

SOD:

Superoxide dismutase

CAT:

Catalase

NDPK:

Nucleoside diphosphate kinase

PAGE:

Polyacrylamide gel electrophoresis

TBARS:

Thiobarbituric acid reacting substances

GST:

Glutathione S-transferase

References

  • Agarwal RP, Robinson B, Parks RE (1978) Nucleoside diphosphate kinase from human erythrocytes. Methods Enzymol 51:376–386

    Article  CAS  PubMed  Google Scholar 

  • Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399

    Article  CAS  PubMed  Google Scholar 

  • Belden WJ, Larrondo LF, Froehlich AC, Shi M, Chen C-H, Loros JJ, Dunlap JC (2007) The band mutation in Neurospora crassa is a dominant allele of ras-1 implicating RAS signaling in circadian output. Genes Dev 21:1494–1505

    Article  CAS  PubMed  Google Scholar 

  • Bölter B, Sharma R, Soll J (2007) Localisation of Arabidopsis NDPK2-revisited. Planta 226:1059–1065

    Article  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantity of protein utilizing the principle of protein–dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Buege JA, Aust SD (1972) Microsomal lipid peroxidation. Methods Enzymol 52:302–310

    Article  Google Scholar 

  • Cheng L, Kellogg EW III, Packer L (1981) Photoinactivation of catalase. Photochem Photobiol 34:125–129

    CAS  PubMed  Google Scholar 

  • Choi G, Yi H, Lee J, Kwon YK, Soh MS, Shin B, Luka Z, Hahn TR, Song PS (1999) Phytochrome signalling is mediated through nucleoside diphosphate kinase 2. Nature 401:610–613

    Article  CAS  PubMed  Google Scholar 

  • Choi G, Kim JI, Hong SW, Shin B, Choi G, Blakeslee JJ, Murphy AS, Seo YW, Kim K, Koh EJ, Song PS, Lee H (2005) A possible role for NDPK2 in the regulation of auxin-mediated responses for plant growth and development. Plant Cell Physiol 46:1246–1254

    Article  CAS  PubMed  Google Scholar 

  • Corpas FJ, Palma JM, Sandalio LM, Lopez-Huertas E, Romero-Puertas MC, Barroso JB, Del Río LA (1999) Purification of catalase from pea leaf peroxisomes: identification of five different isoforms. Free Radic Res 31:S235–S241

    Article  CAS  PubMed  Google Scholar 

  • Cuello F, Schulze RA, Heemeyer F, Meyer HE, Lutz S, Jakobs KH, Niroomand F, Wieland T (2003) Activation of heterotrimeric G proteins by a high energy phosphate transfer via nucleoside diphosphate kinase (NDPK) B and Gβ subunits. J Biol Chem 278:7220–7226

    Article  CAS  PubMed  Google Scholar 

  • Dat J, Inze D, Van BF (2001) Catalase-deficient tobacco plants: tools for in planta studies on the role of hydrogen peroxide. Redox Rep 6:37–42

    Article  CAS  PubMed  Google Scholar 

  • Escobar Galvis LM, Marttila S, Hakansson G, Forsberg J, Knorpp C (2001) Heat stress response in pea involves interaction of mitochondrial nucleoside diphosphate kinase with a novel 86-kilodalton protein. Plant Physiol 126:69–77

    Article  CAS  PubMed  Google Scholar 

  • Feierabend J, Engel S (1986) Photoinactivation of catalase in vitro and in leaves. Arch Biochem Biophys 251:567–576

    Article  CAS  PubMed  Google Scholar 

  • Finan PM, White IR, Redpath SH, Findlay JB, Millner PA (1994) Molecular cloning, sequence determination and heterologous expression of nucleoside diphosphate kinase from Pisum sativum. Plant Mol Biol 25:59–67

    Article  CAS  PubMed  Google Scholar 

  • Foyer CH (1997) Oxygen metabolism and electron transport in photosynthesis. In: Scandalios JG (ed) Oxidative stress and the molecular biology of antioxidant defenses. Cold Spring Harbor Laboratory Press, New York, pp 587–621

    Google Scholar 

  • Foyer CH, Noctor G (2003) Redox sensing and signalling associated with reactive oxygen in chloroplasts, peroxisomes and mitochondria. Physiol Plant 119:355–364

    Article  CAS  Google Scholar 

  • Fukamatsu Y, Yabe N, Hasunuma K (2003) Arabidopsis NDK1is a component of ROS signaling by interacting with three catalases. Plant Cell Physiol 44:982–989

    Article  CAS  PubMed  Google Scholar 

  • Haque ME, Yoshida Y, Hasunuma K (2008) Paraquat-resistant lines in Pisum sativum cv. Alaska: biochemical and phenotypic characterization. Plant Biotechnol Rep 2:21–31

    Article  Google Scholar 

  • Hertwig B, Streb P, Feierabend J (1992) Light dependence of catalase synthesis and degradation in leaves and the influence of interfering stress conditions. Plant Physiol 100:1547–1553

    Article  CAS  PubMed  Google Scholar 

  • Hiromura M, Yano M, Mori H, Inoue M, Kido H (1998) Intrinsic ADP–ATP exchange activity is a novel function of the molecular chaperone, Hsp70. J Biol Chem 273:5435–5438

    Article  CAS  PubMed  Google Scholar 

  • Iigusa H, Yoshida Y, Hasunuma K (2005) Oxygen and hydrogen peroxide enhance light-induced carotenoid synthesis in Neurospora crassa. FEBS Lett 579:4012–4016

    Article  CAS  PubMed  Google Scholar 

  • Jang YP, Zhou J, Nakanishi K, Janet K, Sparrow R (2005) Anthocyanins protect against A2E photooxidation and membrane permeabilization in retinal pigment epithelial cells. Photochem Photobiol 81:529–536

    Article  CAS  PubMed  Google Scholar 

  • Johansson M, MacKenzie-Hose A, Andersson I, Knorpp C (2004) Structure and mutational analysis of a plant mitochondrial nucleoside diphosphate kinase. Identification of residues involved in serine phosphorylation and oligomerization. Plant Physiol 136:3034–3042

    Article  CAS  PubMed  Google Scholar 

  • Kendall AC, Keys A, Turner JC, Lea PJ, Mifflin BJ (1983) The isolation and characterization of catalase-deficient mutant in barley (Hordeum vulgare L.). Planta 159:505–511

    Article  CAS  Google Scholar 

  • Kim Y-H, Lim S, Yang K-S, Kim CY, Kwon S-Y, Lee H-S, Wang X, Zhou Z, Ma D, Yun D-J, Kwak S-S (2009) Expression of Arabidopsis NDPK2 increases antioxidant enzyme activities and enhances tolerance to multiple environmental stresses in transgenic sweet potato plants. Mol Breed 24:233–244

    Article  CAS  Google Scholar 

  • Knox JP, Doge AD (1985) Singlet oxygen and plants. Phytochemistry 24:889–896

    Article  CAS  Google Scholar 

  • Kono Y, Fridovich I (1982) Superoxide radical inhibits catalase. J Biol Chem 257:5751–5754

    CAS  PubMed  Google Scholar 

  • Kowalczyk E, Charyk K, Fijałkowski P, Niedworok J, Błaszczyk J, Kowalski J (2004) Protective influence of natural anthocyanins of Aronia melanocarpa on selected parameters of antioxidative status in experimental intoxication with sulphide-2-chloroethyl-3-chloropropyl. Pol J Environ Stud 13:339–341

    CAS  Google Scholar 

  • Lee B, Yoshida Y, Hasunuma K (2006) Photomorphogenetic characteristics are severely affected in nucleoside diphosphate kinase-1 (ndk-1)-disrupted mutants in Neurospora crassa. Mol Gen Genom 275:9–17

    Article  CAS  Google Scholar 

  • Lee B, Yoshida Y, Hasunuma K (2009) Nucleoside diphosphate kinase-1 regulates hyphal development via the transcriptional regulation of catalase in Neurospora crassa. FEBS Lett 583:3291–3295

    Article  CAS  PubMed  Google Scholar 

  • Leung SM, Hightower LE (1997) A 16-kDa protein functions as a new regulatory protein for Hsc70 molecular chaperone and is identified as a member of the Nm23/nucleoside diphosphate kinase family. J Biol Chem 272:12248

    Article  CAS  Google Scholar 

  • Lledias F, Rangel P, Hansberg W (1998) Oxidation of catalase by singlet oxygen. J Biol Chem 273:10630–10637

    Article  CAS  PubMed  Google Scholar 

  • Lübeck J, Soll J (1995) Nucleoside diphosphate kinase from pea chloroplasts: purification, cDNA cloning and import into chloroplasts. Planta 196:668–673

    Article  PubMed  Google Scholar 

  • Moon H, Lee B, Choi G, Shin S, Prasad DT, Lee O, Kwak SS, Kim DH, Nam J, Bahk J, Hong JC, Lee SY, Cho MJ, Lim CO, Yun DJ (2003) NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants. Proc Nat Acad Sci USA 100:358–363

    Article  CAS  PubMed  Google Scholar 

  • Nishiyama Y, Yamamoto H, Allakhverdiev SI, Inaba M, Yokota A, Murata N (2001) Oxidative stress inhibits the repair of photodamage to the photosynthetic machinery. EMBO J 20:5587–5594

    Article  CAS  PubMed  Google Scholar 

  • Oda K, Hasunuma K (1997) Genetic analysis of signal transduction through light-induced protein phosphorylation in Neurospora crassa perithecia. Mol Gen Genom 256:593–601

    CAS  Google Scholar 

  • Ogura T, Tanaka N, Yabe N, Komatsu S, Hasunuma K (1999) Characterization of protein complexes containing nucleoside diphosphate kinase with characteristics of light signal transduction through phytochrome in etiolated pea seedlings. Photochem Photobiol 69:397–403

    Article  CAS  Google Scholar 

  • Ogura Y, Yoshida Y, Yabe N, Hasunuma K (2001) A point mutation in nucleoside diphosphate kinase results in a deficient light response for perithecial polarity in Neurospora crassa. J Biol Chem 276:21228–21234

    Article  CAS  PubMed  Google Scholar 

  • Richardson AD, Duigan SP, Berlyn GP (2002) An evaluation of non invasive methods to estimate foliar chlorophyll content. New Phytol 153:185–194

    Article  CAS  Google Scholar 

  • Sandalio LM, Daurzom HC, Gomez M, Romero-Puertas MC, del Rio LA (2001) Cadmium induced changes in the growth and oxidative metabolism of pea plants. J Exp Bot 52:2115–2126

    CAS  PubMed  Google Scholar 

  • Shang W, Feierabend J (1999) Dependence of catalase photoinactivation in rye leaves on light intensity and quality and characterization of a chloroplast-mediated inactivation in red light. Photosynth Res 59:201–213

    Article  CAS  Google Scholar 

  • Shao L, Shu Z, Sun S-L, Peng C-L, Wang X-J, Lin Z-F (2007) Antioxidation of anthocyanins in photosynthesis under high temperature stress. J Integr Plant Biol 49:1341–1351

    Article  CAS  Google Scholar 

  • Shen Y, Kim JL, Song PS (2005) NDPK2 as a signal transducer in the phytochrome-mediated light signaling. J Biol Chem 280:5740–5749

    Article  CAS  PubMed  Google Scholar 

  • Sims DA, Gamon JA (2002) Relationships between leaf pigment content and spectral reflectance across a wide range of species, leaf structures and developmental stages. Remote Sens Environ 81:337–354

    Article  Google Scholar 

  • Streb P, Michael-knauf A, Feierabend J (1993) Preferential photoinactivation of catalase and photoinhibition of photosystem II are common early symptoms under various osmotic and chemical stress conditions. Physiol Plant 88:590–598

    Article  CAS  Google Scholar 

  • Tanaka N, Ogura T, Noguchi T, Hirano H, Yabe N, Hasunuma K (1998) Phytochrome-mediated light signals are transduced to nucleoside diphosphate kinase in Pisum sativum L. cv. Alaska. J Photochem Photobiol 45:113–121

    Article  CAS  Google Scholar 

  • Tang L, Kim MD, Yang K-S, Kwon S-Y, Kim S-H, Kim J-S, Yun D-J, Kwak S-S, Lee H-S (2008) Enhanced tolerance of transgenic potato plants overexpressing nucleoside diphosphate kinase 2 against multiple environmental stresses. Transgenic Res 17:705–715

    Article  CAS  PubMed  Google Scholar 

  • Verslues PE, Kim YS, Zhu JK (2007a) Altered ABA, proline and hydrogen peroxide in an Arabidopsis glutamate: glyoxylate aminotransferase mutant. Plant Mol Biol 64:205–217

    Article  CAS  PubMed  Google Scholar 

  • Verslues PE, Batelli G, Grillo S, Agius F, Kim YS, Zhu J, Agarwal M, Katyyar-Agarwal S, Zhu JK (2007b) Interaction of SOS2 with nucleoside diphosphate kinase 2 and catalases reveals a point of connection between salt stress and H2O2 signalling in Arabidopsis thaliana. Mol Cell Biol 27:7771–7780

    Article  CAS  PubMed  Google Scholar 

  • Wang N, Yoshida Y, Hasunuma K (2007a) Loss of catalase-1 (CAT-1) results in decreased conidial viability enhanced by exposure to light in Neurospora crassa. Mol Gen Genom 277:13–22

    Article  CAS  Google Scholar 

  • Wang N, Yoshida Y, Hasunuma K (2007b) Catalase-1 (CAT-1) and nucleoside diphosphate kinase- (NDK-1) play an important role in protecting conidial viability under light stress in Neurospora crassa. Mol Gen Genom 278:235–242

    Article  CAS  Google Scholar 

  • Willekens H, Chamnongpol S, Davey M, Schraudner M, Langebartels C, VanMontagu M, Inze D, VanCamp W (1997) Catalase is a sink for H2O2 and is indispensable for stress defence in C-3 plants. EMBO J 16:4806–4816

    Article  CAS  PubMed  Google Scholar 

  • Yang KA, Moon HJ, Kim GT, Lim CJ, Hong JC, Lim CO, Yun DJ (2003) NDP kinase 2 regulates expression of antioxidant genes in Arabidopsis. Proc Jpn Acad Ser B 79(B):86–91

    Article  CAS  Google Scholar 

  • Yoshida Y, Hasunuma K (2004) Reactive oxygen species affect photo-morphogenesis in Neurospora crassa. J Biol Chem 279:6986–6993

    Article  CAS  PubMed  Google Scholar 

  • Yoshida Y, Hasunuma K (2006) Light-dependent subcellular localization of nucleoside phosphate kinase-1 in Neurospora crassa. FEMS Microbiol Lett 261:64–68

    Article  CAS  PubMed  Google Scholar 

  • Yoshida Y, Ogura Y, Hasunuma K (2006) Interaction of nucleoside diphosphate kinase and catalases for stress and light responses in Nurospora crassa. FEBS Lett 580:3282–3286

    Article  CAS  PubMed  Google Scholar 

  • Yoshida Y, Maeda T, Lee B, Hasunuma K (2008) Conidiation rhythm and light entrainment in superoxide dismutase mutant in Neurospora crassa. Mol Gen Genom 279:193–202

    Article  CAS  Google Scholar 

  • Zhang HH, Young JC, Pease EA, Hangarter RP, McClung CR (1994) Interactions between light and the circadian clock in the regulation of CAT2 expression in Arabidopsis. Plant Physiol 104:889–898

    Article  Google Scholar 

  • Zimmermann S, Baumann A, Jaekel K, Marbach I, Engelberg D, Frohnmeyer H (1999) UV-responsive genes of Arabidopsis revealed by similarity to the Gcn4-mediated UV response in yeast. J Biol Chem 274:17017–17024

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

The authors would like to acknowledge Dr. Nagib Ahsan (National Institute of Crop Science, Tsukuba, Japan), Dr. Atsushi Komamine and Dr. Yosuke Fukamatsu (Kihara Institute for Biological Research, Yokohama, Japan) for their helpful discussions. This work was supported by a grant-in-aid from the Japanese Ministry of Education, Culture and Science, the Japanese Ministry of Agriculture, Forestry and Fishery, the Yamada Science Foundation, and cooperative research support from Nissan Motor Co., Ltd.

Author information

Author notes

  1. Md. Emdadul Haque

    Present address: National Institute of Crop Science (NICS), 2-1-18 Kannondai, Tsukuba, Ibaraki, 305-8518, Japan

Authors and Affiliations

  1. Kihara Institute for Biological Research, Yokohama City University, 641-12 Maioka-cho, Totsuka-ku, Yokohama, 244-0813, Japan

    Md. Emdadul Haque, Yusuke Yoshida & Kohji Hasunuma

Authors
  1. Md. Emdadul Haque
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Yusuke Yoshida
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kohji Hasunuma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Md. Emdadul Haque or Kohji Hasunuma.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Suppl. Fig. 1

a-d Overall phenotypes, plant weights and yield characteristics of the wild-type (WT) and R3-1 plants. WT and R3-1 plants were grown under natural field conditions. When grown to maturity, R3-1 plants produced about a 1.9-fold as many fruits per plant compared to WT (a), 1.9-fold more seeds per plant (b), 1.5-fold more fruit weight per plant (c), and 2.0-fold as much total plant weight (g) as many reproductive nodes and lateral branches before apical arrest (d), as described previously (Haque et al. 2008). Means ± SE were obtained from 15 to 30 plants (PPT 7931 kb)

Suppl. Fig. 2

Effects of RF or MB on seed germination and shoot and root growth in R3-1 plants. After surface-sterilization and imbibitions, pea seeds were grown in paper towels immersed in MS medium contained in glass jars having 0, 200, 400, 800, 1600 or 3200 µM RF for 9 days in an LP-200 Biotron NK system (Nippon Medical & Chemical Instruments Co., Ltd.) with continuous white fluorescent light (155 μmol m−2 s−1) at 23˚C. In a separate experiment, seeds were grown with the MB concentrations listed above in the same chamber with continuous red light (80 μmol m−2 s−1). Germination (%), primary root length (cm), and shoot length (cm) were measured in the WT and R3-1 plants. a-d RF inhibited the growth of WT and R3-1 plants dose dependently, which became critical at 800 µM. The R3-1 plants produced higher levels of germination, shoot growth and root growth in the presence of 800 µM RF than the WT plants, with 3.3-fold more germination (a), 1.9-fold greater root growth (b) and 1.7-fold greater shoot growth (c). WT (deep blue circle) and R3-1 (deep pink circle). d The photographs show WT (top panels) and R3-1 (bottom panels) seedlings treated with 800 µM RF (righthand panels) or untreated (lefthand panels) for each case.e–h The R3-1 plants showed slight differences after MB treatment when the concentration was increased. At 800 µM, there were moderate differences in germination and shoot growth, but not in root growth compared to WT plants; there was 3.4-fold more germination (e), and 1.3-fold greater shoot growth (g). h The photograph shows WT (top panels) and R3-1 (bottom panels) seedlings treated with 800 µM MB (right-hand panels) or untreated (left-hand panels). All values shown are means ± SE of three biological replicates; 10 seedlings were used in each replicate experiment (PPT 760 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Haque, M.E., Yoshida, Y. & Hasunuma, K. ROS resistance in Pisum sativum cv. Alaska: the involvement of nucleoside diphosphate kinase in oxidative stress responses via the regulation of antioxidants. Planta 232, 367–382 (2010). https://doi.org/10.1007/s00425-010-1173-2

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-010-1173-2

Keywords

Search

Navigation