Skip to main content
SpringerLink
Log in

Class-1 hemoglobin and antioxidant metabolism in alfalfa roots

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

Abstract

In the course of nitric oxide (NO) scavenging, hemoglobin (Hb) turnover is linked to antioxidant metabolism and affects the cellular redox level. The influence of Hb presence on the ascorbate-glutathione cycle enzymes and the levels of H2O2 and ascorbate was investigated in alfalfa root cultures transformed to over-express (Hb+) or down-regulate (Hb–) class-1 Hb. Hb+ lines had substantially increased ascorbate levels as well as elevated monodehydroascorbate reductase and ascorbate peroxidase activities. Hb– lines showed significant increases in dehydroascorbate reductase and glutathione reductase activities. The observed changes in ascorbate and ascorbate-glutathione cycle enzymes were pronounced both at high (40 kPa) and low (3 kPa) O2 pressures. Hb– lines had significantly reduced levels of the NO- and H2O2-sensitive enzyme, aconitase, as compared to Hb+ lines. This reduced activity was likely due the higher levels of NO in Hb– lines, as treatment of plant extracts with the NO-donor DEANO also affected aconitase activity. The H2O2 levels were not significantly different amongst the lines and showed no variation with change in oxygen partial pressure. In conclusion, the expression of class-1 Hb improves the antioxidant status through increased ascorbate levels and increased activity of enzymes involved in H2O2 removal.

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

APX:

Ascorbate peroxidase

DEANO:

Sodium 2-(N, N-diethylamino)-diazenolate-2-oxide

DHA:

Dehydroascorbate

DHAR:

Dehydroascorbate reductase

GR:

Glutathione reductase

GSH:

Reduced glutathione

GSSG:

Oxidized glutathione

Hb:

Hemoglobin

MDHA:

Monodehydroascorbate (ascorbate free radical)

MDHAR:

Monodehydroascorbate reductase

MetHb:

Methemoglobin

NO:

Nitric oxide

ROS:

Reactive oxygen species

References

  • Aebi H (1974) Catalase. In: Bergmeyer HU (ed) Methods of enzymatic analysis, vol. 2. Academic Press, NY pp 673–684

  • Arrigoni O, Calabrese G, de Gara L, Bitonti MB, Liso R (1997) Correlation between changes in cell ascorbate and growth of Lupinus albus seedlings. J Plant Physiol 150:302–308

    CAS  Google Scholar 

  • Bartoli CG, Pastori GM, Foyer CH (2000) Ascorbate biosynthesis in mitochondria is linked to the electron transport chain between complexes III and IV. Plant Physiol 123:335–343

    Article  PubMed  CAS  Google Scholar 

  • Beligni MV, Lamattina L (2001) Nitric oxide: a non-traditional regulator of plant growth. Trends Plant Sci 6:508–509

    Article  PubMed  CAS  Google Scholar 

  • Benatti U, Morelli A, Guida L, de Flora A (1983) The production of activated oxygen species by an interaction of methemoglobin with ascorbate. Biochem Biophys Res Communs 111:980–987

    Article  CAS  Google Scholar 

  • Creissen G, Firmin J, Fryer M, Kular B, Leyland N, Reynolds H, Pastori G, Wellburn F, Baker N, Wellburn A, Mullineaux P (1999) Elevated glutathione biosynthetic capacity in the chloroplasts of transgenic tobacco plants paradoxically causes increased oxidative stress. Plant Cell 11:1277–1291

    Article  PubMed  CAS  Google Scholar 

  • de Pinto MC, Tommasi F, de Gara L (2002) Changes in the antioxidant systems as part of the signaling pathway responsible for the programmed cell death activated by nitric oxide and reactive oxygen species in tobacco Bright-Yellow 2 cells. Plant Physiol 130:698–708

    Article  PubMed  CAS  Google Scholar 

  • Dordas C, Hasinoff B, Igamberdiev AU, Manac’h N, Rivoal J, Hill RD (2003) Expression of a stress-induced hemoglobin affects NO levels produced by alfalfa under hypoxic stress. Plant J 35:763–770

    Article  PubMed  CAS  Google Scholar 

  • Foyer C, Halliwell B (1976) The presence of glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta 133:21–25

    Article  Google Scholar 

  • Frey AD, Oberle BT, Farres J, Kallio PT (2004) Expression of Vitreoscilla haemoglobin in tobacco cell cultures relieves nitrosative stress in vivo and protects from NO in vitro. Plant Biotechnol J 2:221–231

    Article  PubMed  CAS  Google Scholar 

  • Guilbaut GG, Kramer DN, Hackley E (1967) A new substrate for fluorimetric determination of oxidative enzymes. Anal Chem 39:271

    Article  PubMed  Google Scholar 

  • Hill RD (1998) What are hemoglobins doing in plants? Can J Bot 76:707–712

    Article  CAS  Google Scholar 

  • Hossain MA, Nakano Y, Asada K (1984) Monodehydroascorbate reductase in spinach chloroplasts and its participation in regeneration of ascorbate for scavenging hydrogen peroxide. Plant Cell Physiol 25:385–395

    CAS  Google Scholar 

  • Igamberdiev AU, Hill RD (2004) Nitrate, NO and haemoglobin in plant adaptation to hypoxia: an alternative to classic fermentation pathways. J Exp Bot 55:2473–2482

    Article  PubMed  CAS  Google Scholar 

  • Igamberdiev AU, Seregélyes C, Manac’h N, Hill RD (2004) NADH-dependent metabolism of nitric oxide in alfalfa root cultures expressing barley hemoglobin. Planta 219:95–102

    Article  PubMed  CAS  Google Scholar 

  • Igamberdiev AU, Bykova NV, Hill RD (2006) Nitric oxide scavenging by barley hemoglobin is facilitated by a monodehydroascorbate reductase-mediated ascorbate reduction of methemoglobin. Planta (submitted)

  • Kampfenkel K, Montagu MV, Inzé D (1995) Extraction and determination of ascorbate and dehydroascorbate from plant tissue. Anal Biochem 225:165–167

    Article  PubMed  CAS  Google Scholar 

  • Klok EJ, Wilson IW, Wilson D, Chapman SC, Ewing RM, Somerville SC, Peacock WJ, Dolferus R, Dennis ES (2002) Expression profile analysis of the low-oxygen response in Arabidopsis root cultures. Plant Cell 14:2481–2494

    Article  PubMed  CAS  Google Scholar 

  • Masuoka N, Kodama H, Abe T, Wang D-H, Nakano T (2003) Characterization of hydrogen peroxide removal reaction by hemoglobin in the presence of reduced pyridine nucleotides. Biochim Biophys Acta 1637:46–54

    PubMed  CAS  Google Scholar 

  • Membrillo-Hernández J, Ioannidis N, Poole RK (1996) The flavohaemoglobin (HMP) of Escherichia coli generates superoxide in vitro and causes oxidative stress in vivo. FEBS Lett 382:141–144

    Article  PubMed  Google Scholar 

  • Møller IM (2001) Plant mitochondria and oxidative stress: Electron transport, NAD(P)H turnover, and metabolism of reactive oxygen species. Annu Rev Plant Physiol Plant Mol Biol 52:561–591

    Article  PubMed  Google Scholar 

  • Moreau S, Puppo A, Davies MJ (1995) The reactivity of ascorbate with different redox states of leghemoglobin. Phytochemistry 39:1281–1286

    Article  PubMed  CAS  Google Scholar 

  • Navarre DA, Wendehenne D, Durner J, Noad R, Klessig DF (2000) Nitric oxide modulates the activity of tobacco aconitase. Plant Physiol 122:573–582

    Article  PubMed  CAS  Google Scholar 

  • Noctor G, Foyer C (1998) Ascorbate and glutathione: keeping active oxygen under control. Annu Rev Plant Physiol Plant Mol Biol 49:249–279

    Article  PubMed  CAS  Google Scholar 

  • Osswald WF, Kraus R, Hippeli S, Benz B, Volpert R, Elstner EF (1992) Comparison of the enzymatic activities of dehydroascorbic acid reductase, glutathione reductase, catalase, peroxidase and superoxide dismutase of healthy and damaged spruce needles (Picea abies (L.) Karst.). J Plant Physiol 139:742–748

    CAS  Google Scholar 

  • Perazzolli M, Dominici P, Romero-Puertas MC, Zago E, Zeier J, Sonoda M, Lamb C, Delledonne M (2004) Arabidopsis nonsymbiotic hemoglobin AHb1 modulates nitric oxide bioactivity. Plant Cell 16:2785–2794

    Article  PubMed  CAS  Google Scholar 

  • Rose IA, O’Connel EL (1967) Mechanism of aconitase action. I. The hydrogen transfer reaction. J Biol Chem 242:1870–1879

    CAS  Google Scholar 

  • Sakamoto A, Sakurao S, Fukunaga K, Matsubara T, Ueda-Hashimoto M, Tsukamoto S, Takahashi M, Morikawa H (2004) Three distinct Arabidopsis hemoglobins exhibit peroxidase-like activity and differentially mediate nitrite-dependent protein nitration. FEBS Lett 572:27–32

    Article  PubMed  CAS  Google Scholar 

  • Sowa AW, Duff SMG, Guy PA, Hill RD (1998) Altering hemoglobin levels changes energy status in maize cells under hypoxia. Proc Natl Acad Sci USA 95:10317–10321

    Article  PubMed  CAS  Google Scholar 

  • Sullivan SG, Stern A (1982) Effects of ascorbate on methemoglobin reduction in intact red cells. Arch Biochem Biophys 213:590–594

    Article  PubMed  CAS  Google Scholar 

  • Verniquet F, Gaillard J, Neuburger M, Douce R (1991) Rapid inactivation of plant aconitase by hydrogen peroxide. Biochem J 276:643–648

    PubMed  CAS  Google Scholar 

  • Yang LX, Wang RY, Ren F, Liu J, Cheng J, Lu YT (2005) AtGLB1 enhances the tolerance of Arabidopsis to hydrogen peroxide stress. Plant Cell Physiol 46:1309–1316

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors are grateful to Doug Durnin for providing excellent technical support. This work was supported by the Natural Sciences and Engineering Research Council of Canada (OGP 4689).

Author information

Authors and Affiliations

  1. Department of Plant Science, University of Manitoba, Winnipeg, MB, R3T 2N2, Canada

    Abir U. Igamberdiev, Maria Stoimenova, Csaba Seregélyes & Robert D. Hill

Authors
  1. Abir U. Igamberdiev
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Maria Stoimenova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Csaba Seregélyes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Robert D. Hill
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Robert D. Hill.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Igamberdiev, A.U., Stoimenova, M., Seregélyes, C. et al. Class-1 hemoglobin and antioxidant metabolism in alfalfa roots. Planta 223, 1041–1046 (2006). https://doi.org/10.1007/s00425-005-0145-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-005-0145-4

Keywords

Search

Navigation