, Volume 218, Issue 3–4, pp 125–133 | Cite as

Nonsymbiotic hemoglobins in rice are synthesized during germination and in differentiating cell types

  • E. J. H. Ross
  • L. Shearman
  • M. Mathiesen
  • Y. J. Zhou
  • R. Arredondo-Peter
  • G. Sarath
  • R. V. Klucas


Nonsymbiotic hemoglobins (ns-Hbs) previously have been found in monocots and dicots; however, very little is known about the tissue and cell type localization as well as the physiological function(s) of these oxygen-binding proteins. We report the immunodetection and immunolocalization of ns-Hbs in rice (Oryza sativa L.) by Western blotting and in situ confocal laser scanning techniques. Ns-Hbs were detected in soluble extracts of different tissues from the developing rice seedling by immunoblotting. Levels of ns-Hbs increased in the germinating seed for the first six days following imbibition and remained relatively constant thereafter. In contrast, ns-Hb levels decreased during leaf maturation. Roots and mesocotyls contained detectable, but low levels of ns-Hbs. Split-seed experiments revealed that ns-Hbs are synthesized de novo during seed germination and are expressed in the absence of any signal originating from the embryo. Immunolocalization of ns-Hbs by confocal microscopy indicated the presence of ns-Hbs primarily in differentiated and differentiating cell types of the developing seedling, such as the aleurone, scutellum, root cap cells, sclerenchyma, and tracheary elements. To our knowledge, this is the first report of the specific cellular localization of these proteins during seedling development.


Nonsymbiotic hemoglobin Plant development Immunodetection Confocal microscopy Seed germination Oryza sativa







Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Andersson CR, Jensen EO, Llewellyn DJ, Dennis ES, Peacock WJ (1996) A new hemoglobin gene from soybean: a role for hemoglobin in all plants. Proc Natl Acad Sci USA 93: 5682–5687Google Scholar
  2. — Llewellyn DJ, Peacock WJ, Dennis ES (1997) Cell-specific expression of the promoters of two nonlegume hemoglobin genes in transgenic legume,Lotus corniculatus. Plant Physiol 113: 45–57Google Scholar
  3. Appleby CA (1984) Leghemoglobins andRhizobium respiration. Annu Rev Plant Physiol 35: 443–478Google Scholar
  4. — (1992) The origin and functions of haemoglobin in plants. Sci Prog 76: 365–398Google Scholar
  5. — Tjepkema JD, Trinick MJ (1983) Hemoglobin in a nonleguminous plantParasponia: possible genetic origin and function in nitrogen fixation. Science 220: 951–953Google Scholar
  6. — Bogusz V, Dennis ES, Peacock WJ (1988) A role for hemoglobin in all plant roots? Plant Cell Environ 11: 359–367Google Scholar
  7. Arredondo-Peter R, Hargrove MS, Sarath G, Moran JF, Lohrman J, Olson JS, Klucas RV (1997) Rice hemoglobins: gene cloning, analysis and oxygen-binding kinetics of a recombinant protein synthesized inEscherichia coli. Plant Physiol 115: 1259–1266Google Scholar
  8. — — Moran JF, Sarath G, Klucas RV (1998) Plant hemoglobins. Plant Physiol 118: 1121–1125Google Scholar
  9. Bogusz D, Llewellyn DJ, Craig S, Dennis ES, Appleby CA, Peacock WJ (1990) Nonlegume hemoglobin genes retain organ specific expression in heterologous transgenic plants. Plant Cell 2: 633–641Google Scholar
  10. Bolognesi M, Bordo D, Rizzi M, Tarricone C, Ascenzi P (1997) Nonvertebrate hemoglobins: structural bases for reactivity. Prog Biophys Mol Biol 68: 29–68Google Scholar
  11. Briggs DE (1972) Enzyme formation, cellular breakdown and the distribution of gibberellins in the endosperm of barley. Planta 108: 351–358Google Scholar
  12. Duff SMG, Wittenberg JB, Hill RD (1997) Expression, purification and properties of recombinant barley (Hordeum spp.) hemoglobin: optical spectra and reactions with gaseous ligands. J Biol Chem 272: 16746–16752Google Scholar
  13. — Guy PA, Nie X, Durnin DC, Hill RD (1998) Haemoglobin expression in germinating barley. Seed Sci Res 8: 431–436Google Scholar
  14. Franche C, Diouf D, Laplaze L, Auguy F, Frutz T, Rio M, Duhoux E, Bogusz D (1998) Soybean (Ibc3),Parasponia andTrema hemoglobin gene promoters retain symbiotic and nonsymbiotic specificity in transgenic casuarinaceae: implications for hemoglobin gene evolution and root nodule symbioses. Mol Plant Microbe Interact 11: 887–894Google Scholar
  15. Fukuda H (1996) Xylogenesis: initiation, progression, and cell death. Annu Rev Plant Physiol Plant Mol Biol 47: 299–325Google Scholar
  16. — Komamine A (1980) Direct evidence for cytodifferentiation to tracheary elements without intervening mitosis in a culture of single cells isolated from the mesophyll ofZinnia elegans. Plant Physiol 65: 61–64Google Scholar
  17. Gardner AM, Martin LA, Gardner PR, Dou Y, Olson JS (2000) Steady state and transient kinetics ofEscherichia coli nitric-oxide dioxygenase (flavohemoglobin). J Biol Chem 275: 12581–12589Google Scholar
  18. Gardner PR, Gardner AM, Martin LA, Salzman AL (1998) Nitric oxide dioxygenase: an enzymic function for flavohemoglobin. Proc Natl Acad Sci USA 95: 10378–10383Google Scholar
  19. Goodman MD, Hargrove MS (2001) Quaternary structure of rice nonsymbiotic hemoglobin. J Biol Chem 276: 6834–6839Google Scholar
  20. Hargrove MS (2000) A flash photolysis method to characterize hexacoordinate hemoglobin kinetics. Biophys J 79: 2733–2738Google Scholar
  21. Hill RD (1998) What are hemoglobins doing in plants? Can J Bot 76: 707–712Google Scholar
  22. Jacobsen-Lyon K, Jensen EO, Jorgensen J, Marcker KA, Peacock WJ, Dennis ES (1995) Symbiotic and nonsymbiotic hemoglobin genes ofCasuarina glauca. Plant Cell 7: 213–233Google Scholar
  23. Jiao J, Echevarria C, Vidai J, Chollet R (1991) Protein turnover as a component in the light/dark regulation of phosphoenolpyruvate carboxylase protein-serine kinase activity in C4 plants. Proc Natl Acad Sci USA 88: 2712–2715Google Scholar
  24. Jones A (2000) Does the plant mitochondrion integrate cellular stress and regulate programmed cell death? Trends Plant Sci 5: 225–230Google Scholar
  25. Jones TJ, Rost TL (1989) The developmental anatomy and ultrastructure of somatic embryos from rice (Oryzasativa L) scutellum epithelium cells. Bot Gaz 150: 41–49Google Scholar
  26. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685Google Scholar
  27. Nie X, Hill RD (1997) Mitochondrial respiration and hemoglobin gene expression in barley aleurone tissue. Plant Physiol 114: 835–840Google Scholar
  28. Nomura T, Kono Y, Akazawa T (1969) Enzymic mechanism of starch breakdown in germinating rice seeds II: scutellum as the site of sucrose synthesis. Plant Physiol 44: 765–769Google Scholar
  29. Penheiter A, Duff SMG, Sarath G (1997) Soybean root nodule acid phosphatase. Plant Physiol 114: 597–604Google Scholar
  30. Radi SH, Maeda E (1987) Ultrastructures of rice scutellum cultured with attached root using two separate media as compared to the intact seedling. Jpn J Crop Sci 56: 73–84Google Scholar
  31. Roberts K, McCann MC (2000) Xylogenesis: the birth of a corpse. Curr Opin Plant Biol 3: 517–522Google Scholar
  32. Seregélyes C, Mustárdy L, Ayaydin F, Sass L, Kovács L, Endre G, Lukács N, Vass I, Kiss GB, Horváth GV, Dudits D (2000) Nuclear localization of a hypoxia-inducible novel non-symbiotic hemoglobin in cultured alfalfa cells. FEBS Lett 482: 125–130Google Scholar
  33. Strózycki PM, Karlowski WM, Dessaux Y, Petit A, Legocki AB. (2000) Lupineleghemoglobin I: expression in transgenicLotus and tobacco tissues. Mol Gen Genet 263: 173–182Google Scholar
  34. Taylor ER, Nie XZ, MacGregor AW, Hill RD (1994) A cereal haemoglobin gene is expressed in seed and root tissues under anaerobic conditions. Plant Mol Biol 24: 853–862Google Scholar
  35. Trevaskis B, Watts RA, Andersson CR, Llewellyn DJ, Hargrove MS, Olson JS, Dennis ES, Peacock WJ (1997) Two hemoglobin genes inArabidopsis thaliana: the evolutionary origins of leghemoglobins. Proc Natl Acad Sci USA 94: 12230–12234Google Scholar
  36. Vinogradov SN, Walz DA, Pohajdak B, Moens L, Kapp OH, Suzuki T, Trotman CN (1993) Adventitious variability? The amino acid sequences of nonvertebrate globins. Comp Biochem Physiol (b) 106: 1–26Google Scholar
  37. Woo H, Hawes MC (1997) Cloning of genes whose expression is correlated with mitosis and localized in dividing cells in root caps ofPisum sativum L. Plant Mol Biol 35: 1045–1051Google Scholar

Copyright information

© Springer-Verlag 2001

Authors and Affiliations

  • E. J. H. Ross
    • 1
  • L. Shearman
    • 1
  • M. Mathiesen
    • 2
  • Y. J. Zhou
    • 2
  • R. Arredondo-Peter
    • 3
  • G. Sarath
    • 1
    • 2
  • R. V. Klucas
    • 1
  1. 1.Department of Biochemistry, George W. Beadle CenterUniversity of Nebraska-LincolnLincolnUSA
  2. 2.Center for Biotechnology, George W. Beadle CenterUniversity of Nebraska-LincolnLincoln
  3. 3.Centro de Investigación sobre Fijación de NitrógenoUniversidad Nacional Autónoma de MéxicoCuernavaca

Personalised recommendations