, Volume 224, Issue 2, pp 246–254 | Cite as

Constitutive arginine-dependent nitric oxide synthase activity in different organs of pea seedlings during plant development

  • Francisco J. Corpas
  • Juan B. Barroso
  • Alfonso Carreras
  • Raquel Valderrama
  • José M. Palma
  • Ana M. León
  • Luisa M. Sandalio
  • Luis A del Río
Original Article


Nitric oxide (NO) is an important signalling molecule in different animal and plant physiological processes. Little is known about its biological function in plants and on the enzymatic source or site of NO production during plant development. The endogenous NO production from l-arginine (NO synthase activity) was analyzed in leaves, stems and roots during plant development, using pea seedlings as a model. NOS activity was analyzed using a novel chemiluminescence-based assay which is more sensitive and specific than previous methods used in plant tissues. In parallel, NO accumulation was analyzed by confocal laser scanning microscopy using as fluorescent probes either DAF-2 DA or DAF-FM DA. A strong increase in NOS activity was detected in stems after 11 days growth, coinciding with the maximum stem elongation. The arginine-dependent NOS activity was constitutive and sensitive to aminoguanidine, a well-known irreversible inhibitor of animal NOS, and this NOS activity was differentially modulated depending on the plant organ and seedling developmental stage. In all tissues studied, NO was localized mainly in the vascular tissue (xylem) and epidermal cells and in root hairs. These loci of NO generation and accumulation suggest novel functions for NO in these cell types.


Nitric oxide Nitric oxide synthase Signalling Pea seedlings Plant development 





confocal laser scanning microscopy


nitric oxide


nitric oxide synthase


4,5-diaminofluorescein diacetate


4-aminomethyl-2′,7′-difluorofluorescein diacetate


NG-nitro-l-arginine methyl ester





AML acknowledges a PhD fellowship (F.P.I.) from the Ministry of Education and Science. This work was supported by the Dirección General de Investigación, Ministry of Education and Science (grants AGL2003-05524 and BFI2002-04440-CO2-01) and Junta de Andalucía (groups CVI 0192 and CVI 0286). Confocal laser scanning microscopy analyses were carried out at the Technical Services of the University of Jaén and special thanks are given to Miss Nieves de la Casa-Adán . The valuable technical help of Mr. Carmelo Ruíz-Torres is also acknowledged.


  1. Barroso JB, Corpas FJ, Carreras A, Sandalio LM, Valderrama R, Palma JM, Lupiáñez JA, del Río LA (1999) Localization of nitric oxide synthase in plant peroxisomes. J Biol Chem 274:36729–36733CrossRefPubMedGoogle Scholar
  2. Beligni MV, Lamattina L (2000) Nitric oxide stimulates seed germination and de-etiolation, and inhibits hypocotyl elongation, three light-inducible responses in plants. Planta 210:215–221PubMedCrossRefGoogle Scholar
  3. Bharathan G, Sinha NR (2001) The regulation of compound leaf development. Plant Physiol 127:1533–1538CrossRefPubMedGoogle Scholar
  4. Bradford MM (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  5. Caro A, Puntarulo S (1999) Nitric oxide generation by soybean embryonic axes. Possible effect on mitochondrial function. Free Rad Res 31:S205–S212CrossRefGoogle Scholar
  6. Corpas FJ, Trelease RN (1998) Differential expression of ascorbate peroxidase and a putative molecular chaperone in the boundary membrane of differentiating cucumber seedling peroxisomes. J Plant Physiol 153:332–338Google Scholar
  7. Corpas FJ, Barroso JB, del Río LA (2001) Peroxisomes as a source of reactive oxygen species and nitric oxide signal molecules in plant cells. Trends Plant Sci 6:145–150CrossRefPubMedGoogle Scholar
  8. Corpas FJ, Barroso JB, del Río LA (2004a) Enzymatic sources of nitric oxide in plant cells—beyond one protein-one function. New Phytol 162:246–248CrossRefGoogle Scholar
  9. Corpas FJ, Barroso JB, Carreras A, Quirós M, León AM, Romero-Puertas MC, Esteban FJ, Valderrama R, Palma JM, Sandalio LM, Gómez M, del Río LA (2004b) Cellular and subcellular localization of endogenous nitric oxide in young and senescent pea plants. Plant Physiol 136:2722–2733CrossRefGoogle Scholar
  10. Correa-Aragunde N, Graziano M, Lamattina L (2004) Nitric oxide plays a central role in determining lateral root development in tomato. Planta 218:900–905CrossRefPubMedGoogle Scholar
  11. Cueto M, Hernández-Perea O, Martín R, Ventura ML, Rodrigo J, Lamas S, Golvano MP (1996) Presence of nitric oxide synthase activity in roots and nodules of Lupinus albus. FEBS Lett 398:159–164CrossRefPubMedGoogle Scholar
  12. Dean JV, Harper JE (1988) The conversion of nitrite to nitrogen oxide(s) by the constitutive NAD(P)H-nitrate reductase enzyme from soybean. Plant Physiol 88:389–395PubMedCrossRefGoogle Scholar
  13. Delledonne M, Xia Y, Dixon RA, Lamb C (1998) Nitric oxide functions as a signal in plant disease resistance. Nature 394:585–588CrossRefPubMedGoogle Scholar
  14. Desikan R, Cheung MK, Bright J, Henson D, Hancock JT, Neill SJ (2004) ABA, hydrogen peroxide and nitric oxide signalling in stomatal guard cells. J Exp Bot 55:205–212CrossRefPubMedGoogle Scholar
  15. Durner J, Wendehenne D, Klessig DF (1998) Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose. Proc Natl Acad Sci USA 95:10328–10333CrossRefPubMedGoogle Scholar
  16. Ferrer MA, Ros Barceló A (1999) Differential effects of nitric oxide on peroxidase and H2O2 production by the xylem of Zinnia elegans. Plant Cell Environ 22:891–897CrossRefGoogle Scholar
  17. Foissner I, Wendehenne D, Langebartels C, Durner J (2000) In vivo imaging of an elicitor-induced nitric oxide burst in tobacco. Plant J 23:817–824CrossRefPubMedGoogle Scholar
  18. Gabaldón C, Gómez Ros LV, Pedreño MA, Ros Barceló A (2005) Nitric oxide production by the differentiating xylem of Zinnia elegans. New Phytol 165:121–130CrossRefPubMedGoogle Scholar
  19. Garcia-Mata C, Gay R, Sokolovski S, Hills A, Lamattina L, Blatt MR (2003) Nitric oxide regulates K+ and Cl channels in guard cells through a subset of abscisic acid-evoked signaling pathways. Proc Natl Acad Sci USA 100:11116–11121CrossRefPubMedGoogle Scholar
  20. Gould KS, Lamotte O, Klinguer A, Pugin A, Wendehenne D (2003) Nitric oxide production in tobacco leaf cells: a generalized stress response? Plant Cell Environ 26:1851–1862CrossRefGoogle Scholar
  21. Gouvêa CMCP, Souza JF, Magalhaes JR, Martins IS (1997) NO-releasing substances that induce growth elongation in maize root segments. Plant Growth Regul 21:183–187CrossRefGoogle Scholar
  22. Guo F-Q, Okamoto M, Crawford NM (2003) Identification of a plant nitric oxide synthase gene involved in hormonal signaling. Science 302:100–103CrossRefPubMedGoogle Scholar
  23. Hampl V, Walters CL, Archer SL (1996) Determination of nitric oxide by the chemiluminescence reaction with ozone. In: Feelisch M, Stamler JS (eds) Methods in nitric oxide research. John Wiley, Chichester, pp 309–318Google Scholar
  24. Kopyra M, Gwózdz EA (2003) Nitric oxide stimulates seed germination and counteracts the inhibitory effect of heavy metals and salinity on root growth of Lupinus luteus. Plant Physiol Biochem 41:1011–1017CrossRefGoogle Scholar
  25. Lamattina L, García-Mata C, Graziano M, Pagnussat G (2003) Nitric oxide: the versatility of an extensive signal molecule. Annu Rev Plant Biol 54:109–136CrossRefPubMedGoogle Scholar
  26. Laszlo F, Evans SM, Whittle BJ (1995) Aminoguanidine inhibits both constitutive and inducible nitric oxide synthase isoforms in rat intestinal microvasculature in vivo. Eur J Pharmacol 272:169–175CrossRefPubMedGoogle Scholar
  27. Leshem YY (2000) Nitric oxide in plants: occurrence, function and use. Kluwer, DordrechtGoogle Scholar
  28. Leshem YY, Wills RBH, Veng-Va Ku V (1998) Evidence for the function of the free radical gas-nitric oxide (NO) as an endogenous maturation and senescence regulating factor in higher plants. Plant Physiol Biochem 36:825–833CrossRefGoogle Scholar
  29. Magalhaes JR, Singh RN, Passos LP (2005) Nitric oxide signaling in higher plants. Studium Press, LLC, Houston, pp 1–347Google Scholar
  30. Nakatsubo N, Kojima H, Kikuchi K, Nagoshi H, Hirata Y, Maeda D, Imai Y, Irimura T, Nagano T (1998) Direct evidence of nitric oxide production from bovine aortic endothelial cells using new fluorescence indicators: diaminofluoresceins. FEBS Lett 427:263–266CrossRefPubMedGoogle Scholar
  31. Navarre DA, Wendehenne D, Durner J, Noad R, Klessig DF (2000) Nitric oxide modulates the activity of tobacco aconitase. Plant Physiol 122:573–582CrossRefPubMedGoogle Scholar
  32. Neill SJ, Desikan R, Clarke A, Hancock JT (2002) Nitric oxide is a novel component of abscisic acid signaling in stomatal guard cells. Plant Physiol 128:13–6CrossRefPubMedGoogle Scholar
  33. Neill SJ, Desikan R, Clarke A, Hancock JT (2003) Nitric oxide signalling in plants. New Phytol 159:11–35CrossRefGoogle Scholar
  34. Orozco-Cardenas ML, Ryan CA (2002) Nitric oxide negatively modulates wound signaling in tomato plants. Plant Physiol 130:487–493CrossRefPubMedGoogle Scholar
  35. Pagnussat GC, Simontacchi M, Puntarulo S, Lamattina L (2002) Nitric oxide is required for root organogenesis. Plant Physiol 129:954–956CrossRefPubMedGoogle Scholar
  36. Ribeiro EA, Cunha FQ, Tamashiro WMSC, Martins IS (1999) Growth phase-dependent subcellular localization of nitric oxide synthase in maize cells. FEBS Lett 445:283–286CrossRefPubMedGoogle Scholar
  37. del Río LA, Corpas FJ, Barroso JB (2004) Nitric oxide and nitric oxide synthase activity in plants. Phytochemistry 65:783–792CrossRefPubMedGoogle Scholar
  38. Rockel P, Strube F, Rockel A, Wildt J, Kaiser WM (2002) Regulation of nitric oxide (NO) production by plant nitrate reductase in vivo and in vitro. J Exp Bot 53:103–110CrossRefPubMedGoogle Scholar
  39. Rodríguez-Serrano M, Romero-Puertas MC, Gómez M, Barroso JB, del Río LA, Sandalio LM (2004) Imaging of ROS and nitric oxide production in pea plants under metal stress. Free Rad Biol Med 36:S139Google Scholar
  40. Shapiro AD (2005) Nitric oxide signaling in plants. Vitam Horm 72:339–398 PubMedCrossRefGoogle Scholar
  41. Simontacchi M, Jasid S, Puntarulo S (2004) Nitric oxide generation during early germination of Sorghum seeds. Plant Sci 167:839–847CrossRefGoogle Scholar
  42. Stöhr C, Ullrich WR (2002) Generation and possible roles of NO in plant roots and their apoplastic space. J Exp Bot 53:2293–2303CrossRefPubMedGoogle Scholar
  43. Takahashi S, Yamasaki H (2002) Reversible inhibition of photophosphorylation in chloroplasts by nitric oxide. FEBS Lett 512:145–148CrossRefPubMedGoogle Scholar
  44. Wojtaszek P (2000) Nitric oxide in plants: To NO or not to NO. Phytochemistry 54:1– 4CrossRefPubMedGoogle Scholar
  45. Xu YC, Zhao BL (2003) The main origin of endogenous NO in higher non-leguminous plants. Plant Physiol Biochem 41:833–838 CrossRefGoogle Scholar
  46. Yamasaki H, Shimoji H, Ohshiro Y, Sakihama Y (2001) Inhibitory effects of nitric oxide on oxidative phosphorylation in plant mitochondria. Nitric Oxide 5:261–270CrossRefPubMedGoogle Scholar
  47. Zhang C, Czymmek KJ, Shapiro AD (2003) Nitric oxide does not trigger early programmed cell death events but may contribute to cell-to-cell signaling governing progression of the Arabidopsis hypersensitive response. Mol Plant Microbe Interact 16:962–972PubMedCrossRefGoogle Scholar
  48. Zottini M, Formentin E, Scattolin M, Carimi F, LoSchiavo F, Terzi M (2002) Nitric oxide affects plant mitochondrial functionality in vivo. FEBS Lett 515:75–78CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Francisco J. Corpas
    • 1
  • Juan B. Barroso
    • 2
  • Alfonso Carreras
    • 2
  • Raquel Valderrama
    • 2
  • José M. Palma
    • 1
  • Ana M. León
    • 1
  • Luisa M. Sandalio
    • 1
  • Luis A del Río
    • 1
  1. 1.Departamento de Bioquímica, Biología Celular y Molecular de Plantas, Estación Experimental del Zaidín (EEZ)Consejo Superior de Investigaciones CientíficasGranadaSpain
  2. 2.Grupo de Señalización Molecular y Sistemas Antioxidantes en Plantas, Unidad Asociada al Consejo Superior de Investigaciones Científicas (EEZ), Área de Bioquímica y Biología MolecularUniversidad de JaénJaénSpain

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