Constitutive arginine-dependent nitric oxide synthase activity in different organs of pea seedlings during plant development
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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.
KeywordsNitric oxide Nitric oxide synthase Signalling Pea seedlings Plant development
confocal laser scanning microscopy
nitric oxide synthase
- DAF-2 DA
- DAF-FM DA
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.
- 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
- 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
- Leshem YY (2000) Nitric oxide in plants: occurrence, function and use. Kluwer, DordrechtGoogle Scholar
- Magalhaes JR, Singh RN, Passos LP (2005) Nitric oxide signaling in higher plants. Studium Press, LLC, Houston, pp 1–347Google Scholar
- 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