The cellular and subcellular localization of endogenous nitric oxide (NO . ) in leaves from young and senescent pea (Pisum sativum) plants was studied. Confocal laser scanning microscopy analysis of pea leaf sections with the fluorescent probe 4,5-diaminofluorescein diacetate revealed that endogenous NO. was mainly present in vascular tissues (xylem and phloem).Green fluorescence spots were also detected in the epidermal cells, palisade and spongy mesophyll cells, and guard cells. ) is a widespread intracellular and intercellular messenger with a broad spectrum of regulatory functions in many physiological processes (Moncada et al., 1991;Ignarro, 2002;Wendehenne et al., 2001;Lamattina et al., 2003;Neill et al., 2003;del Río et al., 2004). In recent years, NO. was reported to be involved in many key physiological processes of plants, such as ethylene emission (Leshem and Haramaty, 1996), response to drought (Leshem, 1996), disease resistance (Delledonne et al., 1998(Delledonne et al., , 2001Durner et al., 1998;Clarke et al., 2000), growth and cell proliferation (Ribeiro et al., 1999), maturation and senescence (Leshem et al., 1998), apoptosis/programmed cell death (Magalhaes et al., 1999;Clarke et al., 2000;Pedroso and Durzan, 2000;Pedroso et al., 2000a;Zhang et al., 2003), and stomatal closure Lamattina, 2001, 2002;Neill et al., 2002a;García-Mata et al., 2003).The application of exogenous NO . to plants has been used as a tool to study how this molecule affects some physiological processes, such as inhibition of certain enzyme activities (Clark et al., 2000;Navarre et al., 2000), cell wall lignification (Ferrer and Ros Barceló , 1999) In animal systems, a considerable attention is being dedicated to this molecule and the enzyme responsible for its production from L-Arg, nitric oxide synthase (NOS; EC 1.14. 13.39;Hemmens and Mayer, 1998; Alderton et al., 2001). On the contrary, in plants comparatively much less is known on the source of NO . production (Neill et al., 2003;Wendehenne et al., 2003;del Río et al., 2004 Article, publication date, and citation information can be found at www.plantphysiol.org/cgi

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.

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