Cellular and Subcellular Localization of Endogenous Nitric Oxide in Young and Senescent Pea Plants

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Abstract
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. In senescent leaves, NO˙ generation was clearly reduced in the vascular tissues. At the subcellular level, by electron paramagnetic resonance spectroscopy with the spin trap Fe(MGD)2 and fluorometric analysis with 4,5-diaminofluorescein diacetate, NO˙ was found to be an endogenous metabolite of peroxisomes. The characteristic three-line electron paramagnetic resonance spectrum of NO˙, with g = 2.05 and aN = 12.8 G, was detected in peroxisomes. By fluorometry, NO˙ was also found in these organelles, and the level measured of NO˙ was linearly dependent on the amount of peroxisomal protein. The enzymatic production of NO˙ from l-Arg (nitric oxide synthase [NOS]-like activity) was measured by ozone chemiluminiscence. The specific activity of peroxisomal NOS was 4.9 nmol NO˙ mg−1 protein min−1; was strictly dependent on NADPH, calmodulin, and BH4; and required calcium. In senescent pea leaves, the NOS-like activity of peroxisomes was down-regulated by 72%. It is proposed that peroxisomal NO˙ could be involved in the process of senescence of pea leaves.