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 a_N = 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.

Low temperature is an environmental stress that affects crop production and quality and regulates the expression of many genes, and the level of a number of proteins and metabolites. Using leaves from pepper (Capsicum annum L.) plants exposed to low temperature (8 °C) for different time periods (1 to 3 d), several key components of the metabolism of reactive nitrogen and oxygen species (RNS and ROS, respectively) were analysed. After 24 h of exposure at 8 °C, pepper plants exhibited visible symptoms characterized by flaccidity of stems and leaves. This was accompanied by significant changes in the metabolism of RNS and ROS with an increase of both protein tyrosine nitration (NO₂‐Tyr) and lipid peroxidation, indicating that low temperature induces nitrosative and oxidative stress. During the second and third days at low temperature, pepper plants underwent cold acclimation by adjusting their antioxidant metabolism and reverting the observed nitrosative and oxidative stress. In this process, the levels of the soluble non‐enzymatic antioxidants ascorbate and glutathione, and the activity of the main NADPH‐generating dehydrogenases were significantly induced. This suggests that ascorbate, glutathione and the NADPH‐generating dehydrogenases have a role in the process of cold acclimation through their effect on the redox state of the cell.