Endogenous Superoxide Production and the Nitrite/Nitrate Ratio Control the Concentration of Bioavailable Free Nitric Oxide in Leaves

Abstract

We have quantitatively measured nitric oxide production in the leaves of Arabidopsis thaliana and Vicia faba by adapting ferrous dithiocarbamate spin tapping methods previously used in animal systems. Hydrophobic diethyldithiocarbamate complexes were used to measure NO interacting with membranes, and hydrophilic N-methyl-D-glucamine dithiocarbamate was used to measure NO released into the external solution. Both complexes were able to trap levels of NO, readily detectable by EPR spectroscopy. Basal rates of NO production (in the order of 1 nmol g ؊1 h ؊1 ) agreed with previous studies. However, use of methodologies that corrected for the removal of free NO by endogenously produced superoxide resulted in a significant increase in trapped NO (up to 18 nmol g ؊1 h ؊1 ). Basal NO production in leaves is therefore much higher than previously thought, but this is masked by significant superoxide production. The effects of nitrite (increased rate) and nitrate (decreased rate) are consistent with a role for nitrate reductase as the source of this basal NO production. However, rates under physiologically achievable nitrite concentrations never approach that reported following pathogen induction of plant nitric-oxide synthase. In Hibiscus rosa sinensis, the addition of exogenous nitrite generated sufficient NO such that EPR could be used to detect its production using endogenous spin traps (forming paramagnetic dinitrosyl iron complexes). Indeed the levels of this nitrosylated iron pool are sufficiently high that they may represent a method of maintaining bioavailable iron levels under conditions of iron starvation, thus explaining the previously observed role of NO in preventing chlorosis under these conditions.The phenomenon of NO production by plants was first reported by Klepper in 1979 (1), significantly earlier than the discovery of NO generation by animals (1985)(1986)(1987). Nevertheless research in the plant NO field has lagged behind the animal field for two important reasons: the absence of a physiological phenomenon elicited by NO and a lack of understanding of the molecular mechanism underpinning any such phenomenon. In animals the role of NO as the endotheliumderived relaxing factor and the discovery of the L-arginine/ nitric-oxide synthase/guanylate cyclase signaling pathway (2, 3) provided the impetus for the explosion of research in this area in the 1990s (4). However, recently there has been a similar expansion of interest in the role of NO in plants.Although a complete molecular model of the physiological role of NO in plants remains to be determined, a number of normal physiological processes are now known to be modulated by NO production. These include: production of the hormone ethylene (5), germination (6), programmed cell death (7, 8), senescence (5), and stomatal closure (9). NO is also involved in pathogen defense, either directly (10) or via production of antimicrobial phytoalexins (11). There are several recent reviews covering the role of NO in plants (12)(13)(14)(15)(16).Although the molecula...

An Iron-induced Nitric Oxide Burst Precedes Ubiquitin-dependent Protein Degradation for Arabidopsis AtFer1 Ferritin Gene Expression

“…These dinitrosyl-iron complexes are relatively stable in contrast to the highly reactive free NO molecule (65,66) and were shown to be potential NO carrier molecules in mammals (62). In plants, interactions of NO with hemes (67-69) or iron-sulfur clusters (70), and S-nitrosylation reactions (71) have been shown, and dinitrosyl-iron complexes have been detected (72). A post-translational modification of a plastidial protein by NO could be involved in the pathway leading to AtFer1 de-repression.…”

Reactive Species and Antioxidants. Redox Biology Is a Fundamental Theme of Aerobic Life

“…(Beckman and Koppenol, 1996;Halliwell et al, 1999;Greenacre and Ischiropoulos, 2001;Alvarez and Radi, 2003). It may happen in plants as well, since they can make both O 2 c 2 and NO c (Vanin et al, 2004), but few data are available.…”

Nitric Oxide Acts Downstream of Auxin to Trigger Root Ferric-Chelate Reductase Activity in Response to Iron Deficiency in Arabidopsis

“…When Fe-deficient roots were treated with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), the accumulation of mRNA for ferric reduction oxidase 1 (FRO1), IRT1, and the basic helix-loop-helix transcription factor (FIT) homolog, FER, was inhibited. Accordingly, the application of the NO donor, S-nitrosoglutathione (GSNO), induced the expression of these same genes (Graziano and Lamattina, 2007a), which coincided with the alleviation of oxidative damage (Sun et al, 2006) and the enhancement of dinitrosyl-Fe complexes formation (Vanin et al, 2004;Graziano and Lamattina, 2007b), which should improve Fe availability inside the plant (Graziano et al, 2002). However, a down-regulation of FRO1, IRT1, and FER in Fe-deficient roots of the tomato mutant fer could not be reversed by the addition of NO, suggesting that the NO acts upstream of FER to initiate adaptations to Fe deficiency (Graziano and Lamattina, 2007a).…”