AtNOS/AtNOA1 Is a Functional Arabidopsis thaliana cGTPase and Not a Nitric-oxide Synthase

Abstract

AtNOS1 was previously identified as a potential nitric-oxide synthase (NOS) in Arabidopsis thaliana, despite lack of sequence similarity to animal NOSs. Although the dwarf and yellowish leaf phenotype of Atnos1 knock-out mutant plants can be rescued by treatment with exogenous NO, doubts have recently been raised as to whether AtNOS1 is a true NOS. Moreover, depending on the type of physiological responses studied, Atnos1 is not always deficient in NO induction and/or detection, as previously reported. Here, we present experimental evidence showing that AtNOS1 is unable to bind and oxidize arginine to NO. These results support the argument that AtNOS1 is not a NOS. We also show that the renamed NO-associated protein 1 (AtNOA1) is a member of the circularly permuted GTPase family (cGTPase). AtNOA1 specifically binds GTP and hydrolyzes it. Complementation experiments of Atnoa1 mutant plants with different constructs of AtNOA1 show that GTP hydrolysis is necessary but not sufficient for the physiological function of AtNOA1. Mutant AtNOA1 lacking the C-terminal domain, although retaining GTPase activity, failed to complement Atnoa1, suggesting that this domain plays a crucial role in planta. cGTPases appear to be RNA-binding proteins, and the closest homolog of AtNOA1, the Bacillus subtilis YqeH, has been shown to participate in ribosome assembly and stability. We propose a similar function for AtNOA1 and discuss it in the light of its potential role in NO accumulation and plant development.Numerous studies have demonstrated that plants, like animals, generate nitric oxide (NO) 3 to regulate a wide range of physiological processes. NO is involved in plant development; it represses flowering (1), reduces seed dormancy (2), and regulates germination (3). NO production has also been detected following different environmental stresses. For example, NO regulates stomata closure in response to abiotic stress (4), and in response to biotic stress, NO participates in induction of plant defenses (5-7).Although NO plays a role as significant in plants as it does in animals, NO synthesis in planta is still a matter of debate (8). Two major routes have been proposed for NO formation in plants. The first one relies on the reduction of nitrite to NO. Several studies demonstrate that nitrate reductase, whose primary function is to catalyze the reduction of nitrate to nitrite, can convert nitrite to NO with low efficiency (9, 10). Nitrite can also be reduced to NO by a plasma membrane-bound nitrite:NOreductase(11),byamitochondrialelectrontransportdependent reductase (12), or nonenzymatically in acidic, reducing environments (13). The second probable NO biosynthetic pathway uses arginine as a substrate, following a reaction similar to that observed for the well characterized animal NOSs. Indeed, several lines of evidence suggest the existence of a mammalian NOS-like enzyme in plants. Application of arginine analogs, inhibitors of animal NOSs, results in a reduction of NO detected in plants (5, 6, 14 -17). Arginine-dependent citrul...

Hunting for Plant Nitric Oxide Synthase Provides New Evidence of a Central Role for Plastids in Nitric Oxide Metabolism

“…For example, the accumulation of plastid-targeted enzymes of the methylerythritol pathway causing fosmidomycin resistance in a recently isolated nos1 allele named resistant to inhibition by fosmidomycin1 (rif1) was unaffected by treatment with an NO donor (Flores-Pé rez et al, 2008). These results and the failure to reproduce the published results on the detection of NOS activity of the recombinant NOS1 protein (Crawford et al, 2006;Zemojtel et al, 2006a;Moreau et al, 2008) and the absence of such activity in bacterial homologs (Sudhamsu et al, 2008) led to the conclusion that NOS1 is not a NOS. However, compelling data on the specific biological role of this protein (renamed NITRIC OXIDE ASSOCIATED PROTEIN1 [NOA1]) have only recently been available.…”

“…Interestingly, YqeH is able to complement the Arabidopsis noa1/rif1 mutant (Flores-Pé rez et al, Sudhamsu et al, 2008), indicating a similar function for bacterial and plant homologs. The Arabidopsis protein was recently demonstrated specifically to bind and hydrolyze GTP, a function that is necessary for complementation of the knockout mutant (Moreau et al, 2008). However, truncated versions of the protein with a functional GTP binding domain but lacking the C-terminal domain failed to complement the mutant, indicating that both domains are required for NOA1/RIF1 function.…”

“…Structural studies of the YqeH protein led to the conclusion that the C-terminal domain harbors a recognition module for peptides and nucleic acids with conserved residues important for RNA binding coupled to GTP hydrolysis (Sudhamsu et al, 2008). Because this module is also found in the Arabidopsis protein, it was proposed that the GTPase activity of YqeH and NOA1/RIF1 might be functionally linked to RNA and/or protein binding (Moreau et al, 2008;Sudhamsu et al, 2008).…”

Nitric Oxide Contributes to Cadmium Toxicity in Arabidopsis by Promoting Cadmium Accumulation in Roots and by Up-Regulating Genes Related to Iron Uptake

“…3B). To further characterize the source of NO during Cd 2+ treatment, similar experiments were performed in the atnoa1 mutant impaired in the expression of the AtNOA1 gene encoding an enzyme initially thought to catalyze NO synthesis from L-Arg (Crawford et al, 2006;Moreau et al, 2008). The mutant atnoa1 was reported to be impaired in NO synthesis or accumulation in response to abscisic acid or lipopolysaccharide treatments (Guo et al, 2003;Zeidler et al, 2004).…”

Oxidation of hydroxylamines to NO by plant cells

“…Indeed, numerous publications have given indirect hints that a "plant NOS" might exist, but the potential NOS gene in Arabidopsis (AtNOS1) has been renamed recently into AtNOA1 (NO-associated 1), in order to make clear that AtNOS 1 has no NOS activity but is somehow involved in regulating NO and NO dependent reactions, possibly via formation of cGMP as secondary messenger. 2 In animals, besides L-arginine, hydroxylamine was considered another substrate for oxidative NO formation in animals. 3 HA was shown to cause vasodilatation just like NO-releasing compounds.…”