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

Magali Moreau,Gyu In Lee,Yongzeng Wang,Brian R Crane,Daniel F Klessig

doi:10.1074/jbc.m804838200

Magali Moreau, Gyu In Lee + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m804838200

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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.

Highlights

AtNOS1 Is Not a Nitric-oxide Synthase—The AtNOS1 fulllength and a deletion variant with the first 101 amino acids removed (⌬101) were expressed in E. coli and tested for their ability to generate nitric oxide (NO) from arginine
The reactions were performed with AtNOS1 fl and ⌬101 in the presence of arginine and NADPH and with or without mammalian nitric-oxide synthase (NOS) cofactors, such as CaM, calcium, or BH4
The NO formation activity of AtNOS1 was reported to be regulated by Ca2ϩ-CaM binding [20]; the rat neuronal NOS (nNOS) isoform was chosen as a positive control in our experiments, since its activity is regulated by Ca2ϩ-CaM binding [43]

Summary

EXPERIMENTAL PROCEDURES

GTP and NADPH were purchased from Roche Applied Science, and (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) came from Shircks Laboratory (Jona, Switzerland). For the expression of the CTD-truncated AtNOA1 in plants, AtNOA1-(1–386) cDNA was amplified and cloned into the plant transformation vector pBIN61HA that encodes AtNOA1-(1–386) protein tagged with an HA epitope at the C terminus [50]. Kd for MantGDP binding to AtNOA1 was determined with 1 ␮M protein at 25 °C in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl supplemented with 200 mM KCl, and 5 mM MgCl2 when indicated. Increasing amounts of MantGDP (up to 2 ␮M final concentration) were added, and the fluorescence was measured (excitation, 356 nm; emission, 450 nm) and corrected with the corresponding signal in the absence of protein. Counts/min were plotted as a function of time for the different GTP concentrations. Curves were fitted to the equation, v0 ϭ (Vmax ϫ [GTP])/(Km ϩ [GTP]) using Origin Pro 7.5 software

RESULTS

Arginine binding

GTP and GDP were able to displace

DISCUSSION

We propose that the elevated amount of ROS observed in the