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S-nitrosoglutathione reductases are low-copy number, cysteine-rich proteins in plants that control multiple developmental and defense responses in Arabidopsis.

Xu S, Guerra D, Lee U, Vierling E - Front Plant Sci (2013)

Bottom Line: S-nitrosoglutathione reductase (GSNOR) is believed to modulate effects of reactive oxygen and nitrogen species through catabolism of S-nitrosoglutathione (GSNO).Six members of the plant specific, ROXY glutaredoxins and three BHLH transcription factors involved in iron homeostasis were strongly upregulated, supporting a role for GSNOR in redox and iron metabolism.Together, these findings indicate GSNOR regulates multiple developmental and metabolic programs in plants and offer insight into putative routes of post-translational GSNOR regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts-Amherst , Amherst, MA, USA ; School of Life Sciences, Lanzhou University , Gansu, China.

ABSTRACT
S-nitrosoglutathione reductase (GSNOR) is believed to modulate effects of reactive oxygen and nitrogen species through catabolism of S-nitrosoglutathione (GSNO). We combined bioinformatics of plant GSNOR genes, localization of GSNOR in Arabidopsis thaliana, and microarray analysis of a GSNOR mutant to gain insights into the function and regulation of this critical enzyme in nitric oxide (NO) homeostasis. GSNOR-encoding genes are known to have high homology across diverse eukaryotic taxa, but contributions of specific conserved residues have not been assessed. With bioinformatics and structural modeling, we show that plant GSNORs likely localize to the cytosol, contain conserved, solvent-accessible cysteines, and tend to be encoded by a single gene. Arabidopsis thaliana homozygous for GSNOR loss-of-function alleles exhibited defects in stem and trichome branching, and complementation with Green fluorescent protein (GFP) -tagged GSNOR under control of the native promoter quantitatively rescued these phenotypes. GSNOR-GFP showed fluorescence throughout Arabidopsis seedlings, consistent with ubiquitous expression of the protein, but with especially high fluorescence in the root tip, apical meristem, and flowers. At the cellular level we observed cytosolic and nuclear fluorescence, with exclusion from the nucleolus. Microarray analysis identified 99 up- and 170 down-regulated genes (≥2-fold; p ≤ 0.01) in a GSNOR mutant compared to wild type. Six members of the plant specific, ROXY glutaredoxins and three BHLH transcription factors involved in iron homeostasis were strongly upregulated, supporting a role for GSNOR in redox and iron metabolism. One third of downregulated genes are linked to pathogen resistance, providing further basis for the reported pathogen sensitivity of GSNOR mutants. Together, these findings indicate GSNOR regulates multiple developmental and metabolic programs in plants and offer insight into putative routes of post-translational GSNOR regulation.

No MeSH data available.


Related in: MedlinePlus

Of nine positionally-conserved ex-zinc cysteines in GSNOR three are solvent-accessible. Three orientations (rotation angles indicated) of a monomer of the Arabidopsis GSNOR dimer are shown with solvent accessible surface (PDB 4JJI, 1.8 Å res., Rfree = 0.223) in yellow. Solvent-accessible ex-zinc cysteines are indicated in blue, and the dimer interface in gray. Images were made in PyMOL.
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Figure 2: Of nine positionally-conserved ex-zinc cysteines in GSNOR three are solvent-accessible. Three orientations (rotation angles indicated) of a monomer of the Arabidopsis GSNOR dimer are shown with solvent accessible surface (PDB 4JJI, 1.8 Å res., Rfree = 0.223) in yellow. Solvent-accessible ex-zinc cysteines are indicated in blue, and the dimer interface in gray. Images were made in PyMOL.

Mentions: GSNOR is remarkably cysteine rich, with a mole percent cysteine of 3.84 % for the Arabidopsis protein, compared to the 1.37 % average for all proteins in the UniProtKB database (2013). Because cysteines can serve as key post-translational regulatory sites being modified by nitrosation, glutathionylation, or reversible oxidation, we analyzed the conservation of the nine extra non-zinc-coordinating cysteine residues (ex-zinc cysteines) in Arabidopsis GSNOR. Four are in all the transcript-supported plant sequences, two are substituted in one, two are substituted in two, and one differs in four organisms (Figure 1, solid and open black arrowheads), yielding an overall conservation of 93.8 % (i.e., on average, each cysteine is present in 17 of the 18 sequences). If additional plant genes are considered for which EST support is lacking, ex-zinc cysteine conservation is still 91.0% (Supp. Figure 1—see Supp. Table 1 for accession codes). Thus, the position and frequency of ex-zinc cysteines are highly conserved in plant GSNORs. We also examined the position of ex-zinc cysteines in the Arabidopsis GSNOR structure (PDB 4JJI, via (PyMOL) and found that three were solvent accessible (Cys-10, Cys-271, and Cys-370, Figures 2A–C), two of which—Cys-10 and Cys-271—are positionally conserved even in the human sequence. The structures of GSNOR from human (1MP0) and tomato (4DL9) showed similar solvent exposure of the homologous ex-zinc cysteines, suggesting these three residues may have conserved functions in regulating GSNOR activity.


S-nitrosoglutathione reductases are low-copy number, cysteine-rich proteins in plants that control multiple developmental and defense responses in Arabidopsis.

Xu S, Guerra D, Lee U, Vierling E - Front Plant Sci (2013)

Of nine positionally-conserved ex-zinc cysteines in GSNOR three are solvent-accessible. Three orientations (rotation angles indicated) of a monomer of the Arabidopsis GSNOR dimer are shown with solvent accessible surface (PDB 4JJI, 1.8 Å res., Rfree = 0.223) in yellow. Solvent-accessible ex-zinc cysteines are indicated in blue, and the dimer interface in gray. Images were made in PyMOL.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3817919&req=5

Figure 2: Of nine positionally-conserved ex-zinc cysteines in GSNOR three are solvent-accessible. Three orientations (rotation angles indicated) of a monomer of the Arabidopsis GSNOR dimer are shown with solvent accessible surface (PDB 4JJI, 1.8 Å res., Rfree = 0.223) in yellow. Solvent-accessible ex-zinc cysteines are indicated in blue, and the dimer interface in gray. Images were made in PyMOL.
Mentions: GSNOR is remarkably cysteine rich, with a mole percent cysteine of 3.84 % for the Arabidopsis protein, compared to the 1.37 % average for all proteins in the UniProtKB database (2013). Because cysteines can serve as key post-translational regulatory sites being modified by nitrosation, glutathionylation, or reversible oxidation, we analyzed the conservation of the nine extra non-zinc-coordinating cysteine residues (ex-zinc cysteines) in Arabidopsis GSNOR. Four are in all the transcript-supported plant sequences, two are substituted in one, two are substituted in two, and one differs in four organisms (Figure 1, solid and open black arrowheads), yielding an overall conservation of 93.8 % (i.e., on average, each cysteine is present in 17 of the 18 sequences). If additional plant genes are considered for which EST support is lacking, ex-zinc cysteine conservation is still 91.0% (Supp. Figure 1—see Supp. Table 1 for accession codes). Thus, the position and frequency of ex-zinc cysteines are highly conserved in plant GSNORs. We also examined the position of ex-zinc cysteines in the Arabidopsis GSNOR structure (PDB 4JJI, via (PyMOL) and found that three were solvent accessible (Cys-10, Cys-271, and Cys-370, Figures 2A–C), two of which—Cys-10 and Cys-271—are positionally conserved even in the human sequence. The structures of GSNOR from human (1MP0) and tomato (4DL9) showed similar solvent exposure of the homologous ex-zinc cysteines, suggesting these three residues may have conserved functions in regulating GSNOR activity.

Bottom Line: S-nitrosoglutathione reductase (GSNOR) is believed to modulate effects of reactive oxygen and nitrogen species through catabolism of S-nitrosoglutathione (GSNO).Six members of the plant specific, ROXY glutaredoxins and three BHLH transcription factors involved in iron homeostasis were strongly upregulated, supporting a role for GSNOR in redox and iron metabolism.Together, these findings indicate GSNOR regulates multiple developmental and metabolic programs in plants and offer insight into putative routes of post-translational GSNOR regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Massachusetts-Amherst , Amherst, MA, USA ; School of Life Sciences, Lanzhou University , Gansu, China.

ABSTRACT
S-nitrosoglutathione reductase (GSNOR) is believed to modulate effects of reactive oxygen and nitrogen species through catabolism of S-nitrosoglutathione (GSNO). We combined bioinformatics of plant GSNOR genes, localization of GSNOR in Arabidopsis thaliana, and microarray analysis of a GSNOR mutant to gain insights into the function and regulation of this critical enzyme in nitric oxide (NO) homeostasis. GSNOR-encoding genes are known to have high homology across diverse eukaryotic taxa, but contributions of specific conserved residues have not been assessed. With bioinformatics and structural modeling, we show that plant GSNORs likely localize to the cytosol, contain conserved, solvent-accessible cysteines, and tend to be encoded by a single gene. Arabidopsis thaliana homozygous for GSNOR loss-of-function alleles exhibited defects in stem and trichome branching, and complementation with Green fluorescent protein (GFP) -tagged GSNOR under control of the native promoter quantitatively rescued these phenotypes. GSNOR-GFP showed fluorescence throughout Arabidopsis seedlings, consistent with ubiquitous expression of the protein, but with especially high fluorescence in the root tip, apical meristem, and flowers. At the cellular level we observed cytosolic and nuclear fluorescence, with exclusion from the nucleolus. Microarray analysis identified 99 up- and 170 down-regulated genes (≥2-fold; p ≤ 0.01) in a GSNOR mutant compared to wild type. Six members of the plant specific, ROXY glutaredoxins and three BHLH transcription factors involved in iron homeostasis were strongly upregulated, supporting a role for GSNOR in redox and iron metabolism. One third of downregulated genes are linked to pathogen resistance, providing further basis for the reported pathogen sensitivity of GSNOR mutants. Together, these findings indicate GSNOR regulates multiple developmental and metabolic programs in plants and offer insight into putative routes of post-translational GSNOR regulation.

No MeSH data available.


Related in: MedlinePlus