Metabolism of S-nitrosoglutathione (GSNO), a major biologically active nitric oxide (NO) species, is catalyzed by the evolutionally conserved GSNO reductase (GSNOR). Previous studies showed that the Arabidopsis GSNOR1/ HOT5 gene regulates salicylic acid signaling and thermotolerance by modulating the intracellular S-nitrosothiol level. Here, we report the characterization of the Arabidopsis paraquat resistant2-1 (par2-1) mutant that shows an anti-cell death phenotype. The production of superoxide in par2-1 is comparable to that of wild-type plants when treated by paraquat (1,1′-dimethyl-4,4′-bipyridinium dichloride), suggesting that PAR2 acts downstream of superoxide to regulate cell death. PAR2, identified by positional cloning, is shown to be identical to GSNOR1/HOT5. The par2-1 mutant carries a missense mutation in a highly conserved glycine, which renders the mutant protein unstable. Compared to wild type, par2-1 mutant has a higher NO level, as revealed by staining with 4,5-diaminofluorescein diacetate. Consistent with this result, wild-type plants treated with an NO donor display resistance to paraquat. Interestingly, the GSNOR1/HOT5/PAR2 protein level, other than its steady-state mRNA level, is induced by paraquat, but is reduced by NO donors. Taken together, these results suggest that GSNOR1/HOT5/PAR2 plays an important role in regulating cell death in plant cells through modulating intracellular NO level.
Nitric oxide (NO) regulates diverse cellular signaling through S-nitrosylation of specific Cys residues of target proteins. The intracellular level of S-nitrosoglutathione (GSNO), a major bioactive NO species, is regulated by GSNO reductase (GSNOR), a highly conserved master regulator of NO signaling. However, little is known about how the activity of GSNOR is regulated. Here, we show that S-nitrosylation induces selective autophagy of Arabidopsis GSNOR1 during hypoxia responses. S-nitrosylation of GSNOR1 at Cys-10 induces conformational changes, exposing its AUTOPHAGY-RELATED8 (ATG8)-interacting motif (AIM) accessible by autophagy machinery. Upon binding by ATG8, GSNOR1 is recruited into the autophagosome and degraded in an AIM-dependent manner. Physiologically, the S-nitrosylation-induced selective autophagy of GSNOR1 is relevant to hypoxia responses. Our discovery reveals a unique mechanism by which S-nitrosylation mediates selective autophagy of GSNOR1, thereby establishing a molecular link between NO signaling and autophagy.
LESION SIMULATING DISEASE1 (LSD1) is an important negative regulator of programmed cell death (PCD) in Arabidopsis (Arabidopsis thaliana). The loss-of-function mutations in LSD1 cause runaway cell death triggered by reactive oxygen species. LSD1 encodes a novel zinc finger protein with unknown biochemical activities. Here, we report the identification of CATALASE3 (CAT3) as an LSD1-interacting protein by affinity purification and mass spectrometrybased proteomic analysis. The Arabidopsis genome contains three homologous catalase genes (CAT1, CAT2, and CAT3). Yeast two-hybrid and coimmunoprecipitation analyses demonstrated that LSD1 interacted with all three catalases both in vitro and in vivo, and the interaction required the zinc fingers of LSD1. We found that the catalase enzymatic activity was reduced in the lsd1 mutant, indicating that the catalase enzyme activity was partially dependent on LSD1. Consistently, the lsd1 mutant was more sensitive to the catalase inhibitor 3-amino-1,2,4-triazole than the wild type, suggesting that the interaction between LSD1 and catalases is involved in the regulation of the reactive oxygen species generated in the peroxisome. Genetic studies revealed that LSD1 interacted with CATALASE genes to regulate light-dependent runaway cell death and hypersensitive-type cell death. Moreover, the accumulation of salicylic acid was required for PCD regulated by the interaction between LSD1 and catalases. These results suggest that the LSD1-catalase interaction plays an important role in regulating PCD in Arabidopsis.
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