Nitric oxide (NO) regulates multiple developmental events and stress responses in plants. A major biologically active species of NO is S-nitrosoglutathione (GSNO), which is irreversibly degraded by GSNO reductase (GSNOR). The major physiological effect of NO is protein S-nitrosylation, a redox-based posttranslational modification mechanism by covalently linking an NO molecule to a cysteine thiol. However, little is known about the mechanisms of S-nitrosylation-regulated signaling, partly due to limited S-nitrosylated proteins being identified. In this study, we identified 1,195 endogenously S-nitrosylated peptides in 926 proteins from the Arabidopsis (Arabidopsis thaliana) by a site-specific nitrosoproteomic approach, which, to date, is the largest data set of S-nitrosylated proteins among all organisms. Consensus sequence analysis of these peptides identified several motifs that contain acidic, but not basic, amino acid residues flanking the S-nitrosylated cysteine residues. These S-nitrosylated proteins are involved in a wide range of biological processes and are significantly enriched in chlorophyll metabolism, photosynthesis, carbohydrate metabolism, and stress responses. Consistently, the gsnor1-3 mutant shows the decreased chlorophyll content and altered photosynthetic properties, suggesting that S-nitrosylation is an important regulatory mechanism in these processes. These results have provided valuable resources and new clues to the studies on S-nitrosylation-regulated signaling in plants.

Nitric oxide (NO) and reactive oxygen species (ROS) are two classes of key signaling molecules involved in various developmental processes and stress responses in plants. The burst of NO and ROS triggered by various stimuli activates downstream signaling pathways to cope with abiotic and biotic stresses. Emerging evidence suggests that the interplay of NO and ROS plays a critical role in regulating stress responses. However, the underpinning molecular mechanism remains poorly understood. Here, we show that NO positively regulates the activity of the Arabidopsis (Arabidopsis thaliana) cytosolic ascorbate peroxidase1 (APX1). We found that S-nitrosylation of APX1 at cysteine (Cys)-32 enhances its enzymatic activity of scavenging hydrogen peroxide, leading to the increased resistance to oxidative stress, whereas a substitution mutation at Cys-32 causes the reduction of ascorbate peroxidase activity and abolishes its responsiveness to the NO-enhanced enzymatic activity. Moreover, S-nitrosylation of APX1 at Cys-32 also plays an important role in regulating immune responses. These findings illustrate a unique mechanism by which NO regulates hydrogen peroxide homeostasis in plants, thereby establishing a molecular link between NO and ROS signaling pathways.

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Methylation and nitric oxide (NO)-based S-nitrosylation are highly conserved protein posttranslational modifications that regulate diverse biological processes. In higher eukaryotes, PRMT5 catalyzes Arg symmetric dimethylation, including key components of the spliceosome. The Arabidopsis prmt5 mutant shows severe developmental defects and impaired stress responses. However, little is known about the mechanisms regulating the PRMT5 activity. Here, we report that NO positively regulates the PRMT5 activity through S-nitrosylation at Cys-125 during stress responses. In prmt5-1 plants, a PRMT5 transgene, carrying a non-nitrosylatable mutation at Cys-125, fully rescues the developmental defects, but not the stress hypersensitive phenotype and the responsiveness to NO during stress responses. Moreover, the salt-induced Arg symmetric dimethylation is abolished in PRMT5/prmt5-1 plants, correlated to aberrant splicing of pre-mRNA derived from a stress-related gene. These findings define a mechanism by which plants transduce stress-triggered NO signal to protein methylation machinery through S-nitrosylation of PRMT5 in response to environmental alterations.

Highlights d ROG1 or CAT3 acts as a transnitrosylase to regulate NO signaling in plants d Cysteine-343 is vital for ROG1 activity d ROG1 transnitrosylates GSNOR1 to regulate its stability d rog1 mutants have reduced sensitivity to NO under normal and stress conditions

Objectives Many cancer cells depend on G2 checkpoint mechanism regulated by WEE family kinases to maintain genomic integrity. The PKMYT1 gene, as a member of WEE family kinases, participates in G2 checkpoint surveillance and probably links with tumorigenesis, but its role in breast cancer remains largely unclear. Materials and Methods In this study, we used a set of bioinformatic tools to jointly analyse the expression of WEE family kinases and investigate the prognostic value of PKMYT1 in breast cancer. Results The results indicated that PKMYT1 is the only frequently overexpressed member of WEE family kinases in breast cancer. KM plotter data suggests that abnormally high expression of PKMYT1 predicts poor prognosis, especially for some subtypes, such as luminal A/B and triple‐negative (TNBC) types. Moreover, the up‐regulation of PKMYT1 was associated with HER2‐positive (HER2+), basal‐like (Basal‐like), TNBC statuses and increased classifications of Scarff, Bloom and Richardson (SBR). Co‐expression analysis showed PKMYT1 has a strong positive correlation with Polo‐like kinase 1 (PLK1), implying they may cooperate in regulating cancer cell proliferation by synchronizing rapid cell cycle with high quality of genome maintenance. Conclusions Collectively, this study demonstrates that overexpression of PKMYT1 is always found in breast cancer and predicts unfavourable prognosis, implicating it as an appealing therapeutic target for breast carcinoma.

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