NO-induced modulation of chromatin structure: S-nitrosothiols affect light-dependent histone acetylation.

NO is an important signaling molecule. However, the molecular mechanism underlying NO functions is not understood yet. The influence of NO on chromatin structure and its consequences on plants’ physiological functions were investigated in this thesis. Tendency towards enhanced histone 3 and 4 acetylation were observed in Arabidopsis seedlings after GSNO and INA treatment, which was likely mediated by inhibition of HDAs activity. HDA6 was identified as one of the NO-sensitive isoform. Furthermore, it was found that NO production is light deependent and therefore might be involved in regulation of histone acetylation during the day. It could be also established that photosynthetic performance might be regulated by NO-induced H3K9ac

Redox-dependent chromatin remodeling: A new function of nitric oxide as architect of chromatin structure in plants.

Nitric oxide (NO) is a key signaling molecule in all kingdoms. In plants, NO is involved in the regulation of various processes of growth and development as well as biotic and abiotic stress response. It mainly acts by modifying protein cysteine or tyrosine residues or by interacting with protein bound transition metals. Thereby, the modification of cysteine residues known as protein S-nitrosation is the predominant mechanism for transduction of NO bioactivity. Histone acetylation on N-terminal lysine residues is a very important epigenetic regulatory mechanism. The transfer of acetyl groups from acetyl-coenzyme A on histone lysine residues is catalyzed by histone acetyltransferases. This modification neutralizes the positive charge of the lysine residue and results in a loose structure of the chromatin accessible for the transcriptional machinery. Histone deacetylases, in contrast, remove the acetyl group of histone tails resulting in condensed chromatin with reduced gene expression activity. In plants, the histone acetylation level is regulated by S-nitrosation. NO inhibits HDA complexes resulting in enhanced histone acetylation and promoting a supportive chromatin state for expression of genes. Moreover, methylation of histone tails and DNA are important epigenetic modifications, too. Interestingly, methyltransferases and demethylases are described as targets for redox molecules in several biological systems suggesting that these types of chromatin modifications are also regulated by NO. In this review article, we will focus on redox-regulation of histone acetylation/methylation and DNA methylation in plants, discuss the consequences on the structural level and give an overview where NO can act to modulate chromatin structure

S-nitrosylation of nuclear proteins: New pathways in regulation of gene expression.

Nitric oxide (NO) is a reactive free radical with pleiotropic function that is not only involved in regulation of plant growth and development, but also in the response to biotic and abiotic stressors. It mainly acts by posttranslationally modifying proteins. The most important mode of action of NO is protein S-nitrosylation, the covalent attachment of an NO group to the thiol side of protein cysteine residues. Other major types of NO-dependent modifications are metal nitrosylation and tyrosine nitration. NO can regulate gene expression at different levels. On one side, it can initiate signalling cascades or modify proteins involved in signal transduction pathways. On the other side, redox-sensitive transcription factors can be also target for S-nitrosylation, and NO can also affect redox-dependent nuclear transport of some proteins. This suggests that NO plays a pivotal role in regulating transcription and/or general nuclear metabolism in plants

Identification of nuclear target proteins for S-nitrosylation in pathogen-treated <em>Arabidopsis thaliana</em> cell cultures.

Nitric oxide (NO) is a significant signalling molecule involved in the regulation of many different physiological processes in plants. One of the most imperative regulatory modes of action of NO is protein S-nitrosylation&mdash;the covalent attachment of an NO group to the sulfur atom of cysteine residues. In this study, we focus on S-nitrosylation of Arabidopsis nuclear proteins after pathogen infection. After treatment of Arabidopsis suspension cell cultures with pathogens, nuclear proteins were extracted and treated with the S-nitrosylating agent S-nitrosoglutathione (GSNO). A biotin switch assay was performed and biotin-labelled proteins were purified by neutravidin affinity chromatography and identified by mass spectrometry. A total of 135 proteins were identified, whereas nuclear localization has been described for 122 proteins of them. 117 of these proteins contain at least one cysteine residue. Most of the S-nitrosylated candidates were involved in protein and RNA metabolism, stress response, and cell organization and division. Interestingly, two plant-specific histone deacetylases were identified suggesting that nitric oxide regulated epigenetic processes in plants. In sum, this work provides a new collection of targets for protein S-nitrosylation in Arabidopsis and gives insight into the regulatory function of NO in the nucleus during plant defense response. Moreover, our data extend the knowledge on the regulatory function of NO in events located in the nucleus

Nitric oxide modulates histone acetylation at stress genes by inhibition of histone deacetylases.

Histone acetylation, which is an important mechanism to regulate gene expression, is controlled by the opposing action of histone acetyltransferases and histone deacetylases (HDACs). In animals, several HDACs are subjected to regulation by nitric oxide (NO); in plants, however, it is unknown whether NO affects histone acetylation. We found that treatment with the physiological NO donor S-nitrosoglutathione (GSNO) increased the abundance of several histone acetylation marks in Arabidopsis (Arabidopsis thaliana), which was strongly diminished in the presence of the NO scavenger 2-4-carboxyphenyl- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. This increase was likely triggered by NO-dependent inhibition of HDAC activity, since GSNO and S-nitroso-N-acetyl-DL-penicillamine significantly and reversibly reduced total HDAC activity in vitro (in nuclear extracts) and in vivo (in protoplasts). Next, genome-wide H3K9/14ac profiles in Arabidopsis seedlings were generated by chromatin immunoprecipitation sequencing, and changes induced by GSNO, GSNO/2-4-carboxyphenyl- 4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide or trichostatin A (an HDAC inhibitor) were quantified, thereby identifying genes that display putative NO-regulated histone acetylation. Functional classification of these genes revealed that many of them are involved in the plant defense response and the abiotic stress response. Furthermore, salicylic acid, which is the major plant defense hormone against biotrophic pathogens, inhibited HDAC activity and increased histone acetylation by inducing endogenous NO production. These data suggest that NO affects histone acetylation by targeting and inhibiting HDAC complexes, resulting in the hyperacetylation of specific genes. This mechanism might operate in the plant stress response by facilitating the stress-induced transcription of genes

Nitric oxide coordinates growth, development, and stress response via histone modification and gene expression.

Nitric oxide (NO) is a signaling molecule with multiple regulatory functions in plant physiology and stress response. In addition to direct effects on transcriptional machinery, NO executes its signaling function via epigenetic mechanisms. We report that light intensity-dependent changes in NO correspond to changes in global histone acetylation (H3, H3K9 and H3K9/K14) in Arabidopsis (Arabidopsis thaliana) wild-type leaves, and that this relationship depends on S-nitrosoglutathione reductase (GSNOR) and histone deacetylase 6 (HDA6). The activity of HDA6 was sensitive to NO, demonstrating that NO participates in regulation of histone acetylation. ChIP-seq and RNA-seq analyses revealed that NO participates in the metabolic switch from growth and development to stress response. This coordinating function of NO might be particularly important in plant ability to adapt to a changing environment, and is therefore a promising foundation for mitigating the negative effects of climate change on plant productivity