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.

The activity of HDA6 was sensitive to NO, demonstrating that NO participates in regulation of histone acetylation.

Chromatin immunoprecipitation sequencing 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.

Nitric oxide NO is a ubiquitous signaling molecule with pleiotropic functions that operates throughout the lifespan of plants. Indeed, NO is involved in several physiological processes, including growth and development, but also in iron homeostasis, as well as biotic and abiotic stress responses, such as to high salinity, drought, ultraviolet-B radiation, high temperature, and heavy metal toxicity Delledonne et al.

NO is a heteronuclear diatomic radical with a half-life of 3—5 s in biological systems, and the multifunctional role of NO is based on its chemical properties, cellular environment, and compartmentalization. Depending to a large extent on its local concentration, which is affected by its rate of synthesis, displacement, and removal, NO has been described as a cytoprotective, signaling, or cytotoxic molecule Floryszak-Wieczorek et al.

Protein S-nitrosation—the covalent attachment of NO to the sulfur group of cysteine residues—is one of the most important NO-dependent protein modifications, and plants respond to many different environmental changes by S-nitrosating a specific set of proteins Romero-Puertas et al.

S-Nitrosated glutathione S-nitrosoglutathione, GSNO has important functions as an NO reservoir, NO transporter, and physiological NO donor that can transfer its NO moiety to protein cysteine residues Hess et al.

Therefore, the level of S-nitrosated proteins corresponds to GSNO levels. The level of GSNO is controlled by the catalytic activity of GSNO reductase GSNOR; EC: 1.

This enzyme catalyzes the degradation of GSNO to oxidized glutathione and ammonium, and in this way, directly regulates the level of GSNO, and indirectly regulates the level of S-nitrosated proteins Liu et al. Loss of GSNOR function results in enhanced levels of low and high molecular S-nitrosothiols SNOs; FeechAn et al.

The pleiotropic phenotype of GSNOR-knock-out mutants backgrounds Columbia and Wassilijewskija and their sensitivity to biotic and abiotic stress clearly demonstrate the importance of this enzyme for plant growth, development, and stress response FeechAn et al.

Using a site-specific nitrosoproteomic approach, several hundred target proteins for S-nitrosation were identified in Arabidopsis gsnor plants Hu et al. These proteins are involved in a wide range of biological processes including chlorophyll metabolism and photosynthesis.

Consistently, gsnor mutants exhibited altered photosynthetic properties, such as increased quantum efficiency of photosystem II PSII photochemistry and photochemical quenching, and decreased nonphotochemical quenching Hu et al.

In several studies, gsnor plants were analyzed on proteome and transcriptome levels to gain insights into the physiological functions of this enzyme Fares et al. Gene transcription can be regulated via modification of transcription factors or via chromatin modifications.

The chromatin structure in eukaryotic organisms is very dynamic and changes in response to environmental stimuli. Chromatin marks are defined modifications on histone tails or DNA, playing key roles in processes such as gene transcription, replication, repair, and recombination Bannister and Kouzarides, DNA methylation is usually associated with long-term silencing of genes, whereas histone modifications contribute to both activation and repression of gene transcription, and are mostly removed after several cell cycles Jaenisch and Bird, ; Minard et al.

In general, the distinct chromatin states that modulate access to DNA for transcription are regulated by multiple epigenetic mechanisms, including DNA methylation, covalent modifications such as methylation and acetylation of core histones, ATP-dependent chromatin remodeling, placement of histone variants, noncoding RNAs, and metabolo-epigenetic effects Schvartzman et al.

Recently, we demonstrated that NO affects histone acetylation by targeting and inhibiting histone deacetylase HDA, EC: 3. Treatment with the physiological NO donor GSNO increased global histone 3 H3 and H4 acetylation.

Chromatin immunoprecipitation sequencing ChIP-seq revealed that several hundred genes displayed NO-regulated histone acetylation. Many of these genes were involved in plant defense response and abiotic stress response, but also in chloroplast function, suggesting that NO might regulate expression of specific genes by modulating chromatin structure Mengel et al.

Arabidopsis contains 18 isoforms of HDAs, divided into three subfamilies: Reduced Potassium Deficiency 3 RPD3 -like, HD-tuins and sirtuins Hollender and Liu, The first subfamily is the largest, and is composed of 12 putative members HDA2, HDA5—10, HDA14—15, HDA17—19 , which, based on structural similarity, can be further divided into three classes.

HDAs of this type are homologous to yeast RPD3 proteins that are ubiquitous in all eukaryotes. All members of this subfamily contain a specific deacetylase domain that is required for their catalytic activity.

The second subfamily contains the HD-tuins HD2 , and was originally found in maize. This type of proteins is plant-specific, although homologous cis—trans prolyl isomerases are also present in other eukaryotes Dangl et al.

The third subfamily of plant HDAs is represented by sirtuins SIR2-like proteins , which are homologous to yeast silent information regulator 2 SIR2; Pandey et al. These HDAs are unique because they require a NAD cofactor for functionality, and unlike RPD3 proteins, they are not inhibited by trichostatin A TSA or sodium butyrate.

Moreover, sirtuins use a wide variety of substrates beyond histones. Interestingly, there were no light intensity-dependent changes in histone acetylation observed in plants with loss of GSNOR gsnor ; FeechAn et al. In vitro measurement of enzyme activities provided evidence that Arabidopsis HDA6 is sensitive to NO.

A ChIP-seq analysis of the H3K9ac mark in wt and mutants gsnor and hda6 under dark and low light LL conditions identified 16, acetylated loci. In plants, both NO and radiation are important regulators of growth, development and stress response.

Probably, the changes in NO emission under these conditions were below the detection limit of the NO analyzer. Light-dependent NO emissions and nitrite and S-nitrosothiol accumulation in Arabidopsis plants.

A—C, NO emission of single Arabidopsis plants. Plants were placed in an Arabidopsis cuvette and NO emission was measured by chemiluminescence using an ultra-high sensitive NO analyzer. Temperature and dark and light conditions were applied as indicated.

D, Experimental setup for treatments with different light conditions. E—G, Determination of S-nitrosothiol and nitrite content after exposure to different light conditions. Total S-nitrosothiol E and nitrite F levels were determined after 4 h.

G, Total S-nitrosothiol levels of wt and gsnor plants were determined at , , , , , and The light period was from to Shaded portions in the graph represent dark conditions. The increase in NO emission was higher in plants with loss of GSNOR activity gsnor ; approximately two-fold in comparison to wt plants Figure 1B.

Increase of temperature alone did not enhance NO emission Figure 1B , while HL intensity in a constant temperature substantially increased NO emission Figure 1C , demonstrating a link between light intensity and NO emission independent of temperature.

As differences in NO emission could result from differences in stomata opening, endogenous SNO and nitrite contents were determined in Arabidopsis leaves.

Plants were grown for 4 weeks under short day, low temperature 22°C , and LL conditions. Afterwards, SNO and nitrite contents were determined. In general, SNO content was higher in gsnor than in wt plants Figure 1E.

In both lines, the SNO level did not significantly increase when plants were transferred to HL intensity. Nitrite content is a frequently used option to infer NO accumulation He et al. The nitrite levels under the different irradiation conditions corresponded to the SNO contents for gsnor and wt plants Figure 1F.

Within the different PPFD levels, wt and gsnor plants did not significantly differ in their nitrite contents, but significantly lower nitrite levels were detected in both wt and gsnor plants when transferred to the dark.

A tendency toward enhanced SNO concentration was observed under light, whereas lower SNO amounts were measured in the dark-treated plants, further confirming a light-dependent accumulation of SNOs. Loss of GSNOR function resulted in slightly higher SNO levels in comparison to wt Figure 1G.

Previously, we demonstrated that exogenously applied NO donors and endogenously induced NO production results in enhanced histone acetylation Mengel et al. Moreover, the H3K9ac level tended to be higher in HL conditions in comparison to D, being close to significance Figure 2C , adjusted P -value 0.

Scavenging of HL-induced NO with carboxyphenyl-4,4,5,5-tetramethylimidazolineoxyloxide cPTIO confirmed the relationship between NO and histone acetylation Supplemental Figure S1.

Thus, the correspondence of histone acetylation to light intensity observed in wt was not present in gsnor plants. Different light conditions lead to altered H3 acetylation in wt and gsnor plants. A, Immunoblot detection of histone modifications.

Four-week-old plants were exposed for 4 h to the indicated light conditions. Histones were extracted and analyzed for the indicated histone marks.

B—D, Quantitative analyses of the immunodetected bands of the different histone marks. Signal intensity was determined with Image J software. Intensities are given relative to the histone acetylation level in wt under D conditions, which was set to 1.

In Mengel et al. However, it is still unclear which of the 18 different Arabidopsis HDAs are sensitive to NO. Members of the RPD3-like subfamily are the most promising candidates since this subfamily includes HDA homologues to human HDA2.

Mammalian HDA2 is S-nitrosated at Cys and Cys in response to NO, resulting in chromatin remodeling in neurons and in dystrophic muscles Colussi et al. Both proteins contain seven conserved Cys, which are located within the HDA domain, including two Cys residues Cys and Cys of AtHDA6 that correspond to the S-nitrosated Cys residues in mammalian HDA2 Figure 3A.

Interestingly, the bioinformatic prediction tool GPS-SNO found that Cys of Arabidopsis HDA6 is a predicted target for S-nitrosation Xue et al.

Structural modeling of Arabidopsis HDA6 using the crystal structure of human HDA2 as template revealed strikingly similar 3D folds of both proteins Figure 3B. In the structural model of Arabidopsis HDA6, Cys, and Cys are located at the same positions as the S-nitrosated Cys and Cys in the 3D structure of human HDA2 Figure 3B , indicating that both proteins exhibit a very similar microenvironment around the substrate binding site.

S-Nitrosation of Arabidopsis HDA6. A, Amino acid sequence alignment of human HDA2 and Arabidopsis HDA6. The alignment was performed using Clustal Omega. Cysteine residues that are S-nitrosated in human HDA2 are marked in red, other conserved cysteines are indicated in yellow.

HDA region is highlighted in gray. B, Structural modeling of Arabidopsis HDA6. The HDA domain of Arabidopsis HDA6 amino acids 18—, Uniprot entry Q9FML2 was modeled using the SwissProt Modeling server with human HDA2 as a template PDB code: 4LXZ.

The 3D models were visualized with Swiss-PdbViewer. Cysteine residues that are S-nitrosylated in human HDA2, as well as the corresponding putative redox-sensitive cysteines of HDA6, are indicated in yellow.

The bound HDAC inhibitor suberanilohydroxamic acid green in human HDA2 indicates its active center, which is highlighted in each enzyme with an orange circle. Recombinant FLAG-HDA6 was produced in Arabidopsis. C, RT-PCR of transgenic 35S:FLAG-HDA6 Arabidopsis lines.

Five 35S:FLAG-HDA6—containing lines, AA21, and A23, were identified. cDNA of wt was used as a negative control. Predicted size of FLAG-HDA6 is around 1, bp.

D, Immunoblot of plant-produced FLAG-HDA6. Total protein TP of the transgenic line A18 and wt was subjected to FLAG resin and recombinant protein was eluted three times E1—E3. TP, flow-through FT , E1-E3, and boiled beads B were analyzed by immunoblotting.

Anti-FLAG-tag antibody , was used for immunodetection. Predicted size of FLAG-HDA6 is 57 kDa. One representative experiment of at least three replicates is shown.

E, Inhibition of FLAG-HDA6 activity by GSNO. The recombinant plant FLAG-HDA6 was incubated with 0. HDA activity was measured using Fluorogenic HDA Activity Assay.

An hda6 cell suspension line was generated to determine whether NO-dependent inhibition of total HDA activity is altered upon the knockout of HDA6.

The hda6 allele also called axe used to generate the cell culture contained an insertion resulting in a premature stop codon and the expression of a nonfunctional, C-terminally truncated version of the HDA6 protein Murfett et al.

Cell cultures exhibited similar growth kinetics and morphology to wt cells. Consistent with previous results Mengel et al. In contrast, GSNO treatment of hda6 cells did not result in an accumulation of acetylated H3 Supplemental Figure S2. TSA treatment did not increase the rate of H3 acetylation either, indicating that HDA6 was the predominant TSA-sensitive HDA isoform in this cell culture system.

These data imply that HDA6 is a promising candidate for a NO-sensitive HDA isoform. To analyze if HDA6 can be S-nitrosated in vitro and if S-nitrosation indeed affects its activity, HDA6 protein was recombinantly produced in Escherichia coli BL21 DE3 cc4—a strain which contains additional chaperones that help to produce proteins with low solubility—as His 6 -HDA6 and GST-HDA6.

However, no deacetylase activity could be measured for His 6 -HDA6 and GST-HDA6. We thus speculate that HDA6 might need certain PTMs or interaction partner s to function as an active HDA. We therefore produced recombinant FLAG-HDA6 in Arabidopsis. The presence of FLAG-HDA6 transcripts in transgenic lines were demonstrated by reverse transcription polymerase chain reaction RT-PCR Figure 3C.

We purified recombinant FLAG-HDA6 protein and confirmed via immunoblot the presence of recombinant FLAG-HDA6, with a predicted size around 55 kDa, in transgenic lines Figure 3D. Activity measurements demonstrated that recombinant FLAG-HDA6 was produced in a catalytically active form Figure 3E.

Taken together, these data imply that HDA6 a promising candidate for a NO-affected HDA isoform. As demonstrated above, exposure to increasing light intensities enhanced NO emissions and SNO accumulation Figure 1. Under HL conditions, the acetylation levels of the analyzed histone marks are very similar between wt and hda6 , while under LL and dark conditions, the acetylation levels tended to be higher in hda6 plants.

These data indicate that like GSNOR activity, HDA6 activity is involved in modulating the chromatin structure, especially in the dark and under LL intensities. Different light conditions lead to altered H3 acetylation in wt and hda6 plants.

B—D, Quantitative analysis of the immunodetected bands of the different histone marks. To identify chromatin regions regulated by GSNOR and HDA6 activities, we performed ChIP-seq using an anti-H3K9ac antibody. H3K9ac is a hallmark of active gene promoters Karmodiya et al. For all samples, the sequence reads aligned well with the Arabidopsis genome, resulting in a total of After peak calling Zhang et al.

Principle component analysis PCA based on all the peaks demonstrated a good clustering of replicates Figure 5A. Principle component 1 shows light to dark effects for all genotypes Figure 5A.

Characteristics of ChIP-seq samples. A, PCA. B, Chromosomal location of H3K9ac peaks averaged for each line. The number of peaks in each kb chromosomal bin of the Arabidopsis genome is shown.

The centromeric and pericentromeric regions of each chromosome are characterized by a very low number of peaks. Black: wt, blue: gsnor , red: hda6.

C, total number of identified peaks for wt, gsnor , and hda6. D, Location of H3K9ac peaks relative to genes. Histogram of distances of peak summits to the closest annotated TSS. The distribution shows a maximum at —bp downstream of the TSS. E, Distribution of H3K9ac peaks according to the genomic region of the summit relative to the closest TSS.

UTR, untranslated region. The highest density of H3K9ac peaks was found along the chromosome arms, whereas centromeric and pericentromeric regions were considerably less enriched in H3K9ac Figure 5B.

The number of H3K9ac peaks was significantly increased in the hda6 mutant compared to wt and gsnor Figure 5, B and C. This hyperacetylation of DNA in hda6 was observed throughout all chromosomes Figure 5B. Most peaks were located —bp downstream of the closest transcription start site TSS; Figure 5D.

In total, we identified 16, H3K9ac peaks. Differences in H3K9ac between LL and D conditions were identified for each genotype e.

All plant lines exhibited light-dependent acetylation changes, with a positive effect preferentially on chloroplast and transport genes, and a negative effect preferentially on stress response and transcription genes Figure 6A; Supplemental Tables S2 and S3.

Peaks exclusively hyperacetylated or hypoacetylated in wt or both mutants could also provide hints to the functions of HDA6 and GSNOR in the context of light stimulus-dependent histone acetylation.

Differential acetylation in mutants. A, B, Overlap of differentially acetylated peaks. Only GO terms from the biological process ontology are shown in the plots.

Each circle corresponds to an enriched GO term and circle size is proportional to the number of differentially acetylated genes C: upregulated, D: downregulated assigned to the GO term. The enriched GO terms are arranged in two dimensions such that their distance reflects approximately how distinct the corresponding sets of differential genes are from each other, that is, neighboring circles share a large fraction of genes.

Each enriched GO term is colored by its membership in the top-level categories, which are grouped into five themes. If a GO term belongs to multiple top-level terms, a pie chart within the circle indicates the relative fraction of each theme.

The total distribution of themes across all enriched GO terms is depicted in the bar plot underneath. Although the response to light already revealed differences between mutants and wt, a direct comparison of mutant and wt H3K9 acetylation under specific conditions will help to identify the basic functions of GSNOR and HDA6.

H3K9ac peaks of gsnor and hda6 were compared to H3K9ac peaks of wt plants under both LL and D conditions e. gsnor LL versus wt LL.

The number of hyperacetylated H3K9ac peaks is higher in gsnor than hda6 plants Figure 6B. Remarkably, six times more H3K9 loci are hyperacetylated in LL in comparison to D conditions for gsnor Figure 6B.

In the hda6 mutant, more H3K9 marks were hypoacetylated in comparison to wt than in gsnor Figure 6B. Interestingly, both mutant lines share many more specifically hyperacetylated and hypoacetylated peaks in LL conditions in comparison to D conditions.

In total, and 1, specifically hyperacetylated and hypoacetylated peaks, respectively, are shared under LL conditions, with highly significant P -values for the overlap 2. To examine, which biological functions are shared by GSNOR- and HDA6-specific changes in chromatin acetylation, a Gene Ontology GO term enrichment analysis was performed for the loci shared by both mutants.

Moreover, both enzyme functions promote acetylation of genes involved in stress response and localization. As H3K9ac is often found in actively transcribed promotors and CDS, we performed RNA-seq using the same experimental setup used for the ChIP-seq experiment.

They share an overlap of 4, upregulated and 4, downregulated genes, which by design, are independent of GSNOR and HDA6 function, and are related to, for example, chloroplast and ribosome functions Figure 7A; Supplemental Table S5.

In contrast, the and genes that are exclusively upregulated and downregulated, respectively, in both mutants, depend on both enzyme functions. Genes, that are exclusively upregulated in wt are enriched in GO terms predominately related to stress response Figure 7A; Supplemental Table S5. This is consistent with the increased acetylation of stress response genes in wt Figure 6A.

Differential gene regulation in mutants. A and B, Overlap of differentially expressed genes. Each circle corresponds to an enriched GO term and circle size is proportional to the number of differentially regulated genes assigned C: upregulated, D: downregulated to the GO term.

See Figure 6 for further details about the plots. To identify common regulatory functions of GSNOR and HDA6 activities in gene expression, the transcriptomes of gsnor and hda6 were directly compared to wt, both under LL and D conditions.

Similar to the acetylation data, in both genotypes, more genes are differentially regulated under LL conditions than under D conditions Figure 7B. Notably, the GO term enrichment results for the LL conditions share some overall trends with the ChIP-seq data Figure 7, C and D ; Supplemental Table S6.

To analyze the influence of H3K9ac on gene expression, ChIP-seq and RNA-seq datasets were integrated at the gene level for both mutants.

Under LL conditions, the two mutants share 23 genes that show hyperacetylation and enhanced expression in comparison to wt plants Table 1 ; Supplemental Table S7.

Under D conditions, only three genes are hyperacetylated and overexpressed in both mutants. One of these genes, AT5G Zinc-binding dehydrogenase family protein , has also shown up under LL conditions and is putatively involved in redox processes in plastids and cytosol.

Gene level integration of ChIP-seq and RNA-seq datasets for both mutants under LL conditions. Comparative visualization of H3K9ac and gene expression.

Wt: black, gsnor : blue, hda6 : red. ChIP-seq and RNA-seq datasets were integrated at the gene level for both mutants to analyze the effect of H3K9ac on gene expression. Genes downregulated and upregulated in both mutants under LL conditions are shown.

Protein functions are given according to the TAIR database. In addition, 65 genes are hypoacetylated and less expressed in both mutants under LL conditions Table 1 ; Supplemental Table S8. NO is an important signaling molecule that is involved in transcriptional regulation of many different physiological processes in plants related to growth and development, abiotic and biotic stress response, and photosynthesis Huang et al.

GSNOR is responsible for controlling SNO homeostasis, and loss of GSNOR function results in enhanced levels of SNO Figure 1, B, E—G. Moreover, light-dependent NO release has been reported for tobacco leaves Planchet et al. NO production and emission under light conditions could be based on light-triggered activation of nitrate reductase activity Riens and Heldt, ; Rockel et al.

However, since the NO emission of nitrite reductase NiR -deficient tobacco leaves still increased in light, other factors besides these reductase activities might also contribute to NO production Planchet et al.

However, this reaction is only observed under low oxygen conditions Gupta et al. NO production in mammals corresponds to the light-triggered expression and activity of NOS Ko et al. Although NOS enzymes have not been found in higher plants yet, it was demonstrated that NO can be produced in the chloroplast via an NADPH-dependent oxidation of l -arginine, which is the substrate of NOS.

It has been shown that l -arginine is one the most common amino acids in chloroplasts and available there in nanomolar concentrations Jasid et al. The synthesis of l -arginine is controlled by the photosynthetic light reaction, suggesting that oxidative NO production might also follow a circadian-like pattern.

Stomata are usually open during the day light and closed at night darkness , which might affect NO emission from leaves. However, light-dependent accumulation of endogenous SNOs and nitrite Figure 1, E—G excluded the possibility that the observed light-dependent NO emission is only due to light-regulated stomata opening.

In addition to the direct modification of metabolic pathways and regulation of gene expression, NO can target the modulation of the chromatin structure, which is a less investigated regulatory mechanism.

Such an association was not observed in gsnor plants Figure 2 , suggesting a regulatory function of GSNOR activity lower SNO level, denitrosation in histone acetylation under these conditions. There are several pieces of evidence indicating that SNO-induced histone acetylation is a result of the inhibition of HDA activity.

Second, stimulation of endogenous NO production also inhibits the catalytic HDA activity in protoplasts Mengel et al. Third, there are hints that the activity of at least some HDA isoforms is redox-regulated. Redox-sensitive Cys residues have been described for Arabidopsis HDA9 and HDA It is suggested that the oxidation of these two HDAs promotes their deactivation and therefore enhances histone acetylation and enables expression of associated genes Liu et al.

Redox regulation of HDAs has already been described in animals and humans. For example, brain-derived neurotropic factor causes NO synthesis and S-nitrosation of human HDA2 at Cys and Cys in neurons. However, in this mammalian system, S-nitrosation of HDA2 does not inhibit deacetylase activity, but causes its release from a CREB-regulated gene promoter.

Oxidation of HDA2 results in enhanced H3 and H4 acetylation at neurotrophin-dependent promoter regions and facilitates transcription of many genes Nott et al.

A different study reported about S-nitrosation of HDA2 in muscle cells of dystrophin-deficient MDX mice Colussi et al. Although NO-sensitive Cys of this enzyme are not identified yet, it was shown that the enzymatic activity of muscle HDA2 is impaired upon treatment with the NO donor DETA-NO.

Furthermore, recombinant mammalian HDA6 and HDA8 have been reported to undergo S-nitrosation resulting in inhibition of their catalytic activities Feng et al.

Moreover, HDA4 and HDA5, as parts of a large protein complex, migrate into the nucleus upon S-nitrosation of protein phosphatase 2A Illi et al.

Based on the studies mentioned above, mammalian HDAs seem to play an important role in redox-signaling 1 directly via NO or ROS production or 2 indirectly by impairing HDA activities. Similar mechanisms seem to be present in plants. Both proteins contain seven Cys residues, which are located within the HDA domain.

The catalytic activity of purified in planta-produced FLAG-HDA6 is partially inhibited by GSNO Figure 3, E and F. Since S-nitrosation of HDA6 could be detected using the biotin switch assay, NO-mediated inhibition of HDA6 activity could be caused by PTM of cysteine residues.

Surprisingly, the activity of GSNO-treated FLAG-HDA6 could not be restored by subsequent treatment with 5 mM DTT. Likely, these quite strong reducing conditions resulted in loss of complex partners important for HDA6 activity or caused structural changes of the HDA6 protein.

Interestingly, HDA6 controls light-induced chromatin compaction in Arabidopsis Tessadori et al. As low- and high-histone acetylation levels correspond to compact and loose chromatin structure, respectively, our data confirm that HDA6 is involved in light-dependent chromatin modulation and imply HDA6 is a promising candidate for a NO-affected HDA isoform.

The proposed mechanism for the deacetylating function of GSNOR and HDA6 in D conditions is shown in Figure 9. Schematic illustration of the regulatory function of NO on histone acetylation in light and dark conditions. As a consequence, histone acetylation and gene transcription are decreased.

In both situations, GSNOR activity is required for fine-tuning the SNO levels. ChIP-seq analysis on H3K9ac was performed to examine the functions of GSNOR and HDA6 on chromatin structure in the dark and under light conditions; 16, H3K9ac sites were found in the chromatin of Arabidopsis leaf tissue.

Peaks mainly resided within gene-enriched areas and were almost depleted from centromeric and pericentromeric regions Figure 5B. This observation is in line with reports from other plant species or other histone acetylation marks.

For example, in the moss Physcomitrium patens , H3K9ac and H3K27ac, and in rice, H3K9ac, showed a strong enrichment in genic regions He et al. The genome-wide profiling of H3K9ac in Arabidopsis revealed that this histone modification is predominantly located within the regions surrounding the TSSs of genes, with a maximum at —bp downstream of the TSS Figure 5D.

This agrees with the distribution of H3K9ac in other plants as well as the distribution of other histone marks, for example, analysis of different histone modification profiles in Arabidopsis revealed that most peaks are localized around bp downstream of the TSSs, whereas peak position, shape, and length are independent of gene length Mahrez et al.

The hda6 mutant displayed more acetylated regions than wt and gsnor throughout all chromosomes Figure 5, B and C. These data suggest that GSNOR and HDA6 function is required to deacetylate these genes under light conditions. Genes related to chloroplast function mainly relate to starch, sulfur and terpenoid metabolism.

As the products of these genes are also required under light conditions, their reduced acetylation is surprising. However, since the acetylation levels of histones are the result of a fine-tuned interplay between acetyltransferases and HDAs, GSNOR and HDA6 are probably required to keep a balanced acetylation level of these genes.

That is in line with the observation that expression of this set of genes does not change substantially in both mutants in comparison wt. The regulatory mechanisms of the deacetylating function of GSNOR under light are unknown.

GSNOR activity lowers the level of GSNO and consequently lowers the level of S-nitrosated proteins. In this way, GSNOR is protecting HDA6 from SNO-dependent inhibition and keeping it active Figure 9.

However, our data do not rule out additional effects of NO, for example, activation of other HDAs or a reduced activity of distinct histone acetyltransferases. To get insight into the regulatory function of SNOs in chromatin modulation during light—dark switch, the S-nitrosylome under these conditions needs to be identified.

In conclusion, according to the results obtained with the gsnor and hda6 genotypes, both enzymes seem to play an important role in the light-dark diurnal regulation of histone acetylation.

In Arabidopsis, 12 SET DOMAIN GROUP SDG -containing histone methyltransferases are present, which are mainly involved in H3K4 and H3K36 methylation.

These marks are marks of active transcription. So far, only a few genes of this gene family have been functionally characterized. SDG25, for instance, is involved in FLOWERING LOCUS C FLC activation and repression of flowering Berr et al. Consequently, high expression of FLC results in a late-flowering phenotype.

Interestingly, in both gsnor and hda6 mutants, histone-lysine N-methyltransferase SETD1B-like protein acetylation and expression were increased, and both mutants displayed a late-flowering phenotype Wu et al. In hda6 , the late-flowering phenotype is likely due to upregulation of FLC expression Yu et al.

In contrast, for gsnor , reduced or unchanged expression of FLC in comparison to wt plants is reported Kwon et al. Besides regulating the flowering time, GSNOR and HDA6 also seem to have important common regulatory functions in brassinosteroid biosynthesis.

The gene encoding the cytochrome P superfamily protein AT3G; DWARF4 was hyperacetylated and more highly expressed in both mutants in comparison to wt plants. DWARF4encodes a 22α hydroxylase that catalyzes a rate-limiting step in brassinosteroid biosynthesis Choe et al.

Brassinosteroids are phytohormones important for plant growth and development as well as for response to environmental stress. Mutants in the brassinosteroid pathway often display a dwarf phenotype Li et al. Interestingly, gsnor displays a dwarf phenotype Holzmeister et al.

Metabolic reprogramming in response to abiotic and biotic stress is governed by a complex network of genes, which are induced or repressed. A large set of stress-related genes is exclusively hyperacetylated in wt under LL versus D conditions Figure 6A.

Various reports have shown that the plant signaling pathways involved in the responses to abiotic and biotic stresses are modulated by different types of photoreceptors, controlling expression of a large fraction of abiotic stress-responsive genes as well as biosynthesis and signaling downstream of stress response hormones Jeong et al.

For example, pathogen inoculations in the morning and midday resulted in higher accumulation of salicylic acid, faster expression of pathogenesis-related genes, and a more pronounced hypersensitive response than inoculations in the evening or at night Griebel and Zeier, The observed plant defense capability upon daytime treatments seems to be attributable to the availability of a long light period during early plant—pathogen interaction rather than to the circadian rhythm.

One might speculate whether, for example, the light-dependent flagellin induced accumulation of salicylic acid Sano et al. We observed light-dependent enrichment of the H3K9ac mark in many stress-related genes in wt in LL versus D comparison Figure 6A.

As H3K9ac is an activating histone mark, these genes might be prepared for expression, and according to the RNA-seq data, many stress-related genes display a higher expression in wt under light in comparison to darkness Figure 7A.

Interestingly, GSNOR, as well as HDA6 function, seems to be involved in regulation of H3K9ac and expression of stress-related genes. Loss of GSNOR and HDA6 activity resulted in relative hypoacetylation and reduced expression of many stress-related genes Figure 6, B and D , suggesting that both enzymes are required to activate these stress-related genes.

Given its HDA function, this means that those stress genes are specifically not targeted by HDA6. The loss of a distinct HDA function could result in activation of other HDAs or reduction of histone acetyltransferase activities, but this is not shown by our data Figure 4. Rather, the increased overall number of acetylated regions might decrease the acetylation intensity at certain sites.

Moreover, other still unknown factors could be involved in regulating histone acetylation. Indeed, indirect gene activating function has also been observed for other HDAs, for example, for HDA5 Luo et al.

Stress-responsive genes, which are hypoacetylated and downregulated under light conditions in both mutants, include OXIDATIVE STRESS 3 AT5G; OXS3. OXS3 is a chromatin-associated factor involved in heavy metal and oxidative stress tolerance Blanvillain et al.

It contains a domain corresponding to a putative N-acetyltransferase or thioltransferase catalytic site. Enhanced stress tolerance of OXS3 overexpression lines and stress-sensitivity of oxs3 mutant indicates a role in stress tolerance. The nuclear localization of this protein supports a function as stress-related chromatin modifier protecting the DNA or altering transcription Blanvillain et al.

Trehalose is a disaccharide composed of two glucose bound by an alpha—alpha 1—1 linkage and is often associated with stress-resistance in a wide range of organisms Fernandez et al. Trehalose accumulation has been observed in plants under different stress situations, such as drought, heat, chilling, salinity, and pathogen attack Fernandez et al.

Moreover, genes involved in detoxification and stress response are induced by exogenous application of trehalose Bae et al. The bZIP transcription factor family protein encodes for AtbZIP63, which is an important node of the glucose—abscisic acid ABA interaction network and may participate in the fine-tuning of ABA-mediated abiotic stress responses Matiolli et al.

The ABA signaling pathway is a key pathway that controls response to environmental stress. Emission of NO and SNO level was higher under light compared to darkness Figure 1, B, C, E—G. Although GSNOR inhibits the activity of recombinant HDA6 Figure 3, E and F , their effects on stress-related genes are probably indirect.

In addition, other HDAs and histone acetyltransferases can be involved in regulation of histone acetylation at stress-responsive genes. It was demonstrated that HDA19 plays an essential role in suppressing SA-biosynthetic genes and PR-genes during unchallenged conditions via deacetylating the corresponding promoters Choi et al.

Furthermore, in Arabidopsis, the plant-specific HD2B binds to genes involved in defense response in untreated plants, whereas after flg22 treatment, it is mainly genes involved in plastid organization that are targeted by HD2B Latrasse et al.

All these observations highlight the importance of a fine-tuned switch between growth and development on one side and stress response on the other side. On the other side, the coordinating function of these enzymes and NO could be a promising target to modify plant metabolism to mitigate the negative effects of a stressful environment on plant performance and productivity.

Moreover, our study shows that, in addition to the known suppressive effects of HDAs, HDA6 has indirect positive effects on transcription and interestingly, GSNOR activity seems to be involved in this process of switching the metabolism from growth and development to stress response.

thaliana wt Col-0, gsnor also called GABI-Kat D11, gsnor , or GSNOR-KO , axe also called hda6 or HDA6-ko were cultivated on soil mixed in ratio with sand. Nuclear proteins were extracted from Arabidopsis cell culture or seedlings according to the protocol of Xu and Copeland, with small modifications.

Approximately 0. The homogenate was centrifuged for 10 min at 1, g and 4°C. The supernatant was discarded, and the pellet was resuspended in 3 mL of NRBT buffer and centrifuged as described above. This step was repeated two more times or until the green color chloroplast contamination was gone.

Triton X was removed from the nuclei pellet by washing in 3 mL of NRB buffer. Two methods were used to break a nuclear envelope and solubilize proteins. Protein concentration was measured using a RC DC protein assay Biorad, Cat No.

The second method was based on the sonication procedure using microtip MS 72 Bandelin, Cat No. Protein concentration was measured using a Bradford reagent Biorad, Cat No. Control treatment was done with 0. After 3 h, the plants were again treated with µM of cPTIO or 0.

Histone proteins were extracted with a Histone Purification Kit Active Motif, Cat No. The extracts were centrifuged at maximal RCF at 4°C for 10 min. Afterwards the supernatants were transferred to PD 10 columns GE Healthcare, Cat No. The proteins were eluted with 3. The eluates were neutralized with one-fourth volumes of 5× neutralization buffer 0.

Columns were prepared by adding µL dH 2 O with a Protease Inhibitor EDTA-free tablet Roche, Cat No. One hundred microliter of purified core histones were added to the column and centrifuged for 2 min at 1, g. An amount of histones was measured by NanoDrop at nm.

After separation of proteins, a gel was either stained for 30 min with Coomassie brilliant blue solution or further used for a western blot. Proteins were transferred to a nitrocellulose membrane Abcam using a semi-dry western blot system.

Prewet membrane and gel were sandwiched between Whatman papers that were presoaked in a transfer buffer. A transfer was performed for 45 min at room temperature RT. An efficient transfer of proteins was determined by staining a membrane with Ponceau S solution Sigma-Aldrich, Cat No. Anti-FLAG-tag antibody , was used to detect plant-produced FLAG-HDA6.

The membrane was washed once with 1× TBS-T and two times with 1× TBS buffer. The signal was developed using Western lightning plus-ECL chemiluminescence substrate PerkinElmer, Cat No. coli strain DH5α, followed by electroporation of Agrobacterium strain GV pMP Transgenic Arabidopsis lines overproducing 35S:FLAG-HDA6 were generated by the floral dip method as described above.

Homozygous lines were selected and used for further studies. Plants expressing recombinant FLAG-HDA6 were harvested three weeks after sowing. For analytical studies, around 4 g of ground material were used.

C supplemented with of the recommended amount of a Protease Inhibitor EDTA-free tablet Roche, Cat No. Extracts were filtrated trough miracloth Millipore, Cat No R , followed by min centrifugation at 6, g and 4°C; 60 µL of FLAG-targeted beads Sigma-Aldrich, Cat No.

A binding of recombinant protein to the beads was performed by rotating for 4 h at 4°C. Afterwards, the resin was centrifuged for 30 s at 8, g , and supernatant was discarded. F by incubating the resin with synthetic peptide and rotating for 30 min at 4°C. The RFU values were used for relative quantification of HDA activity.

HDA activity was also measured according to Wegener et al. The HDA reaction was initiated by adding µM of HDA-substrate Boc-Lys Ac -MCA in 25 µL of HDA buffer, followed by min incubation at 37°C. The mixture was incubated for an additional 20 min at 30°C to ensure the tryptic digestion.

S-Nitrosothiols and nitrite were measured using Sievers Nitric Oxide Analyzer NOA i GE Analytical Instruments. The method is based on reduction of SNOs and nitrite to NO, which is further oxidized by ozone to NO 2 excited state and O 2.

On the way to the ground state, NO 2 emits chemiluminescence that can be measured by photomultiplier. Approximately — mg of plant tissue was homogenized in the same volume of PBS solution, and incubated for 20 min at 4°C while rotating.

Protein extracts were separated from plant debris by centrifugation for 15 min at maximal speed. For the detection of nitrite 20— µL of analyte were injected into triiodide solution. For the detection of SNO content, sulfanilamide was added to protein extracts to scavenge nitrite, and µL of the total sulfanilamide-protein extract were injected triiodide solution.

Every measurement was performed in duplicate. A standard curve was created with sodium nitrite. NO emission was measured from 3. The gas was purified from NO by pulling it through a charcoal column.

Temperature and light were dependent on the experimental setup. This pattern was repeated in total four times. In addition, the NO emission of soil without a plant was measured and subtracted from plant emission.

By this time, plants achieved similar development stage. Later, they were harvested and immediately cross-linked.

Concentration of suitable formaldehyde amount was obtained experimentally. The tubes were put in a desiccator, and vacuum was applied for 10 min. Crosslinking was stopped by adding glycine to each tube with the end concentration of 0. After that, leaves were washed twice with cooled water and dried on paper towels.

For each IP, 20 µL of magnetic beads A were used. Beads of one biological replicate were washed together by pipetting up and down four times with 1 mL buffer RIPA plus protease inhibitor.

Afterwards, beads were suspended in the same volume of RIPA. In the meantime, chromatin isolation steps were performed. After coupling, the AB-coated beads were washed with µL RIPA for three times and resuspended with the same buffer.

Leaves were ground to fine powder with mortar and pestle in liquid nitrogen. A total of 2. The suspension was incubated for 15—20 min at 4°C on a rotation platform, followed by centrifugation at 4°C and 2, g for 20 min.

After that, supernatant was removed, and the pellet was suspended with 3 mL NRBT buffer. First 1 mL of buffer was added, and the pellet was suspended with a pipet tip, and then the remaining 2 mL were added.

Further, the nuclei were extracted using the same procedure as described above. After nuclei were isolated, they were carefully suspended avoiding foam formation with nuclei sonication buffer. Bioruptor Pico ultrasonic bath and Covaris E Evolution were used to shear isolated chromatin for ChIP-qPCR and ChIP-seq, respectively.

To perform DNA shearing for ChIP-qPCR, µL of sonication buffer were added to nuclei and transferred to 1. To perform DNA shearing for ChIP-seq, nuclei were resuspended in µL of sonication buffer and transferred to micro-Tube AFA Fiber Pre-SlitSnap Cap Cat No.

After this, sonicated samples were centrifugated for 5 min at 16, g and 4°C, and the supernatant was used directly for the immunoprecipitation assay or for the detection of shearing efficiency. In total, 50 and 20 µL of sonicated chromatin for ChIP-qPCR and ChIP-seq, respectively, were diluted to µL with sonication buffer.

Decrosslinking was performed by adding 6 µL of 5-M NaCl, and samples were incubated on a thermoblock for 20 min at 95°C and then centrifugated at RT for 2 min g. After that, 2 µL of RNaseA were added and samples were incubated for another 40 min at 37°C and then centrifugated at RT for 2 min g.

DNA was extracted using MinElute PCR purification kit Qiagen, Cat No. DNA was eluted with 11 µL of dH 2 O. Concentration was measured using NanoDrop. Afterwards, the beads were washed two times with 1 mL of the following buffers: low salt buffer, high salt buffer, LiCl buffer and TE buffer.

Each wash step was performed on a rotating platform for 5 min at 4°C. Immunoprecipitated chromatin IP was eluted with µL of elution buffer plus proteinase inhibitor, incubating on a thermoblock for 15 min at g and 65°C.

Elution was performed twice, and bough eluates were mixed together. Decrosslinking was performed by mixing each sample with 10 µL of 5 M NaCl 0. Proteinase K treatment was performed for 2 more hours by adding 2 µL Proteinase K DNA was purified as described above.

The DNA was eluted with 21 µL of dH 2 O for ChIP-qPCR or 15 µL of EB elution buffer Qiagen, Cat No. DNA concentration was measured using Qubit dsDNA HS Assay Kit Cat No. Size selection of fragmented DNA was performed using AMPure XP beads Beckman Coulter, Cat No. A before library preparation.

Twenty-one microliters of magnetic beads 1. Afterwards, beads were placed on a magnetic stand and the supernatant was discarded. DNA was eluted with 12 µL of EB elution buffer by incubating the beads for 3 min. The ChIP-seq reads were aligned against the TAIR10 reference genome assembly for Arabidopsis accessed on May 14, using bowtie After quality-based filtering with samtools Differential analysis between groups was performed based on the DESeq2 method Love et al.

The alignment format conversion required for DiffBind was done with samtools Venn diagrams were made with the R package limma, version 3. test in R version 3. PCA by prcomp and plot functions were employed in R version 3.

Read counts for specific genomic locations were queried by samtools GO term enrichment for lists of differentially regulated genes was computed in R version 3. test and p. adjust with the FDR method.

The GO terms and annotated genes were taken from org. tairGO2ALLTAIRS in the org. db R package, version 3.

The description of the GO term was obtained from the GO. db R package version 3. Significantly enriched GO terms from the biological process ontology were plotted with respect to the first two MDS coordinates, and colored according to their ancestors among the top level biological process terms, which were classified into five broader categories response to stimulus: GO, GO, GO; localization: GO; growth and development: GO, GO, GO, GO, GO, GO, GO; metabolic process: GO; other: GO, GO, GO, GO, GO, GO, GO, GO, GO, GO, GO, GO, GO, GO, GO, GO, GO, GO The visualization was achieved by the R packages ggplot2, version 3.

By this time, plants had achieved a similar development stage. Later, plants were harvested and stored at °C.

In total, four biological replicates were analyzed. RNA was extracted using the innuPREP PLANT RNA Kit Analytik Jena GmbH, Jena Germany according to the manual. Reads were mapped to the TAIR10 reference of Arabidopsis annotated genes www.

org using STAR v2. Stress serves as a potent etiological link to development of several neuropsychiatric diseases such as depression, anxiety, and cognitive impairments. Exposure to stressful stimuli has been found to be associated with activation of nitric oxide synthase and generation of NO which reacts with spontaneous oxygen species to aid formation of active nitrogen radicals.

High concentrations of reactive nitrogen radicals may cause damage to intracellular proteins, in addition to causing impairment to components of the mitochondrial transport chain, leading to cellular energy deficiency.

This may further serve as an etiological link to the development of secondary neurological diseases associated with chronic stress. Also, during stress exposure, pharmacological inhibition of nitric oxide production displays reduction in indicators of anxiety- and depressive-like behavior in animal models.

Therefore, the purpose of this chapter is to present an overview on the role of NO in stress-evoked emergence of secondary neurological disorders like anxiety as well as citing examples where NO has been used as a therapeutic target for the management of stress-induced anxiety-like behavior.