When plants say “NO”: how nitric oxide influences chromatin remodeling Free

Nitric oxide (NO) is a gaseous reactive oxygen species, and while originally discovered to have signaling properties in animals (Palmer et al., 1987), it was also found to play a role in plant defense signaling during the late 1990s (Delledonne et al., 1998). These initial findings suggest that NO as a chemical messenger may be an ancient role for the molecule (Domingos et al., 2015). In plants, NO is not limited to defense and participates in many diverse, sometimes even antagonistic, biological processes, including growth, development, iron homeostasis, response to salinity, response to drought, response to UV-B radiation, temperature fluctuations, as well as heavy metal toxicity (reviewed in Domingos et al., 2015). Despite the far-reaching effects of NO, the half-life of this molecule is a mere 3–5 s; therefore, NO’s role in plant systems is largely determined by where and when it is made by cells.

Glutathione is a well-known and highly conserved reactive oxygen species scavenger, so it comes as no surprise that NO would interact with the antioxidant. By reacting with NO, glutathione becomes S-nitrosoglutathione (GSNO). GSNO can then be used to modify proteins post-translationally through catalysis by GSNO reductase (GSNOR). While prior evidence indicates GSNOR activity plays a role at the protein and transcriptional level (reviewed in Domingos et al., 2015; Hu et al., 2015), it was previously unknown if changes at the chromatin level are also affected by the activities of this enzyme (reviewed in Ageeva-Kieferle et al., 2019). Addressing this gap in knowledge, NO was demonstrated to have global effects on H3 and H4 histone acetylation (Mengel et al., 2017). While intriguing, the specific players that mediate these histone modifications remain unknown.

In this issue of Plant Physiology, Ageeva-Kieferle et al. (2021) set out to discover molecular targets of GSNOR and whether they influence chromatin changes directly. To address this hypothesis, the authors chose light intensity as a means of inducing NO production in Arabidopsis (Arabidopsis thaliana). Initially, the authors examined levels of NO during light treatment in a loss-of-function gsnor mutant. Under high light conditions, compared with dark or low light conditions, gsnor plants displayed higher levels of NO emissions than wild type. To account for differences in stomatal opening, which could release gaseous NO, the authors also measured S-nitrothiol and nitrite levels, well-established proxies for NO (He et al., 2004; Holzmeister et al., 2011). Levels of these metabolites were similarly elevated in gsnor plants under low light conditions alone. These results thus set the stage for determining the molecular underpinnings of NO-mediated response to light to changes at the chromatin level.

The authors next examined whether histone acetylation is modulated by GSNOR activity under changing light intensities. To explore this idea, the authors performed immunoblotting of acetylated histones (H3ac, H3K9ac, and H3K9/14ac) in 4-week-old wild-type and gsnor plants to identify large-scale changes in these histone modifications under dark, low light, and high light conditions. Wild-type plants displayed increased levels of H3ac, H3K9/14ac, and HS3K9ac under both low and high light conditions when compared with dark-treated plants. Interestingly, levels of these acetylated histones did not change significantly in gsnor plants when comparing light and dark treatments. Nearly unchanged global histone acetylation in the gsnor background suggested GSNOR has a regulatory function for chromatin remodeling when plants are subjected to increased light intensity.

The authors previously demonstrated that total histone deacetylase (HDA) activity could be inhibited in vitro by NO donors such as GSNO (Mengel et al., 2017). However, which HDAs in Arabidopsis are regulated by NO is unclear. To complicate matters, Arabidopsis has 18 HDAs that could potentially be regulated by NO. The authors therefore narrowed their search to focus on HDA6, a promising candidate because its closest human ortholog, HDAC2, is post-translationally modified in response to NO, which directly impacts chromatin remodeling (Colussi et al., 2008; Nott et al., 2008). To test their predictions, the authors treated wild-type or hda6 plant cells directly with GSNO. While the wild type showed increased levels of H3ac after GSNO treatment, hda6 mutant cells displayed unchanged levels of H3ac. These results suggest that HDA6 is a promising candidate for modification in response to NO production. Additional biochemical assays demonstrated HDA6 activity could be directly modified, and subsequently inhibited, by treatment with GSNO. The authors concluded HDA6 is a NO-sensitive HDA.

With these data in hand, the authors returned to their initial experimental set-up where increasing light intensity could increase NO emission. The authors observed that, like gsnor mutants, hda6 plants displayed nearly unchanged levels of acetylated histones in response to increasing light intensities. The authors next sought to quantitatively measure chromatin changes for the histone mark H3K9ac via chromatin immunoprecipitation sequencing (ChIP-seq) in wild type, gsnor mutants, and hda mutants under dark and low light conditions. For reference, H3K9ac is a histone mark associated with active promoters and actively transcribed genes. H3K9ac enriched sites, or “peaks,” were significantly more abundant in hda6 mutants than in wild-type and gsnor plants across both experimental conditions. The authors also observed that many hyperacetylated peaks unique to wild type were associated with stress response genes whereas hypoacetylated loci were near genes known to be involved in growth and development. In contrast, the gsnor and hda6 mutants displayed hyperacetylation of genes involved in growth and development whereas genes associated with stress response were hypoacetylated. These results imply that GSNOR and HDA6 function to deacetylate growth genes when plants encounter changes in light conditions.

To complement their ChIP-seq data set, the authors also performed large-scale expression analyses (RNA-seq) to find changes in gene expression related to GSNOR and HDA6 activity. Using the same experimental set-up for their ChIP-seq experiments, the authors observed that genes up-regulated in both mutants were enriched for functions related to growth and development as well as transport and localization. Down-regulated genes in both mutants were enriched for processes related to stress response, but also, somewhat puzzlingly, transport and localization. In the wild type, the authors found similar observations to their ChIP-seq results: growth and development genes were downregulated whereas genes related to stress response were upregulated. For the gsnor and hda6 mutants, genes that displayed both hyperacetylation and increased expression under light conditions contained genes related to brassinosteroid synthesis, cell wall formation, auxin biosynthesis, serine biosynthesis, and histone modification. Genes that were hypoacetylated and had decreased expression during light treatment in the mutants were involved in abiotic and biotic stress response.

Alexandra Ageeva-Keiferle and colleagues were able to link a reactive oxygen species transmitter, NO, directly to changes in chromatin remodeling as well as gene expression. The authors demonstrated that increasing light intensity induced global changes in histone acetylation, but that these changes were not observed in a gsnor mutant. The authors then identified HDA6 as a target for post-translational changes through the actions of GSNOR and NO. Finally, they showed HDA6 is required for deacetylation of growth responsive genes (to “turn them off”) in response to changes in light intensity. Furthermore, many of the chromatin targets identified by the authors appeared to play a role in phytohormone production, suggesting that perception of NO occurs prior to changes in phytohormone signaling. While HDA6 is clearly important for mediating response to NO, it remains to be seen whether any of the other 17 HDAs in Arabidopsis are also directly regulated by NO.

Conflict of interest statement. The author declares no conflicts of interest.

References