Redox regulation of plant HDAC activity in response to climate change – REPHARE

First, antibody-based methods (western blots) will allow detection of Cys sulfenylation (Cys-dimedone adduct detection), disulfide bonds (non-reducing gels), and S-nitrosylation (biotin switch). Second, mass spectrometry (MS) will allow confirming the modifications and identifying Cys residues involved. MS may also reveal Cys S-glutathionylation that might participate in redox regulation of HDACs. Third, using recombinant proteins produced in E coli, in vitro tests will allow examining impact of redox modifications on HDAC activity and identifying redox protein regulators such as thioredoxins (TRXs), glutaredoxins (GRXs) and their reducing systems, NADPH-TRX reductase and glutathione (GSH), respectively (redox proteins already obtained by partner 2). Fourth, tagged HDAC constructs (HDA19-HA, HDA9-HA, and HDA6-HA) will be introduced in redox regulatory gene mutant plants (already available in partner 2 laboratory) and the impact of the mutations on the redox state of tagged HDACs will be examined as mentioned above.

we have tested (using biotin switch) S-nitrosylation (SNO) or S-glutathionylation (SSG) of HDA6, HDA9 and HDA19. All three proteins displayed SNO. The SNO level of HDA19 increases in plants grown at high temperature (27°C) compared with 20°C and under other stresses (SA, 3-AT treatment). In addition, SNO of HDA19 increases when treated the plants with GSNO but disappears when treated with GSNO scavenger cPTIO. All of the three proteins show both cytoplasmic and nuclear localization. Stress enhances HDA19 nuclear localization and SNO of its nuclear fraction.
In addition, we have performed mass spectrometry analysis of HDAC proteins immunoprecipitated from complementation plants revealed at least 4 Cys residues in HDA19, 1 Cys in HDA9 and HDA6. Point mutation of one of the SNO Cys residues in HDA19 reduces SNO levels, mainly of the cytoplasmic fraction of the protein, but showed no effect on the nuclear fraction. Point mutations of SSG Cys residues have also been performed on HDA6. Plants expressing these constructs are under analysis to evaluate the impact of these mutations on HDA6 protein activity, at phenotypic and molecular levels.
We have introduced tagged-HDAC constructs (HDA19-HA, HDA9-HA, and HDA6-HA) into several redox regulatory gene mutant plants (ntra, cat2, gsnor, grxs17, etc). In cat2, gsnor mutants, HDA19 displayed increases of SNO, while in ntra or grxs17 backgrounds, HDA19 SNO was reduced (in ntra) or eliminated (in grxs17).
In the cytoplasm, HDA19 and HDA6 likely exist in a protein complex. MS analysis revealed that a redox related protein is associated with HDA19 in the complex.
Analysis of HDA6-GFP and HDA19-GFP localization under high temperature suggests that both proteins accumulate in cytosolic stress granules (SG) after stress, as shown by colocalization of both fusion proteins with the SG marker PAB2-RFP.

Most of the biological materials have been constructed. Three papers have been published and many preliminary data have been obtained. Importantly, we have identified direct interaction between redox regulators and HDAC, which will allow characterization of precise mechanisms of redox regulation of HDAC activity in response to high temperature.

1. X Cui, Y. Zheng, Y Lu, E. Issakidis-Bourguet, DX Zhou (2021) Metabolic control of histone demethylase activity in plant response to high ambient temperature. Plant Physiology 23;185(4):1813-1828
2. Y Shen, T Lei, X Cui, X Liu, S Zhou, Y Zheng, F Guérard, E Issakidis-Bourguet, DX Zhou (2019) Arabidopsis histone deacetylase HDA15 directly represses plant response to elevated ambient temperature. Plant J 100(5):991-1006. doi: 10.1111/tpj.14492
3. Martins L, Knuesting J, Bariat L, Dard A, Freibert SA, Marchand CH, Young D, Dung NHT, Debures A, Saez-Vasquez J, Lemaire SD, Lill R, Messens J, Scheibe R, Reichheld JP#, Riondet C (2020) Redox modification of the Fe-S glutaredoxin GRXS17 activates holdase activity and protects plants from heat stress. Plant Physiol. Oct 2020, 184 (2) 676-692;

High ambient temperatures due to climate change impact plant growth and survival. Recent data indicate that chromatin modification is an essential process of gene expression reprogramming during plant response to elevated growth temperature. Histone deacetylases (HDAC) that regulate histone acetylation levels were shown to play important roles in plant adaptation to environment. In addition, HDACs are also involved in deacetylation of non-histone proteins such as metabolic enzymes and transcription factors to control their activity. Our preliminary data indicate that Arabidopsis HDAC members play distinct roles in plant response to high ambient temperature. In addition, we found that high ambient temperature alters plant cell redox environment and that cellular redox environment regulates HDAC subcellular localization and deacetylase activity.
The objective of this proposal is to elucidate the molecular mechanisms of interplay between cellular redox and chromatin modification that regulate plant response to high ambient temperature. In particular, the project aims to elucidate how cellular redox environment regulates HDAC activity to control lysine acetylation of histone and non-histone proteins involved in either epigenetic regulation of gene expression or metabolism and/or signaling during plant response to high ambient temperature, and to identify and study implicated redox regulators.
We propose to first identify redox post-translational modifications of Arabidopsis HDACs under normal and high ambient temperature conditions by using biochemical and mass spectrometry approaches. Then, we will examine effects of redox modifications on HDAC enzymatic activity, subcellular localization, and function in plant response to high ambient temperature thanks to plants expressing tagged HDAC proteins in wild-type and mutated versions of redox-sensitive residues. Then, we plan to identify and validate redox regulators involved in modifications of the HDACs by both biochemical and genetic approaches and to study the role of redox regulators in plant response to high temperature. Next, we will study the effects of redox modifications on HDAC epigenetic function in terms of chromatin structure, genome-wide histone modifications, DNA methylation and gene expression by high throughput sequencing and cell biology approaches. Finally, we will investigate the effect of redox modifications on HDAC functions in regulating lysine acetylation of non-histone proteins in order to identify HDAC-regulated key metabolic enzymes and signaling proteins involved in plant response to high ambient temperature.
This project aiming to elucidate redox-epigenetics-metabolism networks in plants will deepen current understanding of how plants adapt or resist to a changing environment. More specifically, the project will decipher the molecular basis of HDAC regulation in response to stress and how thiol modifications modulate their functions. Noteworthy, this study will reveal the function of HDAC-dependent lysine acetylation in regulating activity of non-histone proteins which is largely unknown at present time. We believe that the results obtained from this project will lead to establish a general link and reveal the molecular mechanisms of interplay between redox signaling, epigenetic regulation and plant adaptation to environment. The REPHARE project assembles complementary expertise in the fields of epigenetic regulation and redox signaling from the two partners who have already built a solid basis which will lead the project to success.